Pelotas, 2007
João Rodrigo Gil de los Santos
Construção e avaliação de vacinas de toxina α recombinante de Clostridium perfringens A
UNIVERSIDADE FEDERAL DE PELOTAS Programa de Pós-Graduação em Biotecnologia
Agrícola
Tese
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1
JOÃO RODRIGO GIL DE LOS SANTOS
Construção e avaliação de vacinas de toxina α recombinante de Clostridium perfringens A
Orientador: Carlos Gil Turnes
Pelotas, 2007
Tese apresentada ao Programa de Pós-Graduação em Biotecnologia Agrícola daUniversidade Federal de Pelotas, comorequisito parcial à obtenção do título deDoutor em Ciências (área de conhecimento:Doenças Infecciosas de Animais).
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Dados de catalogação na fonte: Ubirajara Buddin Cruz – CRB-10/901 Biblioteca de Ciência & Tecnologia - UFPel
G463c Gil de los Santos, João Rodrigo
Construção e avaliação de vacinas de toxina α recombi-nante de Clostridium perfringens A / João Rodrigo Gil de los Santos ; orientador Carlos Gil Turnes. – Pelotas, 2007. – 82f. : il. – Tese (Doutorado). Programa de Pós-Graduação em Bio-tecnologia Agrícola. Centro de Biotecnologia. Universidade Federal de Pelotas. Pelotas, 2007.
1.Biotecnologia. 2.Enterite necrótica aviar. 3. Clostridium
perfringens A. 4.Toxina α recombinante. 5.Vacinas. I.Gil Tur-nes, Carlos . II.Titulo.
CDD: 615.372
3
Banca examinadora:
Prof. Dr. Fabricio Rochedo Conceição, Fundação Universidade do Rio Grande
Prof. Dr. Itabajara da Silva Vaz Jr, Universidade Federal do Rio Grande do Sul
Dr. Ricardo Alfredo Soncini, Sadia SA
Prof. Dr. Carlos Gil Turnes, Universidade Federal de Pelotas
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AGRADECIMENTOS
A Deus, por tudo o que Ele me ofereceu.
Ao meu pai, que além de amigo, aconselha-me pelos caminhos da vida e
orienta-me na carreira profissional, estando sempre disposto a ensinar-me.
A minha mãe, cujos conselhos foram essenciais para a conclusão do curso.
A minha futura esposa, Rafaela, cuja compreensão, amizade e amor
sustentaram a realização deste trabalho.
Ao amigo Fabricio Rochedo Conceição, sempre disposto a ensinar-me.
Aos amigos Alceu e Rodrigo, que cuidaram dos animais utilizados nos
experimentos.
A todos que de alguma forma contribuíram.
Muito Obrigado
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RESUMO
GIL DE LOS SANTOS, João Rodrigo. Construção e avaliação de vacinas de
toxina α recombinante de Clostridium perfringens A. 2007. 82 f. Tese
(Doutorado) - Programa de Pós - Graduação em Biotecnologia Agrícola.
Universidade Federal de Pelotas, Pelotas.
A Enterite Necrótica Aviar (ENA) é uma enterotoxemia aguda, causada pelos
Clostridium perfringens A e C, cujo controle baseia-se na adição de antibióticos na
ração. A restrição dessa prática pelo mercado consumidor, que tornou seu controle o
maior desafio para o setor avícola, exigiu a adoção de novas estratégias para o
controle, entre elas a imunização. Vacinas recombinantes vêm despertando grande
interesse entre pesquisadores e empresas do setor. O objetivo deste trabalho foi
elaborar vacinas de toxina α recombinante de C. perfringens (rAT) utilizando como
adjuvantes Al(OH)3 (rAT+Al(OH)3) e subunidade B recombinante da enterotoxina
termolábil de Escherichia coli (rLTB) (rAT+rLTB), e construir e avaliar uma proteína
quimérica contendo rAT fusionada a rLTB (rLTB-AT). A rAT+Al(OH)3 foi inócua e
protetora contra agressão de toxina α nativa (sT) em camundongos, e imunogênica
em frangos de corte, sem afetar a produtividade. A rAT+rLTB demonstrou relação
dose-proteção em camundongos, entanto a rLTB-AT não protegeu camundongos
contra agressão de sT. A rAT demonstrou ser uma alternativa para controlar a ENA.
Palavras-chave: Enterite Necrótica Aviar, Clostridium perfringens A, toxina α
recombinante, vacinas.
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ABSTRACT
GIL DE LOS SANTOS, João Rodrigo. Construction and evaluation of Clostridium
perfringens A recombinant α toxin vaccines. 2007. 82 f. Tese (Doutorado) -
Programa de Pós - Graduação em Biotecnologia Agrícola. Universidade Federal de
Pelotas, Pelotas.
Avian Necrotic Enteritis (NE) is an acute enterotoxaemia caused by Clostridium
perfringens A and C. The control of the disease is based on antibiotics added to
animal feed. The ban of this practice by consumer markets, considered the biggest
challenge to industrial aviculture, demanded the adoption of other alternatives for its
control, among others, immunization with recombinant vaccines. The aim of this work
was to produce and evaluate C. perfringens recombinant α toxin (rAT) vaccines
adjuvanted with either Al(OH)3 (rAT+Al(OH)3 or recombinant B subunit of the heat-
labile enterotoxin of Escherichia coli (rLTB) (rAT+rLTB), and a chimeric protein
containing the α toxin fused to rLTB (rLTB-AT). The rAT+Al(OH)3 was innocuous and
protected mice against a challenge with native α toxin (sT), and it was immunogenic
and did not affect productivity parameters in broilers. The rAT+rLTB showed a dose-
protection relationship in mice, while rLTB-AT did not protect mice against sT
challenge. The rAT could be an alternative for controlling NE.
Key words: Avian Necrotic Enteritis, Clostridium perfringens A, recombinant α toxin,
vaccines.
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SUMÁRIO
1 INTRODUÇÃO 7
2 ARTIGO 1: Enterite Necrótica Aviar 10
3 ARTIGO 2: Evaluation of Clostridium perfringens recombinant α toxin vaccines
in mice 32
Summary 33
Introduction 34
Material and Methods 35
Results 40
Discussion 45
References 47
4 ARTIGO 3: Evaluation of Clostridium perfringens recombinant α toxin vaccines in
broilers 52
Summary 53
Introduction 54
Material and Methods 55
Results 58
Discussion 61
References 64
5 CONCLUSÕES 68
6 ATIVIDADES FUTURAS 68
7 REFERÊNCIAS BIBLIOGRÁFICAS 69
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1 INTRODUÇÃO
Atualmente a carne de frango está em primeiro lugar na produção animal no
Brasil. No primeiro trimestre de 2007 foi abatido mais de 1 bilhão de frangos, o que
representa mais de 2 milhões de toneladas de produto, com um aumento no abate
de 2,1% em relação ao mesmo período de 2006. Comercializaram-se no mercado
externo 697,2 mil toneladas de frangos no primeiro trimestre de 2007, com aumento
de 14,3% com relação ao mesmo período de 2006 e 18,2% no faturamento (IBGE,
2007).
Parte desses índices foi obtida utilizando antimicrobianos adicionados às
rações animais, prática comum e necessária na avicultura industrial, para controlar
doenças e manter ou melhorar índices de produtividade. O Clostridium perfringens
sorotipo A, um dos cinco sorotipos da espécie, agente etiológico da gangrena
gasosa em humanos e animais, de infecções alimentares em humanos (ROBERTS,
1959) e da Enterite Necrótica Aviar (ENA) (PARISH, 1961), é facilmente controlado
por essa prática. Porém, a União Européia baniu, a partir de 2006, a utilização de
antibióticos promotores de crescimento nas rações animais (COUNCIL OF
EUROPEAN UNION, 2003), comprometendo a eficiência dos sistemas intensivos de
produção animal. Uma das doenças mais afetadas por essa medida é a ENA
(KALDHUSDAL & LOVLAND, 2002), um problema freqüente (HOFACRE et al.,
1998) e economicamente importante (ENGSTRÖM et al., 2003) em vários países. A
doença é apontada como um dos maiores desafios para o futuro da avicultura
mundial após a restrição ao uso de antibióticos (PHILLIPS, 2002; SESTI, 2002;
KALDHUSDAL, 2003).
8
A ENA é uma enterotoxemia aguda, que se apresenta em forma clínica ou
subclínica (VAN IMMERSEEL et al., 2004) causada por Clostridium perfringens A e
C. Caracteriza-se por lesões ulcerativas e necrose confluente da mucosa do
intestino delgado e debilidade que se apresenta rapidamente, e afeta principalmente
animais entre duas e cinco semanas de idade, aparecendo subitamente, geralmente
associada à imunossupressão, provocando morte rápida com elevada prevalência
(SCHOCKEN-ITURRINO & ISHI, 2000). Embora os casos de ENA não sejam
reportados às autoridades sanitárias, é conhecido seu impacto negativo na produção
avícola. Estimou-se que nos Estados Unidos o custo dessa doença é de mais de U$
0,05 por animal (VAN DER SLUIS, 2000), podendo o produtor ter prejuízo de até
33% na produção, devido à piora da conversão alimentar, redução do peso vivo e
aumento na condenação de carcaças (LOVLAND & KALDHUSDAL, 2001).
O controle e a prevenção da ENA foram baseados, durante as últimas
décadas, na administração de antibióticos, como promotores de crescimento, na
ração. Entretanto, a restrição a essa prática lançou o desafio de criar novas
estratégias para controlar a doença. Várias alternativas vêm sendo estudadas em
todo o mundo, dentre elas, as vacinas, especialmente as recombinantes, de
produção mais simples e controlável que as tradicionais, vêm despertando grande
interesse entre pesquisadores e empresas do setor. Embora, animais que
sobreviveram à doença desenvolvam imunidade (PRESCOTT, 2000), não há
vacinas eficazes comercialmente disponíveis (KULKARNI et al., 2007).
C. perfringens A produz como antígeno maior a toxina α, uma fosfolipase letal
para camundongos, dermonecrótica e com ação hemolítica, e como antígenos
menores leucocidina, fibrinolisina e hialuronidase, entre outros (STERNE &
WARRACK, 1964). Diversas vacinas baseadas na toxina α nativa ou recombinante
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do C. perfringens foram testadas com resultados variáveis. A clonagem da toxina α
em Escherichia coli (LESLIE et al., 1989) e o desenvolvimento de vacinas com
toxina α recombinante (WILLIAMSON & TITBALL, 1993; SCHOEPE, et al, 2001;
STEVENS et al., 2004) estimularam pesquisadores a utilizar essa tecnologia para
desenvolver vacinas capazes de controlar a ENA.
A fim de induzir imunidade protetora, as vacinas clostridiais necessitam de
adjuvantes tais como hidróxido de alumínio, alume, ou a recentemente desenvolvida
subunidade B recombinante da enterotoxina termolábil de E. coli (rLTB), que provou
ser um eficiente adjuvante de mucosa estimulando respostas sistêmicas e
secretórias (VERWEIJ et al, 1998), e pode ser conjugada a antígenos por fusão
genética (CONCEIÇÃO et al., 2006).
Hipótese
Toxina α recombinante de C. perfringens (rAT) produzida a partir de genes de
cepas autóctones é imunogênica e protege frente à agressão de toxina α nativa.
Objetivos
Produzir rAT a partir de genes de cepas isoladas de surtos ocorridos no
Brasil.
Elaborar vacinas utilizando como adjuvantes Al(OH)3 e rLTB.
Construir e avaliar uma proteína quimérica contendo a rAT fusionada a rLTB.
Avaliar inocuidade e potência das vacinas em camundongos.
Avaliar inocuidade e imunogenicidade das vacinas em frangos de corte.
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2 ARTIGO 1
ENTERITE NECRÓTICA AVIAR
(Revisão submetida à Ciência Rural)
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ENTERITE NECRÓTICA AVIAR
Avian Necrotic Enteritis
João Rodrigo Gil de los Santos1, Fabricio Rochedo Conceição2, Carlos Gil-Turnes3
RESUMO
A Enterite Necrótica Aviar (ENA) é uma enterotoxemia aguda que aparece
subitamente e provoca morte rápida afetando principalmente animais jovens. Embora seu
impacto negativo na produção, devido ao aumento da conversão alimentar e condenação de
carcaças, seja já conhecido, questões relacionadas à etiologia, patogenia e controle desta
importante enfermidade necessitam de maiores esclarecimentos. Nos últimos anos o controle
da ENA baseou-se na aplicação de antibióticos na ração animal, prática banida pelo mercado
consumidor, que exigiu o desenvolvimento de novas estratégias de controle. Esta revisão
aborda informações sobre a etiologia, epizootiologia, patogenia, diagnóstico e controle da
doença, em especial a utilização de probióticos e vacinas como alternativas de controle da
ENA.
Palavras-chave: Enterite Necrótica Aviar, Clostridium perfringens A, probióticos, vacinas.
ABSTRACT
Avian Necrotic Enteritis is an acute enterotoxaemia that appears suddenly producing
rapid deaths, affecting mainly young animals. Although the negative impact of the disease in
poultry production, due to an increase on food conversion and carcass condemnations, is
1Programa de Pós-graduação em Biotecnologia Agrícola, Centro de Biotecnologia. Universidade Federal de Pelotas, Campus Universitário s/n, 96010-900, Pelotas, RS, Brasil. E-mail: [email protected]
2Departamento de Patologia, Fundação Universidade Federal do Rio Grande. 3Faculdade de Veterinária e Centro de Biotecnologia, Universidade Federal de Pelotas.
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known for years, factors related to etiology, pathogeny and control of this important disease
need better clarifications. For long time its control was based on the use of antibiotics in
poultry feed, whose use was banned by several consumer markets, requiring the development
of new control strategies. Informations on the etiology, epizootiology, pathogeny, diagnosis
and control are reviewed, emphasizing the role of probiotics and vaccines as control
alternatives.
Key words: Avian Necrotic Enteritis, Clostridium perfringens A, probiotics, vaccines.
INTRODUÇÃO
No primeiro trimestre de 2007 a carne de frango ocupou o primeiro lugar na produção
animal no Brasil. Nesse período foi abatido mais de 1 bilhão de frangos, representando mais
de 2 milhões de toneladas de carcaças, sendo comercializados no mercado externo 697,2 mil
toneladas, com um aumento de 14,3% com relação ao mesmo período de 2006 (IBGE, 2007).
Parte dos altos índices de produtividade do setor avícola foi obtida com a utilização de
antibióticos na ração animal. Porém, o marcado incremento da resistência bacteriana,
incluindo o C. perfringens (JOHANSSON et al., 2004; MARTEL et al., 2004), à grande
maioria dos antibióticos, foi relacionado a seu uso indiscriminado como promotores de
crescimento. Além disso, C. perfringens oriundo de frangos de corte também pode ser
transmitido a humanos através da cadeia alimentar (VAN IMMERSEEL et al. 2004), como
foi comprovado por SINGH et al. (2005), que isolaram a bactéria de 70,4% de amostras de
carne de frango obtidas em lojas de varejo na Índia. Esses fatos, entre outros, levaram a União
Européia a banir, a partir de 2006, o uso de antibióticos como promotores de crescimento nas
rações animais (COUNCIL OF EUROPEAN UNION, 2003), comprometendo a eficiência
dos sistemas intensivos de produção animal, sustentada em grande parte na utilização de
antibióticos como promotores de crescimento ou como preventivos de doenças infecciosas.
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Uma das doenças mais afetadas por essa medida é a ENA (KALDHUSDAL & LOVLAND,
2002), um problema freqüente (HOFACRE et al., 1998) e economicamente importante
(ENGSTRÖM et al., 2003) em vários países.
A ENA é uma enterotoxemia aguda, que se apresenta em forma clínica ou subclínica
(VAN IMMERSEEL et al., 2004) causada por Clostridium perfringens A e C. Caracteriza-se
por lesões ulcerativas e necrose confluente da mucosa do intestino delgado e debilidade que
se apresenta rapidamente. Afeta principalmente animais jovens, entre duas e cinco semanas de
idade, aparecendo subitamente, geralmente associada à imunossupressão, provocando morte
rápida com elevada prevalência (SCHOCKEN-ITURRINO & ISHI, 2000). Embora os casos
de ENA não sejam reportados às autoridades sanitárias, é conhecido seu impacto negativo na
produção avícola. Estimou-se que nos Estados Unidos o custo dessa doença é de mais de U$
0.05 por animal (VAN DER SLUIS, 2000), podendo provocar prejuízos de até 33% na
produção, principalmente devido ao aumento da conversão alimentar, redução do peso vivo e
aumento na condenação de carcaças devido a Colangio-hepatite (LOVLAND &
KALDHUSDAL, 2001).
O controle e a prevenção da doença foram baseados durante as últimas décadas na
administração de antibióticos na ração, prática comum e necessária para manter e melhorar os
índices de produtividade e competitividade.
ETIOLOGIA
A ENA é causada pelo C. perfringens, bactéria gram positiva, anaeróbia, esporulada e
toxigênica. C. perfringens é classificado em cinco sorotipos em função das toxinas maiores
(HATHEWAY, 1990; PETIT et al., 1999). Os sorotipos A e C, produtores de toxina α e α e β
respectivamente, são considerados os agentes causadores da doença (BABA et al., 1997;
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LOVLAND & KALDHUSDAL, 2001), embora a participação de ambos ainda não seja
totalmente esclarecida. ENGSTRÖM et al. (2003) detectaram por PCR o gene codificador da
toxina α (cpa) exclusivamente em 53 isolados oriundos de intestino e fígado de frangos na
Suécia, indicando que todos eles pertenciam ao sorotipo A. NAUERBY et al. (2003)
encontraram o mesmo resultado em 279 isolados de frangos de corte de 25 granjas da
Dinamarca, assim como JOHANSSON et al. (2004) em isolados de frangos de corte, galinhas
poedeiras e perus, de granjas da Suécia e Dinamarca, utilizando testes bioquímicos e PCR
multiplex. HEIKINHEIMO & KORKEALA (2005) analisaram por PCR multiplex 118
isolados de conteúdo intestinal de frangos de corte, e encontraram que todos possuíam o cpa,
mas não os genes codificadores das toxinas β (cpb), ε (etx), ι (iA) e enterotoxina (cpe), que
caracterizam os outros sorotipos de C. perfringens, indicando que todos os isolados
pertenciam ao sorotipo A. Resultados semelhantes foram encontrados por
GHOLAMIANDEKHORDI et al. (2006), sugerindo que o agente causador da ENA é o C.
perfringens sorotipo A.
EPIZOOTIOLOGIA
C. perfringens é um patógeno oportunista (FIORENTIN, 2006) presente no intestino
das aves e no ambiente, inclusive na água (SARTORI et al., 2006). Coloniza o animal nos
primeiros dias de vida, e causa doença principalmente em animais de duas a cinco semanas de
idade. Coccidiose tem sido considerada como um importante fator predisponente da ENA
(VAN IMMERSEEL et al., 2004). CRAVEN et al. (2001a) encontraram as maiores
concentrações da bactéria em fezes de animais com duas e quatro semanas de vida, decaindo
na sexta semana. Isolaram a bactéria de paredes de aviários, ventiladores, comedouros,
bebedouros, armadilhas para insetos e botas de operadores, com maior freqüência na
15
primavera e verão, demonstrando que a estrutura do aviário e seus equipamentos, assim como
o incubatório, podem ser fontes de infecção de C. perfringens para aves (CRAVEN et al.,
2001b).
Fatores ambientais tais como qualidade da cama, densidade populacional e local de
criação, têm grande importância na multiplicação da bactéria e, conseqüentemente, são
considerados fatores de risco para a ENA. OMEIRA et al. (2006) avaliaram as características
microbiológicas de camas em diferentes sistemas de produção e observaram que poedeiras
(0,25m2/ave) e frangos de corte (0,1m2/ave) em sistema intensivo, apresentaram
concentrações menores de C. perfringens que criações não confinadas (0,33m2/ ave)
sugerindo que animais em contato com o solo ingerem maior número de esporos da bactéria
que animais criados exclusivamente sobre cama em ambiente controlado. McDEVITT et al.
(2006) relacionaram o aumento da incidência de ENA à maior densidade animal, devido ao
aumento da concentração de esporos na cama, associado a sua baixa qualidade (umidade e
altos níveis de compostos nitrogenados), além do risco de dispersão por contato direto ou
aerossol.
Ingredientes da ração também foram relacionados à doença (DEKICH, 1998).
KALDHUSDAL e SKJERVE (1996) comunicaram que o milho atuou como fator de proteção
e, cevada e trigo como fatores de risco para a doença. Observações semelhantes foram feitas
por ANNETT et al. (2002) ao comprovar in vitro que as concentrações da bactéria em meio
tioglicolato contendo sobrenadantes não digeridos de cevada e trigo foram maiores que com
milho, sugerindo que rações a base de cevada e trigo estimulariam a multiplicação do C.
perfringens no trato gastrintestinal das aves.
A concentração e as características das proteínas das rações também foram
consideradas fatores predisponentes da ENA. DREW et al. (2004) comprovaram aumento da
16
concentração de C. perfringens A no íleo e ceco de galinhas quando a concentração de
proteína crua de farinha de peixe foi incrementada de 230 para 400g kg-1, não ocorrendo o
mesmo em animais alimentados com ração a base de proteína de soja, sugerindo que tanto o
nível de proteína crua quanto à fonte protéica utilizados na composição de rações afetam a
multiplicação de C. perfringens no intestino. DAHIYA et al (2005) relacionaram os efeitos
das concentrações de glicina na ração sobre a população de C. perfringens no intestino e as
lesões de ENA em frangos de 28 dias de idade desafiados com o microrganismo, constatando
que as maiores concentrações da bactéria no ceco foram obtidas com rações contendo de 3,3 a
3,9%, e que as lesões intestinais variavam de grau 0 a 4 nos grupos que receberam 3 e 4% de
glicina.
Insetos também poderiam veicular o agente. DHILLON et al. (2004) reportaram um
surto de ENA em galinhas poedeiras de uma granja recém construída. Constataram a presença
de moscas no conteúdo do papo dos animais mortos e nos comedouros, e isolaram C.
perfringens de macerados de moscas capturadas nos galpões afetados. Os autores sugerem
que o surto foi conseqüência da ingestão do C. perfringens presente nas moscas ou suas
secreções, considerando esses insetos vetores mecânicos na transmissão da bactéria.
VITTORI et al. (2007) isolaram C. perfringens de 100% de 40 amostras de besouros adultos
Alphitobius diaperinus (“Cascudinho”) capturados em granjas avícolas industriais de
Descalvado e Sertãozinho, SP, Brasil, sugerindo que o Cascudinho pode ser um vetor
potencial da veiculação do C. perfringens.
PATOGENIA
A ENA é causada pela ação de toxinas produzidas quando, em condições favoráveis,
há rápida multiplicação de C. perfringens no intestino delgado (SCHOCKEN-ITURRINO &
17
ISHI, 2000; THOMPSON et al., 2006). As lesões características da ENA são produzidas pela
toxina α (WILLIAMS, 2005), a qual vem sendo associada com a doença (TITBALL et al.,
1999), sendo considerada o principal fator de patogenicidade da bactéria (DAHIYA et al.,
2006; KEYBURN et al., 2006; THOMPSON et al., 2006). A toxina, que destrói a membrana
celular de enterócitos devido a sua propriedade de fosfolipase C (STERNE & BATTY, 1975),
é uma metalofosfolipase que possui dois domínios, o C-terminal, que penetra na membrana
celular sendo responsável pela fixação da proteína na célula, e o N-terminal, que desempenha
a função enzimática propriamente dita e hidrolisa os fosfolipídios das membranas celulares
separando as porções polar e apolar, formando di-acil-glicerol e ácido fosfatídico, provocando
a lise da membrana celular (SAKURAI et al., 2004).
HOFSHAGEN & STENWIG (1992) demonstraram que C. perfringens isolados de
casos de ENA produziram títulos maiores de toxina α que cepas isoladas de animais sadios.
Reforçando o conceito de que esta toxina é o principal fator de patogenicidade na ENA,
HEIER et al. (2001) observaram que lotes de frangos de corte com altos títulos de anticorpos
maternos anti-toxina α apresentaram menores índices de mortalidade que animais com baixos
títulos. Entretanto, a participação da toxina α de C. perfringens na patogênese foi questionada
por KEYBURN et al. (2006), que desafiaram frangos de corte com bactérias cujos genes de
toxina α foram deletados, observando que as lesões eram similares às produzidas pela bactéria
selvagem.
KULKARNI et al. (2006), procurando identificar antígenos exclusivos a cepas
patogênicas de C. perfringens envolvidos na patogênese, identificaram anticorpos contra seis
proteínas (uma de 190 kDa; Piruvato-ferridoxina-oxidoredutase; Fator G; Perfringolisina O;
Gliceraldeido-3-fosfato dehidrogenase e Bifosfato aldolase), em frangos sobreviventes à
agressão, sugerindo sua participação na patogênese da ENA, ainda que os soros desses
animais também possuíssem anticorpos contra a toxina α. THOMPSON et al. (2006), visando
18
também avaliar a participação de outros antígenos na ENA, compararam as taxas de lesões
produzidas em frangos por cepas de C. perfringens não produtoras e uma cepa produtora de
toxina α, comprovando que não mais de 20% apresentaram lesões nos primeiros frente a
100% nos controles positivo, sugerindo que a toxina desempenha um papel importante na
patogenia da doença.
DIAGNÓSTICO
Em sistemas industriais de produção avícola a identificação da forma sub-clínica da
doença torna-se difícil por não haver testes adequados, ainda que LOVLAND &
KALDHUSDAL (1999) associaram a hepatite por C. perfringens a outras doenças
relacionadas à bactéria, sugerindo que o exame de carcaças no abatedouro poderia auxiliar a
monitorar a ocorrência da ENA. Na forma clínica, porém, é possível fazer o diagnóstico em
função da epidemiologia, frangos de corte com idade de duas a cinco semanas de idade,
intervenção de fatores imunossupressores tais como alterações bruscas de temperatura, falhas
no sistema de arraçoamento, alta densidade populacional, outras doenças, mortalidade alta (de
5 a 15% do lote), curso rápido sem sinais clínicos e alterações patológicas tais como
hiperemia e lesões ulcerativas na mucosa do intestino delgado e cecos, que se apresentam
friáveis, distendidos, com coloração esverdeada escura, fétidos e com gases.
O desenvolvimento de uma ELISA para quantificar toxina α (HALE & STILES,
1999), sugeriu que seria possível diagnosticar as formas clínica e sub-clínica da ENA.
LOVLAND et al. (2003) utilizaram esta técnica para detectar anticorpos anti-toxina α em
frangos de corte, constatando a presença de altos títulos em 59% a 79% dos animais
provenientes de lotes com alta prevalência de lesões hepáticas e intestinais associadas a C.
perfringens, e só em 27% dos animais provenientes de lotes com baixa incidência de lesões.
19
Resultados similares foram obtidos por McCOURT et al. (2005) mediante a utilização de
ELISA para detectar células de C. perfringens e toxina α em conteúdo intestinal de frangos.
Os testes apresentaram uma sensibilidade entre 102 e 106 UFC mL-1 de cepas isoladas de casos
clínicos, e de 60ng mL-1 de toxina. As soroconversões de animais aparentemente saudáveis
foram inferiores a 4 entanto as dos afetados foram superiores a 10. Ainda que os testes
demonstrassem ser eficientes, o fato que as amostras utilizadas para detecção do antígeno
provinham do conteúdo intestinal, inviabilizando a amostragem de animais vivos, impediu a
aplicação dessa metodologia devido à necessidade de sacrificar uma amostra significativa de
animais do lote, inaceitável na avicultura industrial.
CONTROLE
O controle da ENA foi baseado, nas últimas décadas, na administração de antibióticos
na ração (ENGBERG et al., 2000; BRENNAN et al., 2001; KNARREBORG et al., 2002;
BRENNAN et al., 2003). A interdição de seu uso como promotores de crescimento, decretada
pela União Européia (COUNCIL OF THE EUROPEAN UNION, 2003) lançou o desafio de
criar novas estratégias para controlar a doença. Surgiu então o interesse em avaliar métodos
alternativos tais como o uso de prebióticos (TAKEDA et al., 1995; SILVA & NORNBERG,
2003; MCREYNOLDS et al., 2007), enzimas (JACKSON et al., 2003; ZANG et al., 2006),
alimentos funcionais (MITSCH et al., 2004), probióticos (LA RAGIONE et al., 2004;
BARBOSA et al., 2005; KIZERWETTER-SWIDA & BINEK, 2005) e vacinas. Tanto o setor
industrial quanto o acadêmico tem demonstrado grande interesse no uso dos dois últimos para
o controle da ENA.
20
Probióticos
Probióticos são suplementos alimentares compostos de microrganismos vivos que
beneficiam a saúde do hospedeiro através do equilíbrio da microbiota intestinal (FULLER,
1989; KAUR et al., 2002). Sua aplicação na indústria avícola vem sendo amplamente
estudada nos últimos anos, tanto para o controle de doenças quanto por seu efeito na
eficiência alimentar (GIL de los SANTOS & GIL-TURNES, 2005).
HOFACRE et al. (1998) observaram que o produto Aviguard®, constituído por flora
polibacteriana de aves, reduziu a incidência de ENA em frangos de corte. LA RAGIONE &
WOODWARD (2003) comprovaram que a administração de esporos viáveis de Bacillus
subtilis a aves livres de patógenos específicos desafiadas com C. perfringens, reduziu o
numero de patógenos no baço, duodeno, cólon e ceco, e relataram resultados similares com
um probiótico de Lactobacillus johnsonii (LA RAGIONE et al., 2004). TEO & TAN (2005),
por sua vez, demonstraram que Bacillus subtilis inibiu o crescimento de C. perfringens em
cultivo associado. HAGHIGHI et al. (2006) demonstraram que um probiótico comercial
contendo Lactobacillus acidophilus, Bifidobacterium bifidum, e Streptococcus faecalis
estimulou a produção de IgA anti-toxina α de C. perfringens no intestino de pintos não
vacinados.
Vacinas
Aves que sobreviveram à ENA ficaram imunes, sugerindo que a imunidade pode
controlar a doença (PRESCOTT, 2000). LOVLAND et al. (2004) demonstraram pela primeira
vez que progênies de matrizes imunizadas com um toxóide de C. perfringens A apresentaram
títulos de antitoxinas significativamente superiores aos controles, assim como menor
porcentagem de animais com lesões de ENA. A produção industrial de toxóides de C.
perfringens, porém, é um processo laborioso. A clonagem do gene da toxina α em
21
Escherichia coli (LESLIE et al., 1989) e a produção de vacinas contendo toxina α
recombinante (WILLIAMSON & TITBALL, 1993; SCHOEPE et al., 2001; STEVENS et al.,
2004; SCHOEPE, 2006) abriram uma nova perspectiva de produção industrial de antígenos
de Clostridium.
KEYBURN et al. (2007) vacinaram animais com a toxina α nativa e com proteínas
recombinantes secretadas de C. perfringens (piruvato-ferridoxina-oxidoredutase;
gliceraldeido-3-fosfato dehidrogenase; frutose 1,6-bifosfato aldolase e uma proteína
hipotética), comprovando que todas as vacinas apresentaram efeito protetor frente a um
desafio moderado, e que, frente a desafio severo, a toxina α apresentou o melhor resultado,
reafirmando sua importância tanto na patogenia do C. perfringens, quanto na imunidade a
esse patógeno. Entretanto, ao tentarem produzir a toxina α recombinante, concluiram que
pareceria ser tóxica para E. coli. Nosso grupo, porém, logrou produzir toxina α recombinante
em E. coli que protegeu camundongos frente a desafio com mais de 10 DL50 de toxina nativa
e induziu soroconversão em pintos vacinados aos 7 dias de idade (GIL de los SANTOS,
2007).
CONCLUSÃO
A avicultura industrial, que durante várias décadas dependeu da utilização de
antibióticos nas rações animais, necessita manter e melhorar os índices de eficiência alimentar
bem como controlar doenças após a proibição de seu uso como promotores de crescimento. O
controle da ENA é considerado um dos maiores desafios para o setor. Entre as alternativas
para consegui-lo, poderão ser utilizados probióticos, que mantém o equilíbrio da microbiota
do trato gastrintestinal de aves, previnem infecções, reduzem condenações de carcaças e a
mortalidade, melhoram a conversão alimentar, o ganho de peso e a qualidade das carcaças,
22
conservando os índices de produtividade alcançados com a utilização de antimicrobianos, e
vacinas, especialmente as recombinantes, de produção mais simples e controlável que as
tradicionais. Nutrição adequada e o controle de fatores imunossupressores, conjuntamente
com um eficiente programa de biosseguridade, são essências para o êxito do controle da ENA.
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32
3 ARTIGO 2
EVALUATION OF Clostridium perfringens RECOMBINANT α TOXIN
VACCINES IN MICE
(Artigo a ser submetido à Vaccine)
33
Evaluation of Clostridium perfringens recombinant α toxin vaccines
in mice
João Rodrigo Gil de los Santosa, Fabricio Rochedo Conceiçãob*, Carlos Gil-Turnesc
aCentro de Biotecnologia, Universidade Federal de Pelotas, Brazil.
bDepartamento de Patologia, Fundação Universidade Federal do Rio Grande, Brazil.
cFaculdade de Veterinária e Centro de Biotecnologia, Universidade Federal de Pelotas, Brazil.
*Corresponding author: Fundação Universidade Federal do Rio Grande, Departamento de Patologia.
Rua General Osório s/n, FURG - Campus Saúde, Centro, 96200-190, Rio Grande-RS, Brazil. E-mail:
[email protected]. Phone: + 55 53 32330318.
Summary
Clostridium perfringens serotype A is the etiological agent of Avian Necrotic
Enteritis and immunization is one of the most promising alternatives for its control.
Vaccines of C. perfringens recombinant α toxin (rAT) were tested in mice and
compared with a vaccine prepared with standard toxin (sT). Mice were vaccinated at
8 and 11 weeks of age, and their antibodies measured by ELISA. Vaccinated animals
and controls were challenged with 10 LD50 of sT. Western blots showed that rAT
reacted with antibodies elicited by sT and vice-versa. In the potency test rAT+Al(OH)3
was as effective as sT, while the vaccines containing recombinant subunit B of the
Escherichia coli heat-labile enterotoxin (rLTB) as adjuvant showed a dose/protection
relationship, and the chimera did not protect. The recombinant α toxin vaccine is a
candidate to control of Avian Necrotic Enteritis.
34
Key words: Avian Necrotic Enteritis, Clostridium perfringens serotype A,
recombinant vaccines.
1. Introduction
Clostridium perfringens serotype A, one of the five serotypes of the species, is
the etiological agent of gas gangrene in humans and animals, Avian Necrotic
Enteritis (NE) and food poisoning in humans [1], diseases that are successfully
controlled by several antibiotics used therapeutically or prophylactically. The disease
produces high mortality in chicken 2 to 5 weeks old [7], and a reduction in
productivity in older animals estimated in more than U$ 0.05 per animal in the USA
[8], with losses of 33% on the production due mainly to decrease in live weight and
increase in feed conversion and carcass condemnation [9]. Since its discovery [2] it
was controled adding antibiotics to poultry feed, but since 2006 the European Union
[3] banned their use as feed additives, bringing a new challenge to poultry industry
[4, 5, 6].
Various alternatives to NE control were studied all around the world. Although
animals that survived the disease developed immunity [10], there are not effective
vaccines commercially available [11]. C. perfringens serotype A produces the major
antigen α toxin that is lethal for mice, dermonecrotic and has phospholipase and
hemolytic properties, and the minor antigens leucocidin, fibrinolysin and
hyaluronidase, among others [12]. Several vaccines based on native or recombinant
C. perfringens α toxin were tested with variable results. Cloning of α toxin in
Escherichia coli [13] and the development of recombinant α toxin vaccines for other
purposes [14,15,16], stimulated researchers to use this technology in the production
of candidate NE vaccines. In order to induce protective immunity, clostridial vaccines
35
need adjuvants such as aluminum hydroxide, alum or the recently developed
recombinant subunit B of the E. coli heat-labile enterotoxin (rLTB), that proved to be
an efficient mucosal adjuvant stimulating systemic and secretory antibody responses
[17, 18].
The objective of this work was to produce and evaluate vaccines based on
recombinant C. perfringens α toxin to be used in NE control.
2. Material and Methods
2.1. Bacterial strains, culture conditions and DNA extraction
A strain of C. perfringens isolated from a NE field case was grown in Cooked
Meat Medium (CMM) overnight at 37ºC. Genomic DNA was extracted by methods
described by Sambrook and Russel [19]. E. coli Top10F (Invitrogen) was used for
cloning procedures and E. coli BL21 DE3 pLYS (Invitrogen) used for expression of
the recombinant proteins. These strains were grown at 37ºC in Luria-Bertani (LB)
medium with 150 µg mL-1 of ampicilin.
2.2. Cloning, expression and purification of the recombinant proteins
To obtain recombinant α toxin (rAT) and recombinant LTB (rLTB) the α toxin
and LTB genes were amplified by PCR and the products cloned into vector pAE [20],
generating the pAE/AT and pAE/LTB plasmids, respectively. To obtain recombinant
chimeric protein containing the α toxin fused to rLTB (rLTB-AT), the α toxin gene was
cloned in frame with the C-terminal region of the LTB gene into pAE/LTB, generating
36
the pAE/LTB-AT plasmid. All cloning procedures were carried out according to
standard procedures [19]. The expression of the recombinant proteins by E. coli
BL21 DE3 pLYS was induced with 0.4 mM of Isopropyl-ß-D-thiogalactopyranoside
(IPTG), during 3 h, after the absorbance at 600 nm reached 0.6-0.8. Pellets of the
cultures were obtained by centrifugation at 6000×g for 10 min at 4°C, suspended in
lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 15 mM Imidazole and 1 mg mL-1 of
lysozyme) and sonicated. After centrifugation at 10000×g for 30 min at 4°C, the
supernatants were purified by affinity chromatography in a nickel-charged Sepharose
column using the ÄKTAprime chromatography system (GE Healthcare,
Buckinghamshire, UK) following manufacturer’s instructions. The eluted solutions of
purified recombinant proteins were dialyzed overnight in PBS pH 7.4, concentrated
with sugar, quantified with the BCATM Protein Assay Kit (Pierce, Rockford, IL, USA)
following manufacturer’s instructions and the purity was assessed by SDS-PAGE.
2.3. Characterization of recombinant proteins
The denaturated purified recombinant proteins were separated in a 12% SDS-
PAGE and electrotransferred to a 0.45 µm nitrocellulose membrane (Hybond-C
Extra, Amersham Biosciences UK Ltd., Buckinghamshire, UK). After transfer the
membrane was blocked for 1 h with 5% non fat dry milk and incubated by another
hour with 1:2000 peroxidase conjugated anti-6×His MAb (Sigma-Aldrich), 1:2000
rabbit IgG anti-CT (Sigma-Aldrich) or 1:200 anti-α toxin mouse polyclonal serum
produced with C. perfringens phospholipase C type I (sT) (Product Nº P7633, Sigma-
Aldrich Inc., St. Louis, USA). Goat anti-rabbit peroxidase conjugated antibodies
(Sigma) or goat anti-mouse polyvalent immunoglobulins peroxidase conjugate
37
(Sigma) (1:2000) were used as reposted. The immunoreactive protein bands were
visualized with DAB-chromogen substrate (9 mL Tris-HCl 50 mM, 1 mL nickel
sulphate 0.3%, 10 µL of 30% hydrogen peroxide and 6 mg 3.3-diaminobenzidine
tetrahydrochloride). Blue Step Low Range Protein Marker (Amresco Inc., OH, USA)
was used as molecular mass standard.
The lecithovitelinase and dermonecrotic activity of rAT were evaluated by the
method described by Sterne and Batty [21].
2.4. Vaccines and immunization
Isogenic Balb-c male mice were divided in 11 groups of 4 animals each. The
animals received feed and water ad libitum. The proteins and doses injected to the
groups are shown in table 1. The animals were injected twice with an interval of 21
days, intramuscularly, being the first dose at 8 weeks of age.
38
Table 1: Vaccines and doses of each group.
Groups Protein Adjuvant Dose (µg)
1 sT Al(OH)3 25
2 sT Al(OH)3 50
3 sT rLTB 25
4 sT rLTB 50
5 rAT Al(OH) 3 25
6 rAT Al(OH) 3 50
7 rAT rLTB 25
8 rAT rLTB 50
9 rLTB-AT None 25
10 rLTB-AT None 50
Control --- --- ---
The doses of rLTB-AT were adjusted to obtain 25 and 50 µg rAT. The dose of
rLTB (7.3 µg) was the correspondent to the rLTB-AT.
2.5. Antibody response
Serum from each animal was obtained at days 0, 21, 35 and 49 days after the
first dose of vaccine (av). Antibody titers were evaluated by an Enzyme Linked
Immuno-Sorbent Assay (ELISA). Polystyrene microtiter plates (CRAL Artigos para
39
Laboratórios Ltda, Cotia, SP, Brazil) were coated with 0.5 µg of sT or rAT suspended
in carbonate-bicarbonate buffer pH 9.6, incubated overnight at 4ºC. Mouse sera
diluted 1:50 were added in duplicate to the wells and incubated for 1.5 h at 37ºC.
After washing with PBS-T, goat anti-mouse polyvalent immunoglobulins peroxidase
conjugate (Sigma-Aldrich) (1:2000) was added and incubated for 1.5 h at 37ºC. After
washing with PBS-T, OPD-chromogen substrate (1 mL Phosphate-citrate Buffer pH
4.0, 1 µL 30% hydrogen peroxide, 0.4 mg ortophenyle diamine). Absorbancies were
measured in an ELISA spectrophotometer at 492nm (Dynatech MR 700). Titres are
expressed as seroconversions, dividing the mean of the respectives absorbencies by
that of the same group at the day of the first vaccination.
The denaturated sT was separated in a 12% SDS-PAGE and
electrotransferred to a 0.45 µm nitrocellulose membrane (Hybond-C Extra,
Amersham Biosciences UK Ltd., Buckinghamshire, UK). After transfer the membrane
was blocked by 1 h with 5% non fat dry milk and incubated by another hour with
1:200 anti-α toxin mouse polyclonal serum produced with sT, rAT and rLTB-AT. Goat
anti-mouse polyvalent immunoglobulins peroxidase conjugate (Sigma) (1:2000) were
used as regarded. The immunoreactive protein bands were visualized with DAB-
chromogen substrate (9 mL Tris-HCl 50 mM, 1 mL nickel sulphate 0.3%, 10 µL of
30% hydrogen peroxide and 6 mg 3.3-diaminobenzidine tetrahydrochloride).
2.6. Potency test
Vaccinated mice were challenged by the intra-venous route (iv) with 10 LD50 of
sT, in accordance with the requirements for provisional licensing of C. perfringens α
toxin vaccines of the United States Department of Agriculture [22]. LD50 of sT and rAT
40
were determined in Balb-c mice of the same age, inoculated by the iv or
intraperitoneal (ip) routes with decimal dilutions of the respective toxin, and
calculated by the Reed & Muench method.
2.7 Statistical analysis
The significance of the differences of absorbancies means was estimated by
ANOVA using the Statistix software 8 version (Analytical Software, Tallahassee, FL,
USA).
3. Results
3.1. Cloning, expression and purification of the recombinant proteins
All the recombinant proteins were successfully produced in E. coli (Figure 1).
The molecular mass of the constructs were 13.8, 43.7 and 56.5 kDa for the rLTB, rAT
and rLTB-AT, respectively.
Figure 1: 12% SDS-PAGE of rLTB, rAT and rLTB-AT. M, Blue Step Low Range
Protein Marker; 1, rTLB; 2, rAT; 3, rLTB-AT.
120 ―94 ―
47 ―
37 ―
28 ―
19 ―
M 1 2 3 kDa
41
3.2. Properties of rAT
The sequence of rAT showed an identity of 99% or more with C. perfringens α
toxin gene of 100 references (GenBank). The molecular mass of the construct was
43.7 kDa. rAT showed phospholipase and dermonecrotic properties. Mouse LD50 of
sT was 10.4 µg and 22.4 µg (corresponding to 298 µg kg-1 and 628 µg kg-1 of live
weight) when inoculated by the iv or ip routes, respectively, and that of the
recombinant protein was 21.4 µg by the iv route (corresponding to 610 µg kg-1 of live
weight).
3.3. Immune response and potency test
Twenty four h after the first dose of vaccine, 75% of the animals of groups 2
and 3 and 100% of group 4, died. Seroconversions and protection indices in mice 49
days after the first dose of vaccine are shown in table 2. Antibodies elicited by sT
vaccine reacted with rAT and rLTB-AT (Figure 2) and antibodies elicited by rAT and
rLTB-AT vaccines, reacted with sT (Figure 3).
42
Table 2: Seroconversion of mice 49 days after the first dose of vaccine, determined
by ELISA with sT or rAT and protection indices.
Groups Seroconversion*
sT / rAT
Protection
(%)
sT+Al(OH) 3 25 µg 5.2a / 3.5ab 100
rAT+Al(OH)3 25 µg 3.2ab / 2.4a 100
rAT+Al(OH)3 50 µg 1.5ab / 2.7a 100
rAT+rLTB 25 µg 2.0bc / 3.5a 33
rAT+rLTB 50 µg 1.5b / 2.3ab 50
rLTB-AT 25 µg 1.4cd / 2.6b 0
rLTB-AT 50 µg 1.1bc / 1.7ab 0
Control 1.4d / 1.0c 0
*same letters in the same column mean no significant differences of the
absorbencies at α=0.05.
43
Figure 2: Western blot of pool of group 1 sera against sT, rAT and rLTB-AT. M, Blue
Step Low Range Protein Marker; 1, sT; 2, rAT; 3, rLTB-AT; 4, rLTB; 5, 6xHis
recombinant protein (negative control).
Figure 3: Western blot of sera of vaccinated groups against sT. M, Blue Step Low
Range Protein Marker; 1, vaccine 1, 0 av; 2, vaccine 1, 49 av; 3, vaccine 5, 0 av; 4,
vaccine 5, 49 av; 5, vaccine 7, 0 av; 6, vaccine 7, 49 av; 7, vaccine 9, 0 av; 8,
vaccine 9, 49 av.
At day 49 the higher seroconversions against rAT were those of groups 1
(sT+Al(OH)3) and 7 (25 µg of rAT+rLTB), followed by groups 5 and 6 (rAT+Al(OH)3)
and 9 (25µg Chimera) (Figure 4). Using sT as antigen in ELISA, the higher
seroconversions were those produced by vaccine 1 followed by vaccines 5 and 6
(Figure 5). Excluding both groups vaccinated with rAT+Al(OH)3, where a booster
M 1 2 3 4 5kDa
M 1 2 3 4 5 6 7 8kDa
120 ―
94 ―
47 ―
120 ― 94 ―
47 ―
37 ―
28 ―
19 ―
37 ―
44
effect was not produced, in the other groups the booster effect of the second dose
almost doubled the seroconversions.
Mice vaccinated with native or recombinant toxin using Al(OH)3 as adjuvant,
resisted a challenge of 10 LD50 of sT. Animals vaccinated with rAT co-administered
with LTB showed a dose/protection relationship, as shown by 50% protection of the
animals inoculated with 50 µg of antigen and 33% by those inoculated with 25 µg
(Table 2).
0123456
0 21 35 49
Days
Ser
ocon
vers
ion
Control sT+Al(OH)3 25 µg rAT+Al(OH)3 25 µgrAT+Al(OH)3 50 µg rAT+rLTB 25 µg rAT+rLTB 50 µgrLTB-AT 25 µg rLTB-AT 50 µg
Figure 4: Seroconversions of mice determined with rAT.
45
0123456
0 21 35 49
Days
Sero
conv
ersi
on
Control sT+Al(OH)3 25 µg rAT+Al(OH)3 25 µgrAT+Al(OH)3 50 µg rAT+rLTB 25 µg rAT+rLTB 50 µgrLTB-AT 25 µg rLTB-AT 50 µg
Figure 5: Seroconversion of mice determined with sT.
4. Discussion
Since the description of NE by Parish [2], the development of efficient control
measures were intended by several researchers. The use of antibiotics as feed
additives proved to be the more reliable control measure of those tested, but this
practice was questioned for the first time by the Swann Committee [23], that called
attention on the risks it could have for both human and animal populations. Recently,
the European Community [3] banned the use of antibiotics in animal feeds, and
several other countries shall adopt the same restriction in the near future, challenging
the productivity of poultry industry.
Heier et al. [24] reported that flocks with high titres of maternal antibodies
against α toxin had lower mortality during the production period than flocks with low
titres. However, until 2004, when Lovland et al. [25] showed that a C. perfringens A
toxoid adjuvanted with Al(OH)3 induced significant levels of antibodies in breeders
46
that could suggest a protective effect on their progeny, there were no effective
vaccines to control the disease.
Kulkarni et al. [11] reported that an immunization scheme for breeders using
two doses of C. perfringens α toxoid followed by a booster of active toxin, protected
broilers against a severe challenge. The authors used a toxoided version of a
commercially available toxin, and reported that they were not able to obtain
recombinant toxin because the toxin seemed to be toxic for transformed E. coli,
impairing its production by this technology.
The industrial production of C. perfringens α toxin is a laborious process.
Cloning the gene in Escherichia coli [13] and the production of vaccines with
recombinant toxins [14, 15, 16, 26] opened a new perspective to overcome the
difficulties of obtaining this antigen from Clostridium cultures.
We cloned C. perfringens α toxin in E. coli and obtained yields of at least 1475
mouse protecting doses per L from transformed cultures. The recombinant toxin
preserved the lecithinase and dermonecrotic properties of the native toxin. The LD50
of the recombinant toxin was at least twice that of the standard toxin and was
innocuous for mice injected with twice the quantity of standard toxin that killed 100%
of the animals. Western blots of sera from animals vaccinated with the recombinant
toxin reacted with the standard toxin; on the other hand, sera from animals
vaccinated with the standard toxin reacted with the recombinant toxin, showing
antigenic homology between both.
Our results showed the effect of different adjuvants in protection. Mice
vaccinated with Al(OH)3 adjuvanted toxin survived the severe challenge prescribed
by the USDA [22], but when it was co-administered with E. coli LTB was not as
47
effective, showing a relationship between protection and antigen dose. A chimeric
protein containing the α toxin fused with LTB did not protect mice.
Due to the difficulties in reproducing NE experimentally, potency tests consider
the possibility to license provisionally vaccines that elicit antitoxins. The USDA [22]
considers that a vaccine that induces an antitoxin response of at least 4 IU of
antitoxin may be licensed provisionally, and if it does not reach that threshold it may
be licensed if more than 80% of the vaccinated animals survive a challenge of 10
LD50 that kills at least 80% of the controls. The vaccines containing 25 and 50 µg of
our recombinant α toxin adjuvanted with aluminum hydroxide protected mice
challenged by the iv route with 10 LD50 of standard toxin, attaining this requirements
and being candidates to be subjected to field trials.
Acknowledgements
This research was financed by CNPq grant Nº 474509/2004-4 and FAPERGS grant
Nº 05.2329.9. Luciano Pinto did the sequencing; Rodrigo C. Cunha and Alceu G. dos
Santos Jr. assisted in the experiments with animals.
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[2] Parish WE. Necrotic enteritis in the fowl (Gallus gallus domesticus). 1.
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[3] Council of European Union. Council Regulation on the authorization of the
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[9] Lovland A, Kaldhusdal M. Severely impaired production performance in broiler
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[10] Prescott J. Vaccine-Based Control of Necrotic Enteritis of Broiler Chickens.
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[13] Leslie D, Fairweather N, Pickard D, Dougan G, Kehoe M. Phospholipase C and
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Microbiology 1989;3(3):383-392.
[14] Williamson ED, Titball RW. A genetically engineered vaccine against the alpha-
toxin of Clostridium perfringens protects mice against experimental gas gangrene.
Vaccine 1993;11(12):1253-1258.
[15] Schoepe H, Pache C, Neubauer A, Potschka H, Schlapp T, Wieler LH, et al.
Naturally Occurring Clostridium perfringens Nontoxic Alpha-Toxin Variant as a
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Potential Vaccine Candidate against Alpha-Toxin-Associated Diseases. Infection and
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[16] Stevens DL, Titball RW, Jepson M, Bayer CR, Hayes-Schroer SM, Bryant AE.
Immunization with the C-domain of α-toxin prevents lethal infection, localizes tissue
injury, and promotes host response to challenge with Clostridium perfringens. Journal
of Infectious Diseases 2004;190:767-773.
[17] Verweij WR, Haan L, Holtrop M, Agsteribbe E, Brandst R, van Scharrenburg
GJM, Wilschut J. Mucosal immunoadjuvant activity of recombinant Escherichia coli
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[18] Conceição FR, Moreira AN, Dellagostin OA. A recombinant chimera composed
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[21] Sterne M, Batty I. Pathogenic clostridia. London: Butterworths, 1975.
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[22] United States Department of Agriculture. Conditional Licenses for Products
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occurring specific antibodies against Clostridium perfringens alpha toxin in
Norwegian broiler flocks. Avian Diseases 2001;45(3):724-732.
[25] Lovland A, Kaldhusdal M, Redhead K, Skjerve E, Lillehaugal A. Maternal
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alphatoxin. Anaerobe 2006;12(1):44-48.
52
4 ARTIGO 3
EVALUATION OF Clostridium perfringens RECOMBINANT α TOXIN
VACCINES IN BROILERS
(Artigo a ser submetiodo à British Poultry Science)
53
Evaluation of Clostridium perfringens recombinant α toxin vaccines
in broilers
J.R. GIL de los SANTOS1, F.R. CONCEIÇÃO2*, C. GIL-TURNES3
1Centro de Biotecnologia, Universidade Federal de Pelotas, Pelotas, RS, Brazil.
2Departamento de Patologia, Fundação Universidade Federal do Rio Grande, Rio Grande, RS, Brazil.
3Faculdade de Veterinária e Centro de Biotecnologia, Universidade Federal de Pelotas, RS, Brazil.
Summary
Vaccines containing Clostridium perfringens recombinant α toxin (rAT)
adjuvanted with Al(OH)3 or recombinant subunit B of the E. coli heat-labile
enterotoxin (rLTB), and a recombinant chimeric protein of rAT fused to rLTB (rLTB-
AT) were tested in broilers. The immune response and the effect on live weight at 42
days of age were compared to that of controls. The highest seroconversions were
produced in chickens vaccinated with one dose of the rAT adjuvanted with Al(OH)3.
Six groups vaccinated with vaccines containing either Al(OH)3 or rLTB-AT had body
weight higher than the control (α=0.05). We concluded that the recombinant vaccine
is a candidate to control Avian Necrotic Enteritis.
* Corresponding author: Fundação Universidade Federal do Rio Grande, Departamento de Patologia. Rua General Osório s/n, FURG - Campus Saúde, Centro, 96200-190, Rio Grande-RS, Brazil. E-mail: [email protected]. Phone: + 55 53 32330318.
54
INTRODUCTION
Avian Necrotic Enteritis (NE) is an acute enterotoxaemia caused by
Clostridium perfringens that affects animals between two and five weeks of age with
high mortality rates (Van Immerseel et al., 2004) and older animals sub-clinically,
producing hepatic and intestinal lesions, with high economic impact due to a
decrease in live weight and an increase in feed conversion and carcass
condemnations (Lovland and Kaldhusdal, 2001). In USA the cost of the disease was
estimated in more than U$ 0.05 per animal (Van der Sluis, 2000).
During the last decades NE was controlled by antibiotics added to feed. The
European Community banned the use of antibiotics as feed additives (Council of
European Union, 2003), calling attention to the use of probiotics and vaccines, that
appeared as the more promising alternatives for its control. The disease was
appointed as the most important challenge to the poultry industry after the ban of
antibiotics (Sesti, 2002; Phillips, 2002; Kaldhusdal, 2003).
Although animals that survived the disease developed immunity (Prescott,
2000), there are not effective vaccines commercially available (Kulkarni et al. 2007).
Several vaccines based on native or recombinant Clostridium perfringens α toxin
were tested with variable results. Cloning of α toxin, the major antigen of C.
perfringens (Sterne and Warrack, 1964; Titball et al., 1999; Williams, 2005; Dahiya et
al., 2006) in Escherichia coli (Leslie et al., 1989), and the production of recombinant
α toxin vaccines (Williamson and Titball, 1993; Schoepe et al., 2001; Stevens et al.,
2004) stimulated researchers to use this technology in the production of candidate
NE vaccines.
The objective of this work was to evaluate immunogenicity of a the C.
perfringens recombinant α toxin (rAT) in vaccines adjuvanted with Aluminum
55
Hydroxyde and the recombinant E. coli heat-labile enterotoxin subunit B (rLTB). A
recombinant chimeric protein (rLTB-AT) containing the toxin fused to LTB was also
evaluated.
MATERIAL AND METHODS
Vaccines and immunization
Female one-day-old Ross P8 broilers were divided in 13 groups of 5 animals
each, fed commercial feed and water ad libitum throughout the experiment. Vaccines
constructed with a rAT (Gil de los Santos, 2007) were used in the experiment. The
animals were vaccinated intramuscularly with 0.2 mL of the respective vaccine at 7
days of age and those that were revaccinated also at 14 days of age. The vaccines
and doses applied to each group are shown in table 1.
All the experiments with animals were approved by the Federal University of
Pelotas Animal Care Committee, in accordance with Brazilian Federal Laws.
56
Table 1: Vaccines and doses of each group.
Groups Protein Adjuvant Dose (µg) Nº of doses
Control --- --- ---
1 rAT Al(OH)3 25 1
2 rAT Al(OH)3 50 1
3 rAT Al(OH)3 25 2
4 rAT Al(OH)3 50 2
5 rAT rLTB 25 1
6 rAT rLTB 50 1
7 rAT rLTB 25 2
8 rAT rLTB 50 2
9 rLTB-AT None 25 1
10 rLTB-AT None 50 1
11 rLTB-AT None 25 2
12 rLTB-AT None 50 2
The doses of rLTB-AT were adjusted to obtain 25 and 50 µg rAT. The dose of rLTB (7.3 µg)
was the correspondent to the rLTB-AT.
57
Antibody response
Serum from each animal was obtained at days 0, 7, 21 and 35 days after the
first dose of vaccine (av). Antibody titers were evaluated by an Enzyme Linked
Immuno-Sorbent Assay (ELISA). Polystyrene microtiter plates (CRAL Artigos para
Laboratórios Ltda, Cotia, SP, Brazil) were coated with 0.5 µg of standard toxin (sT) or
rAT suspended in carbonate-bicarbonate buffer pH 9.6, incubated overnight at 4ºC.
Mice sera diluted 1:50 were added in duplicate to the wells and incubated for 1.5 h at
37ºC. After washing with PBS-T, goat anti-chicken polyvalent immunoglobulins
peroxidase conjugate (Sigma-Aldrich) (1:2000) was added and incubated for 1.5 h at
37ºC. After washing with PBS-T, OPD-chromogen substrate (1 mL Phosphate-citrate
Buffer pH 4.0, 10 µL of 30% hydrogen peroxide, 0.4 mg ortophenyle diamine) was
added. Absorbancies were measured in an ELISA spectrophotometer at 492 nm
(Dynatech MR 700).
Titres are expressed as seroconversions, dividing the mean of the respective
absorbencies by that of the same group at the day of the first vaccination.
Statistical analysis
The significance of the differences of absorbencies and weight gain means
were estimated by ANOVA using the Statistix software 8 version (Analytical Software,
Tallahassee, FL, USA).
58
RESULTS
Immune response
Seroconversions and weight gain indices in broilers 35 days av are shown in
table 2. Excluding vaccine 11, the higher seroconversions were obtained 35 days av
(Figures 1, 2, 3 and 4). The higher seroconversions were induced by the rAT vaccine
using Al(OH)3 as adjuvant inoculated only once, independently of the amount of
antigen (vaccines 1 and 2). Seroconversions of the animals vaccinated twice were
lower; the revaccination produced a fall in the seroconversions at 42 days of age.
Vaccines using co-administered rLTB as adjuvant, or rLTB-AT induced
seroconversions similar that of the controls.
59
Table 2: Weight gain and seroconversion of broilers 35 days after the first dose of
vaccine, determined with sT or rAT.
Group Nº of doses Weight gain Seroconversion
sT rAT
Control 1720.0de 2.8 2.1
rAT+Al(OH)3 25 µg 1 1877.7abc 3.4 2.2
rAT+Al(OH)3 50 µg 1 1875.0ab 3.5 3.2
rAT+Al(OH)3 25 µg 2 1857.8ab 2.4 2.0
rAT+Al(OH)3 50 µg 2 1640.1e 2.8 2.3
rAT+rLTB 25 µg 1 1816.9bcde 2.2 2.5
rAT+rLTB 50 µg 1 1790.1cde 1.3 1.8
rAT+rLTB 25 µg 2 1824.7bcde 2.0 1.7
rAT+rLTB 50 µg 2 1800.8abcd 2.0 1.9
rLTB-AT 25 µg 1 1861.0abc 1.6 1.4
rLTB-AT 50 µg 1 1677.0de 1.4 1.9
rLTB-AT 25 µg 2 1861.5abc 2.2 2.1
rLTB-AT 50 µg 2 1948.6a 2.2 1.9
*same letters in the same column mean no significant differences at α=0.05.
60
00,5
11,5
22,5
33,5
4
0 7 21 35
Days
Sero
conv
ersi
on Control
Vaccine 1
Vaccine 2
Vaccine 5
Vaccine 6
Vaccine 9
Vaccine 10
Figure 1: Seroconversions of broilers at 35 av vaccinated once determined with sT.
00,5
11,5
22,5
33,5
4
0 7 21 35
Days
Sero
conv
ersi
on Control
Vaccine 1
Vaccine 2
Vaccine 5
Vaccine 6
Vaccine 9
Vaccine 10
Figure 2: Seroconversions of broilers at 35 av vaccinated twice determined with sT.
0
1
2
3
4
0 7 21 35
Days
Sero
conv
ersi
on ControlVaccine 1Vaccine 2Vaccine 5Vaccine 6Vaccine 9Vaccine 10
Figure 3: Seroconversions of broilers at 35 av vaccinated once determined with rAT.
61
0
1
2
3
4
0 7 21 35
Days
Sero
conv
ersi
on ControlVaccine 3Vaccine 4Vaccine 7Vaccine 8Vaccine 11Vaccine 12
Figure 4: Seroconversions of broilers at 35 av vaccinated twice determined with rAT.
Although without statistical significance, the higher seroconversion (3.2)
against rAT was obtained in the chicks vaccinated with one dose of 50 µg
rAT+Al(OH)3. Media were slightly higher when sT (2.3 vs. 2.1) although in
unvaccinated chickens seroconversions detected by sT was 2.8 against 2.1 with rAT.
DISCUSSION
Immunization is one of the strategies that was proposed to control NE in
poultry reared without antibiotics. Recently LOVLAND et al. (2004) showed that
Clostridium toxoids adjuvanted with Al(OH)3 elicited a strong IgG response in
breeders against C. perfringens α toxin that was transferred to their offspring. It is
accepted that this toxin is a main factor in the etiology of NE (Heier et al., 2001;
Lovland et al., 2004; Kulkarni et al, 2007).
Information about the immune response in poultry to C. perfringens α toxin is
scarce. Most of it concerns to experiments in mice that were not validated in poultry.
The main limitation in assessing the effectiveness of vaccines against NE in poultry is
the difficulty to establish a standardized protocol to perform a potency test, due to the
62
variability in the responses to challenge. Several countries defined potency tests to
other clostridial vaccines but not to C. perfringens A vaccines (Pharmaceutical
Society of Great Britain, 1965; United States Departament of Agriculture, 1966). The
USDA (United States Departament of Agriculture, 2002) established a potency test
for these vaccines based on the antibody response against α toxin or on the
resistance to a challenge of 10 DL50 of the toxin, although for the definitive licensing
of the product, field trials are demanded.
Our experiment was designed to compare the immunogenicity and the effect
on productivity of three vaccines containing recombinant α toxin, in broilers.
Considering that C. perfringens A is a member of the normal microbiota of fowls, and
that antibodies against α toxin are normally transferred to the chickens via egg yolk
(Lovland et al., 2004), we opted to vaccinate at seven days of age, revaccinating
some groups one week latter to determine the booster effect of the vaccines, in order
to asses immunity at two to five weeks of age, when the acute form of the disease is
prevalent.
The recombinant α toxin used to prepare the vaccines was previously tested
in mice attaining the requirements of the USDA (Gil de los Santos, 2007). Therefore
we used the same vaccines with the same doses to vaccinate chickens born from
commercial flocks. The immunological response was assessed by ELISA using a
standard toxin and our recombinant toxin, and the results were expressed as
seroconversions (Gil-Turnes et al., 1999; Conceição et al., 2006). Considering that
our recombinant toxin was not detoxified and that it preserves some of the
characteristics of the native toxin, such as lethal, phospholipase and dermonecrotic
properties, we studied the effect it could have on productivity parameters of the
vaccinees.
63
The vaccines containing Al(OH)3 induced the higher seroconversions (3.5), in
agreement with those in mice (GIL de los SANTOS, 2007) and with the results of
Lovland et al. (2004) that used an α toxoid to vaccinate hens. Seroconversions
induced by the other vaccines were very close among them, varying between 1.3 and
2.5. Booster at 14 days of age did not affect seroconversions at 42 days of age. The
booster effect of re-vaccination was not detected. Kulkarni et al. (2007) obtained the
best results of their experiment when they primed chickens with two doses of a
recombinant α toxoid and boosted with the active toxin. Their recombinant α toxoid
did not protect against challenge probably due to modifications on conformational
epitopes of the toxin that may impair the immune response (Kulkarni et al., 2007),
explaining why our recombinant α toxin protected 100% of mice challenged with 10
LD50 of the standard toxin (GIL de los SANTOS, 2007).
The vaccines did not affect body weight at 42 days of age. Six groups
vaccinated with vaccines containing either Al(OH)3 or rLTB-AT had body weight
higher than the control (α=0.05). Among them was that which gave the highest
seroconversion. The vaccine containing recombinant α toxin and Al(OH)3 as adjuvant
was innocuous for chicken, elicited an immune response and did not affect
productivity parameters, being a candidate to be used in the control of NE.
Acknowledgements
This research was financed by CNPq grant Nº 474509/2004-4 and FAPERGS
grant Nº 05.2329.9. Rodrigo C. Cunha and Alceu G. dos Santos Jr. assisted in the
experiments with animals.
64
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5 CONCLUSÕES
- A rAT+Al(OH)3 foi inócua para camundongos e frangos de corte.
- A rAT+Al(OH)3 protegeu camundongos contra desafio com sT.
- Houve relação entre dose e proteção em vacinas rAT+rLTB em camundongos.
- A rLTB-AT não protegeu camundongos contra desafio com sT.
- A rAT foi imunogênica para frangos de corte
- A rAT não provocou alterações na produtividade de frangos de corte.
6 ATIVIDADES FUTURAS
- Avaliar a imunogenicidade de rAT+Al(OH)3 em lotes de frangos de corte de
produção industrial.
- Expressar rAT em Pichia pastoris.
- Avaliar a imunidade passiva de progênies oriundas de matrizes vacinadas com
rAT+Al(OH)3.
- Avaliar rAT como antígeno em ELISA.
69
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