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Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010
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Diagnosis of bovine brucellosis in Mosul city by indirect ELISA and conventional
serological tests
M. S. Rhaymah, K. A. AL-Saad,and O. KH. AL-Hankawe
Department of Internal and Preventive Medicine, College of Veterinary Medicine, University of Mosul, Mosul, Iraq
Abstract
The study was conducted on 126 cattle (94 females and 32 males) of different ages (1->5 years) randomly selected from
July 2007 to August 2008 in Mosul. Indirect ELISA test and other traditional tests (rose Bengal test, tube agglutination test and
2- mercapto-ethanol test) were used to determine the incidence of bovine brucellosis. The highest incidence of disease was
recorded by Indirect ELISA, 23.01%, whereas it was 18.25%, 11.90% and 4.76% by rose Bengal, tube agglutination and 2-
Mercapto-ethanol tests, respectively. The highest incidence was in females in all serological tests and the highest incidence
was in females at the age between 1-3 years whereas in males more than 3 years of age it was 23.07%. The results of tube
agglutination test revealed the titer 1/40 occurred mostly compared with other titers. Six chronic cases were determined by 2-mercapto-ethanol test. The degree of agreement of negative samples with rose Bengal test and indirect ELISA, tube
agglutination, and 2- mercapto-ethanol tests was 94.17%, 100% and 100%, respectively, and by indirect ELISA with rose
Bengal, tube agglutination and 2-mercapto-ethanol tests was 79.31%, 51.72% and 20.68%, respectively.
Available online at http://www.vetmedmosul.org/ijvs
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���� ���� ��������: ���*. 25. Kadohira M, Dermott MCJJ, Shouki MM, Kyule MN. variations in
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32. Kolar J. Diagnosis and control of brucellosis in small ruminants. Prev
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B1.5 )5�$�2(7 %74.6.
Effect of immunization with protoscolices antigens of hydatid cysts on growth of
Echinococcus granulosus in dogs
M. A. Aljawady and M. N. Al-Shahery
Department of Microbiology, College of Veterinary Medicine, University of Mousl, Mosul, Iraq
Abstract
The study was designed to evaluate the immune response of puppies, injected by different protein fractions extracted from
protoscolieces, obtained from ovine hydatid cyst. Indirect heamagglutination revealed a remarkable increase in the antibody
titers for the immunized groups (A1.5, A3, B1.5, and B3) before and after challenge when compared with the control.
Biological variations showed decline in numbers of adult Echinococcus granulosus in the immunized groups when compared
with the control. Other variations proved dropping in numbers of the worms within the same immunized groups. Subsequent
reductions of cestodes were reported which were 83.8%, 81.3%, 78.2% and 74.6% for the groups A3, A1.5, B3, and B1.5,
respectively.
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I���(#� ����D�!�� �� "��� �# �$%���� ���� � &����� ���[.
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Clinical study of foot and mouth disease in feedlot calves in Mosul region
M. A. Abd- Alhameed and M. SH. Rhaymah
Department of Internal and Preventive Medicine, College of Veterinary Medicine, University of Mosul, Mosul, Iraq
Abstract
The affected calves showed clinical signs including presence of vesicles at the bucal cavity and hoof (90.7%), fever (36%), salivation (32.9%), depression (20.5%), Anorexia (19.6%), loss of body weight (7.94%), lamness (15.9%), lesions at the
muzzle (15.3%), presence of the lesions at the feet (6.0%), prostration (4.7%), diarrhea (3.3%), presence of the lesion at the
mouth cavity (3.0%), paralysis of hind quarters (2.2%) and sudden death (1.6%). There was significant increase in the rectal
temperature, respiration rates, and heart rates in the infected animals compared with control group. Recovered animals
appeared some diseases including theileriosis (40.4%), tympany (22.8%), pneumonia (19.9%), foot abscess (14.7%), and
babesiosis (2.2 %). The mortality rate varied between 3.4% to 27.9% in different herds and the mean was 3.3 % in all herds.
There was significant relationship between age of animals and the incidence of the disease.
Available online at http://www.vetmedmosul.org/ijvs * #�)� R:� �� �'(����� :���� ,� #'(� R: ��
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1. Donaldson AI. Foot and mouth disease: the principl features. Irish Vet J. 1987;41:325–327.
2. Kitching RP. Clinical variation in foot and mouth disease: Cattles. Rev
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Medicine: A textbook of the disease of cattle, sheep, goat, pigs, and
horses. 9th ed. London: W.B. Saunders company; 2000.1059–1066p.
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Gazella: Virus Isolation, serology and characterization.(master’s
thesis) Baghadad University; 1988.
?. ������(��� ��:� ��'(��� 8 ,(�()�2-2.( 3��;��� <$�'� #18A���:�� (�(:�� �;� 8&.�� 9�:�� L�� ��$��� �����»o«. ��(�
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������H���� 8� ��� %����� �#$���� �#$����. 9. Gailiunas P, Cottral GE. Presence and persistence of foot and mouth
disease virus in bovine skin. J Bacteriol. 1966;91:2333–238.
10. Kitching RP, Alexanderson S. Clinical variation in foot and mouth
disease: Pigs Rev Sci Tech Int Epiz. 2002;21:513–518.
11. Kitching RP, Hughes GJ. Clinical variation in foot and mouth disease:
Sheep and goats. Rev Sci Tech Int Epiz. 2002;21:505–512.
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Biotyping of Clostridium Perfringens strains isolated from enterotoxemia cases in
sheep using ELISA technique
M. A. Hamad1, N. Habra
2 and A. Kalb Allouz
3
1 Department of Microbiology, College of Veterinary Medicine, University of Mosul, Mosul, Iraq,
2 Department of Microbiology, 3 Department of Animal Diseases, College of Veterinary Medicine, AL-Baath University, Syria
Abstract
The study was designed to determine the types of Clostridium perfringens and their toxins in sheep with suspected
enterotoxemia in Hama province, Syria. For this purpose, 89 bacterial isolates were isolated from 132 samples collected from
dead and some slaughtered sheep with suspected enterotoxemia and diagnosed with classical diagnostic tests and by using enzyme-linked immunosorbent assay (ELISA) technique to determine the types and toxins of C. perfringens. These isolates
appeared as G+ bulged rods with curved ends and were as positive to lecithinase, gelatin hydrolysis and sugar fermented, as
where negative to catalase, oxidase, and indole. Based on the ELISA results all isolates were C. perfringens types A 84 isolate
(94.38 %), D 3 (3.37 %), and C 2(2.25 %). Clostridium perfringens type A was the dominant type in cases of enterotoxemia in
sheep in Hama province detected by ELISA test. The enterotoxaemia causes considerable economic loss to the sheep industry
particularly in Hama province and generally in Syria. Therefore, it is recommended that a proper vaccination schedule against
enterotoxemia should be implemented for sheep flocks in Hama province. These vaccines should provide adequate protective
immunity against all C. perfringens types specially types A and D.
Available online at http://www.vetmedmosul.org/ijvs
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���.��
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٢٢
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��� kappa. ,� E(�()� � ��= �0��*� ���(<� A�2 ���(2*� ,"�� K�� �)����� L>M ,2 34"�� �! �4���� �9��� ���7� ���('�)����$�"���� ���� ���(<� ,� *�� �%�<4( �;)� ���(<�" :����� ,'�;�� �! .
Detection of anti brucella antibodies in lambs and goat kids using rose bengal test
and indirect ELISA in Gugjeli - Ninavah province, Iraq
S. A. Esmaeel, B. A. Mohmmed, S. D. Hassan and Kh. J. Hassain
Department of Internal and Preventive Medicine, Collage of Veterinary Medicine, University of Mosul, Mosul, Iraq
Abstract
The object of the study was to detection of anti-brucella antibodies in lambs and goat kids by using Rose Bengal and
indirect ELISA tests. Sera samples were collected from four flocks, including 135 samples (95 from lambs and 40 from goat
kids). The sera examined by rose Bengal test showed negative result in all of flocks, in percentage was zero %. While the
percentage of anti-brucella antibodies using indirect ELISA test in female and male lambs was (29.34 %, 10 %) respectively,
with total percentage 25.3% and in female and male kids was (55 %, 0.0 %) respectively, with total percentage 27. 5%. The
results showed incompatibility between rose Bengal test and indirect ELISA test with kappa value. The study concluded that
indirect ELISA test can be considerable as a screening and diagnostic test instead of rose Bengal test for detection of anti-
brucella antibodies in lambs and kids.
Available online at http://www.vetmedmosul.org/ijvs
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Reproductive efficiency of rats whose mothers treated with lead acetate during
lactation: role of vitamin E
W. H. Yousif and S. T. Abdullah
Department of Physiology Biochemistry and Pharmacology, College of Veterinary Medicine, University of Mosul, Mosul, Iraq
Abstract
Reproductive efficiency of young male and female rats yielded from mothers treated with lead acetate (10 mg/kg B.W) and
vitamin E as an antioxidant (500 mg/kg) during lactation period was studied. 27 pregnant albino rats aged 4-5 months divided
into 3 groups, the first group given 0.2 ml distilled water through oral intubations (control). The second group given lead
acetate (10 mg/kg B.W), while the 3rd group given lead acetate (10 mg/kg B.W) with vitamin E (500 mg/kg B.Wt). Lead
acetate treatment did not cause significant difference (P>0.05) in young male rats body, testes, epididymal (head, tail), prostate
gland, seminal vesicle weights, number of epidiymal sperms count, percentage of live/dead sperms and sperms abnormalities
accompanied with a significant increase (P<0.05) in seminiferous tubules diameter. On the other hand, lead acetate treatment caused a significant decrease (P<0.05) in the duration of vaginal opening with no significant changes (P>0.05) in ovary and
uterus weights. Lead acetate with vit. E cause significant decrease (P<0.05) in body, testes, epididymal (head, body) weights,
number of epididymal sperm count, percentage of live/dead and sperm abnormalities. Concerning young female rats treated
with lead acetate and vit E showed a significant decrease (P<0.05) in body, uterus weights with a significant increase (P<0.05)
in the duration of vaginal opening. Histopathological study revealed changes in the testes such as desquemination in some
epithelial lining of seminiferous tubules and sertoli cells. Undevelopment of localized area of some seminiferous tubules with
blood vessels congestion in both testes and ovaries with hyperemia in ovarian stroma, increase in follicular cells, decrease in
corpus luteum formation, degeneration of ovarian follicles and increase in ovarian adipose tissue. This study concluded that
lead acetate can be transmitted through mother milk to their pups. The dose of lead acetate (10 mg/kg B.W) was enough to
make bad effects on both male and female reproductive systems. Vitamin E (as an antioxidant) found to have no improving
effect in treatment of lead acetate disturbances on reproductive systems of both sexes.
Available online at http://www.vetmedmosul.org/ijvs
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Extensor and flexor digit synovial sheath, sac and synovial capsule in the distal part
of the limbs in buffalos and camels and its relation of surgical interference
S. AL-sadi1, G. Alosh
2 and A. AL-Omari
3
1 Department of Anatomy, College of Veterinary Medicine, University of Mosul, Mosul, Iraq, 2 Department of Anatomy, 3 Department of Surgery, College of Veterinary Medicine, University of Al-baath, Hama, Syria
Abstract
Sixty one samples of the distal parts of limbs were obtained from different ages of buffalo and camels of both sex to study
the synovial structures to determine the suitable sites for injection of surgical interference. The result showed that extensor
digit synovial sheath was extend between middle or distal part of metacarpal (metatarsal) to the extensor processes and this
formed with synovial capsule dorsal pouches which serve in surgical interference. The flexor digit synovial sheath extended to palmar (planter) between distal extremity of metacarpal (metatarsal) to the middle of second phalanx in buffalo while in camel
it extended to the proximal extremity of second phalanx, that sheath was formed with suspensory ligament and sessamoid bone
palmar or planter pouches which were serve the surgical interference. Fourth synovial bursa observed situated dorsally
������ ��� �� ������ ������ ������� ����� �� �����) �� ���(
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between the extensor digit laterals tendon and capsule of fetlock joint, forms site of injection during surgical interference,
while the other two synovial bursa were located to palmer (planter) between deep flexor tendon and distal sessamoid bone in
buffalo while in camel these bursa were located between deep flexor tendon and cartilage of the second phalanx, these bursa
were served for surgical interference. The synovial capsule which serve the surgical interference through digit cushion these
were shown extended from the claw capsule. The result show that surgical interference was form six pouches in buffalo and
eight pouches in camel, which formed by synovial structures and the tissue associated with them.
Available online at http://www.vetmedmosul.org/ijvs
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Diagnosis of foot and mouth disease by indirect competitive ELISA test in feedlot
calves - in Mosul province
M. A. Abd-Alhameed and M. Sh. Rhaymah
Department of Internal and Preventive Medicine, College of Veterinary Medicine, University of Mosul, Mosul, Iraq
Abstract
Indirect competitive ELISA test used, for detection of non-structural protein (3ABC) in 460 serum samples of infected
animals revealed that 421 samples were positive (91.5%) and 39 samples were negative (8.5%), with 91.5% sensitivity and
28.4% specifity. Blood picture revealed significant (P<0.05) increase in total leukocytic count, mean corpuscular volume, mean corpuscular hemoglobin and the percentage of granulocytes, and significant decrease in the total erythrocytic count and
packed cell volume.
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Evaluation the suitable level of standard concentrated ration for fattening growing
Sharabi calves
N. M. Abdullah1, A. K. Nasser
2 and N. Y. Abou
2
1 Department of Veterinary Public Health, College of Veterinary Medicine, University of Mosul,
2 State of Board of Agriculture Research, Department of Agriculture Research, Nineveh, Mosul, Iraq
Abstract
Three groups of growing Sharabi calves were used in this study to evaluate the best suitable level of concentrate ration.
Each group consisted of four calves between 8-12 months of age, with an average live body weight around 145 kg. They were
fed for 90 days on concentrated standard ration according to NRC, 1984, at three different levels of 2, 3, 4 % of live body
weight, respectively, consisted of 80:20 concentrate to roughage contained 14% crude protein, 72% TDN and 2.4 Mkcal/kg.
ME. The total gain and amount of consumed ration were measured. At the end of the trial, samples of rumen fluid were
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collected from all calves in order to estimate the activity of bacteria and micro organism. Results showed that the average
amount of the daily ration consumed by the three groups of calves were 3.68, 6.91 and 8.12 kg/calves, respectively. There
were significant differences (P<0.05) in average daily gain among the three groups of calves (311, 869 and 1000 gm, for the
three respective treatments). Also the feed conversion ratio was improved for the second and third groups compared with the
first group and they were 7.96, 8.12 and 11.8 kg. ration/kg live body weight gain, respectively. Ration levels showed no effect
on rumen fluid pH but increased the time of methylene blue reduction (more than 15 mints) and sedimentation activity test
(more than 20 minuets) than their normal standard levels. The cost to produce one kg live body weight gain was about the
same for the second and third groups and it was 50% less than first group and they were 3184, 3248 and 4746 I.D.,
respectively. It was concluded that the fattening regime for growing Sharabi calves on concentrated ration given at 4% of live
body weight for three months had more economic profit.
Available online at http://www.vetmedmosul.org/ijvs
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controlled feed intake on performance and carcass characteristics of
feedlot steers and heifers. J Anim Sci. 1993;68:233. 14. Plegge SD. Restricting intake of feedlot cattle, Oklohoma State Univ.
Stillwater. 1987. 15. Zinn RA, Owens FN. Influence of feed inake level on site of digestion
in steers fed a high concentrate diet. J Anim Sci. 1983;56:471-475.
16. Brown MS, Ponce CH, Pulikanti R. Adaptation of beef cattle on high–
concentrate diets: Performance and ruminal metabolism. J Anim Sci.
2006;84:E25- E33.
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$ 7�� = C�� .
The effect of substituting Nigella Sativa Meal as a source of protein in the rations of
local rabbits on their productive performance and carcass traits
N. M. Abdullah and A. A. AL-Kuhla
Department of Veterinary Public Health, College of Veterinary Medicine, University of Mosul, mosul, Iraq
Abstract
Fifteen local male rabbits (6-8 weeks old) were allocated into three nutritional groups. The first group fed standard ration, 5
and 10% of the Nigella Sativa Meal (NSM) were added to the concentrated rations of the 2nd and 3rd groups replacing 36 and
72% of the soybean meal (SBM) protein respectively. The feeding period lasted for eight weeks. Feed consumption and body
weight gain were recorded weekly. At the end of feeding period, all rabbits were slaughtered and carcass traits were studied.
No significant differences were found in total body weight gain and feed conversion rate (475, 502, 478 gm) and (4.8, 4.8, 4.9
kg ration/1 kg wt. gain). Feed cost per 1 kg body gain declined 16% in the 3rd group, which cost 2294 ID, compared with the
1st group (2717) and the 2nd group (2561 ID). No significant differences in all carcass traits were found. Substituting 72% of SBM protein by NSM protein in rabbit ration showed no negative effects on all productive parameters and carcass traits.
Available online at http://www.vetmedmosul.org/ijvs
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2. National Research Council. Nutrient requirements of poultry. 9th
revised , National academy press , Washington. D.C. 1994.
�. X��T $�� ��$9 �, $�& .�5 ��$� �# 4 .��7J(��� A�7J. "(#�� ��� 5 ��� �4-���� ������ �� �. ��+�.
4. Khalifa MM. Nigella seeds oil meal as a protein supplement in broiler
diets.(master's thesis), Face. Agric. Alexandria Univ. 1995.
5. EL-Nattat WS, EL-Kady RI. Effect of different medical plant seeds
residues on the nutritional and reproductive performance of adult male
rabbits. Int J Agric Biol. 2007;9:(3).
6. Zeweil HZ. Evaluation of substituting Nigella seed oil meal for soybean meal on the performance of growing and laying Japanese
quail. Egypt. Poultry Sic. 1996;16:451-477.
7. Nasr AS, Attia AI, Rashwan AA, Abdine AMM. Growth performance
of New Zealand White rabbits as affected by partial replacement of
diet with Nigella sativa or soyaben meals. Egyptian J. Rabbits
Sciences. 1996;6:129-141. 8. Zeweil HS, Ahmed MH, EL-Adawy MM, Zaki B. Evaluation of
substituting Nigella seed meal as a source of protein for soybean meal
in diets of New Zealand white rabbits, 9th World Rabbit congress, June
10-13, 2008, Verona–Italy. �. �P # .�5 ����6�� ..( �� i�� 5 � @�� ..(� �$*�� 5 ��) .
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��D+. 10. AOAC. Official methods of analysis. 16
th ed., Association of Official
Analytical Chemists, Washington DC, 1996.
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Vol. 24, No. 1, 2010
Editor-in-Chief Prof. Dr. Fouad K. Mohammad (BVMS, MS, PhD) Editoria Secretary Dr. Nabeel A. Al-Hussary (BVMS, MSc, PhD) Executive Editorial Board Prof. Dr. Mohammed N. Al-Shahery (BVMS, DSc) Prof. Dr. Samih H. Arslan (BVMS, PhD) Prof. Dr. Fadwa K. Tawfeek (BVMS, MSc, PhD) Dr. Talal H. Hussein (BSc, MSc, PhD) Dr. Tariq S. Qubih (BVMS, MSc, PhD)
Iraqi Journal of Veterinary Sciences is printed and organized by:
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ISSN 1607-3894 (Print), ISSN 2071-1255 (Online)
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Master's thesis: Roguskie JM. The role of Pseudomonas aeruginosa 1244 pilin glycan in virulence [master's thesis]. Pittsburgh (PA): Duquesne University; 2005. 111 p.
Journal article on the Internet: Polgreen PM, Diekema DJ, Vandeberg J, et al. Risk factors for groin wound infection after femoral artery catheterization: a case-control study. Infect Control Hosp Epidemiol [Internet]. 2006 Jan [cited 2007 Jan 5];27(1):34-7. Available from: http://www.journals.uchicago.edu/ICHE/journal/issues/v27n1/2004069/2004069.web.pdf
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Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010
Contents
Research articles Page
Detection of Brucella antigen in the aborted ovine fetal stomach contents using a modified
ELISA test
M. I. Al-Farwachi, B. A. Al- Badrani and Th. M. Al-Nima
1
Effect of bone marrow and low power lasers on fracture healing with destruction of both
periosteum and endosteum in rabbits
M. G. Thanoon and S. M. Ibrahim
5
Concurrent aflatoxicosis and caecal coccidiosis in broilers
A. M. Shareef
11
Molds and mycotoxins in poultry feeds from farms of potential mycotoxicosis
A. M. Shareef
17
Prevalence of intestinal ciliate Buxtonella sulcata in cattle in Mosul
T. M. Al-Saffar, E. G. Suliman, H. S. Al-Bakri
27
Clinical, haematological and biochemical studies of babesiosis in native goats in Mosul
E. G. Sulaiman, S. H. Arslan, Q. T. Al-Obaidi, E. Daham
31
Effect of industrial product IMBO® on immunosuppressed broilers vaccinated with Newcastle
disease vaccine
O. G. Mohammadamin and T. S. Qubih
37
Serodiagnosis of Johne's disease by indirect ELISA in ovine
I. M. Ahmed
41
Effect of treating lactating rats with lead acetate and its interaction with vitamin E or C on
neurobehavior, development and some biochemical parameters in their pups
A. A. Hassan and H. M. Jassim
45
Use of saturated sodium chloride solution as a tissue fixative
A. Al-Saraj
53
Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (1-4)
1
Detection of Brucella antigen in the aborted ovine fetal stomach contents
using a modified ELISA test
M. I. Al-Farwachi*, B. A. Al- Badrani* and Th. M. Al-Nima**
*Department of Internal and Preventive Medicine, College of Veterinary Medicine, University of Mosul,
** Veterinary Hospital in Mosul, Mosul, Iraq
(Received November 17, 2008; Accepted July 13, 2009)
Abstract
This study was conducted on two flocks of sheep suffering from abortion in Mosul city, Iraq. The clinical findings in ewes
were abortion during the 3-4 months of gestation period in the both flocks. The total percentage of abortion was 11.7 %,
whereas stillbirth percentage was 4 %. Brucella spp. was isolated from four (33.3 %) of the 12 samples (stomach contents of
the aborted fetuses). All culture – positive samples had also positive with direct smears. By a modified enzyme-linked
immunosorbent assay (ELISA), Brucella antigens were detected in the fetal stomach contents of 5 samples. The sensitivity and
specificity of the modified ELISA were 100 % and 87.5 % respectively. The test had a good negative predictive value but only
a moderate positive predictive value. Therefore, the test would be useful for confirming the existence of suspect disease.
Comparison of modified ELISA with bacterial isolation demonstrated a close agreement (Kappa value = 0.92). Of the 12
serum samples from aborted ewes, eight samples were positive with Rose Bengle test (66.7 %), more than 10 samples (83.3 %)
were detected by indirect ELISA test. Keywords: Ovine brucellosis, ELISA, Fetus, Stomach contents.
Available online at http://www.vetmedmosul.org/ijvs
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Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (1-4)
2
Introduction
Ovine brucellosis is usually caused by Brucella
melitensis, less frequently by B. abortus, and rarely by B.
ovis (1,2). It is usually manifested by abortion, with
excretion of the organisms in uterine discharges and in milk
(2,3). Diagnosis depends on the isolation of Brucella spp.
from aborted fetuses, milk or from tissues removed at
postmortem examination (1). Alternatively, specific cell-
mediated or serological responses to Brucella antigens can
be demonstrated (3,4).
The demonstration by modified acid-fast or
immunospecific staining of organisms of Brucella
morphology in abortion material or vaginal discharges
provides presumptive evidence of brucellosis, especially if
supported by serological tests (5). Whenever possible, the
species and biovar should be isolated, and identified by
phage lysis or oxidative metabolic tests, or both, and by
cultural, biochemical and serological criteria (5-7). The recently developed polymerase chain reaction (PCR) and
DNA-probe methods provide additional means of detection
(8,9).
No serological test is appropriate for all epidemiological
situations (6). The buffered Brucella antigen tests (Rose
Bengal plate agglutination test and buffered plate
agglutination test) are suitable for screening herds and
individual animals (10). The reactivity of positive samples
should be confirmed by the complement fixation test or by
enzyme-linked immunosorbent assay (ELISA), both of
which can also be used for both screening and confirmation (11,12). The serum agglutination test is inferior to other
tests in specificity and sensitivity, and is not recommended
if other procedures are available (12). Another
immunological test was developed for detection of brucella
antigen in the bovine fetal stomach contents as agar gel
precipitation and counter – immunoelectrophoresis (13).
Counter – immunoelectrophoresis also was used for
detection of brucella antigen in the stomach contents of
aborted buffalo fetuses (14), while coagglutination test was
used for detection of Brucella antigens in aborted ovine
fetal stomach contents (15). The aim of the study to describe a simple and rapid method (Modified ELISA) for
detection of Brucella antigen in aborted ovine fetal stomach
contents.
Materials and methods
The study was conducted in April 2008 on two flocks of
sheep in the Mosul city (Badoosh and Zumar) covering 120
and 52 sheep for each respectively. Complete examination
was performed to all aborted animals and 12 were subjected
to thorough postmortem examination. The two flocks were not vaccinated against brucellosis.
The following specimens were collected for diagnosis
of the disease:
1. Bacterial isolation trials from stomach contents of the
aborted fetuses were made on the modified Brucella agar
medium supplemented with antibiotic (Himedia, India)
and incubated at 37 oC in an atmosphere of 10 % CO2
and in air for 6 days and then Brucella colonies were identified and typed according to conventional methods
(4). Suspected colonies were further identified and
subcultured on Brucella spp. agar slants. We identified
Brucella spp. isolates according to morphologic
characteristics, microscopic appearance. Brucella spp.
isolates were typed according to their CO2 requirement,
H2S production, and growth in the presence of basic
fuchsin and thionin at final concentrations of 20 µg/ml,
as described (4, 5).
2. Direct smear were prepared from aborted fetal stomach
contents, then stained with Koster stain and examined
under light microscope (16). 3. Detection of the brucella antigen from aborted fetal
stomach contents by modified ELISA: Apart of the
samples was heated for 1 hr at 70 oC and centrifuged.
The precipitates were washed twice with phosphate
buffered saline (PBS) and centrifuged. The precipitates
were then used for ELISA. A hyperimmune antibrucella
serum for using in the modified ELISA was prepared in
sheep by four biweekly injections subcutaneously with
live Brucella vaccine (Rev.1 strain of Brucella
melitensis) produced in (CZ Veterinaria, Spain) with the
standard dose of 1×109 colony forming units (CFU) (17). Modified ELISA was done according to the general
principals of immunoblotting (18). Brifly, 20µl of each
of the samples and controls (BPS) were applied to
nitrocellulose membrane (Amersham). The membrane
was blocked in 5 % skim milk in PBS for 45 minutes.
Membrane was washed three times, 5 min each with
PBS- T (PBS containing 0.05 % Tween 20), was placed
in 1: 100 dilution of anti brucella serum in PBS- T and
incubated at room temperature for 1 hr. The membranes
were then incubated in conjugate goat antimouse IgG
horse – radish peroxidase (Svanovir ®, Sweden) diluted 1: 1000 in PBS – T for 1 hr. The same washing
procedure was repeated after incubation with the anti –
sheep peroxidase conjugate (Svanovir ®, Sweden),
followed by reaction with a chromogen – substrate
solution consisting of 30 µl of 30 % H2O2 in 50 ml of
PBS mixed with 30 mg of 4 – Chloro – 1 – naphtol in 10
ml of cold methanol. The results were determined by
observation of violet – stained spots on the membrane.
4. Serum samples: 12 blood samples from all aborted ewes
(12) were collected by venipuncture at 15 days after
abortion. Rose Bengal (RBT), and indirect ELISA were
used to detect antibodies against brucellosis. The Rose Bengal test was performed as described by (19). A
Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (1-4)
3
commercially available indirect ELISA (Brucella – Ab I-
ELISA kit Svanovir ®, Sweden) was used to detect
specific antibodies for Brucella spp. according to
manufacturer´s instruction.
Statistical analysis: The sensitivity, specificity, positive
and negative predictive values were calculated. The Kappa
value was used to evaluate the correlation between modified ELISA and bacterial isolation (20,21)
Results
Clinical examination showed abortion occurred during
the 3-4 months of gestation period in the both flocks. Table
1 illustrate the number of aborted ewes and average of their
gestation period in months. The total percentage of abortion
was 11.7 %, while stillbirth percentage was 4 %.Brucella
spp. was isolated from 4 (33.3 %) of the 12 samples (stomach contents of the aborted fetuses). All culture –
positive samples had also positive results with direct smears
(stained with Koster stain). By modified ELISA, Brucella
antigens were detected in the fetal stomach contents of 5
samples (42.0 %). Other culture – negative samples were
also negative with modified ELISA test (Table 2).
Table 1: Numbers of aborted animals and average of their
gestation period in months.
Gestation
period (Months)
Stillbirth
No. (%)
Abortion
No (%)
Total
lambing Regions
3 - 4 3 (3.6 %) 8 (9.6 %) 120 Badoosh
3 - 3.5 4 (2.0 %) 12 (6.3 %) 52 Zumar
3 - 4 7 (4.0 %) 20 (11.7%) 172 Total
Table 2: Results of the tests used for detection of brucella
antigens in stomach contents of the aborted fetuses.
Modified
ELISA
Direct
smears
Bacterial
culture Samples
- - - 1
+ + + 2
- - - 3
+ + + 4
- - - 5
+ + + 6
- - - 7 - - - 8
- - - 9
+ + + 10
- - - 11
+ - - 12
5 (42.0 %) 4 (33.0 %) 4 (33.0 %) Total
(-) negative results, (+) positive results.
From the results, it appeared that the modified ELISA
has a high sensitivity (100 %), but only moderate
specificity (87.5 %). The test also has a good negative
predictive value(100 %) and moderate positive predictive
value (80 %). Therefore, the test is would be useful for
confirming the existence of suspected disease. Comparison
of modified ELISA with bacterial isolation demonstrated a close agreement (Kappa value = 0.92). Of the 12 serum
samples from aborted ewes, eight samples were positive
with Rose Bengle test (66.7 %), more than 10 samples
(83.3%) were detected by indirect ELISA test.
Discussion Brucellosis is widely regarded as an extremely insidious
disease, demanding the most exhaustive care in diagnosis
and many cases are more difficult to diagnosis than others,
and a few are extremely difficult to pick up, and could
easily be missed altogether (1,2). In this study, Brucella
spp. was isolated from 4 (33.3 %) of the 12 samples (stomach contents of the aborted fetuses). All culture –
positive samples were also positive with direct smears
(stained with Koster stain). By modified ELISA, Brucella
antigens were detected in the fetal stomach contents of 5
samples (42.0 %). Other culture – negative samples were
also negative with modified ELISA test. According to the
results of this study modified ELISA is able to detect
Brucella antigens in fetal stomach content because the
modified ELISA has a high sensitivity (100 % = false
negative rate of 0%), but only moderate specificity (87.5 %
= false positive rate of 12.5%). The test also has a good negative predictive value (proportion of modified ELISA
negative animals which do not have disease = 100 %) and
moderate positive predictive value (proportion of modified
ELISA positive animals which have the disease = 80 %)
(20). Therefore, the test would be useful for confirming the
existence of suspected disease because the test has a few
false positive rate and moderate positive predictive value
(21). Comparsion of modified ELISA with bacterial isolation
demonstrated a close agreement (Kappa value = 0.92).The
Kappa test can be used to measure the level of agreement beyond that which may be obtained by chance. The Kappa
lies within a range between -1 and +1 (20). (20), (21) were
noted that the Kappa value gives no which of the tests is
better and that a good agreement may indicate that both test
are equally good or equally bad.
Acknowledgment
This study was supported by the College of Veterinary
Medicine, University of Mosul, Mosul, Iraq.
Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (1-4)
4
References
1. Radostits OM, Gay CC, Hinchcliff KW, Constable PD. Veterinary
Medicine: A textbook of the diseases of cattle, horses, sheep, pigs and
goats. 10th ed. London: Saunders Elsevier; 2007. 966-994p.
2. Cutler SJ, Whatmore AM, Commander NJ. Brucellosis- new aspects
of an old disease. J Applied Microbiol. 2005;98:1270-1281.
3. Garin-Bastuji B, Blasco JM, Marin C. The diagnosis of brucellosis in
sheep and goats, old and new tools. Small Rum Res. 2006;62:63-70.
4. Forbes BA, Sahm DF, Weissfeld AS. Diagnostic Microbiology. 11th
ed. USA: Elsevier; 2002. 196-201p.
5. Songer JG, Post KW. Veterinary microbiology. Bacterial and fungal
agents of Animal Disease. Missouri: Saunders Elsevier; 2005. 200-
206p.
6. Nielsen K. Diagnosis of Brucellosis by Serology. Vet Microbiol. 2002;
90:447-459.
7. Aggad H. Serological studies of animal brucellosis in Algeria. Assiut
Vet Med J. 2003;49(98):121-130.
8. Fekete A, Bantle A, Halling SM. Detection of Brucella by polymerase
chain reaction in bovine fetal and maternal tissues. J Vet Diag Invest.
1992;11:79-83.
9. Gallien P, Dorn C, Alban G, Staak C, Protz D. Brucella species in
organs of naturally infected cattle by polymerase chain reaction. Vet
Rec. 1998;142:512-514.
10. Baum M, Zamir O, Bergman-Rios R, Katz E, Beider Z, Cohen A,
Banai, M. Comparative evaluation of microagglutination test and
serum agglutination test as supplementary diagnostic methods for
brucellosis. J Clinic Microbiol. 1995;33(8):2166-2170.
11. Blasco JM, Garin-Bastuji B, Marin CM, Gerbier G, Fanlo J, Jimenez
de Bagues MP, Cau C. Efficacy of different rosebengal and
complement fixation antigens for the diagnosis of Brucella melitensis
in sheep and goats. Vet Rec. 1994;134:415-420
12. Minas A, Stournara A, Christodoulopoulos G, Katsoulos PD.
Validation of a competitive ELISA for diagnosis of Brucella
melitensis infection in sheep and goats. The Vet J. 2007;111:102-105.
13. Chand P, Gupta RK, Khanna RN, Sadana IR. Detection of Brucella
antigen in bovine fetal stomach contents by agar gel precipitation and
counter immunoelectrophoresis. Res Vet Sci. 1987;43(1):132-136.
14. Chand P, Khanna RN, Sadana JR. Counter – immunoelectrophoresis
for the detection of Brucella antigen and antibodies in the diagnosis of
brucellosis in buffaloes. J Appl Bacteriol. 1988;64(5):445-449.
15. Erganis O, Kaya O, Hadimli H, Guler L. Rapid diagnosis of ovine
Brucella,Campylobacter and Salmonella infections from fetal stomach
contents by coagglutination test. Small Rum Res. 2002;45:123-127.
16. Coles EH. Veterinary Clinical Pathology. 4th ed. Philadelphia: W.B.
Saunders Company; 1986. 43-79p.
17. Little SF, Ivins BE, Fellows PF, Friedlander AM. Passive protection
by polyclonal antibodies against Bacillus anthracis infection in guinea
pigs. Infect Immunol.1997;65:5171–5175.
18. Golds RA, Kinde TJ, Osborne BA, Kaby J. Enzyme linked
immunosorbent assay: In immunology 5th ed. Philadelphia: W.B.
Saunders Company; 2003. 43-79p.
19. Alton GG, Jones LM Angus RD, Verger JM. Techniques for the
brucellosis laboratory. Paris: INRA;1988.
20. Thrusfield M. Veterinary Epidemiology 2nd
ed. Oxford: Blackwell
Science Ltd; 1995. 95- 112p.
21. Pfeffer D. Veterinary Epidemiology. An Introduction. NewZealand:
Institute of Veterinary, Animal and Biomedical Sciences.Massey
University; 2000. 32-34p.
Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (5-9)
5
Effect of bone marrow and low power lasers on fracture healing with
destruction of both periosteum and endosteum in rabbits
M. G. Thanoon and S. M. Ibrahim
Department of Veterinary Surgery and Theriogenology, College of Veterinary Medicine, University of Mosul, Mosul, Iraq
(Received December 18, 2008; Accepted July 13, 2009)
Abstract
Ten mature rabbits of local breed were used in this study; weighing between 1.5 to 1.75 kg and aged about 1–2 years.
These animals were divided into two equal groups; in group A destruction of both periosteum and endosteum was done one
centimeter from each side of mid-shaft femoral bone fracture, then sufficient amount of autogenously bone marrow was
injected directly at the fracture site after immobilization by intramedullary pin. In group B a similar procedure was achieved as
in group A, but in additional to that He-Ne infrared laser therapy was used for several sessions. The result of radiological
findings indicated that, the fracture healing occurred within group B at fifteen weeks, whereas in group A the healing occurred
at eighteen weeks after operation. The implantation of autologous bone marrow enhanced the fracture healing, whereas using
of combinations of autologous bone marrow and He-Ne infrared laser therapy hastened the healing.
Keywords: Fracture, Bone marrow, Laser, Rabbit. Available online at http://www.vetmedmosul.org/ijvs
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Introduction
Studies suggested that, the periosteum was more
essential than endosteum in callus formation. If there is
destruction of both periosteum and endosteum the results
will be absence of bone formation around the destructed
area (1,2). Bone marrow stem cells residing in the bone
marrow are the progenitors for osteoblasts (3-5).
Autologous bone marrow is a safe, simple and reliable
method for treating delayed and nonunion (6-8). Low
energy laser irradiation has positive effects on bone fracture
healing. The mechanisms by which low-energy laser
irradiation affect on bone healing still not clear (9). He-Ne
low-level energy treatment accelerated the deposition of
Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (5-9)
6
bone matrix and increases vascularization after seven days
of irradiation (10). Direct irradiation of the whole injury
with He-Ne laser on days five, six post-injury altered the
osteoblast and osteoclast cell population (11,12). Studies on
animals were performed on the effect of low-level laser of
fracture healing indicated that, the laser enhanced healing
(13-15). The aim of this study was to investigate the effect
of bone marrow and low-level laser energy on healing of
femoral fracture after destruction of both periosteum and
endosteum in rabbits.
Materials and methods
Ten mature rabbits of both sex of local breed aged 1-2
years and weighing between 1.5-1.75 kg. All rabbits
tolerated on the same manner of condition and housing
along the periods of study. The experimental design divided
into two equal series; Group A: five animals underwent
transverse fracture at the mid-shaft of femur bone then
about one centimeter of both periosteum and endosteum has
been destroyed and treated with sufficient amount of bone
marrow, Group B: five animals underwent the same
conditions of group A in additional to that irradiated with
He-Ne infrared laser for several sessions.
Surgical procedure was performed under general
anesthesia by using protocol of anesthesia include atropine
sulphate 1mg/kg b.w. intramuscularly as a premedication
after fifteen minutes later a mixture of Xylazine and
Ketamine hydrochloride given intramuscularly at a dose of
5 and 20 mg/kg b.w., respectively. The surgical site was
prepared under aseptic technique. Skin incised directly at
the mid-shaft of femoral bone to expose the bone and
osteotomy was performed, about one centimeter of
periosteum and endosteum were destroyed from both sides
of fractured end with bistoury scalpel. The fractured ends
fixed by stainless steel 2.4φ×120 mm intramedullary
pinning. Muscles sutured by simple continuous using catgut
NO. 2/0, then sufficient amount of bone marrow which
aspirated from femoral head of other side (using needle
gauge 18) was injected at the site of fracture in-between the
stitches, after that, suture the fascia and skin. While in
group B in additional to that, animals were exposed to
radiation with He-Ne infrared lasers (the He-Ne wave
length: 632.8 nanometers, frequency: 50-60 Hz were the
wave length and frequency of infrared are 904 nanometers,
700-1200 Hz respectively) applied at the fractured site, in a
series of eight sessions (1-3 , 5, 7, 9, 11 and 13 days) after
operation, the total dose of He-Ne infrared lasers for each
animal was 3.6 joule/cm2.
Post-operative care by using penicillin-streptomycin at a
dose 10000 I.U, 10 mg/kg b.w. injected intramuscularly for
four days. Clinical examination and weekly radiological
studies were performed for the fracture site to determine the
stage of fracture healing.
Results
The results of this study showed that mild inflammation
at the site of operation during the first few days after
operation, theses signs represented by; pain, red, hot,
swelling, but subsided quickly during fifth days in group A
and fourth days in group B. The callus formation was tested
clinically by stress of the fracture site by finger palpation, at
the end of 2nd
week in group A and at the end of 3rd
week
on B. It has noticed that the callus formation around the
fractured area in group A larger than in group B. The callus
disappeared clinically at eight and six weeks in group A
and B respectively. The radiographical finding revealed
that, the healing occurred at the eighteenth and fifteenth
week in group A and B respectively, which characterized
by the invisibility of fracture line and the bone taken about
the normal shape. The periods of fracture healing are
summarized in (Table 1).
Discussion
The periosteum, endosteum and bone marrow provided
cells that proliferate and differentiate into osteoblasts,
chondroblasts and fibroblasts, which contribute to new
bone formation (2,3). It was demonstrated that autologous
bone marrow contains mesenchymal stem cells that are able
to form bone, cartilage and enhancement of the
osteogenesis in fracture healing (5,6).
In our study the destruction of both periosteum and
endosteum at the fracture site and injection of sufficient
amount of autologous bone marrow leaded to healing at
about eighteenth week. This observation was confirmed by
other authors (5,7). Who said that, the autologous bone
marrow enhanced of bone healing. On the other hand this
result agreed with other authors (1,2). Who recorded that,
the periosteum and endosteum are important in fracture
healing, which when both removed at the fracture area
resulting in absence of callus formation.
Low level laser therapy can accelerate the bone
regeneration. This was appeared by altered osteoblast
activity at the fracture site as reflected by alkaline
phosphatase activity (11). Laser irradiation also caused a
significant increase in calcium accumulation at the fracture
site (13). In this study, the results confirm these facts,
which revealed that, the laser therapy enhanced the fracture
healing in-group B when compared with group A. The
destruction of both periosteum and endosteum at the
fracture site then treated with a combination of laser and
autologous bone marrow as in group B, which exhibited
better results than using autologous bone marrow alone by
other authors (6-8). We believed that this combination of
autologous bone marrow and He-Ne infrared laser therapy
may be not mentioned in literature.
Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (5-9)
7
Table 1: Radiographic findings of group A and group B.
Week Group A Group B
1 Slight proliferation of periosteum at a distance from
fracture site. Clear fracture line.
Slight proliferation of periosteum at a distance from
fracture site. Clear fracture line.
2 External callus become active and migrated in to the
fracture site. Clear fracture line.
Slight increase of external callus around the fracture site.
Clear fracture line.
3 Increase internal callus and partially bridged fracture
site. Clear fracture line.
External callus around the fracture site. Visible fracture
line.
5 Thick external callus bridged the fracture site. Still
clear fracture line (fig. 1).
Thin external callus bridged fracture site. Still visible
fracture line.
8 External callus began to absorb. Still visible fracture
line (fig. 2).
Good alignment of the cortex at the fracture site. Bone
almost taken about the normal shape. Disappearance of
fracture line (fig. 3).
10 Thin external callus around the fracture site. Invisible
fracture line.
Thin external callus around the fracture site. The bone
taken about the normal shape (fig. 4).
12 The bone began to taken about the normal shape. Thin
external callus still around the fracture site (fig. 5).
The bone taken the normal shape. Still appear very thin
external callus around the fracture site (fig. 6).
15 Still visible external callus. Bone almost taken the
normal shape.
The bone is normal in shape. Still very thin external
callus around the fractured area (fig. 7).
18 Slight invisible callus. Bone may be taken about the
normal shape.
Fig. 1: Radiographic picture shows thick external callus
bridged the fracture line. Still clear fracture line, five weeks
after operation in group A.
Fig. 2: Radiographic picture shows external callus began to
absorb. Still visible fracture line, eight weeks after
operation in group A.
Fig. 3: Radiographic picture shows bone almost taken the
normal shape. Invisible fracture line, eight weeks after
operation in group B.
Fig. 4: Radiographic picture shows thin external callus
around the fracture site. The bone taken about normal
shape, ten weeks after operation in group B.
Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (5-9)
8
Fig. 5: Radiographic picture shows the bone began to taken
about the normal shape. Thin external callus around the
fracture site, twelve weeks after operation in group A.
Fig. 6: Radiographic picture shows the bone taken the
normal shape. Still appear very thin external callus around
the fracture site, twelve weeks after operation in group B.
Fig. 7: Radiographic picture shows normal shape of bone.
Very thin external callus around the fracture area, fifteen
weeks after operation in group B.
The radiographic findings revealed that, the healing of
the fractured bone is noticed in eighteenth, fifteenth week
in group A and B respectively. These findings may be due
to the He-Ne infrared laser therapy enhancement of
osteogenesis and hasten the healing. This result was agreed
with other authors (11,13). Who said that, the low level
laser therapy can accelerate the osteogenesis. The external
callus was larger in animals of group A than in group B,
this may be due to the He-Ne infrared lasers causes high
activity of the source of osteoblasts and mesenchymal stem
cells of autologous bone marrow for direct formation of
bony material to fill the gap of fracture line instead of
cartilage or fibrocartilage tissue formation as seen in
common stages of fracture healing. The bone return to the
normal feature approximately in eighteenth week in group
A and fifteenth week in group B, this variation of period
between these both groups may be due to the action of
combination of autologous bone marrow and He-Ne
infrared laser which accelerate the osteoblasts and
osteoclasts activity to deposition and resorption of bone
matrix.
In conclusion of this study which revealed that, the
using of autologous bone marrow was enhanced the
fracture healing. While using of combinations of
autologous bone marrow and He-Ne infrared laser therapy
caused more hastened healing.
Acknowledgement
This study was supported by the College of Veterinary
Medicine, University of Mosul.
References
1. Kojimoto H, Yasui N, Goto T, Matsuda S, Shimomura Y. Bone
lengthening in rabbits by callus distraction. The role of periosteum and
endosteum. J Bone Joint Surg Br. 1988;70(4):543-549.
2. Guichet JM, Braillon P, Bodenreider O, Lascombes P. Periosteum and
bone marrow in bone lengthening: a DEXA quantitative evaluation in
rabbits. Acta Orthop Scand. 1998;69(5):527-531.
3. Reynders P. Intra-osseous injection of concentrated autologous bone
marrow in 62 cases of delayed union. Folia Traumat Lovaniensia.
2003;108-116.
4. Porter SE, Hanley EN. The musculoskeletal effect of smoking. J AM
Acad Orthop Surg. 2001;9(1):9-17.
5. Srouji S, Livne E. Bone marrow stem cells and biological scaffold for
bone repair in aging and disease. Mech Ageing Dev. 2005;126(2):281-
287.
6. Sim R, Liang TS, Tay BK. Autologous marrow injection in the
treatment of delayed and non-union in long bones. Singapor Med J.
1993;34(5):412-417.
7. Liang YT, Zhang BX, Lu SB. Experimental study and clinical
application on osteogenesis of percutaneous autogenous bone marrow
grafting in bone defects. Zhongguo Xiu Fu Chong Jian Wai Ke Za
Zhi. 1999;13(3):148-151.
8. Connolly JF, Guse R, Tiedeman J, Dehne R. Autologous marrow
injection as a substitute for operative grafting of tibial nonunions. Clin
Orthop Relat Res. 1991;(266):259-270.
9. Ninomiya T, Miyamoto Y, Ito T, Yamashita A, Wakita M, Nishisaka
T. High-intensity pulsed laser irradiation accelerates bone formation in
metaphyseal trabecular bone in rat femur. J Bone Miner Metab.
2003;21(2):67-73.
10. Garavello I, Baranauskas V, da Cruz-Hofling MA. The effects of low
laser irradiation on angiogenesis in injured rat tibiae. Histol
Histopathol. 2004;19(1):43-48.
11. Yaakobi T, Maltz L, Oron U. Promotion of bone repair in the cortical
bone of the tibia in rats by low energy laser (He-Ne) irradiation. Calcif
Tissue Int. 1996;59(4):297-300.
Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (5-9)
9
12. Barushka O, Yaakobi T, Oron U. Effect of low-energy laser (He-Ne)
irradiation on the process of bone repair in the rat tibia. Bone J.
1995;16(1):47-55.
13. Luger EJ, Rochkind S, Wollman Y, Kogan G, Dekel S. Effect of low-
power laser irradiation on the mechanical properties of bone fracture
healing in rats. Lasers Surg Med. 1998;22(2):97-102.
14. Guzzardella GA, Fini M, Torricelli P, Giavaresi G, Giardino R. Laser
stimulation on bone defect healing: an in vitro study. Lasers Med Sci.
2002;17(3):216-220.
15. Khadra M, Kasem N, Haanaes HR, Ellingsen JE, Lyngstadaas SP.
Enhancement of bone formation in rat calvarial bone defects using
low-level laser therapy. Oral Surg Oral Med Oral Pathol Oral Radijol
Endod. 2004;97(6):693-700.
Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (11-16)
11
Concurrent aflatoxicosis and caecal coccidiosis in broilers
A. M. Shareef
Department of Veterinary Public Health, College of Veterinary Medicine, University of Mosul, Mosul, Iraq
(Received January 27, 2008; Accepted July 13, 2009)
Abstract
A farm of two broiler flocks fed from one day old to 56 days of age a diet naturally contaminated with high level of
Aflatoxin (AF), were suffered from caecal coccidiosis at 35 days of age Aflatoxin levels in feed commodities and mixed feed
were determined using ELISA test. Results of ELISA showed that the average level of corn, soybean and mixed feeds contamination with aflatoxin were 1915, ppb, 229 ppb and 860 ppb respectively. Diagnosis of caecal coccidiosis was
confirmed by clinical signs, post-mortem findings, fecal oocyst excretion per gram, oocyt size, morphological characteristic of
eggs, lesion score and schizonts size. The significance of AF as predisposing factor to coccidiosis infection was discussed.
Keywords: Coccidiosis, Aflatoxin, Broilers.
Available online at http://www.vetmedmosul.org/ijvs
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Introduction
Poultry aflatoxicosis was first reported when 100000
turkey poults in the United Kingdom in 1960 died
following the ingestion of poultry feed containing Brazilian
groundnut cake which led to the discovery of a group of
compounds now called the aflatoxins (1). Soon afterwards,
however, aflatoxicosis became a worldwide problem (2-4).
Mycotoxins are secondary metabolites produced by fungi of various genera when they grow on agricultural products
before or after harvest or during transportation or storage.
Mycotoxins are regularly found in feed ingredients such as
maize, sorghum grain, barley, wheat, rice meal, cottonseed
meal, groundnuts and other legumes. Most are relatively
stable compounds and are not destroyed by processing of
feed and may even be concentrated in screenings. Naturally
occurring toxins present a different problems (5,6).
Mycotoxins cannot be avoided entirely or eliminated from
animal feeds because the molds that create them occur
naturally in grains and other food commodities Aflatoxins
B1, B2, G1, and G2 are mycotoxins that may be produced by three moulds of the Aspergillus species: A. flavus,
parasiticus and A. nomius (7) Aflatoxin B1 is an
immunosuppressive mycotoxin for cell-mediated immunity
Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (11-16)
12
(CMI), humoral immunity, suppression of peritoneal
macrophage phagocytosis and the primary splenic antibody
response (8-11). Aflatoxin has been reported to increase the
severity of avian infectious diseases such as salmonellosis
(12), crop mycosis (13), aspergillosis (14), and coccidiosis
(15).
Avian coccidiosis is the major parasitic disease of poultry with a substantial economic burden on the cost the
industry (16). In-feed medication for prevention and
treatment contributes a major portion of these losses in
addition to mortality, malabsorption, inefficient feed
utilization and impaired growth rate in broilers, and a
temporary reduction of egg production in layers. The
pathogenicity of coccidia depends largely on the successful
replication of developing parasites inside the host.
Theoretical estimates indicate that a single oocyst of a
virulent species such as E. tenella could yield 2,520,000
invasive parasites after the 2nd merogony stage (17). The
profuse bleeding in the ceca is a characteristic feature of E.
tenella infection due to its extensive destruction of the
mucosa with histological lesions (18). In general, young
animals are more susceptible to coccidiosis and more
readily display signs of disease, whereas older chickens are
relatively resistant to infection (19). Young animals which
recover from coccidiosis may later be able to partly
compensate for the loss of body growth, but their growth
potential remains severely compromised. Because the life
cycle of Eimeria parasites is complex and comprised of
intracellular, extracellular, asexual, and sexual stages, host
immune responses are quite diverse and complex. After invasion of the host intestine, Eimeria elicit both
nonspecific and specific immune responses which involve
many facets of cellular and humoral immunity (20,21)
Caecal coccidiosis of broilers has been extensively studied
(22), and as the worlds poultry industry has developed the
disease has continued to be of major economic importance
(23). Interactions occur between chicken caecal coccidiosis
and other diseases, caused by various pathogens (24).
Among those other diseases, aflatoxin intercurrent with
coccidiosis are an increasing health risk to poultry and
poultry coccidiosis due to primary exposure to aflatoxin had been reported previously (25-30). A natural broiler
aflatoxicosis or broiler caecal coccidiosis had been reported
in mosul province (4, 31).
The natural concurrence of these both cases in broilers
is what we are tried to describe in a farm of two broiler
flocks, in Mosul province, Iraq.
Materials and methods
Chicks and diet:
A broiler farm, in Al-Hamdanyea / Mosul province,
reared on letter 8600 and 9650 broilers in two separate house, had been claimed at 35 days of age from mortality
rate of 26% with signs of reduction in feed consumption,
depression, ruffled feathers, closed eyes, stunted growth,
and profuse bloody diarrhea. These birds were fed a
commercial diet, with 22% protein and 3060 kcal/kg
metabolizable energy, without coccidiostatic or Aflatoxin
binder additives.
Pathological examination:
Forty chicks from the two flocks were necropsed for
pathological examination. These birds were scored for cecal
lesions as devised by (32) as follows; Score 0: no gross
lesions; Score1: very few scattered petichiae on the caecal
wall with normal caecal contents; Score 2: lesions are more
numerous, blood in content, wall is somewhat thickened,
normal caecal content; Score 3: large amounts of blood or
caecal cores, caecal wall greatly thickened, little if any fecal
content in ceca; Score 4: cecum is greatly distended, blood
or caseous core, no fecal debris, dead birds; +4.
Ten of these dead chicks were subjected to caecal mucosal scraping for schizont determination. Oocyst size
and shape were determined from about 50 predominant
oocysts. A concentration McMaster technique has been
used for calculation of average fecal oocyst excretion per
gram (33).
Feed sampling: Twenty five feed samples (each 1 kg) were taken from
the broiler farm feed store, five samples of corn, barley,
wheat, soybean and mixed feed according to (34).
Aflatoxin assay:
The levels of aflatoxin contamination of feed samples
were determined by the method of direct competitive
enzyme-linked immunoassay using Neogen extraction kit
(Neogen Corporation). Results of the yield optical densities
of the controls and samples were obtained by using
computerized Neogen Verotex Software program version
2.0.16 (Neogen Corporation).
Results
The most prominent lesions seen in 80 necropsid birds
in both flocks were summarized in (Table 1), in which
pathological changes were mainly seen in the liver and two
caecai. Those in the liver were characterized by creasy,
yellow, ocher discoloration, with scattered areas of sub
capsular hemorrhage. The caecal lesions were characterized
by accumulation with varying quantities of blood and
caseous necrotic materials (Figure 1). From table 1, it is
evident that 5% of necropsed broilers in both flocks show
(+) intensity of liver discoloration. More liver discoloration
intensity (++) were recognized in both flocks with 22.5% in
flock No.1, and 24% in flock No.2. Similar percentage of liver discoloration changes intensity (+++) were noticed in
Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (11-16)
13
both flocks. Higher percentage (37.5%) of liver intensity
(++++) was recorded in flock No. 1, than that of 35% in
flock No. 2. The intensity of sub capsular hemorrhage in
livers of necropsed broiler chicks in both flocks, showed
that only 2.5% of the necropsed birds in flock No. 1 with
sub capsular hemorrhage of (+) was recorded, in
comparison of 10% in flock No. 2. More sub capsular hemorrhage intensity (++) was noticed in flock No. 1
(27.5%), compared with 25% in flock No. 2. Still more
intensity of (+++) in flock No. 1 of 35% was recorded,
compared with 30% in the other flock. The same picture
was continued with the highest sub capsular hemorrhage
intensity of (++++) was also recorded in flock No. 1 with
37.5%, while it was 35% in flock No. 2.
Table 1: Number of necropsed birds, their percentages and
intensity of pathological changes seen in livers of two
broiler flocks.
Broiler flocks
Flock No. 1 Flock No. 2
Liver
lesions
No
. of
nec
rop
sed
bir
ds
%
Inte
nsi
ty
No
. of
nec
rop
sed
bir
ds
%
Inte
nsi
ty
2/40 5 + 2/40 5 +
9/40 22.5 ++ 10/40 5 +
14/40 35 +++ 14/40 35 +++
Dis
colo
rati
on
(Yel
low
och
er)
15/40 37.5 ++++ 14/40 35 ++++
1/40 2.5 + 4/40 10 +
11/40 27.5 ++ 10/40 25 ++
13/40 35 +++ 12/40 30 +++
Su
b c
apsu
lar
hem
orr
hag
e
15/40 37.5 ++++ 14/40 35 ++++
Individual and mean caecal lesion scores (Mean lesion
score, MLS) (MLS = Sum of scores/number of birds) are
presented in Table 2. From table it is clear that the total
mean score lesions in the first flock was 3.5, i.e., higher
than that MLS in the flock N0. 2 (3.05). The distributional
percentages of MLS in both broiler flocks are illustrated in Figure 2. From this table it is clear that score 4 represents
73% in flock No.1, compared with 52% in flock N0.2.
Score 3 in flock No. 2 was about double that in flock
N0.1was about, they were 28 and 16% respectively. Two
percent of score 2 was recorded in both broiler flocks.
Relatively similar percentage for both broiler flocks 1 and
2, and they were 10 and 8% respectively. Nor score lesion
of (0) was reported in flock No. 1, while the score was
higher ten times in flock No.2.
Figure 1: Necropsy liver and caecal lesions of broiler 35
days of age, showing creasy, yellow-ocher discolored liver,
with ballooned two caeci, filled with free blood.
Parasitological findings of both flocks are summarized
in Table 3. The MLS in both flocks was 3.26, with fecal
oocyst excretion per gram X1000 = 636. The morphological
features of examined caecal oocysts were identical for
Eimeria tenella.
Aflotoxin assay of feed commodities taken from both broiler flocks are presented in Table 4. Of these
commodities corn samples were heavily contaminated with
AF. The range of contamination was between 831-3485
ppb, with average concentration of 1915ppb.Soybean
samples were also reported to be contaminated with AF but
at a lower rate than that of corn. Similar rate of
contamination was noted, with range occurred between
220-238ppb, and an average of 229 ppb. No AF
contamination was detected in both wheat and barley
commodities. Mixed feed was consequently contaminated
from the contaminated ingredients made from them, corn
Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (11-16)
14
and soybean. They were contaminated in a rate of 720-1006
ppb, with an average of 860 ppb.
Table 2: Individual and mean total caecal score lesion of
broiler chicks in both flocks.
Broiler flocks
Caecal score
lesion
No. of birds in
flock No. 1
No. of birds in
flock No. 2
0 4
1 4 3
2 1 1
3 6 10
4 29 22
Mean lesion
score(MLS) 140/40 = 3.5 122/40=3.05
Figure 2: Percentages of caecal score lesions in both broiler
flocks.
Table 3: Summary of the parasitological examination of
necropsed birds (Average findings of broilers in both
broiler flocks).
Parameters Results
Mortality 26%
Fecal oocyst excretion per
gram X1000 636
Oocyt size width X length
(µm) 26 X 23
Mophological characteristic
of eggs Broadly ovoid
Mean lesion score for 80
dead birds 262/80 = 3.27
Schizonts maximum in µm 55.0
Table4: Aflatoxin contamination of feed commodities (ppb)
in of both broiler flocks.
Feed commodities
Corn Soybean Wheat Barley Mixed
feed
Sample
No.
Concentrations of AF ppb
1 3485 238 ND � ND 1006
2 3485 233 ND ND 931
3 931 228 ND ND 831
4 831 224 ND ND 753
5 866 220 ND ND 720
Mean 1915 229 ND ND 860
�ND= not detected
Discussion
Aflatoxin was reported to increase susceptibility to
coccidiosis under experimentally prior exposure poultry to
Aflatoxin (25-30,35). To our knowledge, no report is reported here in Mosul province referring to a natural
concurrent secondary caecal coccidiosis due to
contamination of broiler feed commodities with Aflatoxin.
In this study, although anticoccidial agent was not
included in the diet of broiler flocks as prophylactic
measure, but the presence of high natural Aflatoxin
contamination levels (860 ppb) of the mixed feed used in
the feeding of these flocks, could largely impose a great
stress on the birds immune system against coccidial
infection.. This relation is supported by (22), who stated
that 380 ppb AF or more is responsible for a significant reduction in cell mediated immunity (CMI). This effect on
CMI include (a) macrophages, (b) natural killer (NK) cell
lymphocytes and (c) two types of T-derived lymphocyte
population (CD4+and CD8+) (35,36). Aflatoxin causes a
substantial diminution in phagocytic activity by
macrophages (37). Macrophages, CD4+ and NK cell
lymphocytes dominate the CMI reaction upon primary
contact/infection of coccidiosis, while CD8+ cells multiply
particularly upon second contact (reinfection) of coccidiosis
(38). CD4+ and CD8+ cells are cytotoxic and cytolytic, this
means they can kill and lyses parasitic stages during
coccidiosis cycle (39). However, it was reported that specific serum antibodies might also play a part in
protecting chickens against coccidiosis (40) Antibodies,
especially local immunoglobulin A (IgA) in the intestinal
lumen can bind and damage sporozoites, causing loss in the
ability of extra cellular differentiation and thereby
preventing parasite invasion and intracellular development
(41). So, antibodies, although play a minor role as a
protective component against coccidiosis compared with
the role of cell mediated immunity, but it is still negatively
0 1 2 3 4 1 2 3 4
Caecal score lesions Flock 1 Flock 2
Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (11-16)
15
affected by Aflatoxin, since their production was reported
to be decreased by exposing birds to high toxin levels from
300 to 6000 ppb (42). The other possible roles of AF in
predisposing broilers here to coccidial infection is the toxin
reduction effect of vitamin A level in chicken liver and
serum (43). Here, although vitamin A level was not
measured in liver or serum, but it is highly possible that vitamin A deficiency is likely to occur due to feeding
highly AF contaminated diet. Since, it is well known that
disease resistance is a function of cell differentiation, and
one of the primary function of vitamin A is to maintain
proper epithelial tissue differentiation and prevent epithelial
keratinization which occurs in vitamin A deficiency and it
is important for enhancing both cellular and humoral
immunity and enhance phagocytosis activity, and for
maintaining cellularity of lymphoid organs (44,45). The
possible reduction of vitamin A may be related to the high
mortality rate reported here, since vitamin A has been
reported to reduce mortality in chicks infected with coccidial oocyst from E- tenella and E- acervulina (46).
The high coccidial pathogenicity noticed in our study,
expressed by high lesion score of 3.27, could be related, to
high AF diet contamination (15), and the high fecal oocyst
excretion per gram (636000) oocyst/gram could also be
attributed to this contamination. This effect was supported
by (2,28), who confirmed that Aflatoxicosis was
responsible for increased oocyst production and
reproduction potential during coccidiosis. It is evident from
the parasitic parameters examined in this study, that lesion
score was found to be directly proportional to the mortality and oocyst count per gram of faeces/ dropping. Any way, it
should be stressed that other mycotoxins like ochratoxin, T-
2 toxin, and fumonisins may be present or contaminate feed
commodities or mixed feed samples in our study, but the
estimation was tried only with the most effective
immunosuppressant mycotoxins, Aflatoxin. Moreover, no
attempt was carried out to isolate immunosuppressive
viruses like reo virus and Merk disease virus to elucidate
their possible role if they were present in exacerbating
caecal coccidiosis beside aflatoxin on broiler chickens.
From all above, and under field conditions, it seems that mycotoxins and here AF are highly stressful, when fed
for extended period of time to birds on their immune
system, which may be weakened to the point where it
cannot control the organism and set the stage for the
development of coccidiosis. So to prevent Aflatoxicosis and
coccidiosis in poultry, sorbents like sodium bentonite and
hydrated sodium calcium aluminosilicates should be added
to their diet in order to decrease Aflatoxin severity (47), and
by using coccidiostats/coccidiocidal drugs, or by Appling
an alternative system of vaccination against coccidiosis..
Acknowledgement
The author thanks the College of Veterinary Medicine,
Mosul, Iraq, for providing all supports for this study.
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Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (17-25)
17
Molds and mycotoxins in poultry feeds from farms of potential
mycotoxicosis
A. M. Shareef
Department of Veterinary Public Health, College of Veterinary Medicine, University of Mosul, Mosul, Iraq
(Received June 19, 2008; Accepted July 13, 2009)
Abstract
Forty five finished poultry feed samples, collected from different broilers, broiler breeders and layers farms were divided
into two parts, for mycological and mycotoxins examination. In counting of molds, dilute plate technique was used, whereas
feed parts were used for mycotoxin estimation, they were subjected to four standard kits of Aflatoxin, Ochratoxin, T-2 toxin
and Fumonisins. Mold counts were around 105 cfu.g-1 sample. Fourteen mold genera were recovered. From the systematic point of view, 2 genera belonged to Zygomycetes (i.e. Mucor, Rhizopus,), 1 genus belong to Ascomycetes (i.e. Eurotium); the
majority, within so-called mitotic fungi (formerly Deuteromycetes), encompassed 11 genera (i.e. Acremonium, Alternaria,
Aspergillus, Fusarium, Paecilomyces, Penicillium, Scopulariopsis,, Trichothecium, Ulocladium and Aerobasidium). The most
frequent fungi were those from the genus Aspergillus. The concentrations of the four analyzed mycotoxins in the poultry
finished feeds, and the percentages of the recovered mycotoxins, revealed that aflatoxins was recovered in 91.1% of the
examined samples, with a mean value of 179.1µg/kg. The same percentage was found with Ochratoxins, but with lower mean
concentration of 159.4µg/kg. In the third order were Fumonisins mycotoxins were in the third order, and they were recovered
in 51.1% of the tested samples with a mean value of 127µg/kg. In the fourth order was T-2 toxin, with a percentage of 2.2%
and a value of 50.0µg/kg.
Key words: Molds, Mycotoxins, Mycotoxicosis, Poultry feed.
Available online at http://www.vetmedmosul.org/ijvs
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18
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Introduction
Mold occurrence and growth on poultry feeds is one of
the major threats to poultry economic and health. Besides
their negative impacts on nutritional and organoleptic
properties, moulds can also synthesize different
mycotoxins. More than 100.000 fungal species are considered as natural contaminants of agricultural and food
products. However, due to genetical and ecological factors,
relatively few can actually generate mycotoxins (1).
According to Leibetseder (2), 30 to 40 % of existing
moulds can elaborate toxic substances under favorable
conditions. The majority of the toxic species belong to the
genera Aspergillus, Penicillium, Fusarium and Alternaria
(3). The effects of mycotoxins on higher animals include
hepatotoxicity, nephrotoxicity, immunotoxicity,
oncogenesis and genotoxicity (4-6). Despite the great
attention that has been paid to the study of toxigenic moulds and their mycotoxins in various foods and feeds,
little is known about fungal and mycotoxin contamination
of poultry feed in Mosul governorate yet. During the last
two or three decades, the production of mixed poultry feeds
were significantly increased in parallel with the evolution
of poultry industry in the country. Furthermore, it is well
established that contamination of poultry feeds with
mycotoxins may induce sanitary disturbances and mortality
among the birds and secondary contamination of the human
consumer via eggs, poultry meat and giblets (7,8). Variety
of complex and divers clinical signs of potential
mycotoxicosis were always observed in different broiler, broiler breeder and layer farms with potential
mycotoxicosis. Affected flocks showed one or more of the
following symptoms; decreased weight gain; anorexia;
reduced feed conversion efficiency; decreased egg
production; poor egg shell quality ;increased egg blood
spots; spiking mortalities; immunosuppression and failure
of vaccination programs; increased susceptibility to
diseases especially E-coli infection; reduced fertility and
hatchability; visceral hemorrhages; leg weakness and high
percentages of leg deformities; pale bird syndrome; fatty
liver with pale, muddy to yellowish discoloration; increased bruising; enlarged pale kidneys; wet litter; urate deposition
in the body cavities; increased incidence of viral diseases
like Newcastle disease, infectious bursal disease and
inclusion body hepatitis; oral lesions; tibial
dyschondroplasia; gizzard erosions; paralysis; extension of
leg and neck.
The purpose in the present work was to initiate a study
on the toxigenic mycoflora of poultry feed in Ninevah
governorate/ Iraq that It includes: enumeration and
identification of moulds genera naturally contaminating
different kinds of poultry feeds and detection and
quantification of the 4 major mycotoxins in the feed
samples, aflatoxin, ochratoxin A, fumonisins and T-2 toxin.
Materials and methods
Samples The samples of finished poultry feed were delivered to
the College of Veterinary Medicine, University of Mosul
from different broilers, broiler breeders and layers farms.
These farms were located in Nineveh governorate and were
claimed from potential mycotoxicosis, feed samples were
collected during a 2-years-long period from Apr 2005 to
Sep 2007. Random 10 to 30 representative samples of I kg
were collected from several locations within a batch of feed
and combined thoroughly to provide a composite sample of 1 Kg for submission. They were then divided to two parts,
one for mycological examination, and the other for
mycotoxins detection. Feed samples intended for
mycological examination were usually analyzed
immediately upon arrival or, if necessary, they were stored
for 2-3 days in paper bags at room temperature (22-25ºC).
The other parts of feed samples intended for mycotoxins
analysis were stored at -20Co.
Isolation of poultry feed fungi Dilute plate technique was used for isolation of fungi
from the samples (9). General molds count was carried out by weighing 20 g of the poultry mixed feed samples and
their mixing with 180 ml of saline solution (0.85% sodium
chloride) with 0.05% Tween 80 on a horizontal shaker for
ca. 30 minutes. Then, 0.1 ml of appropriate dilutions made
up to 10-5 was applied on Dichloran Rose Bengal
Chloramphenicol agar (DRBC (10, 11). Plates were
incubated at 25oC for 5-7 days. The mold genera were
identified according to (11-15).
Mycotoxins analyses Feed parts intended for mycotoxin estimation were
subjected to four standard kits for aflatoxin, ochratoxin, T-2
toxin and fumonisins using Neogen mycotoxin extraction
kits (Neogen corporation) as follows:
Samples preparation and extraction: Twenty five gram-
samples were collected for analysis. These samples were
finely ground,so that at least 75% of them pass through a 20
mesh. Five gram samples were blended with 25 ml of 70%
Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (17-25)
19
v/v methanol/ water solution for 3 minutes. Extracts were
filtered through a Whattman No.1 filter paper.
Test procedure: All Neogen extraction reagents were
allowed to warm at room temperature (18-30 ºC) before
use. Red marked mixing wells were prepared, one for each
sample plus four red wells for controls. All red- marked
wells prepared were placed in the well holder. An equal number of antibody coated (AB) white wells to those red-
marked wells were also prepared. Hundred µl of conjugate
were transferred to each red-marked mixing well. To those
red wells containing the conjugate another (with new
pipette tips) 100 µl of controls and samples were added by
using a 12-cannel pipettor liquid in wells were mixed and
pipetting it up and down for 3 minutes. After mixing 100 µl
of the (conjugate + samples, or conjugate + controls) were
transferred to AB – coated wells. These wells were moved
back and froth for well mixing the contents in each well for
10-20 seconds without splashing reagents from the wells.
Antibody-coated wells were then incubated at room temperature (18-30 ºC). The contents in AB- coated wells
were shacked out, by filling the wells with deionized water
and dumping them out. This step was repeated 5 times.
Turning the wells upside down and tamping them out on a
paper was carried out until the remaining water has been
removed. Substrate was then added to AB- coated wells, by
using the 12-channel pipettor through pipetting 100 µl of
substrate to these wells. Mixing was done by sliding the
well holder back and froth for 10-20 seconds, followed by
incubation for 3 minutes. Stop solution was poured to these
wells (100 µl) to each, mixing was done by sliding well holder back and froth on a flat surface. Within 20 minutes
after the addition of stop solution. Results were read, using
a micro well reader (ELx00) with a 650 nm filter. More
blue color means less toxins. Results of the yield optical
densities of the controls and samples were obtained by
using computerized Neogen Verotex Softwere Program
version 2.9.16 (Neogen Corporation).
Results
The total number and percentages of enumerated mold
genera in Nineveh finished poultry feed are presented in
Table 1, and the ability to produce appropriate mycotoxins
is shown in Table 2. Total fungal counts were ranged from
0.1 × 101 to 6.5 X 106 cfu.g-1 of feed sample, with an
average 7.2 X 105 cfu.g-1 sample (Table 1. Fourteen mold
genera were recovered during this study.
From the systematic point of view, 2 genera belong to
Zygomycetes (i.e. Mucor, Rhizopus,), 1 genus belong to
Ascomycetes (i.e. Eurotium); the majority, within so-called
mitotic fungi (formerly Deuteromycetes), encompassed 11
genera (i.e. Acremonium, Alternaria, Aspergillus,
Fusarium,, Paecilomyces, Penicillium, Scopulariopsis,,
Trichothecium, Ulocladium and Aerobasidium). The most
frequent fungi were those from the genus Aspergillus,
recovered from 40 samples (88%) with a range of 0.1 X 10 5- 5.3 X 106 and a mean value of 2.6 X 106. In the second
order were the molds from the genus Penicillium and
Mucor, both recovered from 28 samples (64%) with a range
of 0.2 X 105 - 4.4 X 106 and3 X 104 - 2.6 X 105 and a mean
of 2.2 X 106 and 1.4 X 105 respectively. To the third and fourth orders were the molds from the genera Rhizopus, and
Scopulariopsis, recovered from 23 and 22 samples (50%
and 48%) respectively with a range of 1.5 X 104- 2.1 X 105
and 0.2 X 104- 1.8 X 105, and a mean of 1.1 X 105 and 9.1X
105 respectively. The following recovered genera were the
molds of Alternaria and Eurotium, from 20 and 19 samples
with percentages of 45% and 42% and a range of 8 X 10 3 -
1.8 X 1053 and X 10 3 - 1.7 X 105, with a mean of 9.4 X 104
and 8.5 X 104 respectively. In the descending pattern
occurred the molds of the genera Cladosporium and
Fusarium, recovered from 17 and 14 samples (37% and
31%), representing a range between0.2 X 10 3- 9.8 X 104 and 0.4 X 10 3 - 9.2 X 104, and a mean of 4.9X 104 and 4.6
X 104 respectively. From less than 10 samples recovered
the genera of the mitotic molds from Acremonium,,
Paecilomyces, Ulocladium, Aureobasidium, and
Trichothecium, with 11%,7%,7%,2% and 2% respectively,
with an average of 4300,110,70,50 and 25 CFU g-
1respectively.
Table 2 illustrate the frequency of recovered mold
genera out of 45 finished poultry feeds tested through the
study period, of these 41 samples were positives for
aflatoxins and ochratoxins (91.1%).To a lower extent were the positive samples of Fumonisins (51.1%). The lowest
positive sample was that of T-2 Toxin (2.2%). The
mycotoxins concentrations were in general positive
proportion to the number of respective molds producing
them, being highest in aflatoxins and ochratoxins. These
mycotoxins are produced mainly by aspergillus and
penicillium mold genera, the most prominanat mold
enumerated in this study. In a descenging manner were also
the average levels of aflatoxins, ochratoxin A, fumonisins
and T-2 toxin. The highest levels of these mycotoxins were
as follows; aflatoxins: < 475 µg/kg ; ochratoxin A: < 460 µg/kg ; fumonisins: < 350 µg/kg and T-2 toxin: 50 µg/kg.
Table 3 shows the distribution concentration of
different mycotoxins in the tested finished poultry feeds.
Only aflatoxins and ochratoxin A were ranged from 0-500
µg/kg. The highest number of samples contaminated with
aflatoxin were within levels of 200-300 µg/kg. Although
ochratoxin level was also ranged from 0-500 µg/kg, but the
highest levels were occurred in concentrations of 50 and
250 µg/kg. Fumonisins highest levels were occurred in
concentrations between 50-150 µg/kg, and the highest
levels did not exceeds 350 µg/kg. The only one sample
contaminated with T-2 toxin had level of 50 µg/kg.
Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (17-25)
20
Table 1: Frequency and average count of recovered mold genera from 45 Nineveh poultry finished feed samples.
Average
counts cfu g-1 Range
Percentage of mold
genera frequencies
Frequency of Positive
samples Molds
2.6 X 106 0.1 X 10 5- 5.3 X 106 88.8 40 Aspergillus spp.
2.2 X 106 0.2 X 105 - 4.4 X 106 62.2 28 Penicillium spp.
1.4 X 105 3 X 104 - 2.6 X 105 62.2 29 Mucor spp.
1.1 X 105 1.5 X 104- 2.1 X 105 51.1 23 Rhizopus spp.
9.1X 105 0.2 X 104- 1.8 X 105 48.8 22 Scopulariopsis spp.
9.4 X 104 8 X 10 3 - 1.8 X 105 44.4 20 Alternaria spp.
8.5 X 104 3 X 10 3 - 1.7 X 105 42.2 19 Eurotium spp.
4.9X 104 0.2 X 10 3- 9.8 X 104 37.7 17 Cladosporium spp. 4.6 X 104 0.4 X 10 3 - 9.2 X 104 33.3 14 Fusarium spp.
4.3 X 103 0.6 X 10 2 - 8.7 X 103 11.1 5 Acremonium spp.
1.1 X 102 1 X 10 1 - 2.1 X 102 6.6 3 Paecilomyces spp.
7 X 101 2 X 10 1 - 1.2 X 102 6.6 3 Ulocladium spp.
5 X 101 0.2 X 10 1 - 0.8 X 101 2.2 1 Trichothecium spp.
2.5 X 101 0.2 X 10 1 - 0.8 X 101 2.2 1 Aureobasidium spp.
Table 2: Number of tested, positive, percentage and levels of detected mycotoxins in PMFS tested.
Mycotoxins Parameters
Aflatoxins Ochratoxin A Fumonisins T-2 Toxin
No. of samples tested 45 45 45 45
No. of positive samples 41 41 23 1
Percentage positive (%) 91.1 91.1 51.1 2.2 Average level (µg/kg) 179.1 159.4 127 50
Highest level (µg/kg) 475 460 350 50
Figure 4 shows the different AF concentrations (ppb)
in positive AF-PFF samples. It is evident that 24% of the
samples had AF concentration between 201-250 ppb,
followed by 16.6% between 151-200ppb and 11.1%
between 351-400 ppb. The remaining concentrations were
recovered in less than 10% of the samples.
Figure 5 shows the different Ochratoxin concentrations
(ppb) in positive Ochratoxin -PFF samples. From figure it
is clear that 20% of the samples had Ochratoxin up to 50 ppb, followed by 17.7% between 201-250 ppb, 13.3%
between 51-199 and 351-400, 11.1% between 101-150,
while the remaining concentration were occurred in
percentages less than 10%.
Figure 6 shows the different fumonisins concentrations
(ppb) in positive fumonisins -PFF samples. It is evident that
48.8% were negative samples. The positive samples
showed that 22% of them had fumonisins concentrations up
to 50 ppb, and 13.3% between 101-150 ppb, while the
concentrations up to 350 ppb had percentages of less than
10%. No more than 350 ppb of fumonisins mycotoxin was detected.
Figure 7 shows that 97.75 of the tested feed samples
were negative for T-2 toxin, and only one sample with 50
ppb was detected.
Discussion
Both field and storage fungi were recovered in this study
through the examination of poultry finished feed samples
collected from different poultry farms with potential
mycotoxicosis. These molds include members of the genera
Aspergillus, Penicillum, Fusarium, Alternaria and
Cladosporium which could contaminate many agricultural
commodities used in the formulation of poultry finished feed samples like corn, wheat, soybean, barley and others
commodities. These molds are of great importance because
of potential mycotoxin production (16). The total fungal
loads in the analyzed finished feed samples in our study
were around 105 cfug-1 which is higher than that reported in
Slovakia of 103 cfu.g-1 (17), and 104 cfu.g-1 (18). Similar
results were found to those reported in Turky (19), Spain
(20) and from Argentina (21,22). The predominance of
mold genera were to large extent resembles those found in
Saudi Arabia (23), which could be due to some
resemblance in the ecological and climatologically conditions. The main contaminating molds appeared to be
from the genus Aspergillus with a percentage recovery of
91.11%.
Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (17-25)
21
Table 3: Mycotoxins recovered from Nineveh poultry
finished feed.
Sample
No.
Aflatoxins
ppb
Ochratoxins
ppb
Fumonisins
ppm
T-2
toxin
ppb
PFF 1* 57 30 ND ND** PFF 2 189 290 100 ND
PFF 3 254 230 ND ND PFF 4 370 61 ND ND PFF 5 69 291 ND ND
PFF 6 165 242 200 ND PFF 7 ND. 159 250 ND PF F8 250 ND. ND ND
PF F9 350 42 ND ND PF F10 452 460 200 ND PFF 11 54 91 ND ND PFF 12 152 420 ND ND
PF F13 250 223 150 ND PFF 14 340 133 150 ND PF F15 ND. 173 30 ND
PF F16 458 ND. ND ND PFF 17 146 228 ND ND PFF 18 ND 191 150 ND PF F19 240 ND. ND ND
PF F20 36 133 200 ND PFF 12 362 23 350 ND PFF 22 475 214 ND ND
PFF 23 76 38 ND ND PF F24 158 28 150 ND PF F25 370 212 30 ND PF F26 356 273 ND ND
PF F27 235 ND. 20 ND PFF 28 230 19 50 ND PF F29 235 12 130 ND
PFF 30 453 73 20 ND PF F31 ND. 260 ND ND PFF 32 293 142 20 ND
PF F33 250 212 ND ND PF F34 323 57 ND ND PF F35 230 72 ND ND PF F36 196 12 250 ND
PF F37 226 68 ND ND PF F38 180 50 20 ND PFF 39 400 252 250 ND
PFF 40 196 222 10 ND PF F41 188 142 ND ND PFF 42 250 123 20 ND PF F43 219 183 ND ND
PF F44 90 280 150 ND PF F45 22 320 150 50
*PFF=Poultry finished feed from farm 1 ND**=Not detect.
This finding in consistent with (22) and (24) and (25) who
found that the most dominant species isolated of poultry
feed samples belonged to the genus Aspergillus, but not in
consistent with (17), (22) and (25), who found that the most
frequent fungi were those from the genus Penicillium.
Although molds of Aspergillus species are more often soil
fungi or saprophytes but several are important because they produce mycotoxins. Penecilli in our results were in the
second order in counting and percentages among recovered
molds, as they are one of the main contaminants of stored
cereals as well as feeds with worldwide occurrence (26-28)
being producers of toxicologically significant mycotoxins
(29-31). In order, our results were in consistent with (17)
that the third mostly encountered fungi were representatives
of the genus Mucor, who found this genus with frequency
of 44%, followed by the genus Rhizopus. Our results also
revealed that molds from the genus Eurotium were
recovered in 19%, and Fusarium in 14% of the samples.
These results were less than that reported by (17), who found that Eurotium and Fusarium genera were widespread
through the samples they examined and were occurred with
the same frequency of 42%. In this study, the mean of
aflatoxins were recovered at a rate of 91.1%, with an
average of 179.1µg/kg of finished poultry feed samples,
confirming our previous results of aflatoxin contamination
to poultry feed samples (32). Comparing with other Asian
countries, our results were higher than that reported in north
Asia (China, Taiwan and Korea) of 3%, and higher than
south East Asia (Malaysia, Philippines, Thailand and
Vietnam) of 36%, and higher than south Asia (India, Pakistan and Bangladesh) of 53% (33). The second
mycotoxin recovered was Ochratoxin (91.1%), in an
average of 159.4 µg/kg, confirming our previous results of
ochratoxin contamination of poultry finished feeds (34).
Our results were also more than those reported in north
Asia (China, Taiwan and Korea) of 15%, and higher than
south East Asia (Malaysia, Philippines, Thailand and
Vietnam) of 9%, and higher than south Asia (India,
Pakistan and Bangladesh) of 50% (33). Again our high
results here were due to sampling feeds from mycotoxicosis
suffering poultry farms. Ochratoxin has been implicated in significant field outbreaks of mycotoxicosis in poultry (35).
Fumonisins mycotoxins were the third type of mycotoxins
recovered in this study, in a percentage of 53.3% and an
average of 127µg/kg of finished poultry feed samples.
These mycotoxins recovered here in Mosul governorate in
corn samples intended for use in finished poultry feeds
(36). Our results agreed with the percentages reported in
north Asia (China, Taiwan and Korea) of 52%, and with
those of south East Asia (Malaysia, Philippines, Thailand
and Vietnam) of 52%, but higher than hose reported in
south Asia (India, Pakistan and Bangladesh) of 32% (33).
The fourth mycotoxin recovered in this study was T-2 toxin in a percentage of 2% at a rate of 50 µg/kg. This toxin was
Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (17-25)
22
Figure 4:Distribution of different AF
concentrations(ppb) in positive AF- PFF samples
0
5
10
15
20
25
30
1234567891011
Concentrations
%
1=ND 2=0-50ppb 3=51-100ppb 4=101-150ppb 5=151-200ppb 6=201-250ppb
7=351-300ppb 8=301-350ppb 9=351-400ppb 10=401-450 ppb 11=451-500ppb
11.1%
16.6%
24.4%
Figure5: Distribution of different Ochratoxin
concentrations (ppb) in positive Ochratoxin-PFF
samples
0
5
10
15
20
25
1234567891011
Concentrations
%
1=ND 2=0-50ppb 3=51-100ppb 4=101-150ppb 5=151=200ppb 6=201-250ppb
7=351-300ppb 8=301-350ppb 9=351-400ppb 10=401-450 ppb 11=451-500ppb
20%17.7%
13.3% 13.3%
11.1%
Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (17-25)
23
Figure 6:Distribution of different Fumonisins (ppb)
concentrations in positive Fumonisins-PFF in PFF
samples
0
10
20
30
40
50
60
1234567891011
Concentrations
%
1=ND 2=0-50ppb 3=51-100ppb 4=101-150ppb 5=151=200ppb 6=201-250ppb
7=351-300ppb 8=301-350ppb 9=351-400ppb 10=401-450 ppb 11=451-500ppb
48.8%
22.2%
13.3%
Figure 7:Distribution of different T-2 toxin (ppb)
in positive T-2 toxin - PFF samples
0
20
40
60
80
100
120
1234567891011
Concentrations
%
1=ND 2=0-50ppb 3=51-100ppb 4=101-150ppb 5=151=200ppb 6=201-250ppb
7=351-300ppb 8=301-350ppb 9=351-400ppb 10=401-450 ppb 11=451-500ppb
97.7%
2.2%
Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (17-25)
24
also recovered by us through the study performed during
2004-2005 in Mosul governorate (37). Our results were
close to those reported in north Asia (China, Taiwan and
Korea) of 0%, and those found in south East Asia
(Malaysia, Philippines, Thailand and Vietnam) of 1%. In
general our results were agreed with those reported by
Pacin et al., (38) who found that the most recovered mycotoxins from feed associated fungi were ochratoxin A,
aflatoxin and fumonisin. It may be stated that Aspergillus
(including Eurotium), Penicillium and Fusarium are the
typical fungal genera inhabiting poultry feed mixtures. In
fact, they are very important contaminants being renowned
for their ability to form a huge number of various types of
toxic extrolites-mycotoxins (39,40), and that the outcomes
of this study clearly show that finished poultry feeds in
Nineveh governorate represent a rich source of significant
mycotoxin producers, especially those from the
Penicillium, Fusarium and Aspergillus genera. It is possible
that these co-contaminant of estimated mycotoxins in our study, of two carcinogenic mycotoxins, aflatoxin and
fumonisins, and two cancer promoting mycotoxins,
ochratoxin and T-2 toxin, could exert great negative effects
on farms health and productivity than do each of them
singly and of public health concern to the health of
consumer through the food chain by ingestion residual
levels of these toxins in poultry meat and poultry products
(meat and eggs) (41,42). These toxic substances are known
to be either carcinogenic (e.g. aflatoxin B1, fumonisin B1,
Ochratoxin A), neurotoxic (fumonisin B1), nephrotoxic
(ochratoxin A), dermatotoxic (trichothecenes), or immunosuppressive (aflatoxin B1, ochratoxinA and T-2
toxin) (33). the senerginestic effects of these mycotoxins on
poultry productivity and health were well documented.(43-
45).
Acknowledgements
The author thanks the College of Veterinary Medicine,
University of Mosul for providing all supports for this
experiment.
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Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (27-30)
27
Prevalence of intestinal ciliate Buxtonella sulcata in cattle in Mosul
T. M. Al-Saffar*, E. G. Suliman**, H. S. Al-Bakri**
*Department of Internal and Preventive Medicine, **Department of Microbiology, College of Veterinary Medicine,
University of Mosul, Mosul, Iraq
(Received June 11, 2008; Accepted July 13, 2009)
Abstract
The current study was conducted to detect the presence of Buxtonella sulcata (an intestinal ciliate) in faecal samples of
cattle suffering from diarrhea in Mosul city. One hundred and twenty faecal samples were examined, and collected from calves
(44), beef cattle (34) and dairy cattle (42) these animals were divided into two groups those showed diarrhea (86) and (34) had
no symptomatic diarrhea. Direct smear and formalin-ether sedimentation methods were used for detection of this parasite. The
total percentage of infection with Buxtonella sulcata was 24.16%. There was no significant differences in the percentage of
infection and intensity of infection between calves, beef and dairy cattle where as there were significant differences between
diarrheic and non-diarrheic animals.
Keywords: Buxtonella sulcata, Diarrhea, Cattle.
Available online at http://www.vetmedmosul.org/ijvs
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Introduction
Buxtonella sulcata is one of the parasitic protozoa
(Ciliophora) type which inhabited in the colon of the
ruminants and the original role for diarrhea or for
classification has not been fully explained (1-5). Buxtonella
sulcata is similar to Balantidium coli found in the swine
and man, some authors included them into the same genus
(6). However, the classification of Buxtonella sulcata is:
Kingdom: Protozoa, phylum: Ciliophora, Class:
Kinetofragminophora, order: Trichostromatida, Family:
Pyenotrichidae, Genus: Buxtonella (1).
Henrisken (7) reported that percentage of infection
with Buxtonella sulcata in Danish cattle was (71.8%), Fox
(2) recovered Buxtonella sulcata cysts in faeces of adult
British cow from nine commercial dairy farms with a percentage of 44.6% and in Kynoggi-don the prevalence of
Buxtonella sulcata during 1984 and 1994 were 33.6%,
34.5% respectively (8). A higher percentage 38.0%, 21.6%
was reported in a dairy and beef cattle in Costa Rica (9). In
Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (27-30)
28
Germany a more controversial prevalence was reported
which has been range from Zero-73% without any obvious
association with clinical symptoms (10). The first recorded
of infection with Buxtonella sulcata from cattle was in Al
Qadissiyh city (Iraq) and the percentage of infection was
47% (11). The problem of Buxtonellosis in ruminants is not
considered of importance yet, therefore the aim of this study was to diagnose this parasite in faeces of cattle in
Mosul city and to examine the role of this parasite as
etiological agent of diarrhea in calves, dairy and beef cattle.
Materials and Methods
Faecal samples were collected from 120 cattle (44
calves, 42 dairy frezinn cattle, and 34 beef cattle) from
teaching veterinary Medicine College, farms of Agriculture
College and Kogyali village and cattle market in Mosul city
during October 2007 – April 2008, faecal samples were
taken from animals with diarrheal disease (86) and apparently healthy animals (34), samples kept in a clean
plastic container. Direct smear method (12), sedimentation
methods by using ether-formalin (13,14) were used for
detection Buxtonella sulcata. Differentiation of Buxtonella
cyst or trophozoites was based on morphological specific
feature and by microscopic measurement by using ocular
micrometer (2,3,6,8,11). Coproscopical examination for
detection the degree of intensity of infection according to
(15,16) and examination of the 20 microscopical fields. The
data were analyzed statistically by using Z-test, Fisher- test
and χ2 – test (17).
Results
During coproscopical examination of 120 faecal
samples of cattle, infection with Buxtonella sulcata was
appeared in 29 animals with a total percentage of 24.16%
(Table 1), all of these animals infected with Buxtonella
sulcata cysts. These cysts are round or oval in shape,
yellow in color reach 68.6-107.8 µm in diameter with a
mean of 74.58 µm, these cysts surrounded by a two layered capsule (Fig.1). Four positive samples which infected with trophozoites (vegetative forms) with a percentage of
13.79% (Table 1). The vegetative forms were oval, with
diameters of 107.8-137.2X 49-102.9, with a mean of
121.25X94.06 µm. The surface of the cilliate's cell was evenly covered with short cilia, deep syncystoma was at the
anterior pole and the nucleus lies in the centre of the
vegetative form (Fig.2). From Table (1) statistical analysis
showed significant differences between calves and beef
cattle and between calves and dairy cattle while no
significant differences between beef and dairy cattle. From
Table (2) it is evident that there were three degree of
infection, low 51.72%, moderate 24.13% and high 24.13%
according to the number of parasites under high power field (40x hpf) there was no significant differences between
calves, beef and dairy cattle. Buxtonella sulcata cysts were
appeared in 27 animals with diarrhea symptom in a
percentage of 31.39% while those show no diarrhea, the
percentage were 5.88% (Table 3). Statistical analysis
showed significant differences between animals with and
without diarrhea.
Table (1): Incidence of Buxtonella sulcata of cattle in Mosul.
Animals No. of samples No. Of positive
samples Rate of
infection Infection with cyst No.(%)
Infection with Trophozoite No.(%)
Calves (3-8 months) 44 5 11.36 a 5(100.0) 0(0.0) Beef cattle (2-7 years) 34 12 35.29 b 12(100.0) 3(25.0) Dairy cattle (4-8 years) 42 12 28.57 b 12(100.0) 1(8.3) Total 120 29 24.16 29(100.0) 4(13.79)
Rates with different letters have significant difference at P<0.05 according to Z-test between two proportion. Table (2): Distribution and intensity of infection with Buxtonella sulcata of cattle.
Infection Animals No. of +ve samples Low degree* Moderate degree** High degree** Statistical group Calves 5 4(80.0) 1(20.0) 0(0.0) A Beef cattle 12 5(41.6) 3(25.0) 4(33.33) A Dairy cattle 12 6(50.0) 3(25.0) 3(25.0) A Total 29 15(51.72) 7(24.13) 7(24.13)
Groups with same letters have non-significant difference at p<0.05 using Fisher Freeman Halton test. Low degree: 1 cyst/hpf , Moderate degree: 2-4 cyst/hpf , High degree: 5 and more than 5 cyst/hpf
Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (27-30)
29
Figure (1): Cysts of Buxtonella sulcata (10X,40X) by using digital camera.
Table (3): Relationship of Buxtonella sulcata infection with
diarrheal symptoms in cattle by using χ2 – test.
Symptoms No. of
animals
No.of +ve
B.sulcata
% of infection
Animals with diarrhea 86 27 31.39a Animals with normal faeces
34 2 5.88b
Total 120 29 24.16
Groups with different letters have significant differences at P<0.016.
Figure (2): Trophozoites of Buxtonella sulcata (10X,40X)
by using digital camera.
Discussion
In this study, the total percentage of infection with
Buxtonella sulcata was 24.16% and the infection with cysts
appeared in all positive cases while the trophozoites
appeared only in four positive cases from total of all
positive number, while other study accomplished in Iraq by
Ayaiz (11) reported that the percentage of infection was 47%. Other studies which performed in different part of
world such as England, Poland, Costa Rica, Bangalore and
Thailand (2,6,9,18,19) showed a wide differences in
percentage and ranged between 2-87%.The differences in
the percentages of infection could be due to many different
factors, such as environmental conditions, animal, farm
management practices and stress factors. Fox and Jacobs
(2) itself showed that seasonal fluctuations in the
prevalence of the infection and cyst excretion rates were
Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (27-30)
30
related to changes in the diet and opportunities for
transmission, furthermore, the delivery rate may be lead to
an increase in prevalence of infection. The shape and size
of the cysts and trophozoites which observed in this study
are in agreement with those described by (3, 8,11).
The statistical analysis of infection with Buxtonella
sulcata in our study showed a significant differences between the infection of calves, beef and dairy cattle, while
no significant differences was noticed between beef and
dairy cattle, moreover, no significant differences were
observed in the intensity of the infection between calves,
beef and dairy cattle. High rate of infection with Buxtonala
sulcata cysts appeared in animals affected with diarrhea
and which are found to have large number of cysts and
trophozoites (Table 3), the result was in agreement with
(2,6,11,19,20) who reported that B. sulcata can be one of
the causative agent of diarrhea in ruminants. The
pathogenic effect of this parasite has not been found to be
of great interest and it is suggested that more comprehensive studies should be done in order to explain
the cause of diarrhea. In an observations of Tomczuk et al.,
(6) they reported that the pathogensis of B. sulcata may be
compared with the incidence of similar ciliate Balantidium
coli living in the large intestine of pig, man and many other
mammals. In a different studies of Skotarczak and
Zielinski, Urman and Kelky, and Skotarczak (21-24) was
proved that especially significant effect of Balantidium coli
in change in the pH of large intestine content on the
intensively of invasion and the damaging effect on the
mucosa of large intestine results in a secondary bacterial infection and increase in the pathological changes.
In this study, our observation was based on detection of
the B. sulcata cysts in animals which have signs of diarrhea
and those with normal faeces. All examined positive cases
was affected with B. sulcata only, furthermore, no clinical
signs observed other than diarrhea on the affected animals
and no other parasitic agents were diagnosed, this indicates
that B. sulcata can be considered as one of the agents of
unexplained etiology of diarrhea in cattle.
Acknowledgement
This study was supported by the College of Veterinary
Medicine, University of Mosul.
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24. Skotarczak B. Bacterial flora in acute symptom-free balantidiosis.
Acta Parasitol. 1997;42:230-233.
Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (31-35)
31
Clinical, haematological and biochemical studies of babesiosis in native goats in Mosul
E. G. Sulaiman1, S. H. Arslan
2, Q. T. Al-Obaidi
2, E. Daham
2
1Department of Microbiology, 2Department of Internal and Preventive Medicine,
College of Veterinary Medicine, University of Mosul, Mosul, Iraq
(Received March 26, 2008; Accepted July 13, 2009)
Abstract
The study included examination of 175 native goats, 27 were infected with Babesia ovis, B. motasi, B. foliata and B. taylori, (recorded in Mosul for the first time) and 25 were clinically normal and served as control. Results indicated that the percentage of the infection with Babesiosis was 15.42% and the percentage of parasitemia ranged between 3.5-10.4% with a mean 6.95%, infected goats showed signs of loss of appetite, weakness, pale mucous membranes, jaundice, fever, coughing, nasal discharge, recumbency, diarrhea and haemoglobinuria. A statistically significant decrease were recorded in total red blood cells (RBC), haemoglobin concentration (Hb), packed cell volume (PCV) and platelets counts. Anemia was of microcytic hypochromic type. A statistically significant increase in erythrocyte sedimentation rate and significant increase in total white blood cells was recorded due to significant increase in lymphocyte and neutrophile count. Results of the biochemical testes indicated an increase in activity of alanin amino transferase (AST), aspartate amino transferase (ALT), total bilirubin, blood urea nitrogen and icterus index, with a significant decrease in total serum protein, albumin and globulin levels. Results also indicated the presence of Rhipecephalus ticks which were: Rh. sanguineus and Rh. turanicus. Keywords: Babesia, Goats, Hematology.
Available online at http://www.vetmedmosul.org/ijvs
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(� ��> RhipecephalusJ ��� )Rh. turanicus Rh. sanguineus.(
Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (31-35)
32
Introduction
Babesiosis is caused by intraerythrocytic protozoan
parasites of the genus Babesia, the disease which is
transmitted by hard ticks (family: Ixodidae), affect a wide
range of domestic and wild animals and occasionally
humans (1). Babesia occurs seasonally and the peaks of infection were observed in rainy season (2). Babesia forms
can vary as pear-shaped, round and elongated (3) Clinically
Babesiosis is characterized by fever, inappetence, increased
respiratory rate, muscle tremors, anemia, jaundice, body
weight loss, and hemoglobinuria in the final stages (4,5).
Anemia is very common for all infected animals,
hemoglobinuria may not observed in animals infected with
B.ovis (6) Four species of Babesia have been reported from
sheep and goats mainly consisting of one large form (B.
motasi) and three small forms (B. ovis, B. foliata and B.
tyalori) (7), while Friedhoff referred that the Babesiosis in
domestic small ruminants is due to at least three species, namely : B. ovis, B. motasi and B. crassa (8). B. ovis is less
pathogenic than B. motasi for sheep infection and cause
relatively moderate haemolytic anemia (9,10), whereas
Friedhoff considered the B. ovis is the most important
causative agent which transmitted by Rhipicephalus bursa,
R. turanicus, Hyalomma anatolicum excavatum and
probably by R. evertsi evertsi (11), whereas the known
vector of B. motasi are Haemophysalis punctata and R.
bursa and the B. motasi is more pathogenic than B. ovis in
India and northern Africa. In animls affected with Babesia
spp. The studies revealed that decrease in the total erythrocytes counts, haemoglobin concentration,packed cell
volume and platelets counts, and in biochemicals studies
showed increase in activity of alanine amino transferase,
aspartate amino transferase, total bilirubin, blood urea
nitrogen and icterus index, with decrease in total serum
protein. (12-14)
The percentage of infection with B. ovis in goats in Al-
Arich city and El-Hassanah center were 7.0% (15) whereas
the prevalence of B. ovis infection in Awassi sheep in Urfa,
Turkey was 41.02% (16). Little work has been done on
Babesia spp in Mosul, Iraq on goats, hence the present study was taken to determine the occurrence of Babesia spp
together with clinical, haematological and some
biochemical changes.
Materials and methods
A total of 175 goats, 2-5 years of age and from both
sexes were examined in the Teaching Veterinary Hospital
(College of Veterinary Medicine, University of Mosul) and
from other regions in Mosul city, for the possibility of
infection with babesiosis. Clinically normal goats (n=25)
served as control. Careful clinical examination of all suspected animal were carried out.
Blood was collected from jugular vein for
haematological examination by using (Automatic full
digital cell counter, Beckman USA) to get of total red blood
cells (RBC), haemoglobin concentration (Hb), packed cell
volume (PCV), mean corpuscular volume (MCV), mean
corpuscular hemoglobin concentration (MCHC), platelets
counts and total and differential leuckoytes count (TLC & DLC), and westergren method used for get erythrocyte
sedimentation rate (ESR) (17). Thin blood smears were
taken from the vein of the ear and stained with Giemsa
were used to identify the Babesia and the percentage of
parasitemia (18). Blood serum samples were tested spectro-
photometrically for the biochemical changes of alanine
amino transferase, aspartate amino transferase, blood urea
nitrogen and total serum protein, albumin and globulin by
using available kits (Randox, U.K.), total bilirubine using
available kits (Biomerex, France), and Icterus Index by
using potassium dichromate according to (18). Statistical
analysis were done by using t-test (19).
Results
Clinically infected goats showed different signs
graduated from loss of appetite, emaciation, pale mucous
membranes, jaundice, fever, coughing, nasal discharge,
recumbency, diarrhea and haemoglobinuria as well as
presence of ticks (Rhipicephalus sanguineus and Rh.
turanicus) were detected on different parts of the body
(Table 1).
Table (1): Clinical signs of infected goats (n=27) with
babesiosis.
Clinical signs
No. of
infected
goats
%
Pale mucous membranes 24 88.9
Loss of appetite 21 77.8
Haemoglobinuria 21 77.8
Diarrhea 16 59.3
Emaciation 15 55.6
Jaundice 13 48.1
Nasal discharge 10 37 Coughing 9 33.3
Recumbency 7 25.9
Ticks (Rhipicephalus
sanguineus and Rh. turanicus) 15 55.6
Significant increase (P<0.01) were encountered in
body temperature, respiratory and heart rates While,
ruminal contractions were reduced significantly (Table 2).
Examination of blood smear revealed four types of
Babesia in infected goats, which were: B.ovis,, B.taylori,
B.foliata and B. motasi. (Fig1). The number of goats which
Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (31-35)
33
were infected with Babesia spp was (27) with percentage
(15.42%) and the parasitemia ranged between (3.5-10.4%)
in a mean of (6.95%).
Figure (1): four types of babesia in infected goats, which
were: B.ovis (1), B.taylori
(2), B.foliata (3) and B. motas i
(4) in
blood smear.
Table (2): Body temperature, respiratory rates, heart rates
and ruminal contractions of infected goats with babesiosis.
Control Infected
Parameters Mean±S.E. Mean±S.E.
Body temperature oC 39.10±0.51 40.8±0.82**
Respiratory rate/min 26.21±3.82 53.32±7.95**
Heart rate/min 79.81±8.62 112.4±10.90**
Ruminal contraction/5 min 4.09±0.20 2.52±1.5**
** P<0.01, * P<0.05
There was a significant reduction (P<0.01) in the mean
values of TRBC, Hb, PCV, platelets count, anemia was of
Microcytic hypochromic type due to significant reduction
(P<0.01) in the Mean Corpuscular Volume (MCV) and
Mean Corpuscular Haemoglubin Concentration (MCHC).
A statistically significant increase of ESR encountered and
results also indicated a significant increase (P<0.01) in total
leukocytes count due to significant increase in neutrophils
and lymphocytes count (Table 3).
Table (3): Blood parameters of infected goats with babesiosis and control group.
Control Infected Parameters
Mean ± S.E. Mean ± S.E.
RBC x 106 microliter 6.10 ± 0.54 4.21 ± 1.15**
Hb g/100 ml 10.35 ± 2.43 6.93 ± 2.52**
PCV % 32.40 ± 3.22 26.13 ± 4.33**
MCV % 64.37 ± 5.68 61.40 ± 5.46**
MCHC g/100 ml 32.35 ± 4.33 28.31 ± 2.20**
Platelets x 103 microliter 769 ± 157 349 ± 103**
ESR mm/24 h 0.62 ± 0.43 3.94 ± 1.34**
WBC x 103 microliter 7.68 ± 2.35 10.95 ± 3.73**
Neutrophils % 61.33 ± 1.46 61.87 ± 1.02*
Lymphocytes % 37.50 ± 0.93 39.20 ± 1.31**
Monocytes % 0.20 ± 0.03 0.15 ± 0.10 Eosinophils % 0.15 ± 0.03 0.00 ± 0.00
Basophils % 0.04 ± 0.01 0.06 ± 0.01
Parasitemia --- 3.5 – 10.4% ** P<0.01, * P<0.05.
Results of biochemical examination showed a
significant increase (P<0.01) in activity of AST, ALT, as
well as in levels of total bilirubin, BUN and Icterus index,
however significant decrease were recorded in total protein,
albumin and globuline values of infected goats (Table 4).
B. ovis (round
form) at the
margin of the
red cells
(1.66-2.32)
micron with
mean 1.88
micron (1).
B.taylori
(round
appear) under
go several
fission (1.66-
1.19) micron with mean
1.74 micron
(2).
B. foliata.
round lies more
centrally in
red cells
(1.66-1.99)
micron with
mean 1.83
micron (3).
B.motasi
(pyriform stages) (2.9-
4.15) micron
with mean
3.27 micron
(4).
Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (31-35)
34
Table (4): Biochemical parameters of infected goats with
babesiosis and control group.
Control Infected Parameters
Mean ± S.E. Mean ± S.E.
AST U/L 27.13 ± 9.52 77.37 ± 6.94**
ALT U/L 23.65 ± 5.12 29.63 ± 4.59**
Total bilirubin mg/100 ml 0.26 ± 0.10 0.69 ± 0.17**
Total protein g/100 ml 6.70 ± 0.79 3.63 ± 0.53**
albumin g/100 ml 4.07 ± 0.22 2.10 ± 0.21**
Globulin g/100 ml 2.64 ± 0.57 1.27 ± 0.12*
BUN mg/100 ml 32.78 ± 7.64 68.46 ± 12.89**
Icterus index units 2.67 ± 0.39 6.31 ± 1.16**
** P<0.01, * P<0.05.
Discussion
The results of this study showed that the clinical signs
observed in infected goats were in agreement with the
results of other studies carried out by different researchers
concerning ruminants infected with babesiosis (2,3,7). The
cause of pale mucous membrane was development of anemia and decrease in erythrocyte count and haemoglobin
(12), while jaundice due to increase in the total bilirubine
(direct and indirect) and icterus index (18), the
haemoglobinuria may be due to intravascular haemolysis
and high rate of destruction of erythrocytes and
haemoglobinemia (20,21).
Two types of ticks were diagnosed in this study, they
were Rhipicephalus sanguninus and Rh. turanicus and
Friedhoff added that Babesia ovis is transmitted by Rh.
bursa, Rh. turanicus Hyalomma anatolicum excavatun and
probably R. evertsi evertsi (11), while Mazyed and Khalaf
identified Hyalomma anatolicum excavatum and Haemophysalis sulcata in infected goats with B. ovis and
Theileria ovis (15). In general the distribution of the
parasite is correlated with the distribution of tick vector
species (16). Four species of babesia (B. ovis, B. motasi, B.
foliata, B. taylori) were diagnosed in this study, which were
also described by (7,12).
The percentage of infection with Babesia spp was
(15.42%) whereas Mazyed and Khalaf recorded (7%) of
infection with B. ovis in goats (15) and in another study on
goats in a village in south west Nigeria showed (20.4%)
infection with B. motasi (2). Haematological parameters showed relatively
significant decrease in values total Red blood cells,
haemoglobin concentration, packed cell volume and
platelets counts, compared to control group, these results
were in agreement with studies carried out on sheep and
goats by others (2,12,22) and this might be due to
intravascular haemolysis of erythrocyte, increase
erythrocyte phagocytosis by reticloendothelial system and
restricted erythropoitic activity in bone marrow (20,21).
Anemia was microcytic hypochromic type due to
significant reduction in the mean corpuscular volume and
mean corpuscular haemoglubin concentration this was in
agreement with the study of (23) in foals. The increase in
ESR values refers to the correlation between the
sedimentation of RBCs and the intensity of anemia (18),
and Allen added that increase in ESR values due to decrease in Packed cell volume (24). The significant
increase in WBCs was due to increase in lymphocytes, this
was in agreement with (3) in sheep.
Biochemical parameters showed relatively significant
increase in the AST and ALT may be due to indirect
damage of liver, kidney tissue and myocardium, changes
indicated to a possible damage to the liver, kidney tissue
(25), Wright added that increase in the AST due to
distraction of RBCs (26). `
The increase in total bilirubin due to damage of liver
and increase in the indirect bilirubin was due to erythrocyte
haemolysis (18), Babesia also reported to causes nephrosis, renal ischemia, dehydration and some heart diseases that
causes increase in BUN, (26,27). The increase in the icterus
index was due to increase in Total bilirubin in serum and
dehydration (26).
The reduction in total protein, albumin and glubulin
values is might be due to decrease production from the liver
due to direct and indirect effect of parasite, digestive
distribance (diarrhea), loss of appetite and high fever
(28,29).
Acknowledgement
This study was supported by the College of Veterinary
Medicine, University of Mosul.
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Veterinary parasitology. 2nd
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34(3):637-639.
6. Popa E. Ixodid ticks vectors of Babesiosis in animals in Romania.
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7. Soulsby EJL. Helminths, Arthopods and Protozoa of domesticated
animals. 7th
ed. Philadelphia, London, Toronto. 1986.718-719p.
8. Friedhoff KT. Transmission of Babesia, In: Babesiosis of domestic
animals and man. M. Ristic(ed.) CRC. Press, Boca Raton. Florida.
1988.23-52p.
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9. Aytug CN, Alacam E, Gorgul S, Koyun Ve. Keci Hastaliklarive
Yetistiri cillige Istanbul, Tum-Vet Hayvancilik Yayini. 1990;201-202.
10. Imren HY, Sahal M. Veteriner ic Hastaliklari. Ankara. Feryal
Matbacilik San. ve Tic. Ltd. Sti. 1991;207-208p.
11. Friedhoff KT. Tick-borne diseases of sheep and goats caused by
Babesia, Theileria or Anaplasma spp. Parasitologia. 1997;39(2):99-
109.
12. Radostitis OM, Gay CC, Blood DC, Hinchcliff. Veterinary medicine,
A text of the diseases of cattle, sheep, pigs, goats and horse. 9th ed.
Philadelphia: W.B. Saunders Company; 2000.1289-1296p.
13. Arsalan SH. Clinical, haematological and biochemical studies of some
blood protozoa in dogs in ninavah. Iraqi J Vet Sci. 2005;1(19):63-77.
14. Al-Mula GM. Clinical, pathological and therapeutical studies of
Equine Babesiosis in draught horses in mosul. [master's thesis].
Collage of Veterinary Medicine: University of Mosul; 2004.
15. Mazyed SA, Khalaf SA. Studies on Theileria and Babesia infecting
live and slaughtered animals in Al-Arish and El-Hassnah, North Sinai
Governorate, Egypt. J Egypt Soc Parasitol. 2002;32(2):601-610.
16. Emre Z, Duzgun A, Iriadam M, Sert H. Seroprevalence of Babesia
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17. Meyer DJ, Harvy JW. Veterinary laboratory medicine. 2nd
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Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (37-40)
37
Effect of industrial product IMBO® on immunosuppressed broilers
vaccinated with Newcastle disease vaccine
O. G. Mohammadamin* and T. S. Qubih
Department of Pathology and Poultry Diseases, College of Veterinary Medicine, University of Mosul, Mosul, Iraq
(Received May 13, 2009; Accepted October 5, 2009)
Abstract
The effect of IMBO was investigated on humoral immune response to Newcastle disease vaccines in broiler chickens.
Haemagglutination inhibition test and enzyme-linked immunosorbent assay were used to assess the immune response. Results
showed that although IMBO significantly enhanced humoral immune response to live Newcastle disease vaccine, it did not
decrease post virulent NDV challenge mortality.
Keywords: Humoral immunity; Newcastle disease,Vaccine, IMBO.
Available online at http://www.vetmedmosul.org/ijvs
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Introduction
Severe outbreaks of Newcastle disease often occur in
areas of° intensive poultry production, which is reasoned mainly to break down in immunity. Although poor vaccine
quality is one of several possible factors that could lead to vaccination failures (1).The failure of protection usually
results from: (i) mycotoxin and/or drug induced
immunomodulation (ii) cold or heat stress (iii) infectious
agents (iv) malfunctioning of the host defense mechanism
and (v) presence of high titers of maternal antibodies (1-7).
Immunostimulation of a bird may lead to increased
antibody production, increased cellular immune responses,
* Part of MSc thesis submitted by the first auther to the College of veterinary Medicine, Mosul University.
and increased macrophage phagocytic ability which
positively correlates with enhanced resistance to various
viral and bacterial infections (8,9). Probiotics are defined as
direct feed microbials or microbial cell preparations with a beneficial effect on the health and well-being of the host
(10). Probiotic represent one of the most recent examples of
natural substances that influence adaptive immune
responses by activating the innate immune system (11), and
enhancing the systemic antibody.response to some antigens
in chickens (12). Recently, the beneficail effect of Biomin®
C-X (Enterotococcus faecium + prebiotic+cell wall extract)
on humoral immunity to Newcastle disease vaccine of
commercial broilers was studied (13). This experiment was
conductd to investigate the effect of Biomin®IMBO
(Biomin G.T.I. GmbH., Ember AG-Austria; containing Enterotococcus faecium 5×1011 cfu /kg, prebiotic, cell wall
Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (37-40)
38
and algae extracts) as a potential immunostimulator to
enhance humoral immune response to live and killed
Newcastle disease vaccines in broiler chickens.
Materials and methods
A total of 210 day-old Hubbard-Flex broiler chicks were procured from a local supplier. They were reared in cages
in a separate rooms of the animal house, College of
Veterinary Medicine, University of Mosul and fed ad
libitum with a Hubbard-Flex recommended diet. Ambient
temperature, lighting, ventilation and other environmental
conditions fully met the requirements for management of
Hubbard-Flex birds.
Biomin®IMBO
(Biomin G.T.I. GmbH., Ember AG-Austria, it contains
Enterotococcus faecium 5×1011 cfu / kg, prebiotic, cell wall
and algae extracts. IMBO was added to the feed free from antibiotics and administered throughout the study as
recommended by the manufacturer 1.5g/kg.
Drugs Cyclophosphamide (CPA) (Cycloxan® manufaured in
India, Biochem Pharmaceutical Industries LTD) was
procured from a local pharmacy. Day-old chickens of
groups G2 and G5 were given 3 mg per chicken per day for
4 consecutive days intramuscularly into leg muscle (14).
Challenge virus One day before challenge, birds in G1 splited
randomely to two halves; negative control group (G1); left
wthout challeneg and positive control group (G8) which
submitted to challenge. At 39 days of age chickens were
intramuscularly inoculated with virulent field NDV strain
(obtained from the Microbiology Department, College of
Veterinary Medicine, Mosul University). The virus titer
was determined to be 1×106.5 EID50 / 0.1ml.
Sampling On day 7(before vaccination 0, blood samples were
taken from each group to assess the maternal immunity.
Blood samples were taken at weekly intervals after
vaccination and challenge.
Serological Test Antibodies to NDV were quantitified by
hemagglunation inhibition test (HI) using the diluted
serum-constant virus procedure (15) and by indirect
Enzyme-linked Immunosorbent Assay (ELISA).
Experimental design Chickens were randomly divided to 7 groups with 30
birds each. These groups consisted of: 1) Non IMBO + Non
CPA + Non challenge referred to as negative control (G1),
2) IMBO + CPA&L-K (G2), 3) IMBO + Non CPA&L
(G3), 4) IMBO + Non CPA&L-K (G4), 5) Non IMBO +
CPA&L-K (G5), 6) Non IMBO + Non CPA&L (G6), 7)
Non IMBO + Non CPA&L-K (G7). Birds of all groups
except negative and positive controls were vaccinated with
live NDV (Cevac®Vitapest L; CEVA) at seven days old individually by oral route using 1 ml syringe. In addition,
each bird in groups G2,G4, G5, and G7 was
intramuscularly injected with 0.1 ml of killed NDV vaccine
(Cevac®Broiler NDK) at seven days of age. Revaccination
with live ND vaccine LaSota strain (Cevac® NEW
L;CEVA) was done at 21 days of age by spraying.
Results
HI titer serum antibody response According to figure (1), chicks contained maternal
antibody level before vaccination and gradually declined to low levels with time. At 28 days of age, production of
antibody detected in all groups except G1 group. The G2
(vaccine + IMBO+CPA) and G5 (vaccine + CPA) groups
produced significantly (P<0.05) lower levels of antibody in
comparison to other treatment groups indicating that
immunosuppression occurred. Furthermore, no significant
differences were found when the two groups were
compared at different time points post booster vaccination.
The data also revealed that GMT of G3 ( live vaccine +
IMBO) group was significantly (P<0.05) higher than G6
(live vaccine alone) group while GMT of G4 (live& killed vaccine + IMBO) group was statistically (P>0.05) not
different compared with G7 (live& killed vaccines).
Furthermore, when GMTs of G3 and G4 groups were
compared no significant (P>0.05) difference were found.
ELISA titer immune response The results of ELISA test are presented in figure (2). On
day 28, only birds in G3 and G7 groups showed
seroconversions, however their mean titers were not
significantly (P>0.05) different. The data also demonstrated
that the antibody titer of G3 was significantly higher (P<0.05) when compared with titer of G6, meanwhile the
titer of G4 was not significantly (P>0.05) different when
compared with G7. The titers of G2 and G5 groups
remained low and did not differ signficantly.
Mortality The post-challenge test results are shown in Table (1).
The table shows that the protection rate in G2, G3,G4, G5,
G6, G7 and G8 were 28.6, 85.71%, 96.29%, 31.8%, 83.87,
96.66%, and 0% respectively. No significant differences in
mortality were found between probiotic fed and their
corresponding control groups.
Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (37-40)
39
Fig. 1: Geometric mean HI antibody titer (log2) in chickens
with or without IMBO supplementation.
Fig. 2: Mean ELISA antibody titer in chickens with or
without IMBO supplementation.
Table 1: Response to challenge with virulent Newcastle
disease virus in chickens
Groups
No
dead/No
challenged
No
survived/No
challenged
Negative control group
(G1) 0/14
14/14 a
(100)B
IMBO,CPA &L-K vaccine
(G2) 20/28
8/28 c
(28.57)
IMBO&live vaccine
(G3) 4/28
24/28 a
(85.71)
IMBO&L-K vaccine (G4)
1/27 26/27 a
(96.29)
CPA &L-K vaccine
(G5) 15/22
7/22 c
(31.81)
Live vaccine
(G6) 5/31
26/31 a
(83.87)
L-K vaccine
(G7) 1/30
29/30 a
(96.66)
Positive control group
G8) 13/13 0b
Discussion
In this experiment, significantly higher HI and ELISA
titers were seen in birds received live NDV +IMBO(G3).
This is in agreement with finding of (13), however, IMBO
had no effect in birds immunized with live and killed
vaccine G4. The data on the effect of probiotics on immunity are extremely controversial due to the variety of
variables reported (16). More over(17) reported treating
with just one bacterial type may not be as effective and
increasing the types of bacteria in the mix could enhance
the efficacy of probiotic functions. The post-challenge
mortality rates observed in immunosuppressed (G2 and G5
groups) were higher compared with immune-competent
birds (G3, G4, G6 and G7 groups). The protection rate did
not differ significantly in G3 compared with G6 despite
enhancement of humoral immune response which
contradicts preivious report (18). The different results
might be due to twofold increase in titer observed in latter study and in addition, they challenged birds orally with
virulent NDV compared with IM challenge used in our
study, in addition Leghorn male chickens were used
compared to broilers in the present experiment. Under the
conditions of this study, IMBO significantly enhaced
humoral immune response to only live vaccine,and this is in
agreement with (19), but did not restore immunity in
immunosuppressed chickens and did not decrease post
challenge mortality in immunosuppressed and
immunocompetent broiler chickens.
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2007;25:3254-3262.
19. Emad J K and Amjad H E.Study of some Impact of Enterococcus
faecium As Probiotic on chick 2-NewCastle Disease Antibody and
White blood cells.Iraqi J Vet Med 2007;31(1):78-86.
Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (41-43)
41
Serodiagnosis of Johne's disease by indirect ELISA in ovine
I. M. Ahmed
Department of Microbiology, College of Veterinary Medicine, University of Mosul, Mosul, Iraq
(Received August 11, 2008; Accepted October 5, 2009)
Abstract
The study included collection of 92 serum samples from local Awasii breed in Mosul and Karakosh regions, some of them
show clinical signs for John's disease, all samples were assayed using indirect Enzyme-Linked Immunosorbent Assay (ELISA)
to detect antibodies against Mycobacterium avium subsp. paratuberculosis (Map). The results showed that 7/92 (7.6%)
samples were positive for antibodies against (Map), and 7/89 (7.9%) were positive in female and 0/3 (0%) in male.
Keywords: Mycobacterium, Paratuberculosis, Ovine Johne’s disease, Indirect ELISA, Serodiagnosis.
Available online at http://www.vetmedmosul.org/ijvs
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Introduction
Johne's disease is also known as "paratuberculosis"
because it is caused by Mycobacterium avium subspecies
paratuberculosis (Map). It is a contagious disease in dairy
cattle, sheep, goats and other ruminants (1). The clinical
manifestation of paratuberculosis in sheep tends to prevail
at younger age than in cattle. Chronic weight loss is the
primary clinical sign of paratuberculosis in sheep and goats.
Affected sheep will experience progressive weight loss over a period of weeks to months and eventually die (2). Only
10-20% of clinical cases present with diarrhoea or
clumping of faeces in the advanced stage of the disease (3).
Johne’s disease is commonly found in dairy cattle herds and
sheep flocks but identification of individual sheep with
subclinical infections is difficult. Animals apparently are
infected when young but, while shedding the organism via
faeces, these animals may not show clinical symptoms for
several years. Infected animals may have reduced feed
efficiency without obvious clinical signs of disease. (4,5).
In sheep flocks, the fecal culture detects less than 12 % of
clinical cases and requires up to 12 months of incubation,
making it an impractical diagnostic test (6,7). Three
different tests are currently available for measuring
antibodies against Mycobacterium avium subsp. paratuberculosis in the serum of infected animals. These are
the complement fixation test (CFT), the agar gel
immunodiffusion (AGID) test and enzyme-linked
immunosorbent assay (ELISA). ELISA or AGID are still
the main options in live animals (8). Among various
Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (41-43)
42
serological tests for Johne’s disease, ELISA-based tests are
widely used and can be conducted rapidly and require
limited expertise (9).
Materials and methods
A total of 92 blood samples, 3 samples from males and
the others from females were collected from local Awassi
breed sheep that some of them showed clinical signs for
John's disease including emaciation, unresponsive to
dewormers and antibiotics. Appetite was often good, in
spite of weight loss, all animals wer not previously
vaccinated against Johne's disease and they were >2 years
old. The samples were collected through April 2008 from
different regions, 69 samples represent 3 flocks containing
580 sheep from Mosul and 23 samples represent 2 flocks
containing 264 sheep from Karakosh. Samples were submitted to the department of microbiology (College of
Veterinary Medicine, Mosul, Iraq); within 24 h after
bleeding, serum samples were separated and stored at –
20°C until they were assayed (2,10).
A commercial ELISA kit (ID SCREEN®,
Paratuberculosis Indirect) for detection of antibodies
against Mycobacterium avium subsp. paratuberculosis in
ovine serum samples was used, the kit has been supplied
from (ID Vet (innovative diagnostics)-France). The
principle of the test depends on indirect ELISA. Sera were
tested according to the manufacturer's instructions for
ovine, the absorbance reading O.D in all ELISA plate wells were measured at 450 nm using an automated ELISA
reader. ELISA optical density (OD) readings were
transformed to Serum/Positive percentage (S/P%)
according to a specific equation cited by manufacturer. The
sample considered positive if it gives S/P % ≥ 70%, 60% <
S/P % < 70% considered doubtful, S/P % ≤ 60% considered
negative. S/P%=(OD sample-ODNC)/(ODPC-ODNC).
Results
The results showed that 7/92 samples 7.6% were positive for antibodies against Mycobacterium avium
subsp. paratuberculosis, P<0.001. The results are detailed in
table 1. Also the results showed no positive results in males
0/3 (0.0%) when compared with females 7/89 (7.9%). The
distribution of S/P % values for samples, positive control
and negative control are given in figure 1.
Table 1: Positive samples according to Mosul regions
Region No. of samples Positive %
Mosul 69 5 7.3
Karakosh 23 2 8.7
Total 92 7 7.6
Figure 1: Distribution of ELISA S/P % values for (n=92),
positive controls (PC) and negative controls (NC). ID
SCREEN®, Paratuberculosis Indirect ELISA kit, ID Vet
(innovative diagnostics).
Discussion
The aim of this study was to investigate Johne's disease
in sheep by serodiagnosis using ELISA as there are
indications for M. avium subsp. paratuberculosis infection
and unresponsive to dewormers and antibiotics. All sheep
were selected >2 years old as the clinical signs are
commonly not evident until at least 18 months of age (6).
As the cultivation of sheep strains of M. avium subsp.
paratuberculosis using culture media (Herrold's egg yolk
medium) has been extremely difficult to perform, the selection of ELISA in this study was based on the studies
by (6,12,13). Among the antibody tests, ELISA is more
sensitive than AGID and CFT test (14,15). It's performance
is similar in cattle, sheep, and goats (16,17) and can be used
with comparable sensitivity for either milk or serum
samples (18). AGID test is considered less sensitive than
ELISA and CFT (19). Since a strong humoral response
does not occur until the later stage of Johne's disease, the
sensitivity of these 3 tests is the highest for animals with
lepromatous lesions (8,20,21), those with clinical symptoms
(20,22,23), or those that shed large numbers of bacteria
(18,24). Therefore, the main limitation of these antibody tests is their inability to identify animals in early infection
(25,26). Conversely, all of these tests are highly specific,
with false-positive results occurring at low frequency (25).
The ELISA couldn't detect all animals with clinical signs.
Comparative studies of the CFT, AGID test and ELISA
repeatedly show discrepancies in the ability to identify all
infected animals (27,28). This may be due to genetic
variation of the individual animal or the lack of
representation of the entire range of immunodominant
antigens for Mycobacterium avium subsp. paratuberculosis
within a given test (29). There are few studies about ovine Johne's disease in the Arab countries, so there is no
Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (41-43)
43
sufficient data to compare with the results of this study. The
total positivity % (7.6%) is considered high as the disease
now has a virtually worldwide distribution in farmed
ruminants as well as many species of wild ruminants (1,
11), also the high S/P% for the all 7 positive sera indicate
the high levels of antibodies against (Map) in the affected
sheep (20, 22, 23), this mean that we need more investigations to confirm Johne's disease in sheep and also
in other susceptible ruminants. In this study few numbers of
male samples were included, only 3 samples were tested
and all gives negative results 0.0%, because of the breading
style in keeping few numbers of rams for each flock. This
study has resulted in detection of antibodies against
Mycobacterium avium subsp. paratuberculosis in sheep.
The study was done for the first time in Iraq to provide
information about this disease for subsequent studies.
Acknowledgments
This study was supported by College of Veterinary
Medicine, University of Mosul. Mosul, Iraq.
References
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Paratuberculosis. Clin Microbiol Rev 1994;7:328-345.
15. Collins MT. Diagnosis of paratuberculosis. Vet Clin North Am. Food
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17. Dubash K, Shulaw WP, Bech-Nielsen S, Stills HF and Slemons RD.
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between serologic response and pathologic findings in sheep with
naturally acquired paratuberculosis. Am J Vet Res 1997;58:799-803.
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present in sheep with Johne’s disease and cross-react with
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22. Dubash K, Shulaw WP, Bech-Nielsen S, Stills HF, Slemons RD.
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23. Hilbink F, West DM, DeLisle GW, Kittelberger R, Hosie BD, Hutton
J, Cooke MM, Penrose M. Comparison of a complement fixation test,
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Prowsea SJ, Michalskia WP, Butlerb KL and Jonesc SL. A long-term
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avium subsp. paratuberculosis: clinical disease, faecal culture and
immunological studies. Vet Microb. 2004;104:165–178.
27. John PB, Valentina R, Stefania Z, Stefano R, Niyaz A and Leonardo
AS. Antigenic profiles of recombinant proteins from Mycobacterium
avium subsp. paratuberculosis in sheep with Johne’s disease. Vet
Immunol Immunopatho 2008;122:116–125.
28. Darcel C and Logan-Handsaeme B. ELISA testing for antibody to
Mycobacterium paratuberculosis. Can Vet J. 1998;39:335-336.
29. Sugden EA, Stilwell K and Michaelides AA. comparison of
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Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (45-52)
45
Effect of treating lactating rats with lead acetate and its interaction with
vitamin E or C on neurobehavior, development and some biochemical
parameters in their pups
A. A. Hassan and H. M. Jassim
Department of Physiology, Biochemistry and Pharmacology, College of Veterinary Medicine, University of Mosul, Iraq
(Received February 17, 2009; Accepted October 5, 2009)
Abstract
The current study investigated the effect of administration of vitamin E (600mg/ kg diet) concomitantly with lead acetate
(10mg/kg, orally) and vitamin C (100mg/kg, orally) concomitantly with lead acetate (10mg/kg, orally) to the female lactating
rats on the neurobehavioral, landmarks development and some biochemical tests in their pups. Administration of lead acetate
to the female lactating rats caused a significant increase in open field activity test including (the number of squares crossed and
rearing test within 3 minutes), olfactory discrimination test, triglycerides and malondialdehyde brain tissue, with a significant
decrease in glutathione brain tissue and high density lipoproteins in their pups. The present study demonstrated that treatment
of female lactating rats with vitamin C and lead acetate produced a significant decrease in righting reflex test in their pups.
Administration of vitamin E concomitantly with lead acetate to the female lactating rats caused a significant increase in
glutathione level accompanied with a significant decrease in malondialdehyde and triglycerides levels in their pups. The present study showed that treatment of female lactating rats with vitamin E or C with lead acetate produced a significant
decrease in rearing test, whereas a significant increase in high density lipoproteins in their pups. It is concluded that
administration vitamin E or C to the female lactating rats reverse the adverse effects produced by lead acetate on
neurobehavioral. Vitamin E had positive effect on the levels of glutathione, malondialdehyde brain tissue, triglyceride and high
density lipoproteins in their lactating pups.
Keywords: Vitamin E, Vitamin C, Lead acetate, Neurobehavior, Glutathione, Malondialdehyde, Rat pups.
Available online at http://www.vetmedmosul.org/ijvs
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Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (45-52)
46
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Introduction
Lead is an ubiquitous element in the environment, it is
used in many industrial activities including mining, refining
and producing lead – acid batteries (1). Although this heavy
metal is less widely used today, it remains a significant
public health problem. Animals may be exposed to lead via contaminated food or water and fuel additives (2). The
alimentary and respiratory tract are the major routes of lead
entry into the body (3). Once the lead is in the bloodstream,
it is distributed into soft and hard tissues (4). Milk is the
most important food source for newborn, however, also be
a pathway of maternal excretion of toxic elements such as
lead, and these toxins impact most severely on the newborn
at a time of rapid development of the central nervous
system (5). In the nervous system, all neurons and glial
cells form a very large network, integrate all external and
internal stimuli and contributes to the elaboration of
adequate responses (6). Lead enters the brain and selectively deposited in the hippocampus and cortex, as
well as in non-neuronal elements that are important in the
maintenance of the blood brain barrier function. Lead
exposure causes distractibility inability to maintain physical
balance and it affects some complex functions including
learning (7). The neuropathlogical effects of lead include
nervousness, anxiety and symptomatic encephalopathy at
the end (8). Pervious study has shown that the level of lead
in milk are thus similar to those in plasma (5). In rodents
lead mobilized from the skeleton is transferred to the
suckling offspring during lactation (9), and that lactational transfer after current or recent exposure to lead in dams was
considerably higher than placental transfer (10). The main
targets organ of lead toxicity are the red blood cell, central
nervous system, peripheral nerves and the kidney (11).
Recent study reported that lead acetate can be
transmitted through mother milk to their offspring. Lead
acetate make bad effects on the reproductive systems of
both males and females rats. While giving vitamin E as
antioxidant found to have no improving effect in the
treatment of lead acetate disturbances on the reproductive
system of both sexes (12). Lead-induced oxidative stress
contributes to the pathogenesis of lead poisoning for disrupting the delicate prooxidant/antioxidant balance that
exists within mammalian cells. Production of reactive
oxygen species (ROS) is increased after lead treatment in
vitro studies, moreover other studies in vivo suggest that
lead exposure cause generation of ROS and alteration of
antioxidants defense system in animals (13). The aim of present study is to assess the role of
administration of vitamin E & C concomitantly with lead
acetate to the dams during lactation period (21days) on the neurobehavioral, landmarks development and some
biochemical changes in their pups.
Materials and methods
Adult healthy albino rats were obtained from the
animals house of the Veterinary Medical College –
University of Mosul at 3-4 months of age weighing 150-
200g. The rats were mated (3:1 females to male). Pregnant
rats were removed and kept in separated polyproplyne
cages under condition of temperature (22-26 C°) and
lighting (12hours light /12hours dark). The rats were
supplied a standard pellet diet and tap water ad libitum. At
birth dams were separated with their pups from the first day
of parturition.
In this experiment, animals divided into four groups:
Group 1: dams (rats during lactation period) received
normal saline orally as control group. Group2: dams
received lead acetate at (10mg/kg B. Wt.) orally during
lactation period, lead acetate dissolved in distalled water
and given at 1 ml/kg (12). Group3: dams received lead
acetate (10mg/kg B. Wt.) orally with concomitant
administration of vitamin E at (600 mg /kg diet) during lactation period (14) (Shang Hang, China). Group 4: dams
received lead acetate (10mg/kg B. Wt.) orally with
simultaneous administration of vitamin C at (100 mg /kg B.
Wt.) orally during lactation period (15) (Chemical Suppl,
South Australia).
Twenty pups from each groups were selected randomly
for examination. Pups were examined at the 7 day of age
for neural behaviour which included righting reflex, cliff
avoidance and olfactory discrimination tests (16). Central
nervous system activity test were examined at the 21 day
which include onset of movement test, open field activity
test (17). This test measures the general locomotor activity, exploration which include (the number of squares crossed
and rearing test within 3 minutes) and negative geotaxis test
Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (45-52)
47
(16). Sensimotor system activity were examined which
include approach response, touch response, click response
and tail pinch response (18). Landmarks development tests
included opening of ear, appearance of hair and teeth,
opening of eyes, descending time of testis and appearance
time of vaginal opening (16).
Blood was collected from pups at age 21 days for biochemical examination. Total cholesterol, high density
lipoproteins, glucose, alanine aminotransfererase, aspartate
aminotransfererase, and albumin, were measured using
colorimetric assay kit (Syrbio, Syria) and triglyceride was
measured using kit (bioMerieux, France), Pups weights
monitored at birth and at weaning time. Pups were
sacrificed at the end of experiment and the brain was placed
in ice normal saline for glutathione (Moron method) (19)
and malondialdehyde estimation (Gilbert method) (20).
Data were analyzed statistically using one way analysis
of variance. Group differences were determined using
Duncan multiple range test. Data of approach response, touch response, click response and tail pinch response
analyzed statistically using Mann _ whitny _ U _ test.
Statistical significance was considered at (P < 0.05) (21).
Results
In the current study Table 1- revealed that
administration of lead acetate to the dams during lactation
period did not effect the onset of movement and negative
geotaxis tests in their pups compared the pups of control
group. Treatment with vitamin E to the rats receiving lead acetate during lactation period did not effect significantly
the onset of movement and negative geotaxis tests in their
pups, but the treatment with vitamin C to the rats receiving
lead acetate during lactation period produced a significant
increase (P≤0.05) in the onset of movement test. Treatment
with each of vitamin E & C to the rats receiving lead
acetate during lactation period did not effect significantly
the negative geotaxis test in their pups compared with pups
of lead acetate group. Table 1- showed that rats receiving
lead acetate during lactation period cause a significant
increase (P≤0.05) in rearing and the number of squares
crossed tests within 3 minutes in their pups compared with
the pups of the control group. Treatment with vitamin E & C to the rats receiving lead acetate during lactation period
caused no significant changes in rearing test in their pups.
On the other hand, the number of square crossed test was
significantly decrease by vitamin E & C in pups compared
with the lead acetate group.
Table 2 shows that administration of lead acetate to the
rats during lactation period caused a significant increase in
olfactory discrimination (P≤0.05) in their pups compared
with the pups of control group.
Treatment with vitamin E & C to the rats receiving lead
acetate during lactation period did not affect significantly in
olfactory discrimination test in their pups compared with the pups of lead acetate group. No significant changes
between groups in cliff avoidance test. Same Table
demonstrated that administration of lead acetate to the rats
during lactation period did not effect significantly in
righting reflex test in their pups compared with the pups of
control values. Treatment with vitamin E of rats receiving
lead acetate during lactation period did not affect
significantly in righting reflex in their pups compared with
the pups of lead acetate group, but treatment with vitamin C
of rats receiving lead acetate during lactation period cause a
significant decrease (P≤0.05) in righting reflex test in their pups compared with the pups of lead acetate group.
The data of vitamin E & C revealed no significant
differences on sensimotor include (approach, touch, click
and tail pinch responses),landmarks development and
weighing of pups (at 21 days) from rats receiving lead
acetate during lactation period are shown in Table 3,4,5.
Table 1. The onset of movement, open field activity tests and negative geotaxis in suckling pups at(21 days) from dams treated
with lead acetate and their interaction with vitamin E or C for 21 consecutive days (lactation period).
Open field Negative geotaxis/sec square crossed with 3 mins Rearing with 3 min
Onset of movement/ sec
Treatment of dams
a
4.65±0.41 a
11.20±0.51 a
56.75±2.42 ab
2.15±0.13 control
ab
5.80±0.51 b
14.50±0.48 b
65.25±3.48 a
2.05±0.13 Lead acetate (10mg/ kg) orally
b 6.7±0.64
a 11.90±0.46
b 65.20±2.52
ab 2.30±0.14
Lead acetate (10mg/ kg) orally
+ vitamin E (600mg/ kg diet)
ab
6.05±0.29 a
12.05±0.65 ab
58.35±1.96 b
2.55±0.18 Lead acetate (10mg/ kg) orally
+ vitamin C (100mg/kg)
Values were expressed as means ± SE from 20pups per treatment.
Values with different letters are significantly different at (P≤0.05).
Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (45-52)
48
Table 2. The righting reflex,cliff avoidance and olfactory discrimination tests in suckling pups at (7 days) from dams treated
with lead acetate and their interaction with vitamin E or C for 21 consecutive days (lactation period).
Treatment of dams Righting reflex(sec) Cliff avoidance
(sec)
Olfactory discrimination
(sec)
Control b
2.95±0.13
a
9.35±0.57
a
11.75±0.42
Lead acetate (10mg/ kg) orally b
2.90±0.17
a
8.75±0.89
b
23.85±1.33
Lead acetate (10mg/ kg) orally
+ vitamin E(600mg/ kg diet)
ab
2.65±0.18
a
10.25±0.95
b
20.70±1.93
Lead acetate (10mg/ kg) orally
+vitamin C(100mg/kg)
a
2.30±0.11
a
10.30±0.82
b
20.90±1.48
Values were expressed as means ± SE from 20pups per treatment.
Values with different letters are significantly different at (P≤0.05).
Table 3. The approach, touch, click, tail pinch responses in suckling pups at (21 days) from dams treated with lead acetate and
their interaction with vitamin E or C for 21 consecutive days (lactation period).
Treatment of dams Approach response/
score
Touch response/ score
Click response/ score
Tail pinch
response/ score
Control a
1.90±0.02
a
2±0
a
2.60±0.16
a
3±0.1
Lead acetate (10mg/ kg) orally a
1.90±0.02
a
2.05±0.02
a
2.70±0.14
a
2.90±0.12
Lead acetate (10mg/ kg) orally
+ vitamin E (600mg/ kg diet)
a
1.90±0.02
a
2.05±0.02
a
2.70±0.14
a
2.95±0.13
Lead acetate (10mg/ kg) orally
+ vitamin C (100mg/kg)
a
1.95±0.02
a
2±0
a
2.85±0.1
a
3±0.02
Values were expressed as means ± SE from 20 pups per treatment.
Table 4. The landmarks development in suckling pups from dams treated with lead acetate and their interaction with vitamin E
or C.
Treatment of dams
Appearance
of
ear opening
Appearan
ce of hair
Appearan
ce of teeth
Appearance
of eye
Descending
time of testis
Appearance
of vaginal
opening
Control a
2.1±0.12
a
6.4±0.21
a
5.9±0.17
a
16.16±0.2
a
33.5±0.29
a
47.2±0.40
Lead acetate (10mg/ kg) a
2.15±0.13
a
6.7±0.16
a
5.9±0.17
a
17.15±0.26
a
32.8±0.46
a
47.5±0.36
Lead acetate (10mg/ kg)
+ vitamin E (600mg/ kg diet)
a
2.15±0.13
a
6.4±.19
a
6±0.16
a
17.3±0.17 a 33.15±0.37
a
47.0±0.24
Lead acetate (10mg/ kg)
+ vitamin C (100mg/kg)
a
2.1±0.12
a
6.6±0. 2
a
5.9±0.18
a
17.10±0.2
a 33.25±0.33
a
47.4±0.32
Values were expressed as means ± SE from 5 pups per treatment.
In the present study, Table 6 demonstrated that
administration of lead acetate to the rats during lactation
period cause a significant decrease in glutathione level with
increase in malondialdehyde in brain tissue of their pups
compared with the control group. Administration of vitamin
E to the rats receiving lead acetate during lactation period
Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (45-52)
49
produce a significant increased in glutathione and decrease
(P≤0.05)in malondialdehyde levels in their pups compared
with the pups of lead acetate group. No significant
differences in the level of glutathione and malodialdehyde
of the pups when their dams treated with vitamin C and
lead acetate concomitantly during lactation period
compared with the pups of lead acetate group. In same Table the data shows no significant differences in the levels
of alanine aminotransferase, aspartate aminotransferase and
albumin in the pups of all group.
Table 7 demonstrated that administration of lead acetate
alone or concomitantly with vitamin E & C to the rats
during lactation period did not effect significantly glucose
and cholesterol levels in their pups. Administration of lead
acetate to rats during lactation period caused a significant
increase (P≤0.05) in triglyceride level and significant
decrease in high density lipoproteins in their pups
compared with the pups of control value. Treatment with
vitamin E to the rats receiving lead acetate during lactation
period produced a significant decrease (P≤0.05) in
triglyceride and significant increase in high density lipoproteins level in their pups compared with the pups of
lead acetate group. Rats administered lead acetate with
vitamin C during lactation period did not affect triglyceride
level significantly in their pups compared with that of lead
acetate group, on the other hand vitamin C caused a
significant increase (P≤0.05) in high density lipoproteins in
pups compared with the pups of lead acetate group.
Table 5. The body weight in suckling pups from dams treated with lead acetate and their interaction with vitamin E or C for 21
consecutive days (lactation period).
Treatment of dams Weight(g) at 1 day Weight (g)at 21 day
Control a
5.46±0.02 a
25.27±0.19
Lead acetate (10mg/ kg) orally a
5.44±0.02
a
25.31±0.45
Lead acetate (10mg/ kg) orally
+ vitamin E (600mg/ kg diet)
a
5.33±0.10
a
25.06±0.15
Lead acetate (10mg/ kg) orally
+ vitamin C (100mg/kg)
a
5.43±0.02
a
25.13±0.16
Values were expressed as means ± SE from 20 pups per treatment.
Table 6. The glutathione, malondialdehyde brain tissue, alanine amimotraferase, aspartate amimotraferase and albumin in
suckling pups from dams treated with lead acetate and their interaction with vitamin E or C for 21 consecutive days (lactation
period).
Treatment of dams Glutathione
µmlg
Malodialdehy
de nm/g
Alanine
Amimotraferase
Unit/ L
aspartate
amimotraferase
Unit/ L
Albumin
g/dl
Control a
2.73±0.2
b
107±4.19
a
11.54±0.62
a
23.34±1.61
a
3.06±0.17
Lead acetate (10mg/ kg) orally c
1.19±0.17 a
181.8±8.39 a
11.16±0.99 a
22.12±0.99 a 2.97±0.11
Lead acetate (10mg/ kg) orally
+ vitamin E (600mg/ kg diet)
b
1.8±0.15
b
112±6.71
a
10.56±0.54
a
24.92±0.8
a
3.2±0.38
Lead acetate (10mg/ kg) orally
+ vitamin C (100mg/kg)
c
0.99±0.002
a
167.4±9.9
a
11.08±0.47
a
22.6±1.28
a
3.42±0.48
Values were expressed as means ± SE from 5pups per treatment.
Values with different letters are significantly different at (P≤0.05).
Discussion
The present study showed that administration of lead
acetate to rats during lactation period caused a significant
increase in the open field activity test including (rearing and
the number of squares crossed tests within 3 minutes) and
olfactory discrimination test in their pups compared to pups
of the control group.
Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (45-52)
50
The nervous system is the most sensitive target of lead
exposure. Fetuses and young animals are especially
vulnerable to the neurologic effects of lead because their
brains and nervous system are still developing and blood
brain barrier is incomplete (22). Lead neurotoxicity results
in behavioral and neurochemical alteration in neurons as a
result of changes and disruption of main structural components of the blood brain barrier, through primary
injury to astrocytes and to secondary damage of the
endothelial microvasculature (3). There are numerous
studies utilizing experimental animal models on the central
nervous system, These studies have mainly been concerned
with possible effects of lead on certain performance tasks
that might reflects a cognitive function (learning and
memory) or sensorimotor function in the infant animal
exposed to lead very early in life or in utero (23). Some
investigators studied effects of lead on the action of
neurotransmitters using isolated peripheral nerve
preparation, both cholinergic and adrenergic synaptic
evoked transmitter released is inhibited by lead, and this effect is prevented by calcium (23). Lead affects primarily
the metabolism of calcium (24), and inhibits the action of
calcium as a result lead can affect calcium-dependent
processes and interact with proteins including sulfhydryl,
amine, phosphate, and carboxyl groups (21).
Table 7. The glucose, cholesterol, triglyceride and high density lipoproteins in suckling pups from dams treated with lead
acetate and their interaction with vitamin E or C for 21 consecutive days (lactation period).
Treatment of dams glucose
mg/dl
Cholesterol
mg/dl
Triglyceride
mg/dl
High density Lipoprotein
mg/dl
Control a
102±6.5
a
71.3±4.8
b
121±4.8
a
44.1±3.9
Lead acetate (10mg/ kg) orally a
106±3.7
a
91.9±10.4
a
186.8±5.8
b
29.2±3.4
Lead acetate (10mg/ kg) orally
+ vitamin E (600mg/ kg diet)
a
104±4.3
a
71.6±5.9
b
118.2±6.3 a
42.1±2.3
Lead acetate (10mg/ kg) orally
+ vitamin C (100mg/kg)
a
106±3
a
80.5±7.8
a
196±4.8 a
40.9±4.7
Values were expressed as means ± SE from 5pups per treatment.
Values with different letters are significantly different at (P≤0.05).
The nervous system is the most sensitive target of lead
exposure. Fetuses and young animals are especially
vulnerable to the neurologic effects of lead because their
brains and nervous system are still developing and blood
brain barrier is incomplete (22). Lead neurotoxicity results
in behavioral and neurochemical alteration in neurons as a
result of changes and disruption of main structural components of the blood brain barrier, through primary
injury to astrocytes and to secondary damage of the
endothelial microvasculature (3). There are numerous
studies utilizing experimental animal models on the central
nervous system, These studies have mainly been concerned
with possible effects of lead on certain performance tasks
that might reflects a cognitive function (learning and
memory) or sensorimotor function in the infant animal
exposed to lead very early in life or in utero (23). Some
investigators studied effects of lead on the action of
neurotransmitters using isolated peripheral nerve
preparation, both cholinergic and adrenergic synaptic evoked transmitter released is inhibited by lead, and this
effect is prevented by calcium (23). Lead affects primarily
the metabolism of calcium (24), and inhibits the action of
calcium as a result lead can affect calcium-dependent
processes and interact with proteins including sulfhydryl,
amine, phosphate, and carboxyl groups (21).
Neurotoxicity may be a consequence of alterations in
cholinergic function mediated by the enzyme
acetylcholinesterase (AchE) (25).
Maged recorded that lead caused a progressive decrease in the activity of acetylcholinesterase in different brain
regions and spinal cord (26).
The enzyme inhibition is generally reached its
significance after 10 to 20 days of lead acetate intake orally
to the rabbits, such alteration in cholinergic transmission
suggests that lead is able to reach the CNS and exerts its
neurotoxic effect (26). It was supposed that oxidative stress
was one possible mechanism for lead neurotoxicity. lead–
induced oxidative stress might result from accumulation of
5-aminolevulinic acid (ALA), a potential endogenous
source of free radical, induced by inhibition of lead to ALA
dehydratase, overload of ALA seemed to be involved in the neurological disturbances, which leads to inhibition γ- aminobutyric acid (GABA) release from synaptosomes and
blocking GABA receptor (27). Nihei et al (28) have
Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (45-52)
51
reported ALA can cause oxidative stress to rats brain.
Additionally, direct interaction of lead to biological
membranes was to induce lipid peroxidation. Lead-
exposure might also induce decrease in activities of free
radical scavenging enzymes. This mainly attributed to high
affinity of lead to sulfhydryl-groups in these enzymes (29).
The result of the current study showed that administration of lead acetate to the rats during lactation
period caused a significant increase in malondialdehyde and
triglyceride with a significant decrease in glutathione in
their pups compared with the control group. Numerous
reports have documented increased lipid peroxidation
(LPO) and decreased glutathione (GsH) and superoxide
dismutase (SOD) activity in the brain homogenates of lead
treated rats (30). Furthermore, lead exposure led to
depletion of brain glutathione content, superoxide
dismutase activity, and increase in thiobarbituric acid
reactive substances (TBARS), and the activity of
glutathione S-transferase bound enzyme (31). Oxidative damage associated with lead in the brain has been proposed
as a possible mechanism of lead toxicity (31). Some
investigators revealed that lead –treated cultured aortic
endothelial cells caused increase in the production of the
lipid peroxidation products malondialdehyde and enhanced
generation of hydroxyl radical compared with control cells
which is considered as a direct cause of oxidative stress
(33). It should be noted that hydroxyl radicals are primarily
produced from sequential reduction of superoxide radical
and hydrogen peroxide radical (33). Lead-induced oxidative
stress associated with hyperglycemia suggested to contribute in the overproduction of very low density
lipoprotein (VLDL), increasing the burden of triglyceride-
rich lipoproteins on the common lipolytic pathway at the
level of lipoprotein in lipase (34).
In the current study administration vitamin E &C to the
rats receiving lead acetate during the lactation period
ameliorating the effect of lead acetate in their lactating
pups. Recent study carried out effects of lead actate at 600
p.p.m. in drinking water during pregnancy and lactation
caused significant decrease in activities of superoxide
dismutase, glutathione peroxidaes and glutathione reductase in hypothalamus, corpora quadrigemina and corpus striatum
in weaned pups (mouse) (27).
Vitamin E is necessary for the maintenance of normal
neurological structures and function, and play a role in
protecting lipid rich structures such as the brain from free
radical (35). Antioxidant /chelating action represented by
vitamin E improved the enzyme activity in the central
nervous system (25). A major contributor to non-enzymatic
protection of polyunsaturated fatty acid and low density
lipoprotein from oxidation by free radicals against lipid
peroxidation is vitamin E (35). Vitamin E as a lipid soluble,
chain breaking antioxidant (37), plays a major protective role against oxidative stress (38), and prevents the
production of lipid peroxide by scavenging free radicals in
biological membrane (39).
Previous studies have revealed that vitamin E possesses
an antioxidant activity in protecting cells from damage by
highly reactive superoxide free radicals production (35). In
the tissue of vitamin E deficient animals, it is reported that
lipid peroxidation is enhanced suggesting that vitamin E plays a role as physiological antioxidant on its chemical
properties, and prevent oxidation of low density lipoprotein
(40). While vitamin C the most abundant water- soluble
antioxidant in the body acts primarily in cellular fluid of
particular in combating free radical for caused by pollution
furthermore vitamin C help vitamin E to return to its active
form (40). Moreover, Frei (41) found that only ascorbic
acid is protectively enough to intercept oxidant in the
aqueous phase before they can attack and cause detectable
oxidative damage to lipids, as compared to many other
lipophilic and hydrophilic antioxidants. (42) revealed that
administration of vitamin E to rats during lactation period caused a positive effects on lipid profile, glutathione and
malondialdehyde in brain tissue, of their offspring.
Acknowledgements
This study was supported by the College of Veterinary
Medicine, University of Mosul.
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Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (53-58)
53
Use of saturated sodium chloride solution as a tissue fixative
A. Al-Saraj
Department of Dental Basic Sciences, College of Dentistry, University of Mosul, Mosul, Iraq
E.mail : [email protected]
(Received March 4, 2009; Accepted October 5, 2009)
Abstract
The present study was carried out to examine the capability of saturated sodium chloride solution as a fixative agent instead
of formalin which is regarded as a carcinogenic material. For this purpose 3 rabbits were used and their livers, kidneys and
spleens were exposed and removed. Neutral buffered formalin solution, saturated sodium chloride solution and distilled water
were used as fixatives for specimens obtained from the first, second and third rabbits respectively. Routine histological
technique was performed to prepare a stained histological sections for light microscopic examination. The result showed that
the tissue sections which were obtained by using sodium chloride have the same histological features and without any artifacts
when they compared with the results obtained using formalin fixation method. We conclude that the saturated sodium chloride solution can be used as a fixative agent in some circumstances when no any fixative agent is available.
Keywords: Sodium chloride, Formalin, Tissue fixative.
Available online at http://www.vetmedmosul.org/ijvs
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Introduction
As early as 400 BC Hippocrates discussed the biological
effects of mercury and alcohol as fixatives (1). The
objective of fixation is to preserve cells and tissue
constituents in as close a life-like state as possible and to
allow them to undergo further preparative procedures
without change. Fixation arrests autolysis and bacterial
decomposition and stabilises the cellular and tissue
constituents so that they withstand the subsequent stages of
tissue processing. Fixation should also provide for the
preservation of tissue substances and proteins. Therefore, it
is regarded the first step and the foundation in a sequence of
events that culminates in the final examination of a tissue
section (2).
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A large variety of fixatives is now available but no
single substance or known combination of substances has
the ability to preserve and allow the demonstration of every
tissue component. It is for this reason that some fixatives
have only special and limited applications, and in other
instances, a mixture of two or more reagents is necessary to
employ the special properties of each. The selection of an appropriate fixative is based on considerations such as the
structures and entities to be demonstrated and the effects of
short-term and long-term storage (3). Each fixative has
advantages and disadvantages, some are restrictive while
others are multipurpose. Ferdinard Blum has been credited
as the first person to use formaldehyde as a tissue fixative.
Formaldehyde, as 4% buffered formaldehyde (10%
buffered formalin), is the most widely employed universal
fixative particularly for routine paraffin embedded sections
(4).
The aim of the present work is to test the possibility of
using saturated sodium chloride solution as fixative and preserving agent during histological and pathological
procedures and compared with the results obtained by using
conventional formalin fixation.
Materials and methods Three male rabbits were randomly assigned irrespective
of age and weight into the following: First rabbit:
specimens fixed in neutral buffered formalin (positive
control rabbit). Second rabbit: specimens fixed in saturated
sodium chloride solution. Third rabbit: specimens immersed in distilled water (negative control rabbit).
Each rabbit was anaesthetized with chloroform in an air-
tighted jar, then the animal was laid down on dissecting
board. The liver, kidneys and spleen were exposed,
removed and washed by water. Specimens of 5mm
thickness of these organs were excised.
The fixation was made immediately after the removal of
the above mentioned organs for 24 hours by using the
following:
First rabbit: Neutral buffered formalin fixative solution which
composed of: Formaldehyde 37%, 100 ml. Distilled water
900 ml. Sodium phosphate monobasic (NaH2PO3), 4 gm.
Sodium phosphate bibasic (Na2HPO3), 6.5 gm.
Second rabbit: Saturated sodium chloride solution.
Third rabbit: Distilled water.
Then procedure of preparing the paraffin section slides
and staining by Harrie`s Haematoxylin and Eosin (H&E)
was performed to prepare a stained histological sections for
light microscopic examination (5).
Results
Liver: fixed in neutral buffered formalin There were no tissue artifacts such as shrinkage,
precipitate and swelling, therefore the liver appears to be
divided clearly into many lobules, each one is hexagonal in
shape with central vein located at its center. The portal canal which consist of a bile duct, a branch of hepatic artery
and a tributary of the portal vein, all appeared clearly and
enclosed in a common investment of connective tissue (take
pink color). The paranchymal tissue of the liver appeared to
be arranged in one or two cell thickness plates converging
from the periphery to the center of lobule and the sinusoids
are intermingled in between these plates. The cytoplasm
appeared to be acidophilic and the nucleus basophilic and
located at the center of the cells. Some hepatocytes
appeared to contain more than one nucleus, and each
nucleus contains a prominent nucleolus (Fig. 1 and 2).
Liver: fixed in saturated sodium chloride solution The sodium chloride solution was preserve the tissue
and cells without any shrinking or swelling and without
distorting or dissolving cellular constituents. Therefore the
liver lobule architecture is similar to those of the control
group. The central vein being located at the center of the
hexagonal liver lobule. The hepatocytes appeared normal
and arranged in form of plates. The cytoplasm appeared to
be acidophilic while the nuclei basoplilic and some of
hepatocytes were binucleated, this indicates that the tissue
sections were taken the stained in good manner and without any precipitate (Fig. 3 and 4).
Liver: immersed in distilled water There was no section appear under microscope.
Kidney: fixed in neutral buffered formalin The cortex of the kidney showed clear cellular details of
its all structures (renal corpuscles, convoluted and straight
tubules) and stain well by (H &E) stain. There was no
shrinkage or swelling of cells. While the medulla of the
kidney also showed clear cellular details of it all structures (straight portions of tubules, thin segments of Henel`s loop
and collecting tubules). There were no tissue artifacts
(shrinkage, precipitate and swelling) and all of sections
appeared to be stained well (Fig. 5 and 6).
Kidney: fixed in saturated sodium chloride solution Sections taken from kidneys of this rabbit revealed that
the general structure of the kidney is nearly similar to that
of the control group and seemed to be normal i.e. preserved
cortex and medulla architecture without any morphological
changes of the cells such as swelling and shrinkage or any
artifacts. Also the cellular details appeared clearly and stained well (Fig. 7 and 8).
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55
Figure 1: Photomicrograph of the liver of rabbit fixed in
neutral buffered formalin showing hepatocytes (H) and the
sinusoids (S) (H&E X400).
Figure 2: Photomicrograph of the liver of rabbit fixed in
neutral buffered formalin showing the small bile duct (B),
the terminal branch of the portal vein (V) and the terminal
branch of hepatic artery (A) in the portal tract (H&E X400).
Figure 3: Photomicrograph of the liver of rabbit fixed in
sodium chloride solution showing hepatocytes (H) and the
sinusoids (S) (H&E X400).
Figure 4: Photomicrograph of the liver of rabbit fixed in
sodium chloride solution showing the small bile duct (B)
and the terminal branch of the portal vein (V) in the portal
tract (H&E X400).
Figure 5: photomicrograph of the kidney (cortex) of rabbit
fixed in neutral buffered formalin showing the glomerulus
(G), proximal (P) and distal (D) convoluted tubules (H&E
X400).
Figure 6: photomicrograph of the kidney (medulla) of
rabbit fixed in neutral buffered formalin showing the
straight segment of the proximal tubule (SP) and the
straight segment of the distal tubule (SD) (H&E X400).
Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (53-58)
56
Figure 7: photomicrograph of the kidney (cortex) of rabbit
fixed in sodium chloride solution showing the glomerulus
(G), proximal (P) and distal (D) convoluted tubules (H&E
X400).
Figure 8: photomicrograph of the kidney (medulla) of
rabbit fixed in sodium chloride solution showing the
straight segment of the proximal tubule (SP) and the
straight segment of the distal (SD) tubule (H&E X400).
Figure 9: photomicrograph of spleen of rabbit fixed in
neutral buffered formalin showing the white (w) and the red (R) pulps (H&E X100).
Figure 10: photomicrograph of spleen of rabbit fixed in
neutral buffered formalin showing the central artery (A) of
the white pulp and the sinusoids (S) of the red pulp. (H&E
X400).
Figure 11: photomicrograph of spleen of rabbit fixed in
sodium chloride solution showing the white (W) and the red
(R) pulps (H&E X100).
Figure 12: photomicrograph of spleen of rabbit fixed in
sodium chloride solution showing the central artery (A) of
the white pulp and the sinusoids (S) of the red pulp. (H&E
X400)
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Kidney: immersed in distilled water There was no section appear under microscope
Spleen: fixed in neutral buffered formalin Histological section throughout the spleen showed a
well defined cellular structures arranged into white (splenic
nodules and central artery) and red (splenic cords and sinuses) pulps without any artifacts (Fig. 9 and 10).
Spleen: fixed in saturated sodium chloride solution Microscopically, there was a preserved normal
histological sections architecture nearly similar to those of
control group without any morphological (shrinkage or
swelling) changes. The well defined histological structures
indicates well stained and preserved sections (Fig. 11 and
12).
Spleen: immersed in distilled water There was no section appear under microscope.
Discussion
A systematic study of the fixatives began in the latter
half of the 19th century, however, it must be noted that
fixation by itself introduces a major artifact. Much attention
was focused on developing fixatives that would preserve
cells and tissue constituents in as close a life-like state as
possible while allowing them to undergo further preparative
procedures without change (6).
The mechanisms by which fixatives act may be broadly categorized as dehydrants, heat effects, cross-linkers, and
effects of acids and combinations of these. Agents that
combine with proteins are called additives and those that
precipitate proteins are called coagulants. At this time, it is
accepted that no one fixative fulfils all of the aims of cell or
tissue preservation: namely prevention of autolysis and
preservation of physical and chemical properties of the
tissue (7).
In aldehydes include formaldehyde (formalin) the tissue
is fixed by cross-linkages formed in the proteins,
particularly between lysine residues. This cross-linkage does not harm the structure of proteins greatly, so that
antigenicity is not lost. Formalin penetrates tissue well, but
is relatively slow and the standard solution is 10% neutral
buffered formalin. A buffer prevents acidity that would
promote autolysis and cause precipitation of formol-heme
pigment in the tissues (8).
Formalin is used for all routine surgical pathology and
autopsy tissues when (H and E) slide is to be produced. It is
the most forgiving of all fixatives when conditions are not
ideal, and there is no tissue that it will harm significantly.
Most clinicians and nurses can understand what formalin is
and it smells bad enough that they are careful handling it.
However previous workers have indicated that exposure
to formaldehyde has a serious effects and is highly
suspected to be a human carcinogenic (9,10).
A method to overcome the problems of formaldehyde is
to use an alternative fixative that is better suited for the
preservation of tissues.
A study conclude that the cheap saturated table salt solution can be used as an alternative to the formaldehyde
in preserving the bodies of animals (11).
Fixation of skin and lymph node fragments in anhydric
sodium chloride at room temperature for periods of weeks
or months was found to preserve morphological structures
and immunoreactivity (12).
Segments of rat's aorta were harvested, immersed in
pulverized dehydrated sodium chloride, and stored for 1
week to 3 months. Thereafter, they were desalinated and
transplanted. The tensile strength and maximum
intraluminal pressures did not significantly differ from
freshly harvested, transplanted aortic segments (13). Human skin fragments can be preserved in anhydric
sodium chloride at room temperature for periods of weeks
or months and successfully transplanted, retaining normal
morphological structure (14).
The result of the present study showed that the saturated
sodium chloride solution can be used as a fixative for tissue
in pathological or histological procurers. The tissue sections
which obtained have the same histological features when
they compared with the results obtained using conventional
formaldehyde fixation methods. It penetrates tissue but is
relatively slow and prevents autolysis as well as it cause no precipitation of pigment in the tissues nor morphological
changes (swelling or shrinkage) of the cells.
The exact mechanism by which the sodium chloride act
on the tissue is unclear and no one mentioned its precise
action on the tissue. Therefore it need more investigations
and can be classified as unknown mechanism fixative, such
as mercuric chloride, picric acid.
We suggest that this new (cheap and safe) method of
fixation can be considered as an alternative fixative
procedure which can be used in emergency cases when no
any fixative is available (for pathologist, veterinarian, biologist…etc) in such cases for tissue preservation.
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