Combination of monoclonal antibodies improves immunohistochemical diagnosis of Neospora caninum.

Histological analysis is commonly used for a conclusive diagnosis of neosporosis. Immunohistochemistry (IHC) using monoclonal (mAb) and polyclonal (pAb) antibodies can improve diagnosis; however, the use of pAb may induce cross-reactivity with other related parasites. The aims of this study were to compare the performance of mAbs and their combinations with that of pAb in IHC and evaluate the usefulness of mAb to identify Neospora caninum infection in aborted bovine fetal tissues. For this purpose, mAbs targeting NcSRS2 (4.15.15) or NcGRA7 (4.11.5 and 1/24-12) and one pAb collected from a rabbit inoculated with N. caninum tachyzoites were tested by IHC. Artificial standardized tissue sections were prepared as positive controls using homogenized bovine brain spiked with cultured tachyzoites of N. caninum. The numbers of labeled parasites were counted in each positive control section. In addition, four equal proportional combinations of the mAbs were also analyzed in the IHC. Finally, the pAb and the best combination of mAbs obtained in the positive control experiments were tested with tissue sections of naturally-infected cattle. To confirm analytical specificity, mAbs and a pAb were tested with Toxoplasma gondii and Besnoitia besnoiti positive control slides and tissues sections from naturally infected cattle containing Sarcocystis spp. and B. besnoiti antigens. The mAb 4.15.15 detected 57% of the total parasites in sections while 4.11.5 and 1/24-12 were able to detect 49% and 41%, respectively. For the mAb combinations (I: 1/24-12+4.11.5, II: 1/24-12+4.15.15, III: 4.15.15+4.11.5, IV: 1/24-12+4.11.5+4.15.15), the detection capacity was 32.4%, 79.4%, 66.6% and 60.7% for each combination, respectively. The best mAb combination (1/24-12 and 4.15.15) and the pAb serum detected 100% (18/18) of naturally-infected animals. Sarcocystis spp. or B. besnoiti were not detected by mAb combinations in IHC, however the pAb cross-reacted with Sarcocystis spp. cysts. These results confirm the usefulness of mAb application in IHC to N. caninum.

Investigação do gene p53 de frangos expostos às aflatoxinas / Investigation of p53 gene in poultries exposed to aflatoxins

Identificou-se o efeito das aflatoxinas (AFs) sobre o gene p53 de frangos de corte, de linhagem comercial, separados em: grupo experimental, tratado (GT) com ração comercial contendo 2,8ppm de AFs totais durante 21 dias consecutivos, e grupo-controle (GC), sem exposição às AFs. Macroscopicamente, as alterações caracterizaram-se por hepatomegalia e aspecto pálido-amarelado com alguns focos hemorrágicos e, histologicamente, por desarranjo trabecular, pleomorfismo hepatocítico com cariomegalia, degeneração vacuolar intracitoplasmática, necrose com infiltração linfocítica e hiperplasia de ductos biliares. A PCR com os primers GSPT53c-1 com base no gene candidato a p53 (GenBank XM_424937.2) gerou um produto de aproximadamente 350 pares de base. O amplicon sequenciado a partir do DNA dos frangos do GT não apresentou mutação ou deleção, assim como padrão de bandas do PCR-RFLP não foi distinto entre ambos os grupos experimentais e a sequência depositada no banco de genes. Os resultados sugerem que não ocorreu transversão devido à exposição às AFs no fragmento amplificado. Conclui-se que a PCR-RFLP e o sequenciamento do produto da PCR não são ferramentas apropriadas para diagnóstico da exposição de frangos às AFs nas condições experimentais empregadas.

To identify the effects of aflatoxins (AFs), Cobb lineage poultry were separated in an experimental group in which they were treated with commercial ration containing 2.8ppm of total AFs during 21 days (TG) and a control group without AFs exposure (CG). In the liver of poultries exposed to AFs, alterations were microscopically observed, which were characterized by hepatomegaly, a pale yellowish aspect with some hemorrhagic spots, and histologically a trabecullar disarranging pleomorphic hepatocytes with cariomegaly, intracytoplasmatic vacuolar degeneration, necrosis, lymphocytic infiltration and hyperplasia of biliary ducts. The PCR with GSPT53c-1 primers based on p53 candidate gen (GenBank XM_424937.2) generated a product of approximately 350 base pairs. The sequenced amplicon obtained from the DNA of treated poultry did not display any mutation or deletion, and the PCR- RFLP bands patterns were also not distinct in both experimental groups. The results indicated that transversion did not occur in the fragment amplified due to AFs exposure. As a consequence of results obtained with p53 gene (NM_205264.1) we concluded that PCR-RFLP and sequencing of PCR product are not appropriate diagnostic tools for the detection of poultry exposure to AFs, at least in the experimental conditions performed.

Development of fluorogenic probe-based PCR assays for the detection and quantification of bovine piroplasmids.

This paper reports two new quantitative PCR (qPCR) assays, developed in an attempt to improve the detection of bovine piroplasmids. The first of these techniques is a duplex TaqMan assay for the simultaneous diagnosis of Babesia bovis and B. bigemina. This technique is ideal for use in South America where bovids harbour no theilerids. The second technique, which is suitable for the diagnosis of both babesiosis and theileriosis worldwide, involves fluorescence resonance energy transfer (FRET) probes. In FRET assays, Babesia bovis, B. divergens, Babesia sp. (B. major or B. bigemina), Theileria annae and Theileria sp. were all identifiable based on the melting temperatures of their amplified fragments. Both techniques provided linear calibration curves over the 0.1fg/microl to 0.01ng/microl DNA range. The assays showed good sensitivity and specificity. To assess their performance, both procedures were compared in two separate studies: the first was intended to monitor the experimental infection of calves with B. bovis and the second was a survey where 200 bovid/equine DNA samples from different countries were screened for piroplasmids. Comparative studies showed that duplex TaqMan qPCR was more sensitive than FRET qPCR in the detection of babesids.

Trypanosomiasis by Trypanosoma vivax in cattle in the Brazilian semiarid: Description of an outbreak and lesions in the nervous system.

An outbreak of trypanosomiasis by Trypanosoma vivax is reported in the semiarid of Paraíba, Northeastern Brazil from May to August 2002. Sixty-four cows out of 130 were affected; 11 died and the other recovered after treatment with diminazene aceturate. Affected animals had fever, anemia, weight loss, hypoglycemia, increased serum levels of aspartate aminotransferase and, in nine cows, nervous signs. All cows with nervous signs died; six of them recovered after treatment, but the disease relapsed. Six cows aborted and one delivered a calf that died immediately after parturition. Thirty-two out of 100 calves were affected and five died. Nervous signs were not observed in the calves. Gross lesions were thickening of the meninges, enlarged lymph nodes and prominent white pulp of the spleen. The main histological lesion was meningoencephalitis and malacia in the brain of cows with nervous signs. No antibodies against trypanosomes were found in 33 blood samples collected before the outbreak in the affected farm and in 29 samples collected at the same time in two other neighbor farms. Until January 2003, all 89 animals tested had antibodies against T. vivax, suggesting the occurrence of sub clinical infections in cattle without clinical signs. Only two out of 85 serum samples collected on April 2004 were positive for T. vivax antibodies. Data obtained suggested that the semiarid region is non-endemic for trypanosomiasis and that disease occurred due to introduction of the parasite in a susceptible population after an apparent rise in the Tabanus spp. population.

Freqüencia de anticorpos anti-Anaplasma marginale em rebanhos leiteiros da Bahia / Frequency of antibodies against Anaplasma marginale in dairy herds of Bahia State, Brazil

Determinou-se a freqüência de bovinos soro-reagentes para Anaplasma marginale em rebanhos leiteiros das microrregiöes de Jequié, Itabuna e Vitória da Conquista, Estado da Bahia, pelas técnicas de imunoadsorçäo enzimática (ELISA), imunofluorescência indireta (IFI) e teste de conglutinaçäo rápida (TCR). Das 324 amostras de soro bovino analisadas, 96,9 por cento, 97,2 por cento e 91,0 por cento foram positivas para anticorpos contra A. marginale, respectivamente pelo ELISA, IFI e TCR. Todas as regiöes caracterizaram-se por estabilidade enzoótica para a anaplasmose. O desempenho dos três testes sorológicos foi bastante similar na detecçäo de anticorpos contra A. marginale

Comparison between enzyme-linked immunosorbent assay, indirect fluorescent antibody and rapid conglutination tests in detecting antibodies against Babesia bovis.

The performance of an enzyme-linked immunosorbent assay (ELISA), an indirect fluorescent antibody test (IFAT) and a rapid conglutination test (RCT) for the detection of antibodies against Babesia bovis, was evaluated with 462 cattle sera from Bahia State; Brazil. The results showed a concordance of 96.6% between the ELISA and IFAT, 90.5% between the ELISA and RCT, and 91.8% between the IFAT and RCT. Although the prevalence rates determined by ELISA (97.2%) and IFAT (96.8%) were higher than that indicated by the RCT (92.9%), performances of the three serological tests were very similar and characterized the region studied as enzootically stable to B. bovis.

Conservation of merozoite membrane and apical complex B cell epitopes among Babesia bigemina and Babesia bovis strains isolated in Brazil.

Babesia merozoite polypeptides bear surface exposed and neutralization-sensitive B cell epitopes and have been shown to induce partial protection against experimental challenge. Variation in these epitopes has been examined in a limited number of strains. In this study, utilizing strains of Babesia bovis and Babesia begemina from Matto Grosso do Sul in Brazil, we examined the conservation of epitopes bound by monoclonal antibodies developed against Mexico strains of B. bovis and B. bigemina. Apical complex B-cell epitopes, previously shown to be species-specific but common among otherwise antigenically distinct strains, were also conserved between clones of the Mexico strains and the Matto Grosso do Sul strains of each Babesia species. Mexico strain polypeptides bearing these epitopes were recognized by sera from cattle infected with the Matto Grosso do Sul strains. Two distinct epitopes on the B. bovis neutralization-sensitive merozoite surface antigen-1 (MSA-1) were also conserved between the Mexico Mo7 clone and the Matto Grosso do Sul strain, in contrast to previous studies which demonstrated variability among strains. Sera from cattle with B. bovis infections naturally acquired in Matto Grosso do Sul bound Mexico Mo7 MSA-1, demonstrating that conserved MSA-1 epitopes were recognized by the bovine immune system. Similarly, merozoite surface epitopes on the B. bigemina 45 kDa and 55 kDa glycoproteins were conserved on the Matto Grosso do Sul strain of B. bigemina.

Análise de testes de conglutinaçäo rápida para detecçäo de anticorpos contra Babesia bovis e Babesia bigemina / Evaluation of rapid conglutination tests for detection of antibodies against Babesia bovis and Babesia bigemina

Testes de conglutinaçäo rápida foram desenvolvidos para detecçäo de anticorpos contra Babesia bovis e B. bigemina. O primeiro (TCR-B. bovis) apresentou resultados idênticos à imunofluorescência indireta (IFI), na detecçäo de anticorpos resultantes da vacinaçäo com cepas atenuadas de B. bovis, enquanto que o segundo teste de conglutinaçäo (TCR-B. bigemina) divergiu em três dos seis animais na fase inicial da soroconversäo. Aos 28 e 56 dias pós-vacinaçäo (PV) houve coincidência total de resultados. A correlaçäo entre os TCR-B. bovis e IFI no exame dos soros de bovinos de sete estados brasileiros foi de 86,2 por cento, enquanto que a correlaçäo entre TCR-B. bigemina e a prova de imunofluorescência foi de 95,6 por cento. Os resultados demonstraram que os testes de conglutinaçäo podem ser empregados em estudos epidemiológicos com eficiência comparável à imunofluorescência indireta