Trypanosoma sp. infection in Boa constrictor snakes: morphological, hematological, clinical biochemistry, molecular, and phylogenetic characteristics.

There are few reports of Trypanosoma in snakes, as well as little information about its pathogenicity in these animals. Thus, the present study aimed to characterize Trypanosoma found in Boa constrictor snakes, to verify the influence of the parasitism on hematological and clinical biochemistry parameters, and to perform a phylogenetic study of the isolates. Blood samples from sixty-one boas were analyzed for the presence of trypanosomatids and by hematological and clinical biochemistry assays. The flagellates that were found in this analysis were used for cell culture, morphometry, and molecular analysis. Later, molecular typing phylogenetic studies were performed. Nine positive animals (14.75%) were identified by microscopy analysis. The hematological results showed that parasitized animals presented significantly lower levels of packed cell volume, hemoglobin, mean corpuscular volume, and mean corpuscular hemoglobin. In the leukogram, eosinophils and heterophils counts were higher in parasitized animals. Considering the molecular analyses, the isolates presented a higher identity of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the 18S small subunit ribosomal RNA (SSU rRNA) gene fragments with Trypanosoma serpentis. The phylogenetic tree, using the GAPDH, clustered all isolates with T. serpentis and Trypanosoma cascavelli. This is the first description of T. serpentis parasitizing boas and of the clinical changes caused by trypanosomatid infection in snakes.

Sunflower Cake from the Biodiesel Industry in the Diet Improves the Performance and Carcass Traits of Nellore Young Bulls.

We aimed to determine the optimal inclusion level of sunflower cake (0, 90, 180, and 270 g/kg total DM) as a partial replacement of soybean meal and corn ground in young bulls' diets by examining nutrient intake and digestibility, ingestive behavior, nitrogen balance, metabolic serum profile, growth performance, and carcass traits. Thirty-two intact Nellore bulls (BW 374 ± 42.5) were distributed in a completely randomized design. The experiment lasted 90 days. The final BW of the animals was 515.25 ± 24.7. There was a linear decrease effect in the intake of DM, crude protein and nonfibrous carbohydrates, eating and rumination efficiency, N-urinary, N-total excretion, and blood urea nitrogen. Sunflower cake did not affect the NDF digestibility, nitrogen (N)-fecal excretion, blood metabolites, Longissimus lumborum muscle area, or subcutaneous fat deposition. There were linear and quadratic effects on the eating and rumination time, microbial protein production and efficiency, gamma-glutamyl transferase and cholesterol serum concentrations, and muscle carcass tissue. There was a quadratic effect on ether extract intake, final BW, and total gain with the inclusion of sunflower cake in the young bull's diet. The replacement of soybean meal and corn ground with sunflower cake at the level of 90 g/kg of DM in the diet of young bulls is recommended because it reduces the DM intake and digestibility, increases microbial protein synthesis and muscle tissue deposition, and consequently improves the performance, feed efficiency, and carcass traits.

Activity of antifungal drugs and Brazilian red and green propolis extracted with different methodologies against oral isolates of Candida spp.

BACKGROUND: Oral candidiasis is an opportunistic disease caused by fungi of the Candida genus. The occurrence of Candida spp. resistance to the commercial antifungal drugs points to the search for alternative treatments. Propolis has been successfully used in the treatment of infectious diseases for centuries. It has been proposed that an ultrasound pretreatment in the propolis extraction protocol can enhance the concentrations of molecules with antimicrobial activities in the final extract. Thus, this study aimed to compare the antifungal activity against oral Candida spp. isolates of green and red propolis extracts submitted or not to an ultrasound pretreatment before the extraction procedure. METHODS: Candida spp. were isolated from denture stomatitis lesions and identified by sequencing. Oral Candida spp. isolates and reference strains were submitted to broth microdilution assays using commercial antifungals and Brazilian green and red propolis extracts submitted or not to an ultrasound pretreatment. Minimal Inhibitory Concentrations (MIC) and Minimal Fungicide Concentrations (MFC) were determined and biofilm formation interference was evaluated for resistant isolates. RESULTS: C. albicans, Candida tropicalis and Candida dubliniensis were isolated from denture stomatitis lesions. Growth inhibition was observed in all Candida isolates incubated with all green and red propolis extracts. At lower doses, red propolis extracts presented significant antifungal activity. The ultrasound pretreatment did not promote an increase in the antifungal activity of green or red propolis. Three isolates, which were highly resistant to fluconazole and itraconazole, were susceptible to low doses of red propolis extracts. These same three specimens had their biofilm formation inhibted by red propolis ethanolic extract. CONCLUSIONS: Thus, red propolis can be faced as a promising natural product to be used in the auxiliary antifungal therapy of denture stomatitis.

Intestinal changes and performance parameters in ticks feeding on calves immunized with subunits of immunogens against Rhipicephalus microplus.

We describe the intestinal changes and biological parameters of the tick species Rhipicephalus microplus exposed to the immune response of calves vaccinated with two subunits of immunogens. The first group of Bos taurus calves was immunized with a synthetic peptide (SBm7462), whereas the second group received an inoculum for synthetic control. The third group was immunized with a recombinant peptide (rSBm7462); an inoculum was injected into a fourth group of calves for recombinant control. Each formulation was administered to these calves during three times at intervals of 30 days. At 21 days after the last immunization, the calves were challenged using a total of 4500 larvae per animal. Indirect ELISA was realized to identify the kinetics of IgGs from samples of calves studied. Naturally detaching ticks were collected for analyses of biological performance and histological changes in the midgut. We dissected randomly detached ticks. The midgut of each of these ticks was removed and processed routinely for histology, stained with hematoxylin-eosin (H&E) and slow Giemsa. Slides were also subjected to immunohistochemistry. The antibody response showed significant induction of high-affinity IgGs in calves immunized with both peptides in comparison to calves of the control groups. Histological changes included damage of the intestinal epithelium in ticks fed on immunized hosts and intense immunostaining in midgut cells, using the serum of calves immunized with recombinant peptide. There were significant differences in all biological performing parameters of ticks detached from vaccinated calves in comparison with ticks of the control groups. We identified reductions of 87.7 and 93.5% in engorged ticks detached from calves immunized with a synthetic and recombinant peptides, respectively, a 28 and 8.60% lower egg mass in groups immunized with synthetic and recombinant peptides, respectively, and a 38.4% reduction of the value of nutrient index/tick in the group immunized with the recombinant peptide. Our findings show that the immune response induced by small peptides in cattle can modify the digestion and metabolism of ticks fed on vaccinated animals, resulting in changes in tick performance.

NMR insights on nano silver post-surgical treatment of superficial caseous lymphadenitis in small ruminants.

Caseous lymphadenitis (CL), caused by a pathogen of the second class of biosafety - Corynebacterium pseudotuberculosis, is a chronic and severe infectious disease that affects small ruminants and requires long, ineffective treatment which generally leads to animal sacrifice so as to stop the disease spreading. The infected animals suffer the excision of affected superficial lymph nodes and post-surgical treatment with iodine (10% solution in ethanol) and, sometimes, prolonged antibiotic use, but only if the sick animals are of great importance to breeding. Herein, we propose a cheap and easy to apply treatment of CL with excellent results using biogenic silver nanoparticles (AgNP) based technology. AgNP antibacterial properties were investigated in vitro against Corynebacterium pseudotuberculosis cells and in vivo on small ruminants with CL. Treatment of surgical wounds resulting from the excision of superficial CL lesions with a AgNP-based cream was compared to the standard post-surgical treatment method by iodine. Also, the effects of AgNP-based cream treatment were evaluated and compared with the effects of the iodine CL treatment by serum NMR-based metabolomics. Serum samples were collected from 29 animals, 9 sheep and 20 goats, during the treatments and analyzed. All animals showed stable serum metabolomes when iodine or AgNP-based cream effects were compared. The AgNP-based cream treatment showed excellent results, especially in accelerating the healing of wounds, which occurred two to three times faster in comparison with the iodine treatment. AgNP-based cream treatment also prevented CL reappearance and did not cause any side effects on animals. This is the first report on very effective post-surgical treatment of superficial CL in small ruminants based on biogenic silver nanoparticles, which might open up the possibility for a safe veterinary application of AgNP-based cream.

Insight of Genus Corynebacterium: Ascertaining the Role of Pathogenic and Non-pathogenic Species.

This review gathers recent information about genomic and transcriptomic studies in the Corynebacterium genus, exploring, for example, prediction of pathogenicity islands and stress response in different pathogenic and non-pathogenic species. In addition, is described several phylogeny studies to Corynebacterium, exploring since the identification of species until biological speciation in one species belonging to the genus Corynebacterium. Important concepts associated with virulence highlighting the role of Pld protein and Tox gene. The adhesion, characteristic of virulence factor, was described using the sortase mechanism that is associated to anchorage to the cell wall. In addition, survival inside the host cell and some diseases, were too addressed for pathogenic corynebacteria, while important biochemical pathways and biotechnological applications retain the focus of this review for non-pathogenic corynebacteria. Concluding, this review broadly explores characteristics in genus Corynebacterium showing to have strong relevance inside the medical, veterinary, and biotechnology field.

Conjugation-Mediated Horizontal Gene Transfer of Clostridium perfringens Plasmids in the Chicken Gastrointestinal Tract Results in the Formation of New Virulent Strains.

Clostridium perfringens is a gastrointestinal pathogen capable of causing disease in a variety of hosts. Necrotic enteritis in chickens is caused by C. perfringens strains that produce the pore-forming toxin NetB, the major virulence factor for this disease. Like many other C. perfringens toxins and antibiotic resistance genes, NetB is encoded on a conjugative plasmid. Conjugative transfer of the netB-containing plasmid pJIR3535 has been demonstrated in vitro with a netB-null mutant. This study has investigated the effect of plasmid transfer on disease pathogenesis, with two genetically distinct transconjugants constructed under in vitro conditions, within the intestinal tract of chickens. This study also demonstrates that plasmid transfer can occur naturally in the host gut environment without the need for antibiotic selective pressure to be applied. The demonstration of plasmid transfer within the chicken host may have implications for the progression and pathogenesis of C. perfringens-mediated disease. Such horizontal gene transfer events are likely to be common in the clostridia and may be a key factor in strain evolution, both within animals and in the wider environment.IMPORTANCEClostridium perfringens is a major gastrointestinal pathogen of poultry. C. perfringens strains that express the NetB pore-forming toxin, which is encoded on a conjugative plasmid, cause necrotic enteritis. This study demonstrated that the conjugative transfer of the netB-containing plasmid to two different nonpathogenic strains converted them into disease-causing strains with disease-causing capability similar to that of the donor strain. Plasmid transfer of netB and antibiotic resistance was also demonstrated to occur within the gastrointestinal tract of chickens, with approximately 14% of the isolates recovered comprising three distinct, in vivo-derived, transconjugant types. The demonstration of in vivo plasmid transfer indicates the potential importance of strain plasticity and the contribution of plasmids to strain virulence.

Complete Genome Sequence of the Attenuated Corynebacterium pseudotuberculosis Strain T1.

We present here the genome sequence of the attenuated Corynebacterium pseudotuberculosis strain T1. The sequencing was performed with an Ion Torrent Personal Genome Machine platform. The genome is a circular chromosome of 2,337,201 bp, with a G+C content of 52.85% and a total of 2,125 coding sequences (CDSs), 12 rRNAs, 49 tRNAs, and 24 pseudogenes.

Putative virulence factors of Corynebacterium pseudotuberculosis FRC41: vaccine potential and protein expression.

BACKGROUND: Corynebacterium pseudotuberculosis, a facultative intracellular bacterial pathogen, is the etiological agent of caseous lymphadenitis (CLA), an infectious disease that affects sheep and goats and it is responsible for significant economic losses. The disease is characterized mainly by bacteria-induced caseous necrosis in lymphatic glands. New vaccines are needed for reliable control and management of CLA. Thus, the putative virulence factors SpaC, SodC, NanH, and PknG from C. pseudotuberculosis FRC41 may represent new target proteins for vaccine development and pathogenicity studies. RESULTS: SpaC, PknG and NanH presented better vaccine potential than SodC after in silico analyses. A total of 136 B and T cell epitopes were predicted from the four putative virulence factors. A cluster analysis was performed to evaluate the redundancy degree among the sequences of the predicted epitopes; 57 clusters were formed, most of them (34) were single clusters. Two clusters from PknG and one from SpaC grouped epitopes for B and T-cell (MHC I and II). These epitopes can thus potentially stimulate a complete immune response (humoral and cellular) against C. pseudotuberculosis. Several other clusters, including two from NanH, grouped B-cell epitopes with either MHC I or II epitopes. The four target proteins were expressed in Escherichia coli. A purification protocol was developed for PknG expression. CONCLUSIONS: In silico analyses show that the putative virulence factors SpaC, PknG and NanH present good potential for CLA vaccine development. Target proteins were successfully expressed in E. coli. A protocol for PknG purification is described.

Determinação de valores de referência séricos para os eletrólitos magnésio, cloretos, cálcio e fósforo em ovinos das raças Dorper e Santa Inês / Determination of magnesium, chloride, calcium and phosphorus serum reference values for Dorper and Santa Inês sheep breeds

A ovinocultura no Brasil é uma atividade em grande expansão e, com o aumento da demanda mundial por carne ovina, aumentou-se o interesse no monitoramento da sanidade do rebanho, utilizando diversas ferramentas como auxiliares no diagnóstico clínico, tais como os intervalos de referência séricos. Os elementos minerais constituem 2 a 5,5% do corpo dos vertebrados, exercendo diversas funções no organismo. O objetivo deste trabalho foi obter intervalos de referência para os eletrólitos magnésio, fósforo, cloreto e cálcio para ovinos das raças Dorper e Santa Inês. Foram coletados soros de 487 animais clinicamente sadios, sendo 146 da raça Dorper e 341 da raça Santa Inês. Os eletrólitos foram mensurados utilizando-se kits comerciais. Os dados foram analisados quanto à raça, sexo e idade, e os intervalos de referência determinados. Os resultados revelaram diferenças significativas nos intervalos de referência obtidos para os eletrólitos cálcio e magnésio na variável raça, e para o eletrólito fósforo na variável faixa etária e, quando confrontados com valores de referência já publicados, comprovou-se a existência de diferença estatística significativa entre os mesmos em todos os analitos estudados.

The sheep industry in Brazil is an important economic activity, and with the increasing global demand for sheep meat there is a great interest in the monitoring of the herd health, and serum reference ranges are basic tools for veterinary clinical pathology assays. Mineral elements correspond to 2-5.5% of the body of vertebrates, holding different functions in their physiology. The objective of this study was to obtain reference intervals of the electrolytes magnesium, phosphorus, chloride and calcium for the Dorper and Saint Ines sheep breeds. Sera samples were collected from 487 clinically healthy sheep, 146 from Dorper and 341 from Santa Ines breed. Electrolytes were measured using commercial kits. Data were analyzed taking the race, sex and age variables in account, and reference ranges were established. The results revealed significant statistical differences in reference ranges obtained for the electrolytes calcium and magnesium concerning the variable race, and for the electrolyte phosphorus in the variable age and, when compared with reference values already published, proved the existence of significant differences.

Complete Genome Sequence of Corynebacterium pseudotuberculosis Viscerotropic Strain N1.

We present the complete genome sequence of Corynebacterium pseudotuberculosis strain N1. The sequencing was performed with the Ion Torrent Personal Genome Machine system. The genome is a circular chromosome with 2,337,845 bp, a G+C content of 52.85%, and a total of 2,045 coding sequences, 12 rRNAs, 49 tRNAs, and 58 pseudogenes.

Infecção por Corynebacterium pseudotuberculosis em equinos: aspectos microbiológicos, clínicos e preventivos / Corynebacterium pseudotuberculosis infection in horses: clinical, microbiological and prevention aspects

Corynebacterium pseudotuberculosis é o agente causador da linfadenite caseosa em caprinos e ovinos, sendo responsável por significativas perdas econômicas na ovinocaprinocultura mundialmente. Esta bactéria Gram-positiva também infecta equinos, causando desde quadros assintomáticos até infecções sistêmicas, podendo levar o animal a óbito. Especificamente no Brasil, não foram relatados casos de infecção em equinos, mas acredita-se que, devido à convivência de pequenos ruminantes infectados com equinos em diversas propriedades rurais, seja natural que ocorra a infecção desses animais. A presente revisão tem como objetivo fornecer informações sobre a bactéria C. pseudotuberculosis, sobre os aspectos epidemiológicos e clínicos da infecção em equídeos, bem como sobre técnicas de manejo para sua prevenção...

Corynebacterium pseudotuberculosis is the etiologic agent of caseous lymphadenitis in sheep and goats, an infectious disease that is responsible for significant economic losses in small ruminants breeding units worldwide. This Gram-positive bacterium can infect horses, causing symptomatic disease to systemic infections, which can lead to animals' death. Specifically in Brazil, there are no scientific records of equine infections, but it is believed that, due to the maintenance of infected small ruminants in close association to horses in many properties, the infection of horses may be a reality. The present work had the objective to present information on the bacteria C. pseudotuberculosis, on the epidemiological and clinical aspects of the infection in horses, as well as information about breeding procedures that can be adopted in order to prevent the infection...

Corynebacterium pseudotuberculosis cp09 mutant and cp40 recombinant protein partially protect mice against caseous lymphadenitis.

BACKGROUND: Caseous lymphadenitis (CLA) is an infectious disease that affects small ruminants and is caused by Corynebacterium pseudotuberculosis. This disease is responsible for high economic losses due to condemnation and trim of infected carcasses, decreased leather and wool yield, loss of sales of breeding stock and deaths from internal involvement. Treatment is costly and ineffective; the most cost-effective strategy is timely immunisation. Various vaccine strategies have been tested, and recombinant vaccines are a promising alternative. Thus, in this study, different vaccine formulations using a recombinant protein (rCP40) and the CP09 live recombinant strain were evaluated. Five groups of 10 mice each were immunised with saline (G1), rCP40 (G2), CP09 (G3), a combination of CP09 and rCP40 (G4) and a heterologous prime-boost strategy (G5). Mice received two immunisations within 15 days. On day 30 after primary immunisation, all groups were challenged with a C. pseudotuberculosis virulent strain. Mice were monitored and mortality was recorded for 30 days after challenge. RESULTS: The G2, G4 and G5 groups showed high levels of IgG1 and IgG2a; G2 presented significant IgG2a production after virulent challenge in the absence of IgG1 and IgG3 induction. Thirty days after challenge, the mice survival rates were 20 (G1), 90 (G2), 50 (G3), 70 (G4) and 60% (G5). CONCLUSIONS: rCP40 is a promising target in the development of vaccines against caseous lymphadenitis.

Association between haptoglobin and IgM levels and the clinical progression of caseous lymphadenitis in sheep.

BACKGROUND: Sheep caseous lymphadenitis (CLA), caused by Corynebacterium pseudotuberculosis (Cp), is associated with direct economic losses and presents significant zoonotic potential. Despite the importance of the disease, a satisfactory vaccine model has not been developed. Thus, this study aimed to investigate the association between haptoglobin (Hp) and IgM levels and the clinical progression of CLA in primarily infected sheep and in sheep immunized with Cp- secreted antigens adjuvanted with Quillaja saponaria saponins. These animals were kept with CLA-positive sheep to simulate natural exposure that occurs in field conditions. During the experiment, the Hp and IgM levels were monitored for 21 days, and the development of internal CLA lesions was investigated through necropsies on day182 post-immunization. RESULTS: Primarily infected sheep in Group 2 (inoculated with 2x105 Cp virulent strain) had higher Hp values between the first and ninth days post inoculation (PI) than sheep in Group 1 (control; P < 0.05). Immunized animals in Group 3 had significantly higher Hp values between the third and seventh days PI, compared with the control group (P < 0.01). Binary logistic regression (BLR) analysis of primarily infected sheep indicated an association between Hp concentration and CLA clinical progression: animals with high Hp values had 99.9% less risk of having CLA abscesses than animals with low Hp levels (Odds ratio = 0.001, P < 0.05). Both experimental groups had significantly higher IgM titers than the control group around the ninth and eleventh days PI (P < 0.05). The BLR analysis for immunized sheep indicated an association between IgM levels and clinical progression: sheep with high IgM titers had 100.0% less risk of having CLA abscesses than animals with low IgM levels (Odds ratio = 0.000, P < 0.05). CONCLUSIONS: Resistance to C. pseudotuberculosis infection is supported by the early acute phase response, in which up-regulation of Hp and IgM were predictive of a lower risk of CLA lesion development. Because the immunogen used in this study induced a high production of both Hp and IgM, Q. saponaria saponin should be considered a promising candidate in vaccine formulations against sheep CLA.

Maternal immunization with vaccines containing recombinant NetB toxin partially protects progeny chickens from necrotic enteritis.

Avian necrotic enteritis is a major economic and welfare issue throughout the global poultry industry and is caused by isolates of Clostridium perfringens that produce NetB toxin. Previously we have shown that birds directly vaccinated with inactivated C. perfringens type A culture supernatant (toxoid) combined with recombinant NetB (rNetB) protein were significantly protected from homologous and heterologous challenge. In the present study the protective effect of maternal immunization was examined. Broiler breeder hens were injected subcutaneously with genetically toxoided rNetB(S254L) alone, C. perfringens type A toxoid and toxoid combined with rNetB(S254L). Vaccination resulted in a strong serum immunoglobulin Y response to NetB in hens immunized with rNetB(S254L) formulations. Anti-NetB antibodies were transferred to the eggs and on into the hatched progeny. Subclinical necrotic enteritis was induced experimentally in the progeny and the occurrence of specific necrotic enteritis lesions evaluated. Birds derived from hens immunized with rNetB(S254L) combined with toxoid and challenged with a homologous strain (EHE-NE18) at either 14 or 21 days post-hatch had significantly lower levels of disease compared to birds from adjuvant only vaccinated hens. In addition, birds from hens immunized with rNetB(S254L) alone were significantly protected when challenged at 14 days post-hatch. These results demonstrate that maternal immunization with a NetB-enhanced toxoid vaccine is a promising method for the control of necrotic enteritis in young broiler chickens.

Development of an indirect ELISA to detect Corynebacterium pseudotuberculosis specific antibodies in sheep employing T1 strain culture supernatant as antigen / Desenvolvimento de ELISA indireto para detectar anticorpos específicos contra Corynebacterium pseudotuberculosis em ovinos utilizando o supernadante de cultura da cepa T1 como antígeno

Corynebacterium pseudotuberculosis is the etiologic agent of caseous lymphadenitis (CLA), a chronic disease that affects goats and sheep, characterized by granuloma formation in subcutaneous and internal lymph nodes. CLA causes significant economic losses to commercial goat herds. In this study, we aimed to test secreted antigens secreted from T1 strain bacteria grown in brain heart infusion (BHI) broth in an indirect ELISA system to determine the presence of specific immunoglobulins against C. pseudotuberculosis. We analyzed the BHI antigen electrophoretic profile and the recognition pattern by infected sheep sera samples. The ELISA results were compared with multiplex PCR assay and IFN-gamma production. The ELISA was able to discriminate between negative and positive animals, with a sensitivity of 89% and a specificity of 99%, using microbiological isolation as gold standard. When this assay was compared with multiplex PCR and specific IFN-gamma quantification, six discrepant results were found among thirty-two samples. We concluded that the ELISA using antigens secreted from C. pseudotuberculosis T1 strain growth in BHI broth culture can be used for the serodiagnosis of CLA in sheep.

Corynebacterium pseudotuberculosis é o agente etiológico da linfadenite caseosa (LC) uma doença crônica que afeta ovinos e caprinos caracterizada pela formação de granulomas em linfonodos. A LC causa perdas econômicas significativas em criações de pequenos ruminantes. Este trabalho teve como objetivo testar antígenos secretados da cepa T1 da bactéria em um sistema de ELISA indireto para detecção de anticorpos específicos contra C. pseudotuberculosis. O perfil eletroforético do antígeno foi analisado, bem como o padrão de reconhecimento por soros de animais infectados. Os resultados do ELISA foram comparados com ensaio de multiplex PCR e com teste de indução de produção específica de IFN-gama. O ELISA foi capaz de discriminar animais positivos de animais negativos, com sensibilidade de 89% e especificidade de 99%, usando o isolamento microbiológico como padrão ouro. Quando o ensaio foi comparado com o multiplex PCR e a produção específica de IFN-gama, somente seis resultados discrepantes foram encontrados em trinta e duas amostras. Pode-se concluir que o ensaio de ELISA desenvolvido pode ser utilizado com alto grau de confiança para o diagnóstico da LC em ovinos.

Vaccination with recombinant NetB toxin partially protects broiler chickens from necrotic enteritis.

NetB toxin from Clostridium perfringens is a major virulence factor in necrotic enteritis in poultry. In this study the efficacy of NetB as a vaccine antigen to protect chickens from necrotic enteritis was examined. Broiler chickens were immunized subcutaneously with purified recombinant NetB (rNetB), formalin treated bacterin and cell free toxoid with or without rNetB supplementation. Intestinal lesion scores and NetB antibody levels were measured to determine protection after mild oral gavage, moderate in-feed and heavy in-feed challenges with virulent C. perfringens isolates. Birds immunized with rNetB were significantly protected against necrotic enteritis when challenged with a mild oral dose of virulent bacteria, but were not protected when a more robust challenge was used. Bacterin and cell free toxoid without rNetB supplementation did not protect birds from moderate and severe in-feed challenge. Only birds immunized with bacterin and cell free toxoid supplemented with rNetB showed significant protection against moderate and severe in-feed challenge, with the later giving the greatest protection. Higher NetB antibody titres were observed in birds immunized with rNetB compared to those vaccinated with bacterin or toxoid, suggesting that the in vitro levels of NetB produced by virulent C. perfringens isolates are too low to induce the development of a strong immune response. These results suggest that vaccination with NetB alone may not be sufficient to protect birds from necrotic enteritis in the field, but that in combination with other cellular or cell-free antigens it can significantly protect chickens from disease.

Teste de ELISA indireto para diagnóstico sorológico de leishmaniose visceral em canídeos silvestres / Indirect ELISA for the serological diagnosis of visceral leishmaniasis in wild canids

Na América do Sul, alguns canídeos silvestres são considerados reservatórios naturais da Leishmania chagasi. A resposta imunológica desses animais à Leishmania é pouco conhecida, havendo a necessidade de métodos diagnósticos adequados para esse fim. No presente estudo, é descrita a padronização do ensaio imunoenzimático indireto (ELISA) para o diagnóstico sorológico de leishmaniose visceral em canídeos silvestres brasileiros. Foram estudadas amostras de soro e plasma de 12 canídeos cativos: sete lobos-guará (Chrysocyon brachyurus), três raposinhas (Lycalopex vetulus) e dois cachorros-do-mato (Cerdocyon thous). As amostras de um C. brachyurus e uma L. vetulus, cativos em área endêmica para LV, que apresentavam doença clínica e positividade em testes de Imunofluorescência Indireta e Reação em Cadeia de Polimerase, foram utilizadas como controles positivos. Foram comparados os conjugados anti-IgG de cão e proteína A, ambos ligados a peroxidase, cujos testes detectaram quatro (04/12) e três (03/12) C. brachyurus soropositivos para anticorpos anti-Leishmania sp., respectivamente. As médias das densidades ópticas (DOs) das amostras negativas foram nitidamente mais baixas do que as médias das DOs dos positivos tanto no ELISA com anti-IgG de cão (4,8 vezes) como com proteína A (15,5 vezes). Os soros de três C. brachyurus positivos no ELISA indireto foram avaliados por Western blotting e identificaram 22 bandas, sendo imunodominantes as de peso molecular de 19, 22, 24, 45 e 66 kDa. Os testes ELISA com a proteína A e o conjugado anti-IgG de cão apresentaram respectivamente concordância excelente (Kappa = 1; p<0,001) e moderada (Kappa = 0,8; p<0,0015), com o Western blotting. Ambos foram, portanto, considerados adequados a avaliações de triagem de animais cuja resposta humoral de anticorpos indica contato com o parasito, úteis para subsidiar estudos para adequação de metodologias específicas para os canídeos silvestres.

In South America, some wild canids are considered natural reservoirs of Leishmania chagasi. The immunological response of wild canids to Leishmania is not well understood, and the development of diagnostic methods is necessary for such purpose. In the present study, the standardization of an enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of visceral leishmaniasis (VL) in Brazilian species of wild canids is described. Serum and plasma samples from 12 captive wild canids were studied: seven from maned wolves (Chrysocyon brachyurus), three from hoary foxes (Lycalopex vetulus), and two from crab-eating foxes (Cerdocyon thous). Samples from C. brachyurus and L. vetulus, both captive in an endemic area for VL, presenting clinical disease and positivity in Indirect Immunofluorescence Reaction and Polymerase Chain Reaction tests were used as positive controls. The antibody anti-dog IgG and Protein A, both conjugated with horseradish peroxidase, were compared in indirect ELISA tests which detected four (04/12) and three (03/12) seropositive C. brachyurus for anti-Leishmania antibodies, respectively. The ELISA tests were able to clearly distinguish negative from positive samples, as the mean optical density (OD) of the negative samples was 4.8 and 15.5 times lower than those of the positive ones either using anti-dog IgG and Protein A, respectively. Samples from three ELISA - positive C. brachyurus were analyzed by Western blotting and identified immunodominant bands of 19, 22, 24, 45 and 66 kDa, among 22 protein bands detected. The ELISAs with protein A and anti-dog IgG showed respectively excellent (Kappa = 1.0; p<0.001) and moderate (Kappa = 0.8; p<0.0015) agreement with the Western blotting assay. The ELISA tests showed to be adequate for screening studies to identify antibody responses, thus indicating contact with Leishmania infection by wild canids.

High sero-prevalence of caseous lymphadenitis identified in slaughterhouse samples as a consequence of deficiencies in sheep farm management in the state of Minas Gerais, Brazil.

BACKGROUND: Caseous lymphadenitis (CLA), caused by Corynebacterium pseudotuberculosis, is one of the most important diseases of sheep and goats, causing considerable economic losses for herd owners. RESULTS: We assessed the seroprevalence of infection with C. pseudotuberculosis in 805 sheep from 23 sheep farms that supply slaughterhouses in the state of Minas Gerais; we also analyzed management practices that could be associated with CLA occurrence, used on these and nearby farms that also supplied animals to the slaughterhouse (n = 60). The serum samples for assaying CLA infection were taken at the slaughterhouse. Frequency of infection with C. pseudotuberculosis was estimated at 43.7%, and farm frequency was estimated at 100%. Management practices were analyzed through a questionnaire. All farmers (60/60) had extensive/semi-extensive rearing system; 70.0% (42/60) identified sheep individually; 11.7% (7/60) had periodical technical assistance; 41.7% (25/60) disinfected the facilities; 86.7% (52/60) used barbed wire fences and did not implement adequate CLA control measures; only 11.7% (7/60) of breeders reported vaccination against C. pseudotuberculosis; 13.3% (8/60) took note of animals with clinical signs of CLA; 1.7% (1/60) opened and sanitized abscesses, and isolated the infected animals; 10.0% (6/60) knew the zoonotic potential of this disease and 1.7% (1/60) of the farmers culled animals in case of recurrence of abscesses. CONCLUSIONS: It can be concluded that C. pseudotuberculosis infection is widely spread in sheep flocks in Minas Gerais state in Brazil and that there is a lack of good management measures and vaccination, allowing transmission of this infectious agent throughout the production network.

Haptoglobin and fibrinogen concentrations and leukocyte counts in the clinical investigation of caseous lymphadenitis in sheep.

BACKGROUND: Corynebacterium pseudotuberculosis is the etiologic agent of caseous lymphadenitis (CLA), a disease that affects small ruminants and is responsible for economic losses, including condemnation of carcasses and damaged hides. OBJECTIVE: The goal of this study was to determine if serum haptoglobin and plasma fibrinogen concentrations and peripheral blood leukocyte counts are biologic markers of CLA in sheep. METHODS: Blood from 38 clinically healthy Santa-Inês ewes selected and segregated from a commercial flock of 2500 sheep in an area endemic for C. pseudotuberculosis was collected every 30 days for 6 months. An indirect ELISA was used to detect IgM and IgG antibodies against C. pseudotuberculosis. Serum haptoglobin concentration was measured using a hemoglobin-binding assay and plasma fibrinogen concentration by refractometry following heat precipitation. Total leukocyte counts were determined using a hemocytometer, and differential leukocyte counts were performed on smears of peripheral blood. RESULTS: Twenty-one sheep were seropositive at the start of the study; 15 became seropositive during the study. Only 2 sheep were seronegative at the conclusion of the study. Haptoglobin and fibrinogen concentrations and WBC counts were not significantly different for seropositive and seronegative animals. Nine sheep, 5 that were seropositive positive at the start and 4 that became seropositive during the study period, developed abscesses in peripheral lymph nodes. There were 15 animals that became seropositive during the study, and their values did not differ significantly among the 3 phases--seronegative, acute (IgM+/IgG±), and chronic (IgM-/IgG+)--of infection. However, 11 of these sheep did not develop peripheral abscesses and had significantly higher haptoglobin concentrations and lower monocyte counts during the acute phase of the disease than did the 4 sheep that later developed abscesses. CONCLUSION: Serum haptoglobin concentration and monocyte counts may be potential markers for progression of CLA in sheep.