Establishment of a production process for a novel vaccine candidate against Lawsonia intracellularis

BACKGROUND: Lawsonia intracellularis remains a problem for the swine industry worldwide. Previously, we designed and obtained a vaccine candidate against this pathogen based on the chimeric proteins: OMP1c, OMP2c, and INVASc. These proteins formed inclusion bodies when expressed in E. coli, which induced humoral and cellular immune responses in vaccinated pigs. Also, protection was demonstrated after the challenge. In this study, we established a production process to increase the yields of the three antigens as a vaccine candidate. RESULTS: Batch and fed-batch fermentations were evaluated in different culture conditions using a 2 L bioreactor. A fed-batch culture with a modified Terrific broth medium containing glucose instead of glycerol, and induced with 0.75 mM IPTG at 8 h of culture (11 g/L of biomass) raised the volumetric yield to 627.1 mg/L. Under these culture conditions, plasmid-bearing cells increased by 10% at the induction time. High efficiency in cell disruption was obtained at passage six using a high-pressure homogenizer and a bead mill. The total antigen recovery was 64% (400 mg/L), with a purity degree of 70%. The antigens retained their immunogenicity in pigs, inducing high antibody titers. CONCLUSIONS: Considering that the antigen production process allowed an increment of more than 70-fold, this methodology constitutes a crucial step in the production of this vaccine candidate against L. intracellularis.

Occurrence of anti-Toxoplasma gondii and anti-Neospora caninum antibodies in pigs in the State of Pará, Brazil / Ocorrência de anticorpos contra Toxoplasma gondii e Neospora caninum em suínos no estado do Pará, Brasil

Abstract We investigated the occurrence of Toxoplasma gondii and Neospora caninum antibodies in pigs raised in the Northeast of Pará, Brazil. At Study I, convenience sampled 151 pigs at two slaughterhouses, with and without state inspection; and Study II, which assessed 159 pigs with probabilistic sampling from nine pig farms. Serological analysis was performed using indirect fluorescent antibody test for T. gondii and N. caninum with a cutoff of 64 and 50, respectively. Overall, 6.77% pigs were seropositive for T. gondii and 5.16% for N. caninum. In Study I, pigs slaughtered with and without state inspection presented similar occurrence for both coccidia (p>0.05). Study II found an association between N. caninum seropositivity and sludge discarded into the soil, feeding pigs with animal-based protein, subsistence system, and absence of nipple drinkers. No association was found for T. gondii. Pigs from Pará are a potential source of T. gondii infection to humans. To our best knowledge, this is the first study to report anti-N. caninum antibodies in the serum of pigs in Pará State, Brazilian Amazon.

Resumo Foi investigada a ocorrência de anticorpos contra Toxoplasma gondii e Neospora caninum em suínos criados no nordeste do Pará, Brasil. No Estudo I, foram amostrados 151 porcos em dois matadouros, com e sem inspeção estadual. O Estudo II avaliou 159 suínos com amostragem probabilística de nove granjas de suínos. Para sorologia, utilizou-se o teste de imunofluorescência indireta para T. gondii e N. caninum com ponto de corte de 1:64 e 1:50, respectivamente. No geral, 6,77% dos suínos foram soropositivos para T. gondii e 5,16% para N. caninum. No Estudo I, suínos abatidos em matadouros com e sem inspeção estadual apresentaram ocorrência semelhante para ambos os coccídios (p> 0,05). Os animais amostrados de Belém, Benevides, Marituba, Bujaru, Castanhal e Igarapé-Miri foram positivos para T. gondii, enquanto os soropositivos para N. caninum foram encontrados em Belém, Bujaru, Castanhal e Santo Antônio do Tauá. O Estudo II encontrou associação entre soropositividade de N. caninum e esterco descartado no solo, alimentação dos suínos com proteína de origem animal, criação de subsistência e ausência de bebedores tipo "nipple". Não foi encontrada associação para T. gondii. A carne suína apresenta potencial risco de transmissão de T. gondii para os habitantes da região. De acordo com nosso conhecimento, este é o primeiro relato de anticorpos anti-N. caninum em suínos no estado do Pará, Amazônia brasileira.

O papel de imunoglobulinas na nefropatia da leptospirose em suínos / The role of immunoglobulins in nephropathy of leptospirosis in pigs

A leptospirose é uma antropozoonose endêmica em todo o mundo, que afeta o homem e várias espécies de animais domésticos e silvestres. No início da infecção há produção de IgM para o controle da infecção e após alguns dias, IgG são produzidas e provocam lise das leptospiras circulantes. Objetivou-se neste estudo identificar depósitos de antígeno de leptospiras e imunoglobulinas no tecido renal, para avaliar o papel de imunoglobulinas na patogênese da nefropatia da leptospirose em suínos. Foram colhidas 139 amostras de sangue e rim de suínos das cidades de Teresina/PI e Timon/MA, que foram avaliadas pela SAM, imunoistoquímica e PCR. Nefrite intersticial, fibrose, vasculite, tumefação do tufo glomerular e hipercelularidade difusa foram as principais alterações histopatológicas encontradas. A imunoistoquímica detectou antígeno de leptospira em 60 suínos. Depósitos de IgG, IgM e IgA foram observados no endotélio de capilares glomerulares, dos capilares intertubulares e na cápsula de Bowman, com marcação focal, difusa, global e segmentar. A deposição de IgM e IgA foi significantemente maior nos suínos infectados. Estranhamente depósitos de IgG foi significantemente maior nos suínos não infectados, onde não havia presença de antígeno de leptospiras e nem lesão túbulo-intersticial. Concluímos que antígeno de leptospiras no rim de suínos está relacionado a depósitos de IgM e IgA mas não a depósitos de IgG.

Leptospirosis is an endemic worldwide anthropozoonosis, affecting humans and several species of domestic and wild animals. At the beginning of infection is the production of IgM to control the infection and after a few days, IgG is produced and cause lysis of circulating leptospires. The objective of this study was to identify deposits of immunoglobulins and antigens of leptospires in kidney tissue, to assess the role of immunoglobulins in the pathogenesis of leptospirosis nephropathy in pigs. We collected 139 blood samples and kidney of pigs from the cities of Timon/MA and Teresina/PI, to be evaluated by SAM, immunohistochemistry and PCR. Interstitial nephritis, fibrosis, vasculitis; swollen glomeruli hypercellularity and diffuse in a pig were main pathological changes found. Immunohistochemistry leptospira antigen detected in 60 pigs. Deposits of IgG, IgM and IgA were observed in the endothelium of glomerular capillaries, the capillaries intertubulares and the Bowman's capsule, marked focal, diffuse, global and segmental. The deposition of IgM and IgA was significantly higher in infected pigs, strangely deposits of IgG was significantly higher in non-infected pigs, where there was no presence of antigen leptospires nor tubulointerstitial injury. We conclude that Leptospira antigen in porcine kidney relates to deposits of IgM and IgA but not IgG deposits.

Protective efficacy of a high-growth reassortant swine H3N2 inactivated vaccine constructed by reverse genetic manipulation

Novel reassortant H3N2 swine influenza viruses (SwIV) with the matrix gene from the 2009 H1N1 pandemic virus have been isolated in many countries as well as during outbreaks in multiple states in the United States, indicating that H3N2 SwIV might be a potential threat to public health. Since southern China is the world's largest producer of pigs, efficient vaccines should be developed to prevent pigs from acquiring H3N2 subtype SwIV infections, and thus limit the possibility of SwIV infection at agricultural fairs. In this study, a high-growth reassortant virus (GD/PR8) was generated by plasmid-based reverse genetics and tested as a candidate inactivated vaccine. The protective efficacy of this vaccine was evaluated in mice by challenging them with another H3N2 SwIV isolate [A/Swine/Heilongjiang/1/05 (H3N2) (HLJ/05)]. Prime and booster inoculation with GD/PR8 vaccine yielded high-titer serum hemagglutination inhibiting antibodies and IgG antibodies. Complete protection of mice against H3N2 SwIV was observed, with significantly reduced lung lesion and viral loads in vaccine-inoculated mice relative to mock-vaccinated controls. These results suggest that the GD/PR8 vaccine may serve as a promising candidate for rapid intervention of H3N2 SwIV outbreaks in China.

Dietary germanium biotite supplementation enhances the induction of antibody responses to foot-and-mouth disease virus vaccine in pigs

We evaluated the potential ability of germanium biotite (GB) to stimulate the production of antibodies specific for foot-and-mouth disease virus (FMDV). To this aim, we measured the total FMDV-specific antibody responses and IgM production after vaccination against FMD both experimentally and in the field. GB supplementation with FMDV vaccination stimulated the production of anti-FMDV antibodies, and effectively increased IFN-gamma and TNF-alpha levels. These results suggest that GB may be a novel alternative feed supplement that can serve as a boosting agent and an immunostimulator for increasing the efficacy of FMDV vaccination in pigs.

Induction of antibody and interferon-gamma production in mice immunized with virus-like particles of swine hepatitis E virus

Virus-like particles (VLPs) composed of the truncated capsid protein of swine hepatitis E virus (HEV) were developed and immune responses of mice immunized with the VLPs were evaluated. IgG titers specific for the capsid protein of swine HEV were significantly higher for all groups of mice immunized with the VLPs than those of the negative control mice. Splenocytes from mice immunized with the VLPs also produced significantly greater quantities of interferon (IFN)-gamma than interleukin (IL)-4 and IL-10. These newly developed swine HEV VLPs have the capacity to induce antigen-specific antibody and IFN-gamma production in immunized mice.

Comparative evaluation of different immunoassays for the detection of Taenia solium cysticercosis in swine with low parasite burden

Seven swine were experimentally infected with Taenia solium eggs and blood samples from each animal were periodically collected. At the end of the experiment (t140) the animals did not show clinical aspects of cysticercosis or parasites in tongue inspection. All animals were slaughtered and cut into thin slices in searching for cysts. The number of cysts found in each animal varied from 1 to 85. Enzyme-linked immunosorbent assay (ELISA) tests for antibody (Ab) detection and for antigen (Ag) detection were performed, which presented respectively 71 and 57 percent of positivity. By immunoblot (IB), using 18/14(T. crassiceps Ag) or lentil-lectin-purified glycoproteins from T. solium Ag (LLGP) as Ag, five (71 percent) and six (86 percent) animals were positive, respectively. The association between Ag-ELISA with any IB (18/14 or LLGP) allowed the detection of all animals at 140 days post-experimental infection (days p.e.i.). The use of IB 18/14 combined to the Ag-ELISA allowed the detection of all animals since 70 days p.e.i., and the association between IB LLGP and Ag-ELISA allowed the detection of all animals since 112 days p.e.i. While all animals could be considered healthy by conventional screening tests, the use of immunoassays for detecting Ab and Ag showed better accuracy; therefore it would be more useful than usual clinical examination for screening cysticercosis in slightly infected pigs.

Effect of soluble porcine aminopeptidase N on antibody production against porcine epidemic diarrhea virus

A few members of coronavirus group I which includes porcine epidemic diarrhea virus (PEDV) use porcine aminopeptidase N (pAPN) as a cellular receptor. Cellular receptors play an important role in virus attachment and entry. However, the low permissiveness of PEDV to APN-expressing porcine cell lines has made it difficult to elucidate the role of pAPN in vitro. The purpose of this study was to prove whether the treatment of soluble pAPN could enhance the antibody production against PEDV in guinea pigs, rabbits and sows. The animals (20 guinea pigs, 8 rabbits and 20 sows) were divided into 4 groups. Group A was injected intramuscularly (IM) with soluble pAPN at one hour before intramuscular infection of PEDV on the same site, group B for IM simultaneous injection of pAPN and PEDV, and group C for IM injection of PEDV only. Group D served as a control of pAPN treatment or PEDV infection. Antibody production against PEDV was compared among groups at regular intervals. The results suggested that pAPN could enhance the antibody production against PEDV in guinea pigs and rabbits which are free of pAPN, however, the effect of pAPN treatment in sows was not clearly elucidated.

Detecçäo de anticorpos para Toxoplasma gondii em soro de suínos criados e abatidos em frigoríficos da regiäo da grande Porto Alegre-RS, Brasil / Detection of antibodies against Toxoplasma gondii in sera from swine raised and slaughtered in the great Porto Alegre-RS abbattoirs, Brazil

No presente trabalho, objetivou-se contribuir com dados sobre a freqüência de sororeagentes para Toxoplasma gondii em suínos criados e abatidos na Regiäo da Grande Porto Alegre e fornecer subsídios sobre a importância da transmissäo deste protozoário, por suínos. Foram coletadas amostras de 240 suínos em frigoríficos da regiäo. A freqüência de anticorpos anti-Toxoplasma gondii, determinada através da técnica de hemaglutinaçäo indireta, foi de 20 por cento de soros iguais ou superiores a diluiçäo 1:64. Na técnica de imunofluorescência indireta, foram encontrados 33,75 por cento de soro com diluiçäo iguais a 1:16 ou superiores.

Frecuencia de Leptospira interrogans en unidades de producción porcina del altiplano de México / Survey of L. interrogans in pig farms of the high plateau of Mexico

Con el fin de conocer cuáles son las serovariedades más frecuentes del leptospira en explotaciones porcinas tecnificadas y de traspatio de la zona del altiplano de México, se hizo un muestreo serológico de hembras de cuatro o más partos de 17 granjas tecnificadas (114 cerdas) y de 12 explotaciones de traspatio (50 cerdas). Se emplearon 12 antígenos de L. interrogans en la técnica de algutinación microscópica y se consideraron positivos los sueros a partir de la dilución de 1:100. La serovariedad bratislava se encontró en 28 por ciento de las hembras de granjas tecnificadas y explotaciones de traspatio, y panama en 24.5 por ciento y 34 por ciento de las cerdas, respectivamente, y en menor proporción el resto de las serovariedades. De 8 serovariedades detectadas, 7 (87.5 por ciento) se encontraron en granjas tecnificadas y soló 3 (37.5 por ciento) en animales de traspatio; quizá esto fue debido a la mayor densidad poblacional. Probablemente la presencia de bratislava se debe a la importancia de animales. Cuando se compararon estos resultados con los obtenidos de estudios anteriores, se concluyó que en los últimos años bratislava y panama se han convertido en las más frecuentes y es recomendable que se incluyan como antígenos en las vacunas para prevenir adecuadamente la laptospirosis en los cerdos

Control y erradicación de la enfermedad de Aujeszky en un sistema múltiple de tres sitios de producción / Control and eradication of Aujeszky's disease in a multiple site production system

El objetivo del presente trabajo fue controlar y erradicar la enfermedad de Aujeszky (EA) en un sistema múltiple de tres sitios de producción por medio de la vacunación del hato reproductor contra la EA,, la segregación de la descendencia y hacer una valoraicón lineal epidemiológica de los tres sitios de producción mediante el uso de animales centinelas libres de la EA. La seropositividad en los tres sitios fue del 93.67 por ciento de las muestras totales tomadas (1389) en todo el sistema por la prueba de seroneutralización SN y ELISA competitiva g1. Para la erradicación de la EA se vacunó a las hembras con una vacuna con deleción g1+, siendo revacuadas a los 21 días y cada 3 meses durante el primer año posterior al brote al igual que a todo el hato, durante el segundo año se vacunó al pie de cría cada 4 meses. Por otra parte, durante el brote los lechones fueron destetados a los 21 días y llevados a otras instalaciones fuera del sistema y cuando los signos clínicos desaparecieron, los lechones fueron destetados a los 18 días y segregados al sitio 2. En la mayoría de los muestreos serológicos se obtuvo 0 por ciento de seropositividad, excepto en uno de éstos. En este sentido se tiene que los métodos aplicados en el sitio 1 (vacunación, medicación, cerrar la entrada de animales al sitio por un tiempo y la segregación de la descendencia) fueron efectivos para controlar y erradicar la enfermedad de Aujeszky en el sistema, ya que desde que se inició el programa no se presentó un solo caso de la enfermedad en los sitios 2 y 3 del sistema, como tampoco se observaron signos clínicos de la EA en el sitio 1

Production specific antigens from Trichinella spiralis using a continuous elution method and isoelectric focusing.

Immunodominant antigens of 45-53 kDa (one band per fraction) were obtained from excretory/secretory (E/S) and somatic products of infective-larvae of Trichinella spiralis using a continuous-elution method. They were further resolved by isoelectric focusing into different isoforms (45 kDa: pI4.47, 5.09, 5.47 and 5.86; 47 kDa: pI4.72 and 4.97; 53 kDa: pI4.86, 5.11, 5.44 and 5.78). In immunoblotting, the isoforms of pI 5.09, 5.86, 4.97, 5.44 and 5.78 did not cross-react with antisera against Trichuris suis, Metastrongylus apri, Gnathostoma hispidum and Stephanurus dentatus. Hence, they have the potential to serve as specific antigens for the serodiagnosis of trichinellosis.

Avaliaçäo da resposta sorológica de suínos imunizados contra parvovirose suína com uma vacina inativada experimental / Serological evaluation of immunized pigs with an inactivated experimental vaccine against swine parvovirus

Foi estudada a resposta sorológica de leitoas e porcas vacinadas e revacinadas com intervalo de 15 e 21 dias com uma vacina inativada contra parvovirose suína. Os animais vacinados assim como o rebanho de origem eram livres de anticorpos inibidores de hemoaglutinaçäo (HI). Aos 15 dias após a revacinaçäo, 87,5 por cento dos animais vacinados apresentaram títulos HI variando de 1:40 a 1:1280. O intervalo de revacinaçäo e a diferença de títulos HI säo discutidos

Vacinaçäo com Bordetella bronchiseptica e Pasteurella multocida, associada a alteraçöes do manejo, no controle da rinite atrófica dos suínos / Use of a Bordetella bronchiseptica-Pasteurella multocida vaccine in addition to improved management, to control swine atrophic rhinitis

Empregou-se uma vacina preparada com B. bronchiseptica e P. multocida tipo D, toxigênica, para controlar a rinite atrófica em rebanho de 220 matrizes suínas. Aproximadamente 60 por cento dos animais em idade de abate exibiam sinais clínicos da doença antes da vacinaçäo. Lesöes das conchas nasais foram observadas em 70 por cento (56/80) dos animais antes do período experimental e em 77 por cento (55/71) dos näo vacinados, examinados ao final do experimento. O rebanho foi acompanhado durante 12 meses antes e 22 meses após o início da vacinaçäo. Realizaram-se alteraçöes no manejo, reduçäo do plantel de 220 para 180 matrizes e mudanças no esquema de limpeza, desinfecçäo e ocupaçäo dos prédios. As medidas adotadas eliminaram a doença clínica dos leitöes jovens e reduziram (P<0,001) as alteraçöes das conchas nasais, observadas em apenas 15 por cento (39/266) dos leitöes vacinados, a ocorrência de pneumonias e pleuropneumonias, de 22 por cento para 9 por cento dos animais necropsiados e o número de condenados no frigorífico, 11 por cento para 0,8 por cento

Effects of cyclophosphamide on pigs infected and vaccinated with swinepox virus.

Piglets treated with cyclophosphamide (50 mg/kg body wt, iv) before infection/vaccination with swinepox virus showed a strong suppression of humoral immune response. Cell mediated immune response was also affected to some extent and the cyclophosphamide treated piglets did not resist the challenge on 21 day post infection/vaccination and developed mild lesions.

Intense transmission of Japanese encephalitis virus to pigs in a region free of epidemic encephalitis.

Epidemic Japanese encephalitis recurs annually in the northern provinces of Thailand, but in the southern provinces cases of human encephalitis are rare. We investigated transmission of Japanese encephalitis virus (JEV) to pigs in southern Thailand. Blood specimens from one hundred young pigs at abattoirs in three southern provinces were tested for JEV hemagglutination inhibiting (HAI) antibodies. Seventy-four percent were positive. Ten seronegative sentinel pigs were placed at five locations in one southern province. Seven of the ten pigs developed JEV HAI and JEV IgM ELISA antibodies within two weeks of placement. JEV was isolated from all seven seroconverting sentinel pigs from blood specimens collected 3 to 11 days after placement. Fifteen light-trap mosquito collections at the five locations all included known JEV vectors, some in large numbers. We conclude that there is intense transmission of JEV to pigs in southern Thailand despite the rare occurrence of human encephalitis in the same region.

Fatores de virulência em amostras de Escherichia coli enteropatogênicas para suínos isoladas no Brasil / Virulence factors present in strains of porcine enteropathogenic Escherichia coli isolated in Brazil

Trezentas e vinte e três amostras de Escherichia coli, isoladas de suínos com diarréia em Porto Alegre, RS, pertencentes aos sorogrupos 08, 09, 010, 035, 045, 064, 0108, 0115, 0119, 0138, 0139, 0141, 0147, 0149 e 0157, considerados enteropatogênicos para suínos, foram examinadas quanto à presença de fatores de virulência. A enterotoxina termolábil (LT) foi detectada através do teste de imunohemólise passiva em 56 amostras (17,3%), principalmente nos sorogrupos 08 e 0149, que totalizaram 52 amostras (92.85%) das 56 LT+. Amostras pertencentes aos sorogrupos 035, 0141 e 0157 também produziram LT, ao passo que as amostras pertencentes aos demais sorogrupos näo produziram estA enterotoxina. A enterotoxina termoestável, detectável pelo teste do camundongo recém-nascido, (STa), foi encontrada em 33 (10,2%) amostras pertencentes aos sorogrupos 09, 064, 0138, 0141 e 0149. Entre as 122 amostras STa-, examinadas para a detecçäo de enterotoxina termoestável, pelo teste de alça ligada de leitäo de 6 semanas, (STb), 31 (25,4%) foram positivas e pertenceram aos sorogrupos 035, 064, 0108, 0115, 0119, 0139, 0149 e 0157. Ao que se sabe, é a primeira vez que se relata no Brasil a produçäo de STb por amostras de E. coli de origem suína. Quanto às provas de microhemaglutinaçäo manose-resistente com hemácias de cobaia e cavalo, para a detecçäo dos antígenos K88 e K99, respectivamente, observou-se uma frequência maior de positividade entre as amostras LT+. A presença do antígeno 987P, também relatado pela primeira vez em nosso país, foi detectado sorologicamente em 12 (8,75%) das 137 amostras examinadas