Evaluation of HBV-Like Circulation in Wild and Farm Animals from Brazil and Uruguay.

The origin of the hepatitis B virus is a subject of wide deliberation among researchers. As a result, increasing academic interest has focused on the spread of the virus in different animal species. However, the sources of viral infection for many of these animals are unknown since transmission may occur from animal to animal, human to human, animal to human, and human to animal. The aim of this study was to evaluate hepadnavirus circulation in wild and farm animals (including animals raised under wild or free conditions) from different sites in Brazil and Uruguay using serological and molecular tools. A total of 487 domestic wild and farm animals were screened for hepatitis B virus (HBV) serological markers and tested via quantitative and qualitative polymerase chain reaction (PCR) to detect viral DNA. We report evidence of HBsAg (surface antigen of HBV) and total anti-HBc (HBV core antigen) markers as well as low-copy hepadnavirus DNA among domestic and wild animals. According to our results, which were confirmed by partial genome sequencing, as the proximity between humans and animals increases, the potential for pathogen dispersal also increases. A wider knowledge and understanding of reverse zoonoses should be sought for an effective One Health response.

Risk of exposure of farms and subsistence nurseries to contact with wild boar in southern Mato Grosso do Sul / Risco de exposição de granjas e criatórios de subsistência ao contato com javalis no sul do Mato Grosso do Sul

With the advancement of wild boar distribution in the rural environment, its impacts are not limited to health in the pig sector, but the requirements for monitoring and control of the species are requirements laid down by the OIE for the recognition of classical swine fever free zone status. The construction of ecological models of favorability or suitability for the occurrence of pest species are necessary tools for the decision making on priority areas of management aiming at risk management. This work aims to map the level of suitability for the occurrence of wild boar in the southern state of Mato Grosso do Sul, as well as to identify the main risk variables for contact with the wild boar and evaluate the biosecurity measures adopted by commercial farms integrated in the south of the State of Mato Grosso do Sul. To evaluate the risk potential of wild boar for commercial and subsistence swine farming in southern Mato Grosso do Sul, a model of environmental suitability was constructed for this species in the swine producing region. This model considered different environmental strata, being the selection of the layers considered the physiological and behavioral characteristics of the species. In parallel, interviews were carried out in a sample of commercial farms integrating the region to survey the perception of the presence of the invasive species and the biosafety measures adopted. The results of this work indicate that the risk of contact among wild boars and animals reared in closed production systems may be high in the study area and only establishment of appropriate biosecurity measures that consider the characteristics and habits of the boar may prevent the intrusion of this species and contact with domestic swine. The built model can be considered of high reliability and it is recommended to apply it to other areas of the state, being a useful tool for the productive sector, environmental agencies and decision makers.(AU)

Com o avanço da distribuição do javali no ambiente rural, seus impactos não se restringem somente a sanidade suidea, embora as exigências quanto ao monitoramento e controle da espécie sejam exigências previstas pela OIE, para o reconhecimento do status de zona livre de peste suína clássica. A construção de modelos ecológicos de favorabilidade ou adequabilidade para a ocorrência de espécies-praga são ferramentas necessárias para as tomadas de decisão sobre áreas prioritárias de manejo visando gestão de risco. Este trabalho objetiva mapear o nível de adequabilidade para a ocorrência de javalis no sul do Estado de Mato Grosso do Sul, bem como levantar as principais variáveis de risco para o contato com o javali asselvajado e avaliar as medidas de biosseguridade adotadas por granjas comerciais integradas no sul do Estado do Mato Grosso do Sul. Para avaliar o potencial de risco exercido pelos javalis para a suinocultura comercial e de subsistência nesta região foi construído um modelo de adequabilidade ambiental para essa espécie na região produtora de suínos. Esse modelo considerou diferentes estratos ambientais, sendo que para a seleção das camadas consideram-se características fisiológicas e comportamentais da espécie. Em paralelo, entrevistas foram realizadas em uma amostragem de granjas comerciais de integração da região para levantamento da percepção quanto a presença da espécie invasora e as medidas de biossegurança adotadas. Os resultados desse trabalho indicam que o risco de contato entre javalis de vida livre e os animais criados em sistemas de produção fechados pode ser alto na área de estudo e somente estabelecimento de medidas de biosseguridade apropriadas, que considerem as características e hábitos do javali poderá impedir a intrusão dessa espécie e o contato com os suínos domésticos. O modelo construído pode ser considerado de elevada confiabilidade e recomenda-se a sua aplicação para as outras áreas do estado, sendo uma ferramenta útil para o setor produtivo, os órgãos ambientais e os tomadores de decisão.(AU)

Detection of wild animals as carriers of Leptospira by PCR in the Pantanal biome, Brazil.

Leptospiral infection is widespread in wildlife. In this context, wild ecosystems in tropical countries hold a vast biodiversity, including several species that may act as potential reservoirs of leptospires. The Pantanal biome presents highly favorable environmental conditions for the occurrence of leptospirosis, such as high temperatures, constant flooding, and high biodiversity. The purpose of this study was to detect wild animals as carriers of Leptospira sp. using direct methods (PCR and culture) in the Pantanal biome, Brazil. A total of 35 animals were studied, namely Cerdocyon thous, Nasua nasua, Ozotoceros bezoarticus, and Sus scrofa species. Blood for serology (MAT) and urine for bacteriological culturing and PCR was sampled. The most prevalent serogroups were Javanica and Djasiman. Additionally, 40.6% of these animals presented PCR positive reactions. Seroreactivity associated with the high frequency of leptospiral carriers among the different studied species suggests a high level of exposure of the studied animals to pathogenic Leptospira strains. Our results are still limited and the actual role of the studied animals in the epidemiology of leptospirosis in the Pantanal region remains to be elucidated.

Molecular detection and identification of hemoparasites in pampas deer (Ozotoceros bezoarticus Linnaeus, 1758) from the Pantanal Brazil.

Hemoparasites were surveyed in 60 free-living pampas deer Ozotoceros bezoarticus from the central area of the Pantanal, known as Nhecolândia, State of Mato Grosso do Sul, Brazil, through the analysis of nested PCR assays and nucleotide sequencing. Blood samples were tested for Babesia/Theileria, Anaplasma spp., and Trypanosoma spp. using nPCR assays and sequencing of the 18S rRNA, msp4, ITS, and cathepsin L genes. The identity of each sequence was confirmed by comparison with sequences from GenBank using BLAST software. Forty-six (77%) pampas deer were positive for at least one hemoparasite, according to PCR assays. Co-infection occurred in 13 (22%) animals. Based on the sequencing results, 29 (48%) tested positive for A. marginale. Babesia/Theileria were detected in 23 (38%) samples, and according to the sequencing results 52% (12/23) of the samples were similar to T. cervi, 13% (3/23) were similar to Babesia bovis, and 9% (2/23) were similar to B. bigemina. No samples were amplified with the primers for T. vivax, while 11 (18%) were amplified with the ITS primers for T. evansi. The results showed pampas deer to be co-infected with several hemoparasites, including species that may cause serious disease in cattle. Pampas deer is an endangered species in Brazil, and the consequences of these infections to their health are poorly understood.

ELISA based on recombinant MPB70 and P27 for detection of antibodies against Mycobacterium bovis

A tuberculose bovina é uma importante enfermidade causada pela bactéria Mycobacterium bovis. Testes de tuberculinização intradérmica e abate de animais infectados levaram à redução da incidência da tuberculose bovina em muitos países. Entretanto, são necessários métodos maispráticos e eficientes com maior sensibilidade e especificidade. O objetivo do presente estudo foidesenvolver um teste imunoenzimático (ELISA), utilizando as proteínas recombinantes MPB70 e p27 de M. bovis, que possibilitasse detectar anticorpos contra esta bactéria em bovinos. A sensibilidade e especificidade observadas foram, respectivamente, de 88,7por cento e 94,6por cento para o ELISA-MPB70 e de 98,1por cento e 91,9por cento para o ELISA-p27. O uso de testes sorológicos, como o ELISA com MPB70 e p27 recombinantes, juntamente com testes celulares, pode resolver algunsproblemas relacionados ao diagnóstico da tuberculose bovina tais como os resultados inconclusivos e a ausência de detecção de animais anérgicos em estágios avançados da infecção.

Screening of recombinant proteins as antigens in indirect ELISA for diagnosis of bovine tuberculosis.

Bovine tuberculosis is an important infectious disease caused by Mycobacterium bovis, which is responsible for considerable economic losses. This disease constitutes a serious public health problem. Control programs in most countries, including Brazil, are based on the identification and slaughter of infected animals, as defined by the skin tuberculin test, which has its constraints. In the present study, the recombinant proteins CFP-10, ESAT-6, Mb0143, MPB83, PE5, PE13, TB10.4, TB15.3 and a chimera of ESAT-6/MPB70/MPB83 (fusion protein) were tested as ELISA antigens for the diagnosis of bovine tuberculosis. The proteins were produced in Escherichia coli, purified and tested in ELISAs with sera from 126 cattle having tested negative in the comparative intradermal tuberculin test (CITT) and 107 sera from cattle having tested positive in the CITT. Also, 236 sera from two BTB-free beef cattle herds were tested. Among the proteins tested, only the ESAT-6/MPB70/MPB83 chimera demonstrated satisfactory agreement with the CITT (kappa index: 0.688), reflecting in 83.2% sensitivity and 86.5% specificity. The ELISA absorbances of the cattle sera from BTB-free herds showed similar levels to those of CITT positive cattle, probably as the result of successive skin tuberculinizations to define the BTB-free status of the herds. However, the ELISA with the ESAT-6/MPB70/MPB83 chimera was useful to discriminate BTB positive and negative cattle in herds prior to the tuberculin skin test.

Uso do ensaio imunoenzimático e imunoblotting para detecção de imunoglobulinas contra Campylobacter fetus em muco cérvico-vaginal de fêmeas bovinas / The use of enzyme-linked immunosorbent assay and immunoblotting for the detection of Campylobacter fetus immunoglobulins in the cervico-vaginal mucus of female cattle

Foi padronizado um ensaio imunoenzimático do tipo indireto para detecção de imunoglobulina A (ELISA IgA) anti- Campylobacter fetus subp. venerealis em muco cérvico- vaginal bovino utilizando um extrato protéico de Campylobacter fetus subsp. venerealis produzido pelo método de extração ácida pelo tampão de glicina (0,2M; pH2,2). A média dos valores de densidade ótica (DO450) foi de 0,143±0,09. As bandas protéicas dos antígenos de Campylobacter fetus subsp. venerealis e de Campylobacter fetus subsp. fetus melhor reconhecidas pela IgA do muco cérvico- vaginal migraram em 42,6 kDa mas a proteina evidenciada em 93 kDa foi reconhecida exclusivamente pelo Campylobacter fetus subsp. venerealis. Os anticorpos presentes na amostra de muco vaginal testada no “immunoblotting” que apresentou resultado positivo no ELISA IgA, reconheceu antígenos de C. jejuni subsp. jejuni e C. fetus subsp. fetus.

An indirect enzyme-linked immunosorbent assay was developed to detect antigenspecific secretory IgA antibodies to Campylobacter fetus subsp. venerealis in bovine vaginal mucus with a protein extract of the Campylobacter fetus subsp. venerealis by the acid glycine extraction method. Mean optical density measurement (λ=450 nm) was 0.143±0.9. The most immunoreactive protein bands of the Campylobacter fetus subsp. venerealis or Campylobacter fetus subsp. fetus recognized by IgA in immunoblotting, using bovine vaginal mucus samples, migrate at 42.6 kDa. The protein that migrates at 93 kDa was recognized exclusively for C. fetus subsp. venerealis. A positive vaginal mucus sample of a cow from negative herd recognized antigens of C. jejuni subsp. jejuni e C. fetus subsp. fetus.

Prevalência e fatores de risco para a leptospirose em bovinos de Mato Grosso do Sul / Prevalence and risk factors for bovine leptospirosis in Mato Grosso do Sul, Brazil

Foi investigada a prevalência de anticorpos antileptospira em fêmeas bovinas com idade igual ou superior a 24 meses, provenientes de 178 rebanhos de 22 municípios do estado de Mato Grosso do Sul, bem como identificados fatores de risco associados à infecção. Foram analisadas 2.573 amostras de soro sangüíneo por meio do teste de soroaglutinação microscópica perante 10 sorovares de leptospira. Títulos iguais ou superiores a 100 para um ou mais sorovares foram detectados em 1.801 fêmeas (98,8 por cento) de 161 (96,5 por cento) rebanhos. O sorovar Hardjo (65,6 por cento) foi apontado como o mais provável, seguido do sorovar Wolffi (12,3 por cento). Os resultados demonstram que a leptospirose bovina se encontra presente em todos os municípios estudados, com alta prevalência, tanto em animais como em rebanhos. Os fatores de risco identificados neste estudo e associados à infecção por bactérias do gênero lepstopira foram o tipo de exploração pecuária de corte e a raça Zebu.

The prevalence of anti-Leptospira spp. antibodies was estimated for female cattle aged 24 months or older. The sample comprised 178 herds from 22 counties in the state of Mato Grosso do Sul, Brazil. The risk factors associated with the presence of infeccion were investigated. A total of 2,573 blood serum samples were tested against 10 leptospira serovars using the microagglutination test (MAT). Titers of 100 or higher for one or more serovars were detected in 1,801 females (98.8 percent) from 161 herds (96.5 percent). Serovar Hardjo (65.6 percent) was the most frequent, followed by serovar Wolffi (12.3 percent). These results suggest that bovine leptospirosis is widespread in all the counties under study, with a high prevalence both at the animal and the herd level. Beef farms and the Zebu breed were associated to the higher risk of herd infection by leptospiras.

[Epidemiologic survey of Neospora caninum in bovines of rural settlements in Corumbá, MS]. / Levantamento epidemiológico de Neospora caninum em bovinos deassentamentos rurais em Corumbá, MS.

Neospora caninum, is an obligate intracellular parasite, can infect domestic and wild dogs, equines and ruminants. It was described in 1988, and its economic importance is attributed to costs of abortion, the value of the fetuses, artificial insemination and coverage, the decrease in milk production, the increase in the disposal and replacement of animals. In four rural settlements of the municipality of Corumba, MS (Mato Grande, Paiolzinho, Taquaral and Tamarineiro) was used a combination of qualitative methodologies in 37 semi-structured interviews and group dynamics for analysis with participatory rural producers of 657 animals were raised health problems related to production and disease of the herd, and possible variables of risk for the presence of neosporosis. This study evaluated in 392 samples of bovine serum from the settlements of Corumbá, the presence of animals seropositive for N. caninum, to indirect fluorescence antibody test (IFAT), and prevalence weighted for neospora was 9.17%, ranging from 4.96% to 13.38%. The survey of variables of risk to Neospora investigated in the interviews did not indicate any risk factor for the disease.

Investigação epidemiológica da brucelose bovina em um estrato do Estado de Mato Grosso do Sul / Epidemiological investigation of bovine brucellosis in an extract of Mato Grosso do Sul, Brazil

O objetivo deste estudo foi estimar a prevalência da brucelose bovina nos 22 municípios que compõem a região denominada Estrato 1 do Estado de Mato Grosso do Sul, e identificar os fatores de risco associados à infecção. A região amostrada constitui uma área de 70.214,1 km2, que representa 19,7% do Estado. O rebanho de região estudada é de, aproximadamente, 5,7 milhões de cabeças, correspondente a 23% do efetivo de 24,9 milhões de bovinos de Mato Grosso do Sul. Nas 210 propriedades amostradas, no período de dezembro de 2003 a março de 2004, foram colhidas 2.376 amostras de sangue de fêmeas com idade igual ou superior a 24 meses, submetidas a testes diagnósticos em série. A triagem, realizada por meio do teste do antígeno acidificado tamponado, foi seguida pelo teste confirmatório 2-mercaptoetanol. Na mesma ocasião da colheita das amostras, foi preenchido um questionário com informações de identificação, tipo de criação e práticas de manejo. Em animais, a prevalência real foi estimada em 5,6%, e em rebanhos, 37,3%. As variáveis que apresentaram associação, por meio da análise univariada odds ratio (OR) e intervalo de confiança (IC) de 95%, com a soropositividade à brucelose foram: o tipo de exploração corte (OR = 2,82, IC 95% = 1,49-5,34), a raça Zebu (OR = 2,62, IC 95% = 1,40-4,88) e o aborto (OR= 1,83, IC 95% = 1,01-3,33). Os resultados demonstram que, além da brucelose ser prevalente no estrato estudado em Mato Grosso do Sul, o controle da doença pode consistir na adoção de programa com especial atenção à exploração do tipo corte, à raça Zebu e à presença do aborto.

The study aimed to estimate the prevalence of bovine brucellosis in 22 counties which make up the region Extract 1 of the State of Mato Grosso do Sul, Brazil, in order to identify risk factors associated with the infection. The sample region encompasses an area of 70,214.1 km2 and represents 19.7% of the State. The region studied has about 5.7 million head of cattle, corresponding to 23% of the total of 24.9 million cattle in the State of Mato Grosso do Sul. On 210 farms, between December 2003 and March 2004, 2,376 blood samples were collected from cows, aged 24 months or older, for serial diagnostic tests. Screening through the buffered acidified antigen test was confirmed by the 2-mercaptoetanol test. On the occasion of sample collection a questionnaire with information related to identification, kind of cattle and management practices was filled out. In individual animals the real prevalence was estimated at 5.6%, and in the cattle herds at 37.3%. The variables, which presented association through odds ratio (OR), univariate analysis and 95% confidence interval (CI) with serum positivity for brucellosis, were: the exploration of beef cattle (OR = 2.82, 95% CI = 1.49-5.34), Zebu breed (OR = 2.62, 95% CI = 1.40-4.88) and abortion (OR = 1.83, 95% CI = 1.01-3.33). The results shown here demonstrate, despite the prevalence of brucellosis in the extract of Mato Grosso do Sul studied, that the control of the disease may depend on adoption of a program focusing upon the exploration of beef cattle, the Zebu breed and the occurrence of abortion.