Evaluation of long-term immune response in cattle to botulism using a recombinant E. coli bacterin formulated with Montanide™ ISA 50 and aluminum hydroxide adjuvants.

Botulism is a severe disease caused by potent botulinum neurotoxins (BoNTs) produced by Clostridium botulinum. This disease is associated with high-lethality outbreaks in cattle, which have been linked to the ingestion of preformed BoNT serotypes C and D, emphasizing the need for effective vaccines. The potency of current commercial toxoids (formaldehyde-inactivated BoNTs) is assured through tests in guinea pigs according to government regulatory guidelines, but their short-term immunity raises concerns. Recombinant vaccines containing the receptor-binding domain have demonstrated potential for eliciting robust protective immunity. Previous studies have demonstrated the safety and effectiveness of recombinant E. coli bacterin, eliciting high titers of neutralizing antibodies against C. botulinum and C. perfringens in target animal species. In this study, neutralizing antibody titers in cattle and the long-term immune response against BoNT/C and D were used to assess the efficacy of the oil-based adjuvant compared with that of the aluminum hydroxide adjuvant in cattle. The vaccine formulation containing Montanide™ ISA 50 yielded significantly higher titers of neutralizing antibody against BoNT/C and D (8.64 IU/mL and 9.6 IU/mL, respectively) and induced an immune response that lasted longer than the response induced by aluminum, extending between 30 and 60 days. This approach represents a straightforward, cost-effective strategy for recombinant E. coli bacterin, enhancing both the magnitude and duration of the immune response to botulism.

Recombinant vaccine against botulism in buffaloes: Evaluation of the humoral immune response over 12 months.

Botulism is a neuroparalytic intoxication, usually fatal, caused by the botulinum toxins (BoNTs). Vaccination is the best-known strategy to prevent this disease in ruminants. Serotypes C and D and their variants CD and DC are the main types responsible for botulism in bovine and buffaloes in Brazil and cattle in Japan and Europe. Brazil has a herd of approximately 1.39 million buffaloes and is the largest producer in the Western world. This study aimed to assess the humoral immune response of buffaloes during the 12-month period after vaccination against BoNT serotypes C and D with a recombinant vaccine in three different concentrations (100, 200, and 400 µg) of non-purified recombinant proteins (Vrec) and also with a bivalent commercial toxoid (Vcom). Vrec400 was the best vaccine among those tested because it induced higher levels of antibodies and maintained higher levels of antibodies for the longest time, while Vrec200 could be considered the most cost-effective vaccine for large-scale production. None of the vaccines were able to promote continuous immunological protection within the timeframe proposed by the current Brazilian vaccination protocol. Further studies should focus on vaccine adjustments to ensure continued humoral protection against botulism.

Inactivated recombinant Escherichia coli as a candidate vaccine against Clostridium perfringens alpha toxin in sheep.

Clostridium perfringens type A is the causative agent of gas gangrene and gastroenteric ("yellow lamb disease") disease in ruminants, with C. perfringens alpha toxin (CPA) being the main virulence factor in the pathogenesis of these illnesses. In the present study, we have developed recombinant Escherichia coli bacteria expressing rCPA and used it to vaccinate rabbits and sheep. Doses of up to 200 µg of rCPA used for inoculation, induced 13.82 IU.mL-1 of neutralizing antitoxin in rabbits, which is three times higher than that recommended by the USDA (4 IU.mL-1). In sheep, recombinant bacteria induced antitoxin titers of 4 IU.mL-1, 56 days after the first dose. rCPA which was expressed, mainly, in inclusion bodies, was not found to influence the immunogenicity of the vaccine. The recombinant Escherichia coli bacterin, produced simply and safely, is capable of affording protection against diseases caused by C. perfringens CPA. The current findings represent a novel production method for CPA vaccines potentially applicable to veterinary medicine.

Humoral Immune Response Evaluation in Horses Vaccinated with Recombinant Clostridium perfringens Toxoids Alpha and Beta for 12 Months.

In horses, Clostridium perfringens is associated with acute and fatal enterocolitis, which is caused by a beta toxin (CPB), and myonecrosis, which is caused by an alpha toxin (CPA). Although the most effective way to prevent these diseases is through vaccination, specific clostridial vaccines for horses against C. perfringens are not widely available. The aim of this study was to pioneer the immunization of horses with three different concentrations (100, 200 and 400 µg) of C. perfringens recombinant alpha (rCPA) and beta (rCPB) proteins, as well as to evaluate the humoral immune response over 360 days. Recombinant toxoids were developed and applied to 50 horses on days 0 and 30. Those vaccines attempted to stimulate the production of alpha antitoxin (anti-CPA) and beta antitoxin (anti-CPB), in addition to becoming innocuous, stable and sterile. There was a reduction in the level of neutralizing anti-CPA and anti-CPB antibodies following the 60th day; therefore, the concentrations of 200 and 400 µg capable of inducing a detectable humoral immune response were not determined until day 180. In practical terms, 200 µg is possibly the ideal concentration for use in the veterinary industry's production of vaccines against the action of C. perfringens in equine species.

Measurement over 1 Year of Neutralizing Antibodies in Cattle Immunized with Trivalent Vaccines Recombinant Alpha, Beta and Epsilon of Clostridium perfringens.

The alpha (CPA), beta (CPB) and epsilon (ETX) toxins of Clostridium perfringens are responsible for causing diseases that are difficult to eradicate and have lethal potential in production animals. Vaccination of herds is still the best control strategy. Recombinant clostridial vaccines have shown good success at inducing neutralizing antibody titers and appear to be a viable alternative to the conventional production of commercial clostridial toxoids. Research is still needed on the longevity of the humoral immune response induced by recombinant proteins in immunized animals, preferably in target species. The objective of this study was to measure the humoral immune response of cattle immunized with trivalent vaccines containing the recombinant proteins alpha (rCPA), beta (rCPB) and epsilon (rETX) of C. perfringens produced in Escherichia coli at three different concentrations (100, 200, and 400 µg) of each protein for 12 months. The recombinant vaccines containing 200 (RV2) and 400 µg (RV3) yielded statistically similar results at 56 days. They performed better throughout the study period because they induced higher neutralizing antibody titers and were detectable for up to 150 and 180 days, respectively. Regarding industrial-scale production, RV2 would be the most economical and viable formulation as it achieved results similar to RV3 at half the concentration of recombinant proteins in its formulation. However, none of the vaccines tested induced the production of detectable antibody titers on day 365 of the experiment, the time of revaccination typically recommended in vaccination protocols. Thus, reiterating the need for research in the field of vaccinology to achieve greater longevity of the humoral immune response against these clostridial toxins in animals, in addition to the need to discuss the vaccine schedules and protocols adopted in cattle production.

Protective efficacy of recombinant bacterin vaccine against botulism in cattle.

Botulism is a paralytic disease caused by the intoxication of neurotoxins produced by Clostridium botulinum. Among the seven immunologically distinct serotypes of neurotoxins (BoNTs A - G), serotypes C and D, or a chimeric fusion termed C/D or D/C, are responsible for animal botulism. The most effective way to prevent botulism in cattle is through vaccination; however, the commercially available vaccines produced by detoxification of native neurotoxins are time-consuming and hazardous. To overcome these drawbacks, a non-toxic recombinant vaccine was developed as an alternative. In this study, the recombinant protein vaccine was produced using an Escherichia coli cell-based system. The formaldehyde-inactivated E. coli is able to induce 7.45 ± 1.77 and 6.6 ± 1.28 IU/mL neutralizing mean titers against BoNTs C and D in cattle, respectively, determined by mouse neutralization bioassay, and was deemed protective by the Brazilian legislation. Moreover, when the levels of anti-BoNT/C and D were compared with those achieved by the recombinant purified vaccines, no significant statistical difference was observed. Cattle vaccinated with the commercial vaccine developed 1.33 and 3.33 IU/mL neutralizing mean titers against BoNT serotypes C and D, respectively. To the best of our knowledge, this study is the first report on recombinant E. coli bacterin vaccine against botulism. The vaccine was safe and effective in generating protective antibodies and, thus, represents an industry-friendly alternative for the prevention of cattle botulism.

Selection of a Clostridium perfringens type D epsilon toxin producer via dot-blot test.

Clostridium perfringens type D produces enterotoxemia, an enteric disease in ruminants, also known as pulpy kidney disease. Caused by epsilon toxin, enterotoxemia is a major exotoxin produced by this microorganism. Epsilon toxin is also the main component of vaccines against this enteric disorder. In this study, a standardized dot-blot was used to choose strains of C. perfringens type D that are producers of epsilon toxin. Clones producing epsilon toxin were chosen by limiting dilution; after three passages, lethal minimum dose titers were determined by soroneutralization test in mice. These clones produced epsilon toxin 240 times more concentrated than the original strain. The presence of the epsilon toxin gene (etx) was verified by polymerase chain reaction. All clones were positive, including those determined to be negative by dot-blot tests, suggesting that mechanisms in addition to the presence of the etx gene can influence toxin production. The dot-blot test was efficient for the selection of toxigenic colonies of C. perfringens type D and demonstrated that homogeneous populations selected from toxigenic cultures produce higher titers of epsilon toxin.

Immunogenicity of a Bivalent Non-Purified Recombinant Vaccine against Botulism in Cattle.

Botulism is a potentially fatal intoxication caused by botulinum neurotoxins (BoNTs) produced mainly by Clostridium botulinum. Vaccination against BoNT serotypes C and D is the main procedure to control cattle botulism. Current vaccines contain formaldehyde-inactivated native BoNTs, which have a time-consuming production process and pose safety risks. The development of non-toxic recombinant vaccines has helped to overcome these limitations. This study aims to evaluate the humoral immune response generated by cattle immunized with non-purified recombinant fragments of BoNTs C and D. Cattle were vaccinated in a two-dose scheme with 100, 200 and 400 µg of each antigen, with serum sampling on days 0, 56, 120, and 180 after vaccination. Animals who received either 200 or 400 µg of both antigens induced titers higher than the minimum required by the Brazilian ministry of Agriculture, Livestock and Food Supply and achieved 100% (8/8) seroconversion rate. Animals vaccinated with commercial toxoid vaccine had only a 75% (6/8) seroconversion rate for both toxins. Animals that received doses containing 400 µg of recombinant protein were the only ones to maintain titers above the required level up until day 120 post-vaccination, and to achieve 100% (8/8) seroconversion for both toxins. In conclusion, 400 µg the recombinant Escherichia coli cell lysates supernatant was demonstrated to be an affordable means of producing an effective and safe botulism vaccine for cattle.

Humoral Response of Buffaloes to a Recombinant Vaccine against Botulism Serotypes C and D.

Botulism is a fatal intoxication caused by botulinum neurotoxins (BoNTs), which are mainly produced by Clostridium botulinum and characterized by flaccid paralysis. The BoNTs C and D are the main serotypes responsible for botulism in animals, including buffaloes. Botulism is one of the leading causes of death in adult ruminants in Brazil due to the high mortality rates, even though botulism in buffaloes is poorly reported and does not reflect the real economic impact of this disease in Brazilian herds. Vaccination is reported as the most important prophylactic measure for botulism control, although there are no specific vaccines commercially available for buffaloes in Brazil. This study aimed to evaluate the humoral immune response of buffalo groups vaccinated with three different concentrations of recombinant proteins (100, 200, and 400 µg) against BoNTs serotypes C and D as well as to compare the groups to each other and with a group vaccinated with a bivalent commercial toxoid. The recombinant vaccine with a concentration of 400 µg of proteins induced the highest titers among the tested vaccines and was proven to be the best choice among the formulations evaluated and should be considered as a potential vaccine against botulism in buffalo.

Recombinant Alpha, Beta, and Epsilon Toxins of Clostridium perfringens: Production Strategies and Applications as Veterinary Vaccines.

Clostridium perfringens is a spore-forming, commensal, ubiquitous bacterium that is present in the gastrointestinal tract of healthy humans and animals. This bacterium produces up to 18 toxins. The species is classified into five toxinotypes (A-E) according to the toxins that the bacterium produces: alpha, beta, epsilon, or iota. Each of these toxinotypes is associated with myriad different, frequently fatal, illnesses that affect a range of farm animals and humans. Alpha, beta, and epsilon toxins are the main causes of disease. Vaccinations that generate neutralizing antibodies are the most common prophylactic measures that are currently in use. These vaccines consist of toxoids that are obtained from C. perfringens cultures. Recombinant vaccines offer several advantages over conventional toxoids, especially in terms of the production process. As such, they are steadily gaining ground as a promising vaccination solution. This review discusses the main strategies that are currently used to produce recombinant vaccines containing alpha, beta, and epsilon toxins of C. perfringens, as well as the potential application of these molecules as vaccines for mammalian livestock animals.

Clostridium perfringens α and ß recombinant toxoids in equine immunization / Toxóides recombinantes α e ß de Clostridium perfringens na imunização de equinos

Clostridium perfringens is considered one of the main causative agents of superacute enterocolitis, usually fatal in the equine species, due to the action of the ß toxin, and is responsible for causing severe myonecrosis, by the action of the α toxin. The great importance of this agent in the equine economy is due to high mortality and lack of vaccines, which are the main form of prevention, which guarantee the immunization of this animal species. The aim of this study was to evaluate three different concentrations (100, 200 and 400µg) of C. perfringens α and ß recombinant toxoids in equine immunization and to compare with a group vaccinated with a commercial toxoid. The commercial vaccine was not able to stimulate an immune response and the recombinant vaccine was able to induce satisfactory humoral immune response in vaccinated horses, proving to be an alternative prophylactic for C. perfringens infection.(AU)

Clostridium perfringens é considerado um dos principais agentes causadores de enterocolites superagudas, geralmente fatais na espécie equina, devido à ação da toxina ß, além de ser responsável por causar quadros graves de mionecrose, pela ação da toxina α. A grande importância desses agentes na equinocultura, deve-se a elevada mortalidade e a inexistência de vacinas, principal forma de prevenção, que garantam a imunização dessa espécie animal. O objetivo deste trabalho foi avaliar três diferentes concentrações (100, 200 e 400µg) dos toxóides recombinantes α e ß de C. perfringens na imunização de equinos, bem como comparar com um grupo vacinado com um toxóide comercial. A vacina comercial não se mostrou capaz de estimular uma resposta imune e a vacina recombinante foi capaz de induzir resposta imune humoral satisfatória em equinos vacinados, provando ser uma alternativa profilática para infecção por C. Perfringens.(AU)

Vaccination of cattle with a recombinant bivalent toxoid against botulism serotypes C and D.

Cattle botulism is a fatal intoxication caused by botulinum neurotoxins (BoNTs) produced by Clostridium botulinum serotypes C and D resulting in economic losses. Vaccination is the most effective way to control botulism. However, the commercially available vaccines are difficult and hazardous to produce. Neutralizing antibodies against the C-terminal fragment of the BoNT heavy chain (HC) are known to protect against lethal doses of BoNTs. We report the vaccination of cattle with a previously tested recombinant chimera consisting of Escherichia coli heat-labile enterotoxin B subunit and the HC of BoNTs C and D. Vaccinated animals produced neutralizing antibodies against serotypes C and D averaging 5±0 and 6.14±1.06IU/mL, respectively. For BoNT D, the titers were greater than those measured for the commercial vaccine, which induced titers of 5±0 and 2.85±1.35 against the respective serotypes, suggesting that this chimera is effective against cattle botulism.

Vaccination with recombinant Clostridium perfringens toxoids α and ß promotes elevated antepartum and passive humoral immunity in swine.

Due to the increasingly restricted use of antimicrobials in animal production systems, the prevention and control of Clostridium perfringens type A- and C-induced diarrhea in piglets should be based on passive immunization via the prepartum vaccination of sows. Given the current obstacles in the production of conventional clostridial vaccines, the use of recombinant proteins has been considered to represent a promising alternative. In the present study, the neutralizing antibody response of immunized sows and their litters to a bivalent vaccine containing the C. perfringens recombinant toxoids alpha (rTA) and beta (rTB) produced in Escherichia coli was assessed. Rabbits (n=8) and pregnant sows (n=7) were immunized with 200µg of each recombinant antigen using Al(OH)3 as adjuvant. The alpha and beta antitoxin titer detected in the rabbits' serum pool was 9.6 and 20.4IU/mL, respectively. The mean alpha and beta antitoxin titers in the sows' sera were 6.0±0.9IU/mL and 14.5±2.2IU/mL, and the corresponding individual coefficients of variation (CV) were 16.04% and 14.91%, respectively. The mean alpha and beta antitoxin titers in the litters' serum pools were 4.2±0.4IU/mL and 10.9±1.7IU/mL, and the CV between litters was 9.23% and 9.85%, respectively. The results showed that the rTA and rTB proteins produced and tested in the present study induced an immune response and can be regarded as candidates for the development of a commercial vaccine against C. perfringens type A- and C-induced diarrhea in pigs.

Production and evaluation of a recombinant chimeric vaccine against clostridium botulinum neurotoxin types C and D.

Bovine botulism is a fatal disease that is caused by botulinum neurotoxins (BoNTs) produced by Clostridium botulinum serotypes C and D and that causes great economic losses, with nearly 100% lethality during outbreaks. It has also been considered a potential source of human food-borne illness in many countries. Vaccination has been reported to be the most effective way to control bovine botulism. However, the commercially available toxoid-based vaccines are difficult and hazardous to produce. Neutralizing antibodies targeted against the C-terminal fragment of the BoNT heavy chain (HC) are known to confer efficient protection against lethal doses of BoNTs. In this study, a novel recombinant chimera, consisting of Escherichia coli heat-labile enterotoxin B subunit (LTB), a strong adjuvant of the humoral immune response, fused to the HC of BoNT serotypes C and D, was produced in E. coli. Mice vaccinated with the chimera containing LTB and an equivalent molar ratio of the chimera without LTB plus aluminum hydroxide (Al(OH)3) developed 2 IU/mL of antitoxins for both serotypes. Guinea pigs immunized with the recombinant chimera with LTB plus Al(OH)3 developed a protective immune response against both BoNT/C (5 IU/mL) and BoNT/D (10 IU/mL), as determined by a mouse neutralization bioassay with pooled sera. The results achieved with guinea pig sera fulfilled the requirements of commercial vaccines for prevention of botulism, as determined by the Brazilian Ministry of Agriculture, Livestock and Food, Supply. The presence of LTB was essential for the development of a strong humoral immune response, as it acted in synergism with Al(OH)3. Thus, the vaccine described in this study is a strong candidate for the control of botulism in cattle.

Brucellosis in water buffaloes / Brucelose em bubalinos

The domestication of water buffaloes (Bubalus bubalis) originated in India and China and spread throughout the world and represents an important source of food of high biological value. Given the importance and relevance of brucellosis for buffalo production, this article reviews the history, etiopathogenesis, epidemiology, clinical signs, anatomopathological findings, diagnosis and control of the disease, focusing on data from studies on water buffaloes performed in different countries and the Brazilian Amazon biome.(AU)

A domesticação do búfalo (Bubalus bubalis) ocorreu particularmente na Índia e China, difundindo-se pelo mundo, gerando fontes de alimento de alto valor biológico. Diante da importância e relevância da brucelose para a bubalinocultura este trabalho tem por objetivo fazer uma revisão acerca do histórico da doença, etiopatogenia, fatores epidemiológicos, sinais clínicos, achados anatomopatológicos, diagnóstico e o controle, com enfoque nos dados obtidos em estudos em bubalinos no mundo e no Bioma Amazônico brasileiro.(AU)

Type C waterborne botulism outbreaks in buffaloes (Bubalus bubalis) in the Amazon region / Surtos de botulismo hídrico tipo C em búfalos (Bubalus bubalis) na região amazônica

Botulism is a poisoning caused by botulinum neurotoxins (BoNTs). BoNTs serotypes C and D are involved in botulism outbreaks in cattle in several countries. Despite the high number of buffaloes worldwide, the real impact of botulism in buffaloes is not known, because it is not a notifiable disease in Brazil and only few studies have evaluated the occurrence of the disease in buffaloes. Those studies did not conduct diagnostic tests to confirm the presence of BoNTs. The objective of the present study was to describe three outbreaks of botulism in buffaloes in the Brazilian Amazon region considering epidemiological and clinical data as well as laboratory diagnosis to confirm the presence of BoNTs. The results of the bioassay were negative in the tissues and in feed samples, but positive for BoNT C in water samples. Confirmation of the occurrence of botulism in buffaloes allows the implementation of preventive strategies in susceptible herds. Waterborne botulism in buffaloes is prevented by ensuring the constant circulation of water collections and restricting the presence of dead animals and bones in order to prevent the accumulation of organic matter and the development of anaerobic conditions, which might favor the replication of Clostridium botulinum. Another measure that can be adopted is the shading of the pasture, in order to maintain the thermal comfort for the buffaloes and to avoid the excess of permanence of them in the water pools.(AU)

Botulismo é uma intoxicação causada por neurotoxinas botulínicas (BoNTs). Os sorotipos C e D de BoNTs estão envolvidos em surtos de botulismo em bovídeos em vários países. Apesar do elevado número de búfalos em todo o mundo, o real impacto do botulismo em búfalos não é conhecido; pois não é uma doença de notificação obrigatória no Brasil e poucos estudos avaliaram a incidência desta doença em búfalos. Além disso, estes estudos não realizaram testes diagnósticos para confirmar a presença de BoNTs. O objetivo do presente estudo foi descrever três surtos de botulismo em búfalos na região amazônica brasileira, considerando dados epidemiológicos e clínicos, bem como o diagnóstico laboratorial para confirmar a presença de BoNTs. Os resultados do bioensaio em camundongos foram negativos em todos os tecidos e nas amostras de alimentos testados; no entanto foram positivos para BoNT C nas amostras de água. A confirmação da ocorrência de botulismo em búfalos permite a implementação de estratégias preventivas nos rebanhos. O botulismo hídrico nos búfalos pode ser prevenido assegurando-se que coleções de água fossem mantidas limpas, sem a presença de animais mortos e ossadas no seu interior e não permitindo o acúmulo de matéria orgânica e condições de anaerobiose favoráveis à multiplicação de Clostridium. botulinum. Outra medida que pode ser adotada é o sombreamento das pastagens, a fim de manter o conforto térmico dos búfalos e assim evitar o excesso de sua permanência dentro das fontes de água.(AU)

Teores de cobre, zinco e ferro no fígado de búfalos (Bubalus bubalis) com paratuberculose / Copper, zinc and iron content in the liver of buffaloes (Bubalus bubalis) with paratuberculosis

Com o objetivo de realizar um estudo dos teores de cobre (Cu), zinco (Zn) e ferro (Fe) em búfalas com paratuberculose (PTB) foram utilizadas 13 búfalas, das raças Murrah, Mediterrâneo e seus mestiços acima de três anos de idade, pertencentes a duas propriedades localizadas nos municípios de São Luiz e São Mateus, no Estado do Maranhão. Os animais foram selecionados de acordo com a presença de sinais clínicos sugestivos de paratuberculose, caracterizados por estado nutricional regular a ruim, diarreia crônica líquida a semi-líquida, desidratação, edema submandibular, anestro prolongado, mastites e verminose gastrintestinal. Foi realizada biópsia retal em todos os animais, para detecção de Mycobacterium avium subsp. paratuberculosis (Map) por meio da qPCR, e exames histopatológicos (HE e Ziehl-Neelsen)...

In order to study copper, zinc and iron concentration in the liver of buffaloes with paratuberculosis (PTB), 13 buffalo cows above three years of age of the Murrah and Mediterranean races or their crosses were used. They originated from two farms in the municipalities of São Luís and São Mateus, state of Maranhão, Brazil. The animals were selected according to clinical signs of paratuberculosis, as diarrhea, dehydration and submandibular edema. Rectal biopsies for detection of Mycobacterium avium subsp. paratuberculosis (Map) through PCR in real time (qPCR) and Ziehl-Neelsen (ZN) stain were performed on all animals...

Infecção transplacentária e intrauterina por Brucella abortus em búfalos (Bubalus bubalis) / Placenta and intrauterine infection by Brucella abortus in buffaloes (Bubalus bubalis)

O objetivo deste trabalho foi verificar a presença de Brucella abortus e as lesões causadas por esse agente nos anexos fetais e nos fetos de búfalas. Para isso, 20 búfalas em diversos meses de gestação, sorologicamente positivas para brucelose, foram submetidas ao abate sanitário. A idade fetal foi determinada através de exames ultrassonográficos associados à mensuração dos fetos durante a necropsia. Do útero fechado desses animais foram coletadas amostras para histopatologia e qPCR. A partir do segundo mês de gestação foi possível detectar a presença de DNA de B. abortus em líquido amniótico, líquido alantoide e em útero e, a partir do quinto mês, na placenta, coração, baço, rim, pulmão, intestino, fígado e linfonodos dos fetos. Os principais achados anatomopatológicos foram placentite fibrinopurulenta necrótica e endometrite supurativa crônica...

The objective of this study was to detect Brucella abortus and injuries caused by the bacteria in fetal membranes and fetuses. Twenty buffaloes serologically positive for brucellosis were used and subjected to stamping for collection of material from the closed uterus of several months gestation. Fetal age was determined by ultrasound examination and the size of fetuses was measured at necropsy. The samples were subjected to histopathology and qPCR. From the second month of pregnancy on it was possible to detect the presence of B. abortus DNA in amniotic fluid, allantoic liquid and uterus, and from the fifth month on in placenta, heart, spleen, kidney, lung, intestine, liver and lymph nodes of the fetuses. The main pathological findings were fibrinous suppurative necrotic placentitis, and chronic endometritis...

Potency against enterotoxemia of a recombinant Clostridium perfringens type D epsilon toxoid in ruminants.

Enterotoxemia, a disease that affects domestic ruminants, is caused mainly by the epsilon toxin from Clostridium perfringens type D. Its eradication is virtually impossible, control and prophylaxis are based on systematic vaccination of herds with epsilon toxoids that are efficient in inducing protective antibody production. The use of recombinant toxins is one of the most promising of these strategies. This work evaluates the potency of a Cl. perfringens type D epsilon toxoid expressed by Escherichia coli administered to goats, sheep, and cattle. The etx gene was cloned into the pET-11a plasmid of E. coli strain BL21 to produce the recombinant toxin. Rabbits (n=8), goats, sheep, and cattle (n=5 for each species) were immunized with 0.2mg of the insoluble recombinant protein fraction to evaluate vaccine potency of the epsilon toxoid studied. Antibody titers were 40, 14.3, 26, and 13.1 IU/mL in the rabbit, goat, sheep, and cattle serum pools, respectively. The epsilon toxoid produced and tested in this work is adequate for immunization of ruminants against enterotoxemia.