Detection of icaA, icaD, and bap genes and biofilm production in Staphylococcus aureus and non-aureus staphylococci isolated from subclinical and clinical bovine mastitis / Detecção dos genes icaA, icaD e bap e produção de biofilme por Staphylococcus aureus e estafilococos não-aureus isolados de mastite clínica e subclínica bovina

Algumas espécies de Staphylococcus causam infecções crônicas intramamárias e podem levar à formação de biofilme. No presente estudo, levantou-se a hipótese de que as espécies de Staphylococcus isolados da mastite bovina são capazes de formar biofilme in vitro associado à presença dos genes icaA, icaD ou bap. Um total de 200 isolados de Staphylococcus, sendo 100 Staphylococcus aureus de casos de mastite subclínica e 100 estafilococos não aureus (ENA) de casos de mastite subclínica e clínica, obtidos em duas fazendas leiteiras, no estado de São Paulo, foram avaliados quanto à capacidade de produzir biofilmes in vitro. A presença de icaA, icaD e bap foi confirmada por PCR, e a produção de biofilme em ágar vermelho congo (Congo Red Agar - CRA) e em teste de microplaca (Microtiter Plate - MtP) foi avaliada nos isolados de S. aureus e ENA. Os resultados mostraram a presença dos genes icaA, icaD e bap em S. aureus, mas não em ENA. A produção de biofilme pode estar associada à presença de outros fatores ou genes que estimulam a produção de biofilme in vitro. O ensaio de MtP serve como um modelo quantitativo para o estudo da aderência de espécies de estafilococos associados à mastite bovina.(AU)

Ocorrência, patógenos e fatores de risco para mastite subclínica em cabras leiteiras / Occurrence, pathogens and risk factors for subclinical mastitis in dairy goats

Milk samples from 257 goats and 513 half udders of 10 dairy goat farms were analyzed by microbiological culture, to evaluate the sanitary management, and epidemiological questionnaire results. Multivariate analysis of risk factors for subclinical mastitis in goats, with the adjusted Odds Ratio for subclinical mastitis was performed. The prevalence of subclinical mastitis in goats by microbiological culture was 43.6%, and clinical mastitis was 5.84%. From 178 positive teats by microbiological culture, the most prevalent pathogens were coagulase-negative staphylococci - CNS (70%), S. aureus (13.5%), S. intermedius (7.9%), and Enterobacteriaceae (4%). The risk factors analysis revealed significant association between the disease and the flock size (OR= 3.33; P= 0.0268), and the farm was a confounding factor, being kept in the final statistical model. Despite the non-identification of all the factors associated with subclinical mastitis in herds and the existence of confounding factors that hinder statistical analyzes, the study of risk factors is important for the improvement of disease control and prevention programs in dairy goat, and it can be used as a tool in the reduction of environmental and contagious pathogens such as staphylococci that were the most identified pathogens causing caprine mastitis in the study.(AU)

Identification of subclinical mastitis caused by Mycoplasma spp. from screenings of bulk tanks / Identificação de mastite subclínica causada por Mycoplasma spp. de triagem de tanques de expansão

Mastitis caused by Mycoplasma spp., regardless of species, are considered highly contagious pathogens and, usually was not responsive to antimicrobial therapy. Five dairy herds, comprising 489 animals and 1,956 mammary glands, were used in this study. Milk samples were obtained from bulk tanks and subjected to polymerase chain reaction (PCR) for the identification of Mollicutes, Mycoplasma spp., and Mycoplasma bovis. Moreover, individual samples from cases of clinical and subclinical mastitis in quarters of the dairy herds' animals that yielded a positive PCR upon bulk tank analysis were subjected to molecular analysis. Only one bulk tank was positive for class Mollicutes by PCR. All positive samples classified as mastitis teats had their DNA extracted and tested by PCR for both class Mollicutes and M. bovis. Of these, two (2.08%) were positive for Mycoplasma genus, although none was positive for M. bovis. This result suggests that the PCR of bulk tanks is a viable tool in monitoring and preventing mastitis infections caused by Mycoplasma spp.(AU)

Mastites bovinas causadas por Mycoplasma spp., independentemente da espécie causadora, são consideradas de alta contagiosidade e geralmente não responsivas à terapia antimicrobiana. Cinco propriedades leiteiras foram utilizadas neste estudo, totalizando 489 animais e 1956 quartos mamários. As amostras de leite foram obtidas de tanques de expansão e submetidas à reação em cadeia da polimerase (PCR) para pesquisa de Mollicutes, Mycoplasma spp. e Mycoplasma bovis. Apenas um tanque de uma propriedade foi positivo na PCR para a classe Mollicutes. Amostras individuais de casos de mastite subclínica provenientes de propriedade com tanque positivo também foram submetidas à análise molecular; dessas, duas delas (2,08%) foram positivas para a classe Mollicutes e para o gênero Mycoplasma, entretanto nenhuma foi positiva para a espécie Mycoplasma bovis. Isso sugere que a PCR de tanques de expansão de propriedades leiteiras demonstra ser uma ferramenta viável no monitoramento e na prevenção de infecções por Mycoplasma spp.(AU)

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)

Beta-lactamase detection in Staphylococcus aureus and coagulase-negative Staphylococcus isolated from bovine mastitis / Detecção de beta-lactamase em Staphylococcus aureus e Staphylococcus coagulase negativa isolados de mastite bovina

The objectives of the study were to evaluate the presence/production of beta-lactamases by both phenotypic and genotypic methods, verify whether results are dependent of bacteria type (Staphylococcus aureus versus coagulase-negative Staphylococcus - CNS) and verify the agreement between tests. A total of 200 bacteria samples from 21 different herds were enrolled, being 100 CNS and 100 S. aureus. Beta-lactamase presence/detection was performed by different tests (PCR, clover leaf test - CLT, Nitrocefin disk, and in vitro resistance to penicillin). Results of all tests were not dependent of bacteria type (CNS or S. aureus). Several S. aureus beta-lactamase producing isolates were from the same herd. Phenotypic tests excluding in vitro resistance to penicillin showed a strong association measured by the kappa coefficient for both bacteria species. Nitrocefin and CLT are more reliable tests for detecting beta-lactamase production in staphylococci.

Os objetivos do presente estudo foram avaliar a presença/produção de beta-lactamases por ambos os métodos fenotípicos e genotípicos, verificar se os resultados são dependentes do tipo de bactéria (Staphylococcus aureus contra Staphylococcus coagulase negativa - CNS) e verificar a concordância entre os testes. Um total de 200 amostras bactérianas oriundas de 21 rebanhos distintos foram incluídos, sendo 100 CNS e 100 S. aureus. A presença/detecção de beta-lactamase foi realizada por diferentes testes (PCR, teste trevo (clover leaf test) - CLT, disco Nitrocefin e resistência in vitro à penicilina). Os resultados de todos os testes não foram dependentes do tipo de bactérias (CNS ou S. aureus). Vários isolados de S. aureus produtores de beta-lactamase eram de um mesmo rebanho. Testes fenotípicos excluindo resistência in vitro à penicilina mostraram uma forte associação medida pelo coeficiente kappa para ambas as espécies de bactérias. Nitrocefina e CLT são testes mais confiáveis para detectar a produção de beta-lactamase em estafilococos.

Occurrence and risk factors associated with canine leptospirosis

Leptospirosis is a globally distributed emerging zoonosis. Dogs are commonly affected and although other serovars can cause canine leptospirosis, Leptospira interrogans serovar Canicola is primary found in these animals. A retrospective study was conducted using a database of 1195 dogs tested for Leptospira infection from 2003 to 2010 at the Laboratory of Zoonosis Diagnosis at the Veterinary Hospital of São Paulo State University (UNESP) in Botucatu, São Paulo state, Brazil. The seroprevalence of infected dogs was 20.08 percent (240/1195), and the most prevalent serovars were Canicola (6.7 percent), Copenhageni (5.0 percent), Icterohaemorrhagiae (2.9 percent), Autumnalis (2.9 percent), Pyrogenes (2.8 percent), Pomona (2.0 percent), Hardjo (2.0 percent), Australis (1.8 percent), Bratislava (1.6 percent), Cynopteri (1.4 percent), Grippotyphosa (1.3 percent) and Djasiman (1.0 percent). By univariate analysis, the variables age and breed were not statistically related to the infection, while gender and season were. The effects of gender were also noticeable related to serovars Australis, Canicola and Hardjo. In multivariate analysis, the level of significance (p-value) of season was suppressed by gender, indicating possible collinearity between those two variables.

Isolation of Staphylococcus epidermidis from inflamed upper respiratory tract of an orange-spined hairy dwarf porcupine (Sphiggurus villosus)

The orange-spined hairy dwarf porcupine (Sphiggurus villosus) is a rodent species common in most parts of South America, and little is known about the pathologies that can afflict it. A specimen was delivered at the Wildlife Research and Medical Center (CEMPAS), School of Veterinary Medicine and Animal Husbandry, UNESP, Botucatu, SP, Brazil. The animal showed intense apathy, with purulent secretion in the nasal cavity and fracture of the lumbar spine. Due to the unfavorable prognosis, the porcupine was euthanized and microbiological culture of nasal discharge showed Staphylococcus epidermidis. The antimicrobial resistance test revealed sensitivity to all tested antimicrobials (ampicillin, oxacillin, tetracycline, penicillin G, neomycin, cephalexin, gentamicin, enrofloxacin, ciprofloxacin, cotrimoxazol, cefoxitin and cephalothin). This bacterium is part of the nasal flora of humans and other animals, and may cause infection under certain conditions. In the present study, the infection and colonization by S. epidermidis was the probable cause of the inflammatory process. The sensitivity to all tested antimicrobials suggests that this strain has not been previously exposed to such drugs.

Estudo dos métodos de rotina diagnóstica de mastite no leite de éguas / Study of routine diagnosis methods of mastitis in mares

Routine diagnosis methods used in bovine mastitis were studied in 55 mares in lactation. The findings of strip cup test, California Mastitis Test-CMT, electronic somatic cell count-CCS, microbiological culture, and in vitro antimicrobial susceptibility profile of isolates were discussed. Streptococcus spp., Staphylococcus spp, and enterobacteria were the most common microorganisms isolated in health and CMT-positive mammary glands. Staphylococcus aureus and Arcanobacterium pyogenes were identified in two mares presenting clinical mastitis. Mean somatic cell count of eight mares without presence of microorganisms in milk was 247.57x10³/mL and 1.621,86x10³/mL in 47 mares with positive microbiological culture. Moderate concordance (63.8 percent) between positive reactions in CMT (1 to 3+) and microbiological culture was observed. Amicacin (78.9 percent), ceftiofur (74.7 percent), sulpha-trimetoprim (69,0 percent) and norfloxacin (69.0 percent), were the most effective drugs, while resistance of isolates was mainly observed against penicillin (64.8 percent), gentamycin (35.2 percent), azithromycin (35.2 percent), enrofloxacin (28.2 percent), and florfenicol (28.2 percent).

Interactions between environment, wild animals and human leptospirosis: [review]

Leptospirosis, a worldwide distributed zoononis caused by bacteria of the genus Leptospira (antigenically classified into serovars), may be direct or indirectly transmitted through infected urine or environment. Several domestic and wild animals are leptospirosis reservoirs. The disease presents occupational character since it is widely reported in professionals that work in humid environments - such as sewage workers and fishermen - and in places where rodents or susceptible animals are found, like slaughterhouses and veterinary clinics. In developing countries, outbreaks are related to lack of sanitation, overcrowding in inadequate housing and climatic conditions. In developed countries, sporadic cases occur in aquatic recreational activities including swimming and triathlon. The diagnosis of leptospirosis is complex due to the variety of symptoms, disease severity and the lack of techniques that are able to early detect the infection. Thus, leptospirosis causes numerous public health problems and educational activities are very important to its control.

Leptospiral antibodies in wild boars (Sus scrofa) bred in Brazil

In the recent years, the wild boar (Sus scrofa) trade has increased in Brazil. The present study aimed to detect Leptospira spp. infection in 308 blood samples from wild boars bred in São Paulo state, Brazil. The microscopic agglutination test (MAT) was performed using 29 serovars. Sixty three (63; 20.45 percent) animals tested positive against the serovars Hardjo (29/63; 46.0 percent), Copenhageni (11/63; 17.4 percent), Pomona (8/63; 12.7 percent), Pyrogenes (4/63; 6.3 percent), Wolfii (4/63; 6.3 percent), Autumnalis (3/63; 4.7 percent), Icterohaemorraghiae (2/63; 3.1 percent), and Hardjo-miniswajezak (1/63; 15.8 percent). These results indicate that captive wild boars were infected with Leptospira spp. and may represent a source of infection to humans and other animals.

Comparison of different diagnostic tests in dogs uninfected and naturally infected with visceral leishmaniasis

Uninfected dogs (n = 10) and those naturally infected with leishmaniasis (n = 10) were subjected to several diagnostic tests, namely: hemoculture, polymerase chain reaction (PCR) of hemoculture, indirect immunofluorescence (RIFI), cytological examination of lymph node aspirate, culture of lymph node aspirate and PCR of lymph node aspirate. RIFI - followed by PCR of lymph node aspirate culture - presented more positive results in infected dogs than in uninfected ones. In infected animals, RIFI was more effective than PCR of lymph node aspirate culture. There was no statistical difference in positivity between RIFI and hemoculture; lymph node aspirate culture/cytological examination of lymph node aspirate and PCR of hemoculture; and between PCR of lymph node aspirate culture and PCR of hemoculture. All infected and uninfected animals had positive and negative results in at least one test. In conclusion, the association of several tests improves the efficacy of canine visceral leishmaniasis diagnosis.

Anticorpos para Leptospira spp., Toxoplasma gondii e Neospora caninum em cães errantes albergados em canil privado / Antibodies to Leptospira spp., Toxoplasma gondii, and Neospora caninum in mongrel dogs kept in private kennel

The serological profile of 300 mongrel dogs of various ages and gender were investigated. Animals were captured in the streets and afterwards directed to a private kennel in Avaré city (SP) to search for leptospirosis, toxoplasmosis, and neosporosis. Blood samples were obtained from jugular or cephalic vein for the obtention of sera. The microscopic agglutination test (MAT) was used to leptospirosis. MAT detect the prevalence of 9.3%. The most frequent reactant serovars were Bratislava (35.7%), Cynopteri (17.9%), Autumnalis (14.3%), and Copenhageni (10.7%), besides 7.1% to others serovars: Icterohaemorrhagiae, Canicola, and Hardjo. The modified agglutination test used for the diagnosis of toxoplasmosis showed 26% of positive animals, with titers varying from 16 to 256, with 16 in 3.3%, 64 in 13.7%, and 256 in 9% of the samples. To canine neosporosis, it was used the indirect fluorescent antibody test, and two animals (0.7%) demonstrated antibodies with titers 25 and 100. The results show the participation of the animals in the epidemiological chain of the researched diseases.

Associação entre a presença de anticorpos anti-Leishmania sp. e anti-Neospora caninum em cães de Bauru, SP / Association between the presence of anti-Leishmania sp. and anti-Neospora caninum antibodies in dogs from Bauru, Brazil

The survey analyzed 100 samples of serum collected from dogs at the Center for Zoonoses Control of Bauru, randomly chosen. In the study, 65 percent of the samples were positive for leishmaniasis and 14 percent for neosporosis. The association between the presence of antibodies by the reaction of indirect immunofluorescent antibody test (IFAT) in the detection of anti-Leishmania and anti-Neospora antibodies was examined by the Fischer Exact Test (P=0.41), indicating no association between the results for Leishmania sp. and Neospora caninum (α=0.05). The absolute frequencies of the IFAT in the detection of antibodies anti-Leishmania and anti-Neospora caninum were analyzed using the Spearmann correlation coefficient for Leishmania and N. caninum titters, r=0.0975 and P=0.33, which did not indicate significant correlation between the titters for both pathogens.

Detection of Toxoplasma gondii in the milk of experimentally infected Wistar female rats

The milk of experimentally infected rats was investigated for the presence and possible transmission of Toxoplasma gondii. Wistar (Rattus norvegicus) female rats were divided into three groups and orally inoculated with bradyzoites. Polymerase chain reaction (PCR) was employed to detect the parasite in the milk. Transmission to the offspring was verified by indirect immunofluorescence antibody test (IFAT), modified agglutination test (MAT), bioassay tests and PCR. Rat milk samples were PCR-positive, pups were serum-reactive to T. gondii and tissue samples also presented positive DNA results through PCR.

Performance of IFAT, ELISA, direct parasitological examination and PCR on lymph node aspirates for canine visceral leishmaniasis diagnosis

Canine visceral leishmaniasis (CVL) is endemic in numerous Brazilian regions. The greatest difficulty in controlling the disease is the diagnostic limitation. In the present study, the most common tests employed for visceral leishmaniasis diagnosis were compared: immunofluorescence antibody test (IFAT), immunoenzymatic assay (ELISA), direct parasitological examination and polymerase chain reaction (PCR). Samples of lymph node aspirates and blood were collected from 100 dogs that lived in an endemic area (Bauru city, São Paulo state) and from 100 negative controls from a non-endemic area (Botucatu city, São Paulo state). Specificity of both IFAT and PCR was 100 percent whereas ELISA was 99 percent. Sensitivities were 97.77, 93.33 and 91.11 percent respectively for IFAT, ELISA and PCR.

Cell-mediated immune response to Leishmania chagasi experimental infection of BALB/c immunosuppressed mice

Leishmaniasis, a zoonosis of worldwide distribution, presents a significant impact on immunosupressed patients. This study aimed to evaluate Leishmania chagasi infection in BALB/c mice immunosuppressed with dexamethasone. Spleen cells stimulated or not with L. chagasi were cultured for cytokine quantification (IFN-gama, IL-2, IL-4 and IL-10) by sandwich ELISA. Parasite loads in the spleen and liver were determined by means of culture microtitration. Immunosuppressed groups showed statistically lower spleen weight and CD4-cell percentage in blood on the day of infection and produced Th1 and Th2 cytokines on other days of the study. The other infected groups, weather immunosupressed or not, also produced Th1 and Th2 cytokines. Parasite loads in the spleen and liver were not statistically different among the groups. It was concluded that L. chagasi infection was not affected by dexamethasone-induced immunosuppression, probably due the reversible effect of the treatment.

Leptospira spp. antibodies in captive coatis (Nasua nasua Storr, 1780) (Carnivora: Procyonidae)

Leptospirosis is a worldwide infection that affects many species, including wild animals. The present study aimed to detect Leptospira spp. antibodies in 17 captive coatis (Nasua nasua) by the microscopic agglutination test (MAT). Nine (52.94 percent) animals tested positive and the following serovars were identified: Copenhageni (22.22 percent), Shermani (22.22 percent), Andamana (11.11 percent), Wolfii (11.11 percent) and Pyrogenes (11.11 percent). Two samples presented coagglutination, one (11.11 percent) for Hebdomadis and Wolfii, and another (11.11 percent) for Hebdomadis, Hardjo and Wolfii. The current study revealed the presence of the infection even in animals without any clinical signs, reinforcing the possibility that wild animals in captivity may be infected by leptospire serovars, thus enabling reservoirs.

Leptospira spp. and Toxoplasma gondii antibodies in vampire bats (Desmodus rotundus) in Botucatu region, SP, Brazil

The destruction of natural ecosystems has caused several problems to humans and other animals; herein we investigate the close relationship among vampire bats, humans and domestic animals. Toxoplasma gondii and Leptospira spp. infections are two worldwide zoonoses that provoke serious damage to animals. To determine the prevalence of bats seropositive for toxoplasmosis and leptospirosis in the Botucatu region, 204 serum samples of vampire bats (Desmodus rotundus) were tested for T. gondii antibodies by modified agglutination test (MAT-t) and for Leptospira spp. by microscopic agglutination test (MAT-l). No animal was tested positive for T. gondii while leptospiral positivity was 7.8 percent for Pyrogenes, Shermani and Javanica serovars, with titers varying from 100 to 1,600. Thus, it was verified that D. rotundus does not play a relevant role in toxoplasmosis epidemiology. However, these bats can be important in the maintenance of Leptospira spp. in the environment.(AU)

Experimental infection with Leishmania chagasi in immunosuppressed Balb/c mice: cytokines and parasite burdens

The immune response in leishmaniasis may result in a polarization of the T lymphocyte subpopulation, altering cell phenotype and resulting in immune protection or disease exacerbation. Leishmania may persist in the body either during asymptomatic infections or after treatment, which represents high risk under immunosuppression. The objective of this study was to evaluate the effect of infection with immunosuppression by dexamethasone associated with pentoxifylline on animal weight, spleen weight, spleen and hepatic parasitic load and immunopathology, as well as the IFN-ã and IL-10 production in spleen cell culture of Balb/c mice infected with Leishmania chagasi. The infection did not cause body weight gain in animals, but both the weight and size of the spleen were increased. The immunosuppression using dexamethasone associated with pentoxifylline affected body weight gain and spleen weight and size in both infected and non-infected animals. The immunosuppression did not significantly alter the course of the splenic or hepatic parasite burden. Dexamethasone and pentoxifylline significantly affected cytokine production, but did not influence the Th1/Th2 ratio in infected animals.(AU)