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.

Immunization against porcine epidemic diarrhea virus and vaccine development / 生物工程学报

Porcine epidemic diarrhea (PED) is a major disease of pigs that inflicts heavy losses on the global pig industry. The etiologic agent is the porcine epidemic diarrhea virus (PEDV), which is assigned to the genus Alphacoronavirus in the family Coronaviridae. This review consists of five parts, the first of which provides a brief introduction to PEDV and its epidemiology. Part two outlines the passive immunity in new born piglets and the important role of colostrum, while the third part summarizes the characteristics of the immune systems of pregnant sows, discusses the concept of the "gut-mammary gland-secretory IgA(sIgA) axis" and the possible underpinning mechanisms, and proposes issues to be addressed when designing a PEDV live vaccine. The final two parts summarizes the advances in the R&D of PEDV vaccines and prospects future perspectives on prevention and control of PEDV, respectively.

Protection against Toxoplasma gondii cysts in pigs immunized with rROP2 plus Iscomatrix / Proteção contra cistos de Toxoplasma gondii em suínos imunizados com rROP2 associado à Iscomatrix

Abstract This study aimed to evaluate the humoral immune response in pigs immunized intranasally and intramuscularly with recombinant Toxoplasma gondii rROP2 protein in combination with the adjuvant Iscomatrix. Twelve mixed breed pigs divided into three groups (n=4) were used, G1 received recombinant ROP2 proteins (200 µg/dose) plus Iscomatrix, G2 received PBS plus Iscomatrix, and G3 as the control group. The intranasal (IN) and intramuscular (IM) routes were used. Animals were challenged orally with VEG strain oocysts and treated on day three after challenge. Fever, anorexia, and prostration were the clinical signs observed in all animals. All the G1 animals produced antibodies above the cut-off on the day of the challenge, while the G2 and G3 remained below the cut-off. Better partial protection against parasitemia and cyst tissue formation was observed in G1 than G3. The protection factors against tissue cyst formation were 40.0% and 6.1% for G1 and G2, respectively, compared to G3. In conclusion, there were not systemic antibody responses in pigs with IN immunization with rROP2+Iscomatrix; however, after IM immunization, those animals produced higher titers than animal controls. We associated these results with partial protection obtained against parasitemia and tissue cysts formation.

Resumo O objetivo deste estudo foi avaliar a resposta imune humoral em suínos imunizados pelas vias intranasal e intramuscular com proteínas recombinantes rROP2 do Toxoplasma gondii associadas ao adjuvante Iscomatrix. Doze suínos cruzados divididos em 3 grupos (n=4) foram utilizados. O G1 recebeu proteína recombinante ROP2 (200mg/dose) associada ao adjuvante Iscomatrix; o G2 recebeu PBS associado ao Iscomatrix; e o G3 foi o grupo controle. As vias intranasal (IN) e intramuscular (IM) foram utilizadas. Os animais foram desafiados por via oral com a cepa VEG e tratados no dia três após o desafio. Febre, anorexia e prostração foram os sinais clínicos observados em todos os animais. Todos os animais do G1 produziram anticorpos acima do ponto de corte no dia do desafio, enquanto os animais do G2 e G3 permaneceram abaixo do ponto de corte no desafio. Proteção parcial contra parasitemia e formação de cistos teciduais foram observadas nos suínos do G1 comparados ao G3. Os fatores de proteção contra a formação de cistos teciduais foram 40,0% e 6,1% no G1 e G2, respectivamente, comparados com o G3. Como conclusão, não houve estimulação da resposta imune humoral sistêmica nos suínos após as imunizações IN com rROP2+Iscomatrix. Estes animais, porém, após a imunização IM, produziram títulos de anticorpos mais altos que os animais controles. Esses resultados foram associados a uma proteção parcial contra a parasitemia e formação de cistos teciduais.

Efficacy of leptospiral commercial vaccines on the protection against an autochtonous strain recovered in Brazil

Abstract In swine and bovines, leptospirosis prevention and control is carried out via vaccination of susceptible animals using bacterins. However, the efficiency of leptospirosis vaccines has been questioned. This work aimed to investigate the potency of five leptospirosis vaccines sold commercially in Brazil, challenging the animals with one autochthonous strain of Leptospira, Canicola serovar, denoted LO4, isolated from swine. The standard protocol was followed, and renal carriers of Leptospira were identified among the surviving animals by culture and PCR. Of the five vaccines tested, only two proved effective. None of the surviving animals was positive by culture; however, one animal was positive by PCR. Three of the five vaccines sold commercially in Brazil for the immunization of swine or bovines failed the test of the efficacy to protect the vaccinated animals following challenge with an autochthonous Leptospira strain, Canicola serovar. The two vaccines provided protection against the renal carrier state in the surviving animals. The criteria used to produce leptospirosis bacterins sold commercially in Brazil must be reviewed. The industry should support researches on leptospiral vaccinology to improve the quality of the present vaccines and discover new immunogenic strains, because it is known that vaccination is one of the most important tools to increase the reproduction rates in livestock.

Assessing PCV2 antibodies in field pigs vaccinated with different porcine circovirus 2 vaccines using two commercial ELISA systems

Porcine circovirus type 2 (PCV2) is the primary causative agent for post-weaning, multisystemic, wasting syndrome. Consequently, serologic detection of and vaccination against PCV2 are important for the swine industry. Among several serological tests, the enzyme-linked immunosorbent assay (ELISA) is commonly used to measure anti-PCV2 antibody levels. In the present study, we used two commercial ELISA systems to comparatively evaluate anti-PCV2 antibodies in field pigs treated with three different PCV2 vaccines. Among a total of 517 serum samples, the results of the two ELISAs were fully concordant for 365 positive and 42 negative samples, indicating 78.7% agreement. In addition, the Pearson coefficient (0.636) indicated a moderate correlation between data from the two ELISAs. Results from the farms with pigs vaccinated with the three different PCV2 vaccines demonstrated that most of the vaccinated animals underwent seroconversion. However, the increase and duration of antibody titers varied depending on the vaccine, the presence of maternal antibodies, and the vaccination program. PCV2 serologic status and anti-PCV2 antibody levels of herds from this study could be utilized to determine the best timing for vaccination and assessing vaccination compliance.

Evaluación del impacto de un programa de control de la teniasis-cisticercosis (Taenia solium) / Evaluation of the impact of a control program against taeniasis-cysticercosis (Taenia solium)

Objetivo. Evaluar el impacto de un programa de control de la teniasis-cisticercosis por Taenia solium con fines de erradicación, basado en educación de la comunidad y vacunación de cerdos. Material y métodos. Se estimó la prevalencia de cisticercosis porcina por medio de la palpación de lengua, ultrasonido y presencia de anticuerpos en suero, antes de iniciar el programa y tres años después, en tres regiones del estado de Guerrero. Resultados. Se observó una reducción significativa en la prevalencia de cisticercosis porcina de 7 a 0.5% y de 3.6 a 0.3%, estimadas por examen de lengua y ultrasonido, respectivamente (p<0.01), y una disminución no significativa de la seroprevalencia de 17.7 a 13.3%. Conclusiones. La reducción de la prevalencia de teniasis-cisticercosis comprueba la efectividad del programa para prevenir la infección. La presencia sostenida de anticuerpos es compatible con continuos contactos con Taenia solium u otros helmintos relacionados, y señala la necesidad de mantener las intervenciones para lograr su erradicación.

Objetive. The impact of a control program is evaluated to eventually eradicate taeniasis-cysticercosis (Taenia solium) based on education and vaccination of pigs. Materials and methods. The prevalence of porcine cysticercosis was estimated using tongue inspection, ultrasound and determination of antibodies, before and three years after the application in three regions of the state of Guerrero. Results. A significant reduction in the prevalence of porcine cysticercosis of 7 to 0.5% and 3.6 to 0.3% estimated by tongue examination or ultrasound respectively (p<0.01) and a no significant decrease in seroprevalence from 17.7 to 13.3% were observed. Conclusions. The reduction of the prevalence of taeniasis-cysticercosis establishes the program's effectiveness in preventing infection. The sustained presence of antibodies, compatible with contact of Taenia solium or other related helminths, underlines the importance of maintaining interventions to achieve eradication.

Epidemiología y control de la cisticercosis en el Perú / Epidemiology and control of cysticercosis in Peru

La neurocisticercosis, infección del sistema nervioso humano por el estadio larvario de la Taenia solium, es una causa importante de epilepsia y otras manifestaciones neurológicas en el Perú y en la mayoría de países en desarrollo. Desde 1987, el Grupo de Trabajo en Cisticercosis en Perú ha desarrollado una serie de estudios epidemiológicos que han llevado a estimar el impacto y entender la transmisión de la Taenia solium, y que posteriormente se aplicaron al diseño y ejecución de un programa de control en Tumbes, en la costa norte del país. En este artículo se revisan los principales hallazgos epidemiológicos, así como las líneas generales del programa de eliminación y las herramientas utilizadas. Los avances en el control de la teniasis/cisticercosis en nuestro país abren el camino hacia su eliminación y eventual erradicación.

Neurocysticercosis, the infection of the human central nervous system by the larval stage of the cestode Taenia solium, is an important cause of epilepsy and other neurological manifestations in Peru and most developing countries. Since 1987, the Cysticercosis Working Group in Peru has performed a series of epidemiological studies which led to estimate the impact and to better understand the transmission of Taenia solium. This information was later applied to the design and execution of a control program in Tumbes, in the Northern Coast of Peru. This paper reviews the main epidemiological findings, as well as the conceptual framework of the elimination program and the tools used. Advances in the control of taeniasis/cysticercosis in our country open the road towards its elimination and potential eradication.

Erradicação da doença de Aujeszky em Santa Catarina: importância da condição sanitária das leitoas de reposição / Aujeszky's diasease eradication in Santa Catarina State: relevance of sanitary status of replacement gilts

A doença de Aujeszky (DA) é uma infecção causada por um herpesvírus, o vírus da DA (VDA), primariamente em suínos. Esta doença está presente no Estado de Santa Catarina (SC) desde 1984. Devido ao impacto da DA no mercado exportador de carne suína, no comércio de reprodutores e nas perdas de produtividade, um programa de erradicação, financiado por uma parceria entre a indústria e a associação de produtores tem tido sucesso em eliminar gradualmente a DA de rebanhos suínos de SC. O último caso de DA no estado foi identificado em julho de 2004. Durante o processo de despovoamento/repovoamento, foi detectado um rebanho suíno positivo para o VDA localizado no oeste de SC. Estudos de rastreabilidade da origem daqueles animais indicaram que a fonte era uma granja que distribuía reprodutores ilegalmente sem certificação sanitária. Este suinocultor mantinha um sistema de integração que incluía 40 diferentes produtores para quem eram comercializados reprodutores e/ou suínos para terminação. Testes de soroprevalência detectaram anticorpos anti-VDA em 12 daqueles rebanhos. Devido a sua localização dentro do raio de 2,5km do foco inicial, uma outra granja, onde havia uma central de inseminação artificial que distribuía sêmen suíno para outras 5 granjas do mesmo proprietário, foi testada e também resultou positiva. Os objetivos deste artigo são descrever as condições sanitárias frente ao VDA naquelas granjas que receberam suínos ou sêmen suíno daqueles suinocultores, as medidas para controlar e eliminar o VDA dos rebanhos positivos e a situação atual decorrente deste trabalho. Além disso, o artigo busca alertar que medidas de vigilância ativa e normas sanitárias para comércio e distribuição de material genético devem ser seguidas, caso contrário, a DA pode recrudescer e tornar-se fora de controle como ocorria antes do início deste programa.

Aujeszky's disease (AD) is an herpesvirus infection, caused by the pseudorabies virus (PRV), primarily in swine and present in Santa Catarina State (SC) since 1984. Due to the impacts of AD in the pork export, breeder's trade and productivity losses, an eradication program, financed by industry and swine producers association, has successfully eradicated the AD of swine herds in SC State. The last case of AD in the State was identified in July of 2004. During the depopulation/repopulation process, a PRV positive swine herd located in the west region of SC State was detected. Traceability studies of the origin of those animals indicated that the source was a swine farm which illegally distributed breeders without sanitary certification. This swine producer maintained an integration system which included 40 different producers, to whom were commercialized breeders and/or finishers. Serum-prevalence tests detected PRV antibodies in 12 of those herds. Due to the location into the 2.5km of radius from the initial outbreak, another swine farm, which had an artificial insemination center that distributed swine semen to another 5 herds of the same owner was tested positive as well. The objectives of this paper are to describe the sanitary status related to AD on the farms which have received pigs or swine semen from these swine producers, the measures to control and eliminate ADV from positive herds and the outcome of this work. Beyond that, to alert that measures of active surveillance and sanitary rules for commerce and distribution of genetic materials must be properly fulfilled, otherwise, AD can reactivate and become out of control as occurred before the eradication program.

Monitoramento das patologias em suínos no período de crescimento / Monitoring of pathologies in growing swines

Determinou-se, por meio de um sistema de monitoramento de suínos em fase de crescimento e em matadouro, a presença de patologias como indicadoras de problemas na população de referência. O estudo foi realizado em uma criação intensiva de ciclo completo, durante nove semanas. Foram determinadas as prevalências dos diagnósticos clínico-patológicos obtidos durante um período de nove semanas. Utilizaram-se cinco grupos de 43 leitões, com pesos de até 19kg, considerados de baixa performance. Estimaram-se, por intervalos de confiança de 95 por cento, as prevalências acumuladas em cada monitoramento. Obteve-se correlação de 0,99 (P<0,0001) entre as prevalências anuais com as nove semanas de estudo, mostrando correspondência com o achado em matadouro. Amostras em semanas alternadas de três grupos de 43 animais são bons indicadores das patologias que afetam os suínos em crescimento, mesmo aquelas de baixa freqüência ou muito letais.

The prevalence of different pathologies as an indicator of health problems in a particular population by means of slaughterhouse monitoring of growing pigs was determined. The work was carried out in an indoor intensive farrow to finish swine farm during 9 weeks. The prevalence of diseases was determined by means of clinical and pathological diagnosis. For the slaughterhouse inspection, five groups of 43 pigs each weighting up to 19kg, considered as poor performers, were put together. These animals were sent to the slaughterhouse and were observed for the presence or absence of skin and joint lesions of arms and legs and also lesions of serous membranes and parenchimal organs. An estimative with a confidence interval level of 95 percent was performed on cumulated prevalence in each monitoring session for 43, 86, 129, 172 and 215 animals. Annual prevalence from clinical and pathological diagnosis and 9-week-period had correlations (r=0.99; P<0.0001) and correspondence with slaughterhouse observations. In conclusion, samplings in alternate weeks of three groups of 43 pigs seem to be a good indicator of pathologies affecting growing swines, even those of very low prevalence or highly lethal.

Hacia el desarrollo de una vacuna en contra de la cisticercosis porcina basada en la paramiosina de Taenia solium

La paramiosina de Taenia solium (TPmy) es un antígeno inmunodominante de la cisticercosis humana y porcina. Se trata de una proteína de 100 kDa con una estructura alfa-hélice superenrollada asociada al músculo y a estructuras tegumentarias del cisticerco. La TPmy tiene la propiedad de unirse al C1q e inhibir la cascada del complemento. La TPmy probablemente se une al C1q a través sus dominios tipo colágena y podría estar relacionada con una estrategia parasitaria para modular la respuesta inmune del huésped. En el hombre y en el ratón, la respuesta inmune humoral en contra de la TPmy está preferentemente dirigida hacia el extremo carboxilo terminal mientras que el extremo amino terminal de la TPmy induce una respuesta protectora celular de tipo Th1. Ensayos de protección en el modelo murino de cisticercosis en ratones inmunizados con fragmentos recombinantes de TPmy revelaron que el extremo amino terminal induce alrededor de 60% de protección en contra de un reto intraperitoneal con cisticercos de Taenia crassiceps. Ensayos preliminares de protección por inmunización génica revelaron que el extremo amino terminal de la TPmy clonado en un vector plasmídico con un promotor de citomegalovirus induce alrededor de 79% de protección, junto con plásmidos para la expresión de IL-12, sugiriendo que este tipo de inmunización con TPmy puede resultar en el desarrollo de una vacuna eficaz y económica en contra de la cisticercosis.

Taenia solium paramyosin (TPmy) is a prominent 100 kDa antigen in human and porcine cysticercosis. TPmy is an α-helical coiled coil protein present in muscle and tegumentary structures of T. solium cysticerci. TPmy has the property of binding C1q resulting in inhibition of the complement cascade. TPmy probably binds C1q through its collagen-like domains and could be involved in a parasite strategy to modulate host immune response. Humoral immune response against TPmy is preferentially directed against carboxyl terminal end in humans and mice, whereas amino terminal end of TPmy preferentially induces a Th1-related cellular immune response. Protection studies in murine model of cysticercosis showed that the amino terminal end fragment of TPmy induces approximately 60% protection against an i.p. challenge with Taenia crassiceps cysts when mice are immunized with recombinant fragments of TPmy. Initial protection studies using genetic immunization showed that amino terminal end fragment of TPmy cloned into a plasmid expression vector with a cytomegalovirus promoter, together with IL-12-expressing plasmids induced 79% protection, suggesting that this kind of TPmy-immunization might result in development of a cost-effective vaccine against cysticercosis.

Vaccination against Taenia solium cysticercosis

Taenia solium is a parasite that causes human cysticercosis. Its life cycle includes the adult stage, the egg and the larval stage. Human cysticercosis is a disease related to underdevelopment, the main clinical manifestation is neurocysticercosis. Control measures include mass cestocidal treatment aimed to cure possible taeniosis cases. Although useful it has certain disadvantages, such as the generation of symptomatology in occult neurocysticercosis. Alternatively, health education has been shown to be highly effective since people become aware of the importance of human and porcine cysticercosis and the possibility of eliminating it. Nevertheless it has to be implemented by knowledgeable people. On the other hand, the life cycle can be controlled by avoiding swine cysticercosis. This review describes the studies performed to vaccinate pigs against T. solium and indicate that short time perspectives are very encouraging for the production of an optimal vaccine

Passive immunization of piglets against enterotoxigenic colibacillosis by vaccinating dams with K88ac pili bearing bacterins.

Day-old-piglets were passively immunized by vaccinating the pregnant sows with K88ac enterotoxigenic Escherichia coli (ETEC) vaccine. High level of ETEC specific antibodies was excreted in colostrum (3733.33 +/- 1152.13) and maintained a detectable level (100.00 +/- 0.00) up to 21 day post partum (DPP). The IgG was the predominant immunoglobulin followed by IgA and IgM. Piglets born of vaccinated dam (group A) and unvaccinated dam (group B) were challenged in 7 day of age. Clinical and faecal scores were significantly (P < 0.01) low in group A than that of group B. Piglets of group A developed mild diarrhoea (33.33%), while all the control piglets developed profuse diarrhoea and 3 of these died before 14 day of challenge infection.

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

La caracterización de los antígenos de superficie del nemátodo parásito Trichinella spiralis: estudio de su participación en los mecanismos de protección y su utilidad en el diagnóstico de la triquinosis / Charaterization of surface antigens of the parasite nematode Trichinella spiralis: mechanisms of protection and use in diagnosis of trichinosis

Entre los aspectos más importantes en el estudio de la triquinosis, se encuentra el desarrollo de los métodos de diagnóstico específicos y confiables que permitan la detección oportuna de la infección, así como la identificación y caracterización de antígenos del parásito que previenen la infección. En este contexto, la caracterización de los antígenos de superficie esticosoma y de excreción secreción de la larva muscular de Trichinella spiralis, ha sido muy relevante, dada su alta inmunogenicidad prácticamente en todos los huéspedes hasta ahora estudiados. Así, durante el desarrollo de este trabajo se ha logrado identificar y caracterizar las moléculas de estos dos compartimentos de la larva muscular de T. spiralis que se han empleado tanto en la detección específica de la infección por T. spiralis (particularmente en cerdos), como en los ensayos para evaluar su potencial en la inducción de protección modelos murinos

Prevalência e avaliaçäo de estratégia de controle da doença de Aujezsky em suínos na Zona da Mata, Minas Gerais, Brasil / Aujeszky in swine-prevalence and evaluation of control methods in Minas Gerais, Brazil

Testes de micro-soroneutralizaçäo para doença de Aujeszky, realizados em 1245 soros, provenientes de 45 granjas de suínos da Zona da Mata, Estado de Minas Gerais, apresentaram resultados negativos. A regiäo foi considerada livre da doença

Control of Taenia solium taeniasis and cysticercosis by focus-oriented chemotherapy of taeniasis.

The mortality, mortality, and disability due to Taenia solium neurocysticercosis are common and serious health problems in some regions, including several Asian countries. Neurocysticercosis in man and cysticercosis in pigs can be controlled by treatment, in man with a single, low (2 1/2 or 5 mg/kg) and safe dose of praziquantel. The problem which remains is the identification of a Taenia solium taeniasis foci. The definition of a focus is: T. solium-infected or suspected case; household with recent case of epilepsy in family or cysticercosis in pigs; group of houses or a village with high rate of cysticercosis in pigs. In a rural area a focus can be easily identified by tracing infected pigs to their place of origin, or in vivo tongue examination. All people with suspected T. solium taeniasis in a focus should be treated.