Identification of single nucleotide polymorphisms in the prion protein gene in Santa Ines and Dorset sheep / Identificação de polimorfismos de nucleotídeos únicos em ovinos Santa Inês e Dorset através do gene da proteína priônica

Scrapie is a transmissible spongiform encephalopathy (TSE) that affects sheep and goats and results from accumulation of the abnormal isoform of a prion protein in the central nervous system. Resistance or susceptibility to the disease is dependent on several factors, including the strain of infecting agent, the degree of exposure, and the presence of single nucleotide polymorphisms (SNPs) in the prion protein gene. The most important polymorphisms are present in codons 136, 154, and 171. SNPs have also been identified in other codons, such as 118, 127, 141, 142, and 143. The objective of this study was to investigate the genotypic profile of Santa Ines (n=94) and Dorset (n=69) sheep and identify polymorphisms in the prion protein gene using real-time PCR techniques and sequencing. We analyzed SNPs in 10 different codons (127, 136, 138, 140, 141, 142, 143, 154, 171, and 172) in Santa Ines sheep. Classification of the flock into risk groups associated with scrapie revealed that approximately 68% of the Santa Ines herd was considered at moderate risk (group 3), and the most frequent haplotype was ARQ/ARQ (47.8%). For Dorset sheep, 42% of the herd was considered at moderate risk (group 3), 40% at low risk (group 2), and 12% at very low risk (group 1). These findings improve our understanding of the genotype breed and further highlight the importance of genotyping and identification of polymorphisms in Brazilian herds to assess their effects on potential infections upon exposure to the sheep prion.(AU)

Scrapie é uma encefalopatia espongiforme transmissível que afeta ovinos e caprinos, resultante do acúmulo de uma isoforma anormal da proteína priônica no sistema nervoso central. A resistência ou susceptibilidade está relacionada a diversos fatores, tais como, a cepa do agente infectante, o grau de exposição e o polimorfismo de nucleotídeo único (SNPs) do gene da proteína priônica. Os principais polimorfismos estão presentes nos códons 136, 154 e 171. SNPs também são identificadas em outros códons, tais como, 118, 127, 141, 142, e 143. O objetivo do trabalho foi descrever o perfil genotípico de um rebanho da raça Santa Inês (n=94) e um rebanho da raça Dorset (n=89) para identificar potenciais polimorfismos através da técnica de PCR em tempo real e sequenciamento. Os achados no rebanho Santa Inês indicaram a presença de polimorfismos de nucleotídeos únicos em 10 códons diferentes (127, 136, 138, 140, 141, 142, 143, 154, 171 e 172). A classificação do rebanho, quanto aos grupos de risco associados ao scrapie, relevaram que aproximadamente 68% dos ovinos foram considerados do grupo de risco moderado (grupo 3), onde o haplótipo mais frequente foi ARQ/ARQ (47,8%). Para os ovinos da raça Dorset, 42% do rebanho foi considerado do grupo de risco moderado (grupo 3), 40% do grupo de risco baixo (grupo 2) e 12% do grupo de risco muito baixo. Os dados encontrados contribuem para o conhecimento do genótipo das raças, destacando a importância de trabalhos que relatam os polimorfismos genéticos para a identificação de rebanhos brasileiros, bem como o seu impacto a infecções com exposição ao príon ovino.(AU)

Direct detection of Mycobacterium tuberculosis complex in bovine and bubaline tissues through nested-PCR.

Post-mortem bacterial culture and specific biochemical tests are currently performed to characterize the etiologic agent of bovine tuberculosis. Cultures take up to 90 days to develop. A diagnosis by molecular tests such as PCR can provide fast and reliable results while significantly decreasing the time of confirmation. In the present study, a nested-PCR system, targeting rv2807, with conventional PCR followed by real-time PCR, was developed to detect Mycobacterium tuberculosis complex (MTC) organisms directly from bovine and bubaline tissue homogenates. The sensitivity and specificity of the reactions were assessed with DNA samples extracted from tuberculous and non-tuberculous mycobacteria, as well as other Actinomycetales species and DNA samples extracted directly from bovine and bubaline tissue homogenates. Regarding the analytical sensitivity, DNA of the M. bovis AN5 strain was detected up to 1.5 pg by nested-PCR, whereas DNA of M. tuberculosis H37Rv strain was detected up to 6.1 pg. The nested-PCR system showed 100% analytical specificity for MTC when tested with DNA of reference strains of non-tuberculous mycobacteria and closely-related Actinomycetales. A clinical sensitivity level of 76.7% was detected with tissues samples positive for MTC by means of the culture and conventional PCR. A clinical specificity of 100% was detected with DNA from tissue samples of cattle with negative results in the comparative intradermal tuberculin test. These cattle exhibited no visible lesions and were negative in the culture for MTC. The use of the nested-PCR assay to detect M. tuberculosis complex in tissue homogenates provided a rapid diagnosis of bovine and bubaline tuberculosis.

Direct detection of Mycobacterium tuberculosis complex in bovine and bubaline tissues through nested-PCR

Post-mortem bacterial culture and specific biochemical tests are currently performed to characterize the etiologic agent of bovine tuberculosis. Cultures take up to 90 days to develop. A diagnosis by molecular tests such as PCR can provide fast and reliable results while significantly decreasing the time of confirmation. In the present study, a nested-PCR system, targeting rv2807, with conventional PCR followed by real-time PCR, was developed to detect Mycobacterium tuberculosis complex (MTC) organisms directly from bovine and bubaline tissue homogenates. The sensitivity and specificity of the reactions were assessed with DNA samples extracted from tuberculous and non-tuberculous mycobacteria, as well as other Actinomycetales species and DNA samples extracted directly from bovine and bubaline tissue homogenates. Regarding the analytical sensitivity, DNA of the M. bovis AN5 strain was detected up to 1.5 pg by nested-PCR, whereas DNA of M. tuberculosis H37Rv strain was detected up to 6.1 pg. The nested-PCR system showed 100% analytical specificity for MTC when tested with DNA of reference strains of non-tuberculous mycobacteria and closely-related Actinomycetales. A clinical sensitivity level of 76.7% was detected with tissues samples positive for MTC by means of the culture and conventional PCR. A clinical specificity of 100% was detected with DNA from tissue samples of cattle with negative results in the comparative intradermal tuberculin test. These cattle exhibited no visible lesions and were negative in the culture for MTC. The use of the nested-PCR assay to detect M. tuberculosis complex in tissue homogenates provided a rapid diagnosis of bovine and bubaline tuberculosis.

Prevalência sorológica e molecular de Babesia bovis e Babesia bigemina em búfalos (Bubalus bubalis) na Ilha de Marajó, Pará / Molecular and serological prevalence of Babesia bovis and Babesia bigemina in water buffaloes (Bubalus bubalis) on Marajo Island, State of Pará, Brazil

The aim of the study was to estimate the prevalence of Babesia bovis and Babesia bigemina in water buffaloes of the Marajó Island, State of Pará, Brazil. We used an indirect enzyme-linked immunosorbent assay (iELISA), with total antigen containing proteins outer surface, and polymerase chain reaction (qPCR), involving the use of SYBR Green based on amplification of a small fragment of the cytochrome b gene. The prevalence of positive animals in iELISA to B. bovis B. bigemina and mixed infection was 24.87% (199/800), 20.75% (166/800) and 18.75% (150/800), respectively. Using the PCR, the presence of B. bovis was detected in 15% (18/199) and B. bigemina in 16% (19/199) of animals, and of these, 58% (11/19) presented co-infected by the two agents. The results show a low prevalence of antibodies anti-B. bovis and anti-B. bigemina in water buffaloes from Marajó Island. However, it was observed that the agents of bovine babesiosis circulate in buffaloes, and these may act as reservoirs.

O objetivo do estudo foi testar a prevalência sorológica e molecular de Babesia bovis e Babesia bigemina em búfalos da Ilha de Marajó, Pará. Foi utilizado ensaio de imunoadsorção enzimático indireto (iELISA) com antígeno total contendo proteínas de superfície externa e reação em cadeia da polimerase (qPCR), envolvendo o uso de SYBR Green com base na amplificação de um pequeno fragmento de gene do citocromo b. A prevalência de animais positivos no ELISA para B. bovis, B. bigemina e para infecção mista foi de 24.87% (199/800), 20.75% (166/800) e 18.75% (150/800), respectivamente. Na PCR foi detectado a presença de B. bovis em 15% (18/199) e de B. bigemina em 16% (19/199) dos animais, sendo que destes, 58% (11/19) apresentavam-se co-infectados pelos dois agentes. Os resultados mostram uma baixa prevalência de anticorpos anti-B. bovis e anti-B. bigemina em búfalos da Ilha do Marajó. Porém, observou-se que os agentes da babesiose bovina circulam em búfalos, podendo estes atuar como reservatórios.

Freqüência de anticorpos homólogos anti-Borrelia burgdorferi em eqüinos na mesorregião metropolitana de Belém, Estado do Pará / Occurrence of homologous anti-Borrelia burgdorferi antibodies in equines in the metropolitan mesorregion of Belém, State of Para, Brazil

Espiroquetas transmitidas por carrapatos são microrganismos de ampla distribuição geográfica e acometem animais silvestres, domésticos e seres humanos. Procedeu-se a análise sorológica de 300 soros de eqüinos onde 58 animais eram do município Ananideua, 61 eram de Belém, 131 de Castanhal e 50 eram do município de Santa Izabel do Pará para Borrelia burgdorferi através do teste ELISA indireto. Não foram observadas diferenças significativas (P < 0,05) entre os municípios, nem quanto à raça, sexo e função dos animais. Um total de 80 (26,7 por cento) animais foram positivos para B. burgdorferi com os títulos de 1:800, 72 (90 por cento) eqüinos; 1:1.600, 6 (7,5 por cento) eqüinos; e 1:3.200, 2 (2,5 por cento) eqüinos. Os resultados observados foram similares aos descritos nos EUA, onde foram relatadas freqüências de soropositivos variando entre 7 e 75 por cento em eqüinos assintomáticos. A presença de anticorpos homólogos contra B. burgdorferi em eqüinos na mesorregião metropolitana de Belém é indicativo da ampla distribuição do agente e da possibilidade de ocorrerem casos humanos deste agente na região.

Spirochaetes transmitted by ticks are microorganisms of worldwide distribution, which infect wild, domestic animals and human beings. A total of 300 equine sera from four municipalities: Ananideua (58), Belém (61), Castanhal (131), and Santa Izabel do Pará (50), were evaluated for Borrelia burgdorferi by an Elisa test. There were no significant differences (P<0.05) among municipalities, breed, sex or husbandry. A total of 80 (26.7 percent) horses were B. burgdorferi positive with titles of 1:800, 72 (90 percent) horses, 1:1.600, 6 (7.5 percent) horses, and 1:3.200, 2 (2.5 percent) horses. The results were similar to those in the USA, where related frequencies ranged from 7 to 75 percent in asymptomatic seropositive horses. The presence of anti-B.burgdorferi homologous antibodies in horses from four municipalities in the metropolitan mesorregion of Belém suggests the possibility of occurrence of human cases in the region.

Frequency of antibodies to Babesia bigemina, B. bovis, Anaplasma marginale, Trypanosoma vivax and Borrelia burgdorferi in cattle from the Northeastern region of the State of Pará, Brazil.

Babesiosis, anaplasmosis, and trypanosomosis are relevant diseases, potentially causing morbidity in cattle, leading to economic losses. Borreliosis is import as a potential zoonosis. The objective of this study was to determine, by indirect enzyme-linked immunosorbent assay (ELISA), the frequency of seropositive cattle to Babesia bigemina, B. bovis, Anaplasma marginale, Trypanosoma vivax and Borrelia burgdorferi in cattle from the Northeastern region of Pará, Brazil. Sera samples from 246 female adult cattle from municipalities of Castanhal and São Miguel do Guamá were used. Crude antigens ELISAs were used to detect antibodies to all agents, except to A. marginale, to which an indirect ELISA with recombinant major surface 1a protein (MSP1a) antigen was used. Overall frequencies of seropositive animals were: B. bigemina--99.2%; B. bovis--98.8%; A. marginale--68.3%; T. vivax--93.1% and B. burgdorferi--54.9%. The frequencies of seropositive cattle to B. bovis and B. bigemina suggest a high rate of transmission of these organisms by tick in the studied region, which can be classified as enzootically stable to these hemoprotozoans. The low frequency of seropositive cattle to A. marginale may be attributed to a lower sensitivity of the recombinant antigen ELISA utilized or a distinct rate of inoculation of this rickettsia by ticks, as compared with Babesia sp. transmission. The high frequency of seropositive cattle to T. vivax indicates that this hemoprotozoan is prevalent in herds from the Northeastern region of Pará. The rate of animal that showed homologues antibodies to B. burgdorferi indicates the presence of the tickborne spirochaetal agent in the cattle population in the studied region.

Frequency of antibodies to Babesia bigemina, B. bovis, Anaplasma marginale, Trypanosoma vivax and Borrelia burdgorferi in cattle from the northeastern region of the state of Pará, Brazil / Freqüência de anticorpos para Babesia bigemina, B. bovis, Anaplasma marginale, Trypanosoma vivax e Borrelia burgdorferi em bovinos do nordeste do Estado do Pará, Brasil

Babesiosis, anaplasmosis, and trypanosomosis are relevant diseases, potentially causing morbidity in cattle, leading to economic losses. Borreliosis is import as a potential zoonosis. The objective of this study was to determine, by indirect enzyme-linked immunosorbent assay (ELISA), the frequency of seropositive cattle to Babesia bigemina, B. bovis, Anaplasma marginale, Trypanosoma vivax and Borrelia burgdorferi in cattle from the Northeastern region of Pará, Brazil. Sera samples from 246 female adult cattle from municipalities of Castanhal and São Miguel do Guamá were used. Crude antigens ELISAs were used to detect antibodies to all agents, except to A. marginale, to which an indirect ELISA with recombinant major surface 1a protein (MSP1a) antigen was used. Overall frequencies of seropositive animals were: B. bigemina - 99.2 percent; B. bovis - 98.8 percent; A. marginale - 68.3 percent; T. vivax - 93.1 percent and B. burgdorferi - 54.9 percent. The frequencies of seropositive cattle to B. bovis and B. bigemina suggest a high rate of transmission of these organisms by tick in the studied region, which can be classified as enzootically stable to these hemoprotozoans. The low frequency of seropositive cattle to A. marginale may be attributed to a lower sensitivity of the recombinant antigen ELISA utilized or a distinct rate of inoculation of this rickettsia by ticks, as compared with Babesia sp. transmission. The high frequency of seropositive cattle to T. vivax indicates that this hemoprotozoan is prevalent in herds from the Northeastern region of Pará. The rate of animal that showed homologues antibodies to B. burgdorferi indicates the presence of the tickborne spirochaetal agent in the cattle population in the studied region.

A babesiose, a anaplasmose e a tripanossomose são enfermidades relevantes, potencialmente causadoras de morbidade em bovinos, levando a perdas econômicas. A borreliose assume importância como zoonose potencial. O objetivo desse estudo foi determinar, por meio do ensaio de imunoadsorção enzimática (ELISA), a freqüência de anticorpos para Babesia bigemina, B. bovis, Anaplasma marginale, Trypanosoma vivax e Borrelia burgdorferi em bovinos da região nordeste do Estado do Pará, Brasil. Amostras de soro de 246 vacas dos municípios de Castanhal e São Miguel do Guamá foram usadas. ELISAs com antígeno bruto foram utilizados para detector anticorpos contra todos os agentes, exceto para A. marginale, para o qual um ELISA indireto com proteína principal de superfície 1a (MSP1a) foi usado. As freqüências de bovinos soropositivos foram: B. bigemina - 99,2 por cento; B. bovis - 98,8 por cento; A. marginale - 68,3 por cento; T. vivax - 93,1 por cento and B. burgdorferi -54,9 por cento As freqüências de bovinos soropositivos para B. bovis e B. bigemina sugerem uma alta taxa de transmissão desses organismos por carrapatos, na região estudada, a qual pode ser classificada com sendo de estabilidade enzoótica para os hemoparasitos. A baixa freqüência de bovinos soropositivos para A. marginale pode ser atribuída a uma menor sensibilidade do ELISA com antígeno recombinante, ou uma menor taxa de inoculação da riquétsia pelos carrapatos, quando comparada àquelas observadas para Babesia sp. A alta freqüência de bovinos soropositivos para T. vivax indica que esse hemoprotozoário é prevalente em rebanhos do nordeste do Estado do Pará. O percentual de animais com anticorpos homólogos para B. burgdorferi indica a presenças deste espiroquetídeo transmitido por carrapatos na população de bovinos da região estudada.