PESQUISA VETERINÁRIA BRASILEIRA

Abstract in English:

ABSTRACT.- Leal D.C., Madruga C.R., Matos P.F., Souza B.M.P.S. & Franke C.R. 2011. Evaluation of PCR and multiplex PCR in relation to nested PCR for diagnosing Theileria equi. Pesquisa Veterinária Brasileira 31(7):575-578. Departamento de Produção Animal, Escola de Medicina Veterinária, Universidade Federal da Bahia, Av. Adhemar de Barros 500, Ondina, Salvador, BA 40110170, Brazil. E-mail: franke@ufba.br Conventional PCR (PCRTeq) for diagnosing Theileria equi and multiplex PCR (M/PCRTeq-Bc) for diagnosing T. equi and Babesia caballi were comparatively evaluated with nested PCR (N/PCR-Teq) for diagnosing equine piroplasmosis. In DNA sensitivity determinations, in multiple dilutions of equine blood that had tested positive for T. equi, PCR-Teq and N/PCR-Teq detected hemoparasite DNA in the larger dilutions (1:128), but did not differ significantly from the M/PCRTeq-Bc (1:64). In analyses on equine serum tested by ELISA, there was high agreement between this serological test and PCR-Teq (k = 0.780) and moderate agreement with N/PCR-Teq (k = 0.562) and M/PCRTeq-Bc (k = 0.488). PCR-Teq found a higher frequency of T. equi both in extensively and intensively reared horses, but this was not significant in relation to N/PCR-Teq (P>0.05), and both PCRs indicated that there was an endemic situation regarding T. equi in the population of horses of this sample. PCR-Teq was only significantly different from M/PCR-Teq-Bc (P<0.05). PCR-Teq presented high sensitivity and specificity, comparable to N/PCR-Teq, but with the advantage of higher speed in obtaining results and lower costs and risks of laboratory contamination. This accredits PCR-Teq for epidemiological studies and for determinations on affected horses.

• Theileria equi

Abstract in Portuguese:

RESUMO.- Leal D.C., Madruga C.R., Matos P.F., Souza B.M.P.S. & Franke C.R. 2011. Evaluation of PCR and multiplex PCR in relation to nested PCR for diagnosing Theileria equi. Pesquisa Veterinária Brasileira 31(7):575-578. Departamento de Produção Animal, Escola de Medicina Veterinária, Universidade Federal da Bahia, Av. Adhemar de Barros 500, Ondina, Salvador, BA 40110170, Brazil. E-mail: franke@ufba.br Uma PCR convencional (PCRTeq) para diagnóstico de Theileria equi e PCR multiplex (M/PCRTeq-Bc) para diagnóstico T. equi e Babesia caballi foram avaliadas comparativamente a nested PCR (N/PCR-Teq) no diagnóstico de piroplasmose equina. Na determinação da sensibilidade com DNA em diluições múltiplas de sangue de equino positivo para T. equi, as PCR-Teq e N/PCR-Teq detectaram DNA do hemoparasito nas diluições maiores (1:128), mas não diferiu significativamente da M/PCRTeq-Bc (1:64). Na análise com soros de equinos testados por ELISA houve uma concordância elevada entre esse teste sorológico e a PCR-Teq (k =0,780) e moderada com N/PCR-Teq (k = 0.562) e M/PCRTeq-Bc (k = 0.488). A PCR-Teq determinou freqüência maior de T. equi tanto nos equinos de criação extensiva como na intensiva, entretanto essa não foi significativa com relação N/PCR-Teq (P>0.05), e ambas PCRs indicaram que há uma situação endêmica para T. equi na população de equinos que constaram da amostragem. A PCR-Teq diferiu significativamente apenas para a M/PCRTeq-Bc (P<0.05). A PCR-Teq apresentou alta sensibilidade e especificidade comparável a N/PCR-Teq, mas com a vantagem de maior rapidez na obtenção dos resultados e menor custo e risco de contaminação no laboratório. Isso credencia a PCR-Teq para estudos epidemiológicos e para determinação de equinos portadores.

Abstract in English:

ABSTRACT.- Lange C.C., Brito M.AV.P., Brito J.R.F., Arcuri E.F., Souza G.N., Machado M.A., Domingues R. & Salimena A.P.S. 2011. [Identification of Staphylococcus strains isolated from bovine mastitis by PCR and 16S rDNA sequencing.] Uso de PCR e sequenciamento do rDNA 16S para identificação de bactérias do gênero Staphylococcus isoladas de mastite bovina. Pesquisa Veterinária Brasileira 31(1):36-40. Embrapa Gado de Leite, Rua Eugênio do Nascimento 610, Juiz de Fora, MG 36030-330, Brazil. E-mail: clange@cnpgl.embrapa.br The objective of this study was to identify the species of 100 isolates of Staphylococcus from mastitis in dairy cows from herds located in the state of Minas Gerais, Brazil. PCR reactions were carried out using specific primers described previously for S. aureus (femA gene), S. intermedius (16S rDNA) and S. hyicus (16S-23S rDNA spacer region). In addition, products of amplification of variable regions of the 16S rDNA gene of the strains were sequenced. According to the results of the PCR, 83 strains were identified as S. aureus, 13 as S. intermedius, two as S. hyicus and two isolates were not identified. The sequencing of 16S rDNA was applied to 23 strains identified by PCR amplifications: six S. aureus and the strains identified as S. intermedius (n=13), S. hyicus (n=2) or not identified (n=2). The sequencing of 16S rDNA confirmed the six strains as S. aureus. The others 17 strains were identified as S. chromogenes (13 isolates) and S. hyicus (four isolates). Each sample was related to a specie according to the smallest E-value and highest similarity (³ 99%). The identification of S. hyicus and S. chromogenes was accomplished only by 16S rDNA sequencing.

• Staphylococcus aureus S. chromogenes S. hyicus

Abstract in Portuguese:

RESUMO.- Lange C.C., Brito M.AV.P., Brito J.R.F., Arcuri E.F., Souza G.N., Machado M.A., Domingues R. & Salimena A.P.S. 2011. [Identification of Staphylococcus strains isolated from bovine mastitis by PCR and 16S rDNA sequencing.] Uso de PCR e sequenciamento do rDNA 16S para identificação de bactérias do gênero Staphylococcus isoladas de mastite bovina. Pesquisa Veterinária Brasileira 31(1):36-40. Embrapa Gado de Leite, Rua Eugênio do Nascimento 610, Juiz de Fora, MG 36030-330, Brazil. E-mail: clange@cnpgl.embrapa.br O objetivo deste trabalho foi identificar espécies de Staphylococcus (n=100) isoladas de mastite em rebanhos bovinos do Estado de Minas Gerais. Para esta finalidade foram utilizadas reações de PCR empregando oligonucleotídeos iniciadores descritos anteriormente para amplificar genes específicos de S. aureus (femA), S. intermedius (rDNA 16S) e S. hyicus (rDNA 16S-23S) e o sequenciamento do rDNA 16S. De acordo com as reações de PCR, 83 isolados foram identificados como S. aureus, 13 isolados como S. intermedius, dois como S. hyicus e dois isolados não foram identificados. Foram submetidos ao sequenciamento do rDNA 16S seis isolados identificados como S. aureus e os 17 restantes. Os seis isolados identificados como S. aureus confirmaram essa identificação. Dos outros 17 isolados, 13 foram identificados como S. chromogenes e quatro como S. hyicus, com similaridade igual ou superior a 99%. Baseando-se nos resultados da reação de PCR do gene femA e do sequenciamento do rDNA 16S, foram identificados 83 S. aureus, 13 S. chromogenes e quatro S. hyicus. Neste estudo os oligonucleotídeos iniciadores empregados na reação de PCR para S. intermedius não foram específicos, pois amplificaram também S. chromogenes; e os empregados na reação de PCR para S. hyicus não foram sensíveis, pois falharam na identificação de dois isolados de S. hyicus. A identificação definitiva das duas últimas espécies somente foi possível pelo sequenciamento do rDNA 16S.

Abstract in English:

ABSTRACT.- Gerber P.F., Pinto F.F., Heinemann M.B. & Lobato Z.I.P. 2010. Detection and dynamics of porcine circovirus type 2 in semen using conventional and real time PCR. Pesquisa Veterinária Brasileira 30(11):918-920. Laboratório de Pesquisa em Virologia Animal, Departamento de Medicina Veterinária Preventiva, Universidade Federal de Minas Gerais, Avenida. Antônio Carlos 6627, Belo Horizonte, MG 30123-970, Brazil. E-mail: ziplobato@vet.ufmg.br The dynamics of porcine circovirus type 2 (PCV2) shedding in semen of naturally infected boars was studied. Semen was collected serially each 15 or 20 days during 62 days from 5 boars from a herd and from 11 boars from an artificial insemination center. All boars were positive for PCV2 DNA by nested polymerase chain reaction of raw semen in at least two sampling dates, and most of them had detectable shedding in all sampling dates. Real-time quantitative PCR was performed in 23 samples. All samples showed low amounts of PCV2 DNA, ranging from 98 to 652 PCV2 copies/mL. No differences between the frequencies of PCV2 DNA shed in semen were found considering herds and age of boars. PCV2 shedding in the semen can occur continuously or intermittently up to 60 days in naturally infected boars at 12 to 42 months old in absence of PCV2 clinical signs. These results demonstrate sporadic and long-term shedding patterns of low amounts of PCV2 DNA in semen from naturally infected boars.

• Porcine circovirus type 2 circovírus suíno tipo 2

Abstract in Portuguese:

RESUMO.- Gerber P.F., Pinto F.F., Heinemann M.B. & Lobato Z.I.P. 2010. Detection and dynamics of porcine circovirus type 2 in semen using conventional and real time PCR. [Deteccão e dinâmica de excreção do circovírus porcino tipo 2 no semen pelo PCR convencional e PCR em tempo real.] Pesquisa Veterinária Brasileira 30(11):918-920. Laboratório de Pesquisa em Virologia Animal, Departamento de Medicina Veterinária Preventiva, Universidade Federal de Minas Gerais, Avenida. Antônio Carlos 6627, Belo Horizonte, MG 30123-970, Brazil. E-mail: ziplobato@vet.ufmg.br

Abstract in English:

ABSTRACT.- Santos S.B., Nascimento E.R., Faccini J.L.H., Barreto M.L., Almeida J.F., Pereira V.L.A. & Campos C.A.M. 2010. [Detection of Mycoplasma mycoides cluster by indirect immunoperoxidase (IPI) and PCR-REA in the ear canal of bovines.] Detecção do Grupo Mycoplasma mycoides por imunoperoxidase indireta (IPI) e PCR-REA em conduto auditivo de bovinos. Pesquisa Veterinária Brasileira 30(5):465-469. Departamento de Saúde Coletiva Veterinária e Saúde Pública, Universidade Federal Fluminense, Rua Vital Brazil Filho 64, Niterói, RJ 24230-340, Brazil. E-mail: elmiro@vm.uff.br Mycoplasma mycoides cluster (MMC) was diagnosed by polimerase chain reaction-restriction endonuclease analysis (PCR-REA) and indirect immunoperoxidase (IPI), both, carried out in flushing from external ear canal, collected from bovine at slaughter time in the State of Rio de Janeiro, southeastern, Brazil. A total of 60 bovines were randomly selected. Sterile syringes (60mL) loaded with buffer solution (PBS, pH 7.2) were used for the ear canal flushing. The obtained samples were stored in glycerol (1:2) and frozen at -20oC until use. These specimens were diluted up to 10-5, inoculated in liquid and solid modified Hayflick´s media and incubated at 37oC for 2-3 days. The plates were kept in a microaerophilia condition and examined every two days under a stereomicroscope for the presence of typical colonies “fried-egg”. In this study, 35 strains selected in agreement with their biochemistry and physiologic proprieties, were used. From the 60 cultivated samples, 48 (80.00%) were positive for Mycoplasma spp. Under IPI the prevalence obtained for MMC was 20.0% (12/60) while by PCR-REA it was 41.7% (25/60). The IPI typing of these isolates resulted in 58.3% (7/12) for M.mycoides mycoides LC and 41.7% (5/12) for M. capricolum. PCR-REA for MMC was confirmed by the amplicon size of 785bp, compatible with this group. The Kappa value for the association between these two tests was 0.14 (p>0.05). After restriction analysis with AluI in all MMC strains the fragments size obtained were of 81, 98, 186 and 236bp, but not of 370bp that is compatible with Mycoides mycoides mycoides SC of bovine type. The presence of mycoplasmas species in the ear canal of asymptomatic bovines represent a risk of subsequent propagation of Mycoplasma spp. among bovine herds in Brazil.

• Mycoplasma mycoides

Abstract in Portuguese:

RESUMO.- Santos S.B., Nascimento E.R., Faccini J.L.H., Barreto M.L., Almeida J.F., Pereira V.L.A. & Campos C.A.M. 2010. [Detection of Mycoplasma mycoides cluster by indirect immunoperoxidase (IPI) and PCR-REA in the ear canal of bovines.] Detecção do Grupo Mycoplasma mycoides por imunoperoxidase indireta (IPI) e PCR-REA em conduto auditivo de bovinos. Pesquisa Veterinária Brasileira 30(5):465-469. Departamento de Saúde Coletiva Veterinária e Saúde Pública, Universidade Federal Fluminense, Rua Vital Brazil Filho 64, Niterói, RJ 24230-340, Brazil. E-mail: elmiro@vm.uff.br O Grupo Mycoplasma mycoides (GMM) foi diagnosticado por PCR-REA e imunoperoxidase indireta (IPI) em amostras de lavados de conduto auditivo de bovinos no Estado do Rio de Janeiro, Brasil. 60 bovinos foram selecionados aleatoriamente. As lavagens foram feitas com uso de seringas estéreis contendo um volume de 60 mL de solução salina tamponada (PBS pH 7.2). As amostras obtidas foram estocadas em glicerol (1:2) e congeladas a -20oC até uso. Estas amostras foram diluídas até 10-5 e repicadas em meio Hayflick modificado, sólido e líquido, sendo incubados a 37oC por 48-72 horas. As placas foram mantidas em microaerofilia e observadas diariamente, para visualização das colônias típicas em “ovo-frito”. Das 60 amostras cultivadas, 48 (80,00%) foram positivas para Mycoplasma spp. A prevalência obtida para o GMM na IPI foi de 20,0% (12/60) enquanto na PCR-REA foi de 41,7% (25/60). Das cepas tipificadas pela IPI 58,3% (7/12) foram M. mycoides subsp. mycoides LC e 41,7% (5/12) foram M. capricolum. Na PCR-REA o grupo M. mycoides foi confirmado pela visualização de um amplicon de 785bp, compatível com este grupo. O valor encontrado no teste Kappa para associação entre estes testes foi de 0,14 (P>0,05. Na clivagem do produto da PCR com a enzima de restrição AluI, de cepas de referências e dos isolados de ouvido os fragmentos obtidos foram de 81, 98, 186 e 236pb, mas não de 370pb, que é específica para o agente da Pleuropneumonia Contagiosa Bovina. A presença de espécies de micoplasmas no conduto auditivo de bovinos assintomáticos representa um risco para propagação de Mycoplasma spp. entre rebanhos bovinos no Brasil.

Abstract in English:

ABSTRACT.- Asano K.M, Souza S.P., Silva S.O.S., Richtzenhain L.J. & Brandão P.E. 2009. Rapid detection of bovine coronavirus by a semi-nested RT-PCR. Pesquisa Veterinária Brasileira 29(11):869-873. Departamento de Medicina Veterinária Preventiva e Saúde Animal, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, Av. Prof. Dr. Orlando Marques de Paiva 87, São Paulo, SP 05508-270, Brazil. E-mail: karen.asano@gmail.com Bovine coronavirus (BCoV) is a member of the group 2 of the Coronavirus (Nidovirales: Coronaviridae) and the causative agent of enteritis in both calves and adult bovine, as well as respiratory disease in calves. The present study aimed to develop a semi-nested RT-PCR for the detection of BCoV based on representative up-to-date sequences of the nucleocapsid gene, a conserved region of coronavirus genome. Three primers were designed, the first round with a 463bp and the second (semi-nested) with a 306bp predicted fragment. The analytical sensitivity was determined by 10-fold serial dilutions of the BCoV Kakegawa strain (HA titre: 256) in DEPC treated ultra-pure water, in fetal bovine serum (FBS) and in a BCoV-free fecal suspension, when positive results were found up to the 10-2, 10-3 and 10-7 dilutions, respectively, which suggests that the total amount of RNA in the sample influence the precipitation of pellets by the method of extraction used. When fecal samples was used, a large quantity of total RNA serves as carrier of BCoV RNA, demonstrating a high analytical sensitivity and lack of possible substances inhibiting the PCR. The final semi-nested RT-PCR protocol was applied to 25 fecal samples from adult cows, previously tested by a nested RT-PCR RdRp used as a reference test, resulting in 20 and 17 positives for the first and second tests, respectively, and a substantial agreement was found by kappa statistics (0.694). The high sensitivity and specificity of the new proposed method and the fact that primers were designed based on current BCoV sequences give basis to a more accurate diagnosis of BCoV-caused diseases, as well as to further insights on protocols for the detection of other Coronavirus representatives of both Animal and Public Health importance.

• Coronavírus bovino diarréia

Abstract in Portuguese:

RESUMO.- Asano K.M, Souza S.P., Silva S.O.S., Richtzenhain L.J. & Brandão P.E. 2009. Rapid detection of bovine coronavirus by a semi-nested RT-PCR. [Detecção rápida do Coronavírus Bovino (BCoV) por meio de uma semi-nested RT-PCR.] Pesquisa Veterinária Brasileira 29(11):869-873. Departamento de Medicina Veterinária Preventiva e Saúde Animal, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, Av. Prof. Dr. Orlando Marques de Paiva 87, São Paulo, SP 05508-270, Brazil. E-mail: karen.asano@gmail.com O Coronavírus bovino (BCoV) pertence ao grupo 2 do gênero Coronavirus (Nidovirales: Coronaviridae) e é agente causador de enterites tanto em bezerros como em bovinos adultos, bem como de doença respiratória em bezerros. O presente estudo teve por objetivo desenvolver uma semi-nested RT-PCR para a detecção do BCoV com base em seqüências representativas e recentes do gene do nucleocapsídeo, região conservada do genoma dos coronavírus. Três primers foram desenhados, a primeira amplificação com um fragmento esperado de 463pb e a segunda (semi-nested) com um fragmento esperado de 306pb. A sensibilidade analítica foi determinada pela diluição do BCoV cepa Kakegawa (título HA: 256) na base de 10 em água ultra-pura tratada com DEPC, em soro fetal bovino (SFB) e em uma suspensão fecal negativa para o BCoV, onde foram encontrados resultados positivos até a diluição de 10-2, 10-3 e 10-7, respectivamente. Este resultado sugere que a quantidade total de RNA na amostra influencia na precipitação dos pellets pelo método de extração utilizado. Quando se utiliza amostra fecal, a grande quantidade de RNA total funciona como carreadora do RNA do BCoV, demonstrando elevada sensibilidade analítica e ausência de possíveis substâncias inibidoras da PCR. O protocolo final da semi-nested RT-PCR foi aplicado a 25 amostras fecais de vacas adultas, previamente avaliadas por uma nested RT-PCR RdRp utilizada como teste de referência, resultando em 20 e 17 amostras positivas para o primeiro e segundo teste, respectivamente. Os resultados dos dois sistema de diagnóstico apresentaram concordância substancial (kappa: 0,694). A elevada sensibilidade e especificidade do novo método proposto e o fato de que os primers foram desenhados baseados em sequências atuais do BCoV, oferecem bases para o diagnóstico mais acurado de infecções causadas pelo BCoV, assim como para novas perspectivas em protocolos de detecção de outros Coronavírus de importância tanto em saninade animal quanto em saúde pública.

Abstract in English:

ABSTRACT.- Schatzmayr H.G., Simonetti B.R., Abreu D.C., Simonetti J.P., Simonetti S.R., Costa R.V.V., Gonçalves M.C.R., Gerhardt M.., Silva M.E.V., Farias-Filho J.C. & Barth O.M. 2009. Animal infections by vaccinia-like virus in the state of Rio de Janeiro: An expanding disease. Pesquisa Veterinária Brasileira 29(7):509-514. Laboratório de Morfologia e Morfogênese Viral, Instituto Oswaldo Cruz, Fiocruz, Avenida Brasil 4365, Rio de Janeiro, RJ 21040-900, Brazil. E-mail: hermann@ioc.fiocruz.br In the present study we investigated the presence of infections by vaccinia-like viruses in dairy cattle from 12 counties in the state of Rio de Janeiro in the last 9 years. Clinical specimens were collected from adult animals with vesicular/pustular lesions mainly in the udder and teats, and from calves with lesions around the nose and mouth. A plaque reduction neutralization test (PRNT) was applied to search for antibodies to Orthopoxvirus; the vesicular/pustular fluids and scabs were examined by PCR, electron microscopy (EM) and by inoculation in VERO cells for virus isolation. Antibodies to Orthopoxvirus were detected in most cases. The PCR test indicated a high nucleotide homology among the isolates and the vaccinia viruses (VACV) used as controls. By EM, typical orthopoxvirus particles were observed in some specimens. The agents isolated in tissue culture were confirmed as vaccinia-like viruses by EM and PCR. The HA gene of the vaccinia-like Cantagalo/IOC virus isolated in our laboratory was sequenced and compared with other vaccinia-like isolates, showing high homology with the original Cantagalo strain, both strains isolated in 1999 from dairy cattle. Antibodies to Orthopoxvirus were detected in one wild rodent (genus Akodon sp.) collected in the northwestern region of the state, indicating the circulation of poxvirus in this area. Nonetheless, PCR applied to tissue samples collected from the wild rodents were negative. Vesicular/pustular lesions in people in close contact with animals have been also recorded. Thus, the vaccinia-like virus infections in cattle and humans in the state seem to be an expanding condition, resulting in economic losses to dairy herds and leading to transient incapacitating human disease. Therefore, a possible immunization of the dairy cattle in the state should be carefully evaluated.

• Orthopoxvirus infections PCR neutralization test electron microscopy state of Rio de Janeiro

Abstract in Portuguese:

ABSTRACT.- Schatzmayr H.G., Simonetti B.R., Abreu D.C., Simonetti J.P., Simonetti S.R., Costa R.V.V., Gonçalves M.C.R., Gerhardt M.., Silva M.E.V., Farias-Filho J.C. & Barth O.M. 2009. Animal infections by vaccinia-like virus in the state of Rio de Janeiro: An expanding disease. Pesquisa Veterinária Brasileira 29(7):509-514. Laboratório de Morfologia e Morfogênese Viral, Instituto Oswaldo Cruz, Fiocruz, Avenida Brasil 4365, Rio de Janeiro, RJ 21040-900, Brazil. E-mail: hermann@ioc.fiocruz.br In the present study we investigated the presence of infections by vaccinia-like viruses in dairy cattle from 12 counties in the state of Rio de Janeiro in the last 9 years. Clinical specimens were collected from adult animals with vesicular/pustular lesions mainly in the udder and teats, and from calves with lesions around the nose and mouth. A plaque reduction neutralization test (PRNT) was applied to search for antibodies to Orthopoxvirus; the vesicular/pustular fluids and scabs were examined by PCR, electron microscopy (EM) and by inoculation in VERO cells for virus isolation. Antibodies to Orthopoxvirus were detected in most cases. The PCR test indicated a high nucleotide homology among the isolates and the vaccinia viruses (VACV) used as controls. By EM, typical orthopoxvirus particles were observed in some specimens. The agents isolated in tissue culture were confirmed as vaccinia-like viruses by EM and PCR. The HA gene of the vaccinia-like Cantagalo/IOC virus isolated in our laboratory was sequenced and compared with other vaccinia-like isolates, showing high homology with the original Cantagalo strain, both strains isolated in 1999 from dairy cattle. Antibodies to Orthopoxvirus were detected in one wild rodent (genus Akodon sp.) collected in the northwestern region of the state, indicating the circulation of poxvirus in this area. Nonetheless, PCR applied to tissue samples collected from the wild rodents were negative. Vesicular/pustular lesions in people in close contact with animals have been also recorded. Thus, the vaccinia-like virus infections in cattle and humans in the state seem to be an expanding condition, resulting in economic losses to dairy herds and leading to transient incapacitating human disease. Therefore, a possible immunization of the dairy cattle in the state should be carefully evaluated.

Abstract in English:

ABSTRACT.- Buim M.R., Mettifogo E., Timenetsky J., Kleven S. & Ferreira A.J.P. 2009. Epidemiological survey on Mycoplasma gallisepticum and M. synoviae by multiplex PCR in commercial poultry. Pesquisa Veterinária Brasileira 29(7):552-556. Departamento de Patologia, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, São Paulo, SP 05508-900, Brazil. E-mail: ajpferr@usp.br Mycoplasmas are important avian pathogens, which cause respiratory and joint diseases that result in large economic losses in Brazilian and world-wide poultry industry. This investigation regarding the main species of mycoplasmas, Mycoplasma gallisepticum (MG) and M. synoviae (MS), responsible for the above mentioned conditions, was carried out through PCR Multiplex analysis. One thousand and forty-six (1,046) samples of tracheal swabs and piped embryos were collected from 33 farms with laying hens, breeders, broilers or hatchery, located in the Brazilian states of São Paulo, Paraná and Pernambuco, where respiratory problems or drops in egg production had occurred. The MG and MS prevalence on the farms was 72.7%. These results indicated (1) high dissemination of mycoplasmas in the evaluated farms, with predominance of MS, either as single infectious agent or associated with other mycoplasmas in 20 farms (60.6%), and (2) an increase of MS and decrease of MG infection in Brazilian commercial poultry.

• Mycoplasma gallisepticum M. synoviae PCR epidemiology chicken

Abstract in Portuguese:

ABSTRACT.- Buim M.R., Mettifogo E., Timenetsky J., Kleven S. & Ferreira A.J.P. 2009. Epidemiological survey on Mycoplasma gallisepticum and M. synoviae by multiplex PCR in commercial poultry. Pesquisa Veterinária Brasileira 29(7):552-556. Departamento de Patologia, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, São Paulo, SP 05508-900, Brazil. E-mail: ajpferr@usp.br Mycoplasmas are important avian pathogens, which cause respiratory and joint diseases that result in large economic losses in Brazilian and world-wide poultry industry. This investigation regarding the main species of mycoplasmas, Mycoplasma gallisepticum (MG) and M. synoviae (MS), responsible for the above mentioned conditions, was carried out through PCR Multiplex analysis. One thousand and forty-six (1,046) samples of tracheal swabs and piped embryos were collected from 33 farms with laying hens, breeders, broilers or hatchery, located in the Brazilian states of São Paulo, Paraná and Pernambuco, where respiratory problems or drops in egg production had occurred. The MG and MS prevalence on the farms was 72.7%. These results indicated (1) high dissemination of mycoplasmas in the evaluated farms, with predominance of MS, either as single infectious agent or associated with other mycoplasmas in 20 farms (60.6%), and (2) an increase of MS and decrease of MG infection in Brazilian commercial poultry.

Abstract in English:

ABSTRACT.- Ecco R., Lazzari M.A. & Guedes R.M.C. 2009. [Enzootic pneumonia in wild boars (Sus scrofa).] Pneumonia enzoótica em javalis (Sus scrofa). Pesquisa Veterinária Brasileira 29(6):461-468. Departamento de Clinica e Cirurgia Veterinárias, Escola de Veterinária, Universidade Federal de Minas Gerais, Av. Antônio Carlos 6627, Cx. Postal 567, Belo Horizonte, MG 31270-901, Brazil. E-mail: ecco@vet.ufmg.br The aim of this paper is to describe the clinical, epidemiological, pathological, bacteriological and immunohistochemical aspects of a pneumonia outbreak in a wild pig farm in the Distrito Federal, Brazil. Ninety wild pigs died in a period of five months, and 63 of these had pulmonary lesions. Clinically, the pigs presented reduced growth rate, anorexia, lethargy, cough and dyspnea, especially after they were moved. High body temperature (40oC in average) was verified in some animals. Auscultation revealed moderate pulmonary crepitation and stertors. Pulmonary gross lesions were typical of lobular bronchopneumonia. Lung lesions were characterized by ventral-cranial consolidation in the majority of the cases. The color of affected pulmonary areas varied from diffuse dark red to mosaic pattern (dark red lobule intercalate by grayish lobule) or diffusely grayish. The majority of the lungs had mucopurulent exsudate in the bronchial lumen that also drained from the parenchyma cut surface. Upon microscopy, the changes were characterized by purulent and histiocytic bronchopneumonia with necrotic foci. In some animals, there was BALT hyperplasia associated with perivascular and peribronchial plasma cells and lymphocytes infiltration in most of these cases. Bordetella bronchiseptica and Streptococcus spp. were the most frequently isolated bacteria. Immunohistochemistry evaluation demonstrated Mycoplasma hyopneumoniae on the luminal surface of bronchial and bronchiolar epithelial cells, and the DNA of bacteria was detected by PCR. This is the first report of bronchopneumonia in wild boars associated with M. hyopneumoniae infection.

• Wild pigs bronchopneumonia pathology bacteriology PCR immunohisto-chemistry Mycoplasma hyopneumoniae

Abstract in Portuguese:

ABSTRACT.- Ecco R., Lazzari M.A. & Guedes R.M.C. 2009. [Enzootic pneumonia in wild boars (Sus scrofa).] Pneumonia enzoótica em javalis (Sus scrofa). Pesquisa Veterinária Brasileira 29(6):461-468. Departamento de Clinica e Cirurgia Veterinárias, Escola de Veterinária, Universidade Federal de Minas Gerais, Av. Antônio Carlos 6627, Cx. Postal 567, Belo Horizonte, MG 31270-901, Brazil. E-mail: ecco@vet.ufmg.br The aim of this paper is to describe the clinical, epidemiological, pathological, bacteriological and immunohistochemical aspects of a pneumonia outbreak in a wild pig farm in the Distrito Federal, Brazil. Ninety wild pigs died in a period of five months, and 63 of these had pulmonary lesions. Clinically, the pigs presented reduced growth rate, anorexia, lethargy, cough and dyspnea, especially after they were moved. High body temperature (40oC in average) was verified in some animals. Auscultation revealed moderate pulmonary crepitation and stertors. Pulmonary gross lesions were typical of lobular bronchopneumonia. Lung lesions were characterized by ventral-cranial consolidation in the majority of the cases. The color of affected pulmonary areas varied from diffuse dark red to mosaic pattern (dark red lobule intercalate by grayish lobule) or diffusely grayish. The majority of the lungs had mucopurulent exsudate in the bronchial lumen that also drained from the parenchyma cut surface. Upon microscopy, the changes were characterized by purulent and histiocytic bronchopneumonia with necrotic foci. In some animals, there was BALT hyperplasia associated with perivascular and peribronchial plasma cells and lymphocytes infiltration in most of these cases. Bordetella bronchiseptica and Streptococcus spp. were the most frequently isolated bacteria. Immunohistochemistry evaluation demonstrated Mycoplasma hyopneumoniae on the luminal surface of bronchial and bronchiolar epithelial cells, and the DNA of bacteria was detected by PCR. This is the first report of bronchopneumonia in wild boars associated with M. hyopneumoniae infection.

Abstract in English:

ABSTRACT.- Cavallini Sanches E.M., Pacheco S.M., Cericatto A.S., Melo R.M., Colodel E.M., Hummel J., Bianchi S.P., Spanamberg A., Santurio J.M. & Ferreiro L. 2009. Detection of Pneumocystis in lungs of bats from Brazil by PCR amplification. Pesquisa Veterinária Brasileira 29(6):469-473. Setor de Micologia Veterinária, Faculdade de Veterinária, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves 9090, Porto Alegre, RS 90540-000, Brazil. E-mail: cavallini.sanches@ufrgs.br Pneumocystis has been isolated from a wide range of unrelated mammalian hosts, including humans, domestic and wild animals. It has been demonstrated that the genome of Pneumocystis of one host differs markedly from that of other hosts. Also, variation in the chromosome and DNA sequence of Pneumocystis within a single host species has been observed. Since information about the occurrence and nature of infections in wild animals is still limited, the objective of this work was to detect the presence of Pneumocystis sp. in lungs of bats from two states from Brazil by Nested-PCR amplification. The bats, captured in caves and in urban areas, were obtained from the Program of Rabies Control of two States in Brazil, Mato Grosso and Rio Grande do Sul, located in the Mid-Western and Southern regions of the country, respectively. DNAs were extracted from 102 lung tissues and screened for Pneumocystis by nested PCR at the mtLSU rRNA gene and small subunit of mitochondrial ribosomal RNA (mtSSU rRNA). Gene amplification was performed using the mtLSU rRNA, the primer set pAZ102H - pAZ102E and pAZ102X - pAZY, and the mtSSU rRNA primer set pAZ102 10FRI - pAZ102 10R-RI and pAZ102 13RI - pAZ102 14RI. The most frequent bats were Tadarida brasiliensis (25), Desmodus rotundus (20), and Nyctinomops laticaudatus (19). Pneumocystis was more prevalent in the species Nyctinomops laticaudatus (26.3% = 5/19), Tadarida brasiliensis (24% = 6/25), and Desmodus rotundus (20% = 4/20). Besides these species, Pneumocystis also was detected in lungs from Molossus molossus (1/11, 9.1%), Artibeus fimbriatus (1/1, 100%), Sturnira lilium (1/3, 33.3%), Myotis levis (2/3, 66.7%) and Diphylla ecaudata (1/2, 50%). PCR products which could indicate the presence of Pneumocystis (21.56%) were identified in DNA samples obtained from 8 out of 16 classified species from both states (5 bats were not identified). This is the first report of detection of Pneumocystis in bats from Brazil.

• Pneumocystis sp. bats Nested-PCR

Abstract in Portuguese:

ABSTRACT.- Cavallini Sanches E.M., Pacheco S.M., Cericatto A.S., Melo R.M., Colodel E.M., Hummel J., Bianchi S.P., Spanamberg A., Santurio J.M. & Ferreiro L. 2009. Detection of Pneumocystis in lungs of bats from Brazil by PCR amplification. Pesquisa Veterinária Brasileira 29(6):469-473. Setor de Micologia Veterinária, Faculdade de Veterinária, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves 9090, Porto Alegre, RS 90540-000, Brazil. E-mail: cavallini.sanches@ufrgs.br Pneumocystis has been isolated from a wide range of unrelated mammalian hosts, including humans, domestic and wild animals. It has been demonstrated that the genome of Pneumocystis of one host differs markedly from that of other hosts. Also, variation in the chromosome and DNA sequence of Pneumocystis within a single host species has been observed. Since information about the occurrence and nature of infections in wild animals is still limited, the objective of this work was to detect the presence of Pneumocystis sp. in lungs of bats from two states from Brazil by Nested-PCR amplification. The bats, captured in caves and in urban areas, were obtained from the Program of Rabies Control of two States in Brazil, Mato Grosso and Rio Grande do Sul, located in the Mid-Western and Southern regions of the country, respectively. DNAs were extracted from 102 lung tissues and screened for Pneumocystis by nested PCR at the mtLSU rRNA gene and small subunit of mitochondrial ribosomal RNA (mtSSU rRNA). Gene amplification was performed using the mtLSU rRNA, the primer set pAZ102H - pAZ102E and pAZ102X - pAZY, and the mtSSU rRNA primer set pAZ102 10FRI - pAZ102 10R-RI and pAZ102 13RI - pAZ102 14RI. The most frequent bats were Tadarida brasiliensis (25), Desmodus rotundus (20), and Nyctinomops laticaudatus (19). Pneumocystis was more prevalent in the species Nyctinomops laticaudatus (26.3% = 5/19), Tadarida brasiliensis (24% = 6/25), and Desmodus rotundus (20% = 4/20). Besides these species, Pneumocystis also was detected in lungs from Molossus molossus (1/11, 9.1%), Artibeus fimbriatus (1/1, 100%), Sturnira lilium (1/3, 33.3%), Myotis levis (2/3, 66.7%) and Diphylla ecaudata (1/2, 50%). PCR products which could indicate the presence of Pneumocystis (21.56%) were identified in DNA samples obtained from 8 out of 16 classified species from both states (5 bats were not identified). This is the first report of detection of Pneumocystis in bats from Brazil.

Abstract in English:

ABSTRACT.- Scarpelli L., Lopes W.D.Z., Migani M., Bresciani K.D.S. & Costa A.J. 2009. Toxoplasma gondii in experimentally infected Bos taurus and Bos indicus semen and tissues. Pesquisa Veterinária Brasileira 29(1):59-64. Departamento de Medicina Veterinária Preventiva, Faculdade de Ciências Agrárias e Veterinárias, Universidade Estadual Paulista, Via de Acesso Prof. Paulo Donatto Castellani s/n, Jaboticabal, SP 14884-900, Brazil. E-mail: wdzlopes@hotmail.com Eighteen young steers were inoculated with Toxoplasma gondii and randomly distributed into three groups of six animals each: GI, 2.5x105 “P” strain oocysts, GII, 5.0x106 “RH” strain tachyzoites, and GIII (Control). Clinical, serological and parasitemia exams were realized. Parasite investigation by bioassay and PCR was realized on semen and fragments of skeletal musculature, lymph nodes, brain, retina, spleen, liver, lung, testicle, epididymis and seminal vesicle. Blood and semen samples were collected on days -2, -1, 1, 3, 5, 7, 14 and weekly thereafter, up to postinfection day (PID) 84. The inoculated steers (GI and GII) presented hyperthermia from PID 3 to 16. Antibodies against T. gondii were detected through the indirect fluorescence antibody test (IFAT) on PID 5 (1:16) in both inoculated groups (oocysts and tachyzoites), reaching peaks of 1:4096 on PID 7. Parasitemia outbursts occurred in all infected bovines, principally from PID 7 to 28, independent of the strain and inoculate used. Bioassays revealed the presence of parasites in semen samples of animals infected with oocysts (GI) and tachyzoites (GII) on several experimental days between PID 7 and 84. Tissue parasitism by T. gondii was diagnosed by bioassay and the PCR technique in several organ and tissue fragments. These findings suggest the possibility of sexual transmission of T. gondii in the bovine species.

• Cattle PCR semen Toxoplasma gondii Apicomplexa

Abstract in Portuguese:

ABSTRACT.- Scarpelli L., Lopes W.D.Z., Migani M., Bresciani K.D.S. & Costa A.J. 2009. Toxoplasma gondii in experimentally infected Bos taurus and Bos indicus semen and tissues. Pesquisa Veterinária Brasileira 29(1):59-64. Departamento de Medicina Veterinária Preventiva, Faculdade de Ciências Agrárias e Veterinárias, Universidade Estadual Paulista, Via de Acesso Prof. Paulo Donatto Castellani s/n, Jaboticabal, SP 14884-900, Brazil. E-mail: wdzlopes@hotmail.com Eighteen young steers were inoculated with Toxoplasma gondii and randomly distributed into three groups of six animals each: GI, 2.5x105 “P” strain oocysts, GII, 5.0x106 “RH” strain tachyzoites, and GIII (Control). Clinical, serological and parasitemia exams were realized. Parasite investigation by bioassay and PCR was realized on semen and fragments of skeletal musculature, lymph nodes, brain, retina, spleen, liver, lung, testicle, epididymis and seminal vesicle. Blood and semen samples were collected on days -2, -1, 1, 3, 5, 7, 14 and weekly thereafter, up to postinfection day (PID) 84. The inoculated steers (GI and GII) presented hyperthermia from PID 3 to 16. Antibodies against T. gondii were detected through the indirect fluorescence antibody test (IFAT) on PID 5 (1:16) in both inoculated groups (oocysts and tachyzoites), reaching peaks of 1:4096 on PID 7. Parasitemia outbursts occurred in all infected bovines, principally from PID 7 to 28, independent of the strain and inoculate used. Bioassays revealed the presence of parasites in semen samples of animals infected with oocysts (GI) and tachyzoites (GII) on several experimental days between PID 7 and 84. Tissue parasitism by T. gondii was diagnosed by bioassay and the PCR technique in several organ and tissue fragments. These findings suggest the possibility of sexual transmission of T. gondii in the bovine species.