Retrospective serological survey for influenza in horses from Brazil.

Equine influenza (EI) virus is one of the most economically important pathogens of respiratory diseases of horses worldwide. Despite availability of vaccines for control of EI, the highly contagious nature and variability properties of the virus mean global outbreaks occur. Thus, continuous surveillance programs, including seroprevalence studies of disease in different countries, may contribute to better control of the disease. In this study, the seroprevalence of equine influenza in 850 horses from Brazil was investigated. The serodiagnosis was based on the single radial hemolysis (SRH) assay using influenza A/equine/Richmond/1/2007 (H3N8) antigen. Antibodies against A/equine/Richmond/1/07 (H3N8) were detected in 44.7% (380/850, 95% CI: 41.4-48.1%) of horses. Seroprevalence was significantly lower (p = 0.001) in younger animals (< 5 years, 38.6%) than in "adult" animals (5-14 years, 52.1%). There was also a significant relationship between the year of sampling and seroprevalence (p < 0.0005). The mean SRH antibody value was 42.0 mm2 (range 4-238.9 mm2), with the majority of horses (95.3%) having an SRH value ≤ 150 mm2, which is considered an insufficient level for protection of equine hosts against influenza infections and potential virus shedding. These findings indicate the need to reinforce preventive/control measures against equine influenza in Brazil.

Abortion and fetal death in bitches due anemia caused by vector-borne diseases / Abortamento e morte fetal em cadelas devido anemia causada por doenças transmitidas por vetores

Doenças infecciosas são as maiores responsáveis por falhas reprodutivas (FR) em cadelas, causando aborto, morte fetal e natimortalidade. Este estudo teve como objetivo investigar a associação entre agentes infecciosos, FR inexplicáveis e anemia em cadelas. Todas as amostras maternas e fetais foram negativas para a presença dos principais agentes infecciosos causadores de FR: herpes vírus canino 1, Neospora caninum, Brucella spp. e B. canis, enquanto agentes como o de Leishmania spp., parvovírus canino, Ehrlichia canis e Anaplasma platys foram encontrados em sangue materno. Coinfecções de A. platys/E. canis e A. platys/Leishmania spp. foram diagnosticadas. Os resultados indicam que os animais com anemia causadas por doenças transmitidas por vetores podem ser mais suscetíveis a sofrerem FR do que animais com valores hematológicos normais.(AU)

Performance of microbiological, serological, molecular, and modified seminal plasma methods in the diagnosis of Brucella abortus in semen and serum of bovine bulls.

Brucellosis remains as a major infectious disease of domestic animals and is considered a re-emerging zoonosis in several countries. B. abortus infections in bulls are related to reproductive tract infections, although infected animals show transient serological titers or nonreactor status. Thus, diagnosis of bovine brucellosis based exclusively on serological tests probably underestimates B. abortus infections in bulls. In this scenario, three hundred thirty-five serum samples from reproductively mature bovine bulls were subjected simultaneously to standard serodiagnosis using the rose Bengal test (RBT), 2-mercaptoethanol (2-ME), complement fixation (CFT), and fluorescence polarization assay (FPA). Furthermore, conventional semen plasma agglutination (SPA) and modified 2-ME, FC and, FPA were carried out in all bulls replaing serum by seminal plasma. Semen from all bulls was also analyzed for sperm viability, microbiological culture in Farrell media, and polymerase chain reaction (PCR). Only eight (2.38%) semen samples were considered improper for reproduction services (necrospermia and azoospermia), although none of these animals was positive in any of the diagnosis methods used. Five bulls (1.49%) were simultaneously positive in conventional RBT, 2-ME, SPA, modified 2-ME, microbiological culture in Farrell media, and in PCR for B. abortus strain 19. Two (1.67%) bulls were positive in PCR for B. abortus field strains and negative in all other tests, although semen was considered viable to reproduction service. The identification of B. abortus B19 strain in serum and semen of bulls occurred probably due to improper vaccination of males or infection by B19 strain shedding by vaccinated females that could to contaminated environment of farms. In addition, detection of B. abortus field strains only using PCR in bulls without sperm viability abnormalities indicate the need for including molecular methods to improve diagnosis of the disease in bovine bulls.

Pre-Multidrug-Resistant Mycobacterium tuberculosis Infection Causing Fatal Enteric Disease in a Dog from a Family with History of Human Tuberculosis.

This report describes a fatal case of a pet dog with major enteric signs owned by a family that has experienced cases of pulmonary tuberculosis (TB) in the household. Clinical and epidemiological aspects, imaging data, microbiological, haematological and histopathological examinations were assessed to diagnosis of disease. gyrB-RFLP, spoligotyping and MIRU-VNTR allowed molecular detection of M. tuberculosis strain from S family. The resazurin microtiter assay indicated that all isolates were resistant to isoniazid, ethambutol, ciprofloxacin, ofloxacin, streptomycin and amikacin. The public health concerns related to canine tuberculosis and risk of the dissemination by pets of M. tuberculosis pre-multidrug-resistant (PMD) to isoniazid, ethambutol and other first-line drugs used in human therapy of TB are discussed. We believe this to be the first report of PMD M. tuberculosis infection in a dog presenting mainly enteric manifestation, confirmed as S lineage by molecular methods, owned by a family in which TB has spread in the household for generations.

Soropositividade para Mycoplasma hyopneumoniae em suínos abatidos em frigoríficos da região central do estado de São Paulo / Seropositivity for Mycoplasma hyopneumoniae in pigs at a slaughterhouse in the Central region of São Paulo

Mycoplasma hyopneumoniae is the etiologic agent of enzootic pneumonia in pigs and causes large economic losses in the swine industry. There is little data on the positivity of this disease in Brazil. The objective of this study was to evaluate the seropositivity for this agent in 200 serum samples collected from pigs in a slaughterhouse located in the central region of São Paulo. A high percentage (52%) of positivity was found indicating the presence of the agent and the need to implement control measures.

Serology for Brucella abortus in cart horses from an urban area in Brazil / Sorologia para Brucella abortus em cavalos de carroça de área urbana do Brasil

Avaliou-se a infecção por Brucella abortus em cavalos de carroça de Curitiba e São José dos Pinhais-PR. Um total de 123 amostras foi submetido ao teste do antígeno tamponado acidificado (ATA), soroaglutinação lenta em tubos (SAL) e prova do 2-mercaptoetanol (2-ME) para confirmação dos resultados. Oito (6,5%) equinos foram positivos para o ATA e um animal permaneceu positivo ao teste confirmatório. Existem evidências da presença de brucelose entre os cavalos de carroça.

Species-specific nested PCR as a diagnostic tool for Brucella ovis infection in rams / Nested PCR espécie-específica para o diagnóstico da infecção por Brucella ovis em carneiros

The aim of the present study was to evaluate a species-specific nested PCR based on a previously described species-specific PCR for detection of B. ovis in semen and urine samples of experimentally infected rams. The performance of the species-specific nested PCR was compared with the results of a genus-specific PCR. Fourteen rams were experimentally infected with the Brucella ovis REO 198 strain and samples of semen and urine were collected every week up to 180 days post infection. Out of 83 semen samples collected, 42 (50.6%) were positive for the species-specific nested PCR, and 23 (27.7%) were positive for the genus-specific PCR. Out of 75 urine samples, 49 (65.3%) were positive for the species-specific nested PCR, whereas 11 (14.6%) were genus-specific PCR positive. Species-specific nested PCR was significantly more sensitive (P<0.001) than the genus-specific PCR in semen and urine from experimentally infected rams. In conclusion, the species-specific nested PCR developed in this study may be used as a diagnostic tool for the detection of B. ovis in semen and urine samples from suspected rams.

O presente estudo objetivou avaliar uma técnica de nested PCR espécie-específica delineada a partir de PCR espécie-específica descrita anteriormente para detecção de B. ovis em sêmen e urina de carneiros infectados experimentalmente. O desempenho da nested PCR espécie-específica foi comparado com os resultados de uma PCR gênero-específica. Quatorze carneiros foram infectados experimentalmente com Brucella ovis REO 198 e amostras de sêmen e de urina foram colhidas semanalmente até 180 dias após a infecção. De 83 amostras de sêmen, 42 (50,6%) foram positivas pela nested PCR espécie-específica, e 23 (27,7%) foram positivas pela PCR gênero-específica. De 75 amostras de urina, 49 (65,3%) foram positivas pela nested PCR espécie-específica, enquanto 11 (14,6%) foram positivas em PCR gênero-específica. A técnica de nested PCR espécie-específica foi significativamente mais sensível (P<0,001) do que a PCR gênero-específica no sêmen e na urina de carneiros infectados experimentalmente. Em conclusão, a nested PCR espécie-específica desenvolvida neste estudo pode ser utilizada como ferramenta de diagnóstico para detecção de B. ovis em sêmen e urina de carneiros suspeitos.

Canine distemper virus and Toxoplasma gondii co-infection in dogs with neurological signs / Co-infecção pelo vírus da cinomose canina e Toxoplasma gondii em cães com sinais neurológicos

O presente estudo relata a ocorrência de co-infecção entre o vírus da cinomose canina (CDV) e Toxoplama gondii em cães com sinais neurológicos. Amostras de soro e tecido nervoso (pos-mortem) de 21 cães, suspeitos de cinomose canina foram analisadas pela Reação de Imunofluorecência indireta (RIFI) para pesquisa de anticorpos contra T. gondii e N. caninum e por RT-PCR para CDV. Dezessete (80,9 por cento) cães foram positivos para o CDV pela RT-PCR e 8 (38,1 por cento) foram positivos para anticorpos contra T. gondii. Sete cães (41,1 por cento) apresentaram-se positivos para ambos agentes, caracterizando processo de co-infecção. Somente 1 (4,7 por cento) cão foi soropositivo para N. caninum (RIFI=100), entretanto este mesmo animal foi positivo para T. gondii (RIFI=4096) e para CDV (RT-PCR).

Serological profile of buffalo (Bubalus bubalis) female calves vaccinated with standard Brucella abortus strain 19 vaccine using rose bengal, 2-mercaptoethanol and complement fixation tests.

The serological profiles of 21 female buffaloes vaccinated between 3 and 8 months of age using Brucella abortus strain 19 (S19) were evaluated by rose bengal (RBT), 2-mercaptoethanol (2ME) and complement fixation (CFT) tests. The serum strains were collected in day zero, 15, 30, 45, 60th days and subsequently to each 30 months, until 720th day after vaccination. No animal showed reaction in day zero. In 15th day above 95% of animals revealed reaction in all tests. All the animals presented absence of reactions in CFT, RBT and 2ME tests at 270, 300 and 360 days after vaccination, respectively. Our finding highlighted early response in CFT compared than other conventional agglutination tests. None of animals presented oscillation of titers or reactions in any test after 360 day of study, which enables the use of these tests after this period without interference of antibodies from S19 vaccine origin between 3 and 8 months in buffalo heifers.

Vaccine immune response and interference of colostral antibodies in calves vaccinated against rabies at 2, 4 and 6 months of age born from antirabies revaccinated females.

Considering the high prevalence of rabies in cattle, we aimed to evaluate the interference of colostral antibodies transferred to calves after birth and the benefit of administering an antirabies vaccination in two-month-old calves compared to vaccinating at 4 and 6 months of age. Calves born from females revaccinated against rabies during the third trimester of pregnancy were studied. Forty-eight hours after parturition, blood samples from dams and offspring were collected, and antirabies neutralizing antibody titers were analyzed using the Rapid Focus Fluorescent Inhibition Test. We found that all calves had similar titers of antibodies transferred through the colostrum. Furthermore, none of the calves presented a satisfactory serological response after the first vaccination, but all had an appropriate response after revaccination. This study demonstrates that antirabies vaccination should be recommended for calves at two months of age in endemic and epizootic situations.

Adaptation and evaluation of polymerase chain reaction for Brucella ovis detection in semen, urine and organs of rams experimentally infected / Adaptação e avaliação da reação em cadeia da polimerase para detecção de Brucella ovis em sêmen, urina e órgãos de carneiros infectados experimentalmente

O objetivo do estudo foi adaptar e avaliar a PCR para detecção de Brucella ovis e comparar os resultados com aqueles obtidos por cultivo microbiológico do sêmen, urina e dos órgãos de carneiros infectados experimentalmente. Dos 31 animais infectados experimentalmente, amostras de PCR do sêmen apresentaram maior sensibilidade (21,6 por cento) do que o cultivo (8,0 por cento). Em amostras de urina, a sensibilidade das técnicas foi semelhante (10,1 por cento para a cultivo e 12,7 por cento para PCR). PCR detectou a presença do agente em 21,5 por cento das amostras testadas, enquanto os órgãos de cultivo detectaram em apenas 3,3 por cento das amostras. PCR detectou um maior número de amostras positivas do que o cultivo microbiológico.

Rabies virus in a pregnant naturally infected southern yellow bat (Lasiurus ega)

Current knowledge on bat lyssavirus infections in their native hosts is limited and little is known about the virulence, virus dissemination and transmission among free-living insectivorous bats. The present study is a brief description of rabies virus (RABV) dissemination in tissues of a naturally infected pregnant southern yellow bat (Lasiurus ega) and its fetuses, obtained by reverse-transcriptase polymerase chain reaction (RT-PCR). The RT-PCR was positive in samples from the brain, salivary gland, tongue, lungs, heart, kidneys and liver. On the other hand, the placenta, three fetuses, spleen, intestine and brown fat tissue tested negative. This research demonstrated the absence of rabies virus in the fetuses, thus, in this specific case, the transplacentary transmission was not observed.

Comparison of a PCR assay in whole blood and serum specimens for canine brucellosis diagnosis.

The performance of a serum PCR assay was compared with that of a blood PCR assay for the diagnosis of canine brucellosis caused by Brucella canis in 72 dogs. The dogs were classified into three groups (infected, non-infected and suspected brucellosis) according to the results of blood culture and serological tests. The sensitivities of blood PCR and serum PCR were, respectively, 97.14 per cent and 25.71 per cent. The specificities of both were 100 per cent. In the group of dogs with suspected brucellosis, three were positive by blood PCR and none was positive by serum PCR. Serum PCR showed little value for the direct diagnosis of canine brucellosis as the assay had low diagnostic sensitivity and fewer positive dogs were detected by this test than by blood culture, blood PCR, rapid slide agglutination test (RSAT) and RSAT with 2-mercaptoethanol.

Importance of antirabies revaccination for an adequate antirabies protection in bovine newborns.

The transfer of antirabies immunoglobulins in cows that were prime vaccinated and cows that were revaccinated against rabies correlated to the serum titers in their offspring was evaluated. The results demonstrated that revaccination against rabies during pregnancy induces neutralizing antibody titers at a protective level that are transferred directly to calves through colostrum and reinforce the importance of revaccination for improved colostral antibody transfer and offspring protection against rabies.

Comparison of infection by brucella spp: in free-ranging and captive wild animals from São Paulo State, Brazil

The aim of the current study was to evaluate the infection rate by Brucella spp. in wild and in captive animals. Serum samples from 121 animals (94 free-ranging and 27 captive) of different mammal species were evaluated. Sera were submitted to rose Bengal test (RBT) for screening and serum agglutination tests (SAT) and 2-mercaptoethanol test (2-ME) for confirmatory results. Nine animals (five free-ranging and four captive) tested positive in RBT, but negative in the confirmatory tests. Several domestic animal diseases that have control programs are not focused on wild reservoirs, such as brucellosis in Brazil. The study of new reservoirs in wildlife is essential to prevent emerging diseases.

Infecção em cão por Brucella abortus: relato de caso / Brucella abortus infection in dog: case report

Brucella abortus infection is reported in a dog from a rural area that presented at clinical evaluation left testicular enlargement and right testicular decrease. Serum resulted negative to rapid agglutination test and agar gel immunodifusion with Brucella ovis antigen but positive to buffered plate agglutination test, tube agglutination test and 2- Mercapthoetanol with B. abortus antigen. Brucella isolation was negative in blood, testicular material, semen and urine. Brucella DNA was detected in PCR from urine and blood

A polymerase chain reaction for detection of Brucella canis in vaginal swabs of naturally infected bitches.

A PCR assay for the detection of Brucella canis in canine vaginal swab samples was evaluated, comparing its performance with that of bacterial isolation, serological tests, and a blood PCR assay. One hundred and forty-four female dogs were clinically examined to detect reproductive problems and they were tested by the rapid slide agglutination test, with and without 2-mercaptoethanol (2ME-RSAT and RSAT, respectively). In addition, microbiological culture and PCR were performed on blood and vaginal swab samples. The results of the vaginal swab PCR were compared to those of the other tests using the Kappa coefficient and McNemar test. Of the 144 females that were examined, 66 (45.8%) were RSAT positive, 23 (15.9%) were 2ME-RSAT positive, 49 (34.02%) were blood culture positive, 6 (4.1%) were vaginal swab culture positive, 54 (37.5%) were blood PCR positive, 52 (36.2%) were vaginal swab PCR positive, and 50.69% (73/144) were positive by the combined PCR. The PCR was able to detect as few as 3.8 fg of B. canis DNA experimentally diluted in 54 ng of canine DNA, extracted from vaginal swab samples of non-infected bitches. In addition, the PCR assay amplified B. canis genetic sequences from vaginal swab samples containing 1.0 x 10(0) cfu/mL. In conclusion, vaginal swab PCR was a good candidate as a confirmatory test for brucellosis diagnosis in bitches suspected to be infected, especially those negative on blood culture or blood PCR; these animals may be important reservoirs of infection and could complicate attempts to eradicate the disease in confined populations.

Avaliação do teste do anel em leite na vigilância epidemiológica da brucelose bovina em rebanhos e em laticínios / Evaluation of the ring test in an epidemiological surveillance of bovine brucellosis in herds and dairies

Analisaram-se 464 amostras individuais de leite e 54 amostras de leite de latões, oriundos de leite dos mesmos animais, por meio do teste do anel do leite (TAL) visando à sua aplicação no diagnóstico individual e de rebanhos da brucelose bovina. Foram também avaliadas 464 amostras de soro sangüíneo por meio de provas do antígeno tamponado acidificado (ATA), soroaglutinação lenta em tubos (SAL) e 2-mercaptoetanol (2-ME), todas para brucelose. Cento e vinte e três amostras (26,5 por cento) testadas pelo TAL apresentaram resultados positivos. Dessas, 30 resultaram positivas ao ATA, 28 ao ATA, à SAL e ao 2-ME e 18 à SAL. Das amostras positivas ao TAL, 95 pertenciam a animais sorologicamente negativos ao 2-ME, caracterizando 77,2 por cento (95/123) das reações falso-positivas; dos resultados negativos ao TAL, 4 pertenciam a animais sorologicamente positivos, caracterizando 1,2 por cento (4/341) de reações falso-negativas no TAL individual. Das 54 amostras de leite de latões analisadas pelo TAL, 17 foram consideradas positivas, das quais uma foi caracterizada como falso-positivo, pois todos os animais que a compunham foram negativos ao 2-ME. De 37 latões considerados negativos ao TAL, três continham leite de animais positivos ao 2-ME, caracterizando 8,1 por cento de falso-negativos. O TAL individual demonstrou elevado percentual de resultados falso-positivos, enquanto o TAL em amostras de leite obtidas em latões detectou 84,2 por cento de latões contaminados e 75 por cento de rebanhos infectados.

The ring test (RT) was analyzed regarding its application for the individual and herd bovine brucellosis diagnoses. Individual samples of milk from 464 cows and 54 composite samples of milk bucket from these animals were evaluated. The results were analyzed considering the serological results obtained by the rose bengal (RBT), tube agglutination (TAT) and 2-mercaptoethanol (2-ME) tests. From the 464 individual milk samples analyzed by the RT, 123 (26.5 percent) presented positive results. From those, 30 were positive only to the RBT, 28 by the RBT/TAT/2-ME and 18 by the TAT. From the 123 positive samples by the RT, 95 came from the 2-ME seronegative animals, characterizing 77.2 percent of false-positive reactions and 4/341 negative reactions came from the seropositive animals, characterizing 1.2 percent of false-negative reactions in the individual RT. From the 54 samples of compositive milk analyzed by the RT, 17 were positive. From these positive samples, one was considered false positive since all the animals that composed it were negative by the 2-ME. Three of the composite milk samples that were negative to RT(3/37) were constituted by milk from positive animals, characterizing 8.1 percent of false-negative results by the RT. The individual RT showed high percentage of false-positive results, while the RT in samples from bucket detected 84.2 percent of the contaminated milk and 75 percent of the infected herds.

A polymerase chain reaction for the detection of Brucella canis in semen of naturally infected dogs.

The objective was to evaluate a PCR assay for the detection of Brucella canis in canine semen, comparing its performance with that of bacterial isolation, serological tests and PCR assay of blood. Fifty-two male dogs were examined clinically to detect reproductive abnormalities and their serum was tested by the rapid slide agglutination test, with and without 2-mercaptoethanol (2ME-RSAT and RSAT, respectively). In addition, microbiological culture and PCR assays were performed on blood and semen samples. The findings of the semen PCR were compared (Kappa coefficient and McNemar test) to those of blood PCR, culture of blood and semen, RSAT, and 2ME-RSAT. Nucleic acid extracts from semen collected from dogs not infected with B. canis were spiked with decreasing amounts of B. canis RM6/66 DNA and the resulting samples subjected to PCR. In addition, semen samples of non-infected dogs were spiked with decreasing amounts of B. canis CFU and the resulting suspensions were used for DNA extraction and amplification. Of the 52 dogs that were examined, the following tests were positive: RSAT, 16 (30.7%); 2ME-RSAT, 5 (9.6%); blood culture, 14 (26.9%); semen culture, 11 (21.1%); blood PCR, 18 (34.6%); semen PCR, 18 (34.6%). The PCR assay detected as few as 3.8 fg of B. canis DNA experimentally diluted in 444.9 ng of canine DNA (extracted from semen samples of non-infected dogs). In addition, the PCR assay amplified B. canis genetic sequences from semen samples containing as little as 1.0 x 10(0) cfu/mL. We concluded that PCR assay of semen was a good candidate as a confirmatory test for the diagnosis of brucellosis in dogs; its diagnostic performance was similar to blood culture or blood PCR. Furthermore, the PCR assay of semen was more sensitive than the 2ME-RSAT or semen culture. Examination of semen by PCR should be included for diagnosis of brucellosis prior to natural mating or AI; in that regard, some dogs that were negative on serological and microbiological examinations as well as blood PCR were positive on PCR of semen.

Diagnosis of canine brucellosis: comparison between serological and microbiological tests and a PCR based on primers to 16S-23S rDNA interspacer.

A pair of primers directed to 16S-23S rDNA interspacer (ITS) was designed directed to Brucella genetic sequences in order to develop a polymerase chain reaction (PCR) putatively capable of amplifying DNA from any Brucella species. Nucleic acid extracts from whole-blood from naive dogs were spiked with decreasing amounts of Brucella canis RM6/66 DNA and the resulting solutions were tested by PCR. In addition, the ability of PCR to amplify Brucella spp. genetic sequences from naturally infected dogs was evaluated using 210 whole-blood samples of dogs from 19 kennels. The whole-blood samples collected were subjected to blood culture and PCR. Serodiagnosis was performed using the rapid slide agglutination test with and without 2-mercaptoethanol. The DNA from whole blood was extracted using proteinase-K, sodium dodecyl sulphate and cetyl trimethyl ammonium bromide followed by phenol-chloroform purification. The PCR was capable of detecting as little as 3.8 fg of Brucella DNA mixed with 450 ng of host DNA. Theoretically, 3.8 fg of Brucella DNA represents the total genomic mass of fewer than two bacterial cells. The PCR diagnostic sensitivity and specificity were 100%. From the results observed in the present study, we conclude that PCR could be used as confirmatory test for diagnosis of B. canis infection.