Prevalence of bovine mastitis-related pathogens, identified by mass spectrometry in flies (Insecta, Diptera) captured in the milking environment.

Diptera (Insecta) are able to transmit approximately 200 pathogenic microorganisms to humans and animals, causing more than 65 diseases, including bovine mastitis, which constitutes a major cause of economic losses in the dairy industry. In this scenario, 217 adult specimens of Diptera were collected from nine farms and identified to the family and/or species level. Of the 11 families recorded, Muscidae was the most prevalent (152 out of 217; 70%). All Diptera specimens were subjected to microbiological culture using conventional and selective media, and isolates were then identified at the species level by mass spectrometry. In total, 275 microorganisms were identified, with a predominance of pathogens related to environmental bovine mastitis (166/275 = 60·4%), that is, Enterococcus species (70/275 = 25·4%) and Escherichia coli (49/275 = 17·8%). Nontraditional agents related to bovine mastitis (called miscellaneous) were detected in 28% (77/275), as well as microorganisms with well-known zoonotic behaviour (e.g. Bacillus cereus). This is study contributes with knowledge of diversity of microorganisms carried by Diptera in the dairy environment, including pathogens associated with environmental and contagious bovine mastitis, and agents with human relevance. To our knowledge, a three-part chromogenic selective medium used to microbial culture of milk on farms was used for the first time to identification of pathogens in Diptera.

Species diversity and antimicrobial resistance patterns of Enterococcus spp. isolated from mastitis cases, milking machine and the environment of dairy cows.

The objectives were to study the diversity of Enterococcus spp. isolated from mastitis cases, milking equipment and the environment of dairy cows; and to determine in vitro resistance of isolates to antimicrobials that are relevant to human and animal health. Ten dairy farms were visited to collect samples from mastitis cases, faeces, bedding, aisles, water and milking equipment. Identification of Enterococcus at the species level and antimicrobial resistance testing was performed by MALDI-TOF and the disk-diffusion method, respectively. Of 365 isolates, Enterococcus hirae was the most prevalent, being more likely to be isolated from faeces than from milk (odds ratio (OR) = 39·2), liners (OR = 5·4) or bedding (OR = 2·2). Enterococcus saccharolyticus was the most prevalent in milk samples. The chances of isolating Enterococcus faecalis from milk were higher than from aisles (OR = 12·5), faeces (OR = 5·3), bedding (OR = 3·6) or liners (OR = 3·0). The odds of isolating Enterococcus faecium from faeces were higher than from liners (OR = 7·3), bedding (OR = 2·5) or aisles (OR = 2·4). Of 360 tested isolates, 1·9, 0·3 and 0·6% were resistant to penicillin, vancomycin and teicoplanin, respectively. Our results suggest that Enterococcus species can occupy specific ecological niches on dairy farms and pose a risk to public and animal health.

Antimicrobial resistance, biofilm production and invasion of mammary epithelial cells by Enterococcus faecalis and Enterococcus mundtii strains isolated from bovine subclinical mastitis in Brazil.

Members of the genus Enterococcus are among the most relevant etiologic agents of bovine clinical and subclinical mastitis, a major problem for the dairy industry. In Brazil, clonal diversity, and multidrug resistance profiles related to bovine infections need further investigation. In this study, 11 bacterial strains recovered from mastitis subclinical cases detected in different farms of São Paulo, Brazil, were identified as Enterococcus faecalis (n = 8) and Enterococcus mundtii (n = 3) by biochemical testing and MALDI-TOF mass spectrometry. Pulsed-field gel electrophoresis categorized the enterococcal isolates into two main clusters (A and B) with similarity ranging from 85 to 100%. The isolates were shown to be resistant tetracycline (73%), erythromycin (73%), quinupristin-dalphopristin (64%), norfloxacin (9%), fosfomycin (9%) and linezolid (9%). Moreover, seven strains (64%) were considered multidrug-resistant. All the isolates were able to produce biofilms when grown in milk for 24 h: 54·54% were classified as moderate producers and 45·45% were weak producers. Interestingly, only two strains (Ef17 and Em42) remained as moderate biofilm producers after 48 h incubation. Moreover, all isolates showed no ability to form biofilm in tryptic soy broth (TSB) after 24 and 48 h incubation. In addition, cytoskeleton components were partially involved in E. faecalis and E. mundtii entry to epithelial cells as demonstrated by induction of actin stress fibre. In conclusion, enterococci isolates recovered from bovine subclinical mastitis were resistant to several classes of antibiotics, showing the ability to form biofilms in milk and invade mammary epithelial cells, suggesting an advantageous feature in mammary gland colonization during mastitis development. In addition, they can spread along the food chain by different routes and eventually constitute a possible threat for public health, including E. mundtii specie.

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.

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)

Influence of pathogens causing clinical mastitis on reproductive variables of dairy cows.

In dairy cattle, mastitis is a disease of the mammary gland caused by pathogens such as bacteria, viruses, fungi, and algae. Mastitis causes economic losses to dairy farms as well as public health concerns. The reproductive efficiency of commercial dairy herds has important implications for the economic success of dairy operations and is strongly associated with the health status of cows. Mastitis has previously been linked with decreased fertility of dairy cows, but the effect of specific pathogens on the severity of fertility reduction is still unclear. In this study, cows diagnosed with mastitis caused by major pathogens (Staphylococcus aureus, Streptococcus agalactiae, Escherichia coli, Klebsiella spp., Mycoplasma spp., and environmental Streptococcus) needed more artificial inseminations (AI) than did cows with mastitis caused by minor pathogens (coagulase-negative Staphylococcus and Corynebacterium spp.) and healthy cows. Cows diagnosed with mastitis, independent of what pathogen was causing mastitis, had more days open compared with nonmastitic cows. The percentage of cows that successfully established pregnancy at first AI was greater for the control group than for the major pathogens group but not significantly different from the minor pathogens group. Pregnancy loss was lower in the control group than in the major pathogens group; however, there was no difference compared with the minor pathogen group. Mastitis caused by gram-negative bacteria decreased the percentage of pregnancy per first AI and increased days open and pregnancy loss compared with the control group. Cows with mastitis caused by gram-positive bacteria also had increased days open compared with control cows. This study shows that different mastitis-causing bacteria can affect the fertility of cows differently. Mastitis events caused by major pathogens and gram-negative bacteria were associated with the greatest decrease in reproductive efficiency.

Genotyping of long term persistent Staphylococcus aureus in bovine subclinical mastitis.

Bovine mastitis affects dairy cattle worldwide and Staphylococcus aureus is one of the most common microorganisms involved in subclinical and chronic disease. Superantigens, such as enterotoxins contribute to S. aureus persistence and pathogenicity in this disease. Subclinical and chronic mastitis cases were diagnosed and S. aureus isolates from sub-clinical cases were investigated for carriage of virulence and antibiotic resistance genes that may contribute to long-term carriage and infection. Over a 12-month period, 116 S. aureus strains were recovered from 68 cows with subclinical mastitis. Classical enterotoxin genes (sea-see) were detected in 24.1% of isolates, and pvl and tsst-1 were identified in 3.4% and 46.6% the isolates, respectively. 18.1% that were persistent isolates were identified and characterized by pulsed field gel electrophoresis (PFGE), MLST, spa typing. Four isolates were methicillin-resistant S. aureus (MRSA) and belonged to SCCmec type I. Molecular typing showed that the agrI group was the most frequent, and a rare isolate was positive for both agrI and agrIII groups. Molecular characterization revealed the persistence of the spa type t10856 (ST133, clonal complex CC133, agr I), in a single animal for nine months and the persistence t605 (ST126, CC126) colonizing four animals for four months. These strains have been described recently in other herds in the same region, indicating their transmissibility and clonal expansion. We conclude that animals with subclinical mastitis are an important and somewhat overlooked reservoir for transmission within and between herds, and may carry virulence and antibiotic resistance genes contributing to persistent colonization, hinder the control of mastitis and may cause risks to the public health.

Putative virulence factors of extra-intestinal Escherichia coli isolated from bovine mastitis with different clinical scores.

We investigated the genes kpsMTII, iucD, sfaDE, afaBC, papA and papC, (proposed to be involved in extra-intestinal pathogenic Escherichia coli-ExPEC), phylogroup classification and the in vitro swimming and swarming motility in 50 E. coli isolated from bovine mastitis with different clinical severity scores (mild, moderate and severe). The aforementioned genes were detected in 12 (n = 12/50; 24·0%) isolates. kpsMTII and iucD were the most frequent genes identified in six (n = 6/50; 12·0%) and four (n = 4/50; 8·0%) of the isolates, respectively. In only one (n = 1/50; 2·0%) isolate, more than one gene was simultaneously identified: iucD and kpsMTll were detected whereas sfaDE and afaBC were not detected. Mild, moderate and severe clinical signs were observed in 40·0% (n = 20/50), 28·0% (n = 14/50) and 32·0% (n = 16/50) of the cases. Commensal phylogroups B1 (n = 19/50; 38·0%) and A (n = 19/50; 38·0%) were prevalent; whereas pathogenic phylogroups B2 and D were observed in only 10·0% (n = 5/50). Swarming and swimming motilities were observed in 90·0% (n = 45/50) and 68·0% (n = 34/50) of the isolates, respectively; and there was a significant association (P = 0·0036) between swarming motility and severe clinical cases (score 3). To the best of our knowledge, this is the first study where clinical severity of bovine mastitis cases and the genes proposed to classify ExPEC were assessed in relation to swarming and swimming motility. SIGNIFICANCE AND IMPACT OF THE STUDY: Escherichia coli is classified as extra-intestinal (ExPEC) when strains contain at least two of the genes kpsMTII, iucD, sfaDE, afaBC and papA and/or papC. We investigated in vitro motility and the presence of these genes in 50 E. coli isolated from bovine mastitis with different clinical scores (mild, moderate and severe). Clinical severity was not associated with the genes studied. Swarming motility was associated with severe cases (score 3) of clinical mastitis. Results of this study contribute to a better understanding of the factors that determine the severity of clinical mastitis.

Molecular epidemiology of methicillin-susceptible Staphylococcus aureus (MSSA) isolated from milk of cows with subclinical mastitis.

Bovine mastitis has been a concern for dairy herd for decades. The adaptation capacity of one of the main species responsible for this disease, Staphylococcus aureus (S. aureus), plays a pivotal role in this issue. The aim of this study was to establish a molecular and phenotypic profile of 285 S. aureus strains isolated from milk of subclinical mastitis cows from 18 different farms in São Paulo State using spa typing, multilocus sequence typing (MLST), pulsed field gel electrophoresis (PFGE), agr cluster (I, II, III and IV) typing, PCR for genes including enterotoxins (sea, seb, sec, sed, see, seg, seh, sei), toxic shock syndrome toxin (tsst-1), and Panton-Valentine leucocidin (pvl), as well as in vitro resistance assays for 12 antibiotics. The results showed a wide variety of strains with a high toxigenic potential; concomitantly, sec, seg and seh were prevalent. In addition, we observed a predominance of the spa types t605 (ST 126, CC126) and t127 (ST1, CC1) and the unusual presence of t321 causing bovine mastitis, which has been previously reported only in swine. The most frequent ST were ST126 (70.5%) and ST1 (10.5%). Regarding PFGE, we observed four major groups and six profile patterns. The highest resistance was observed for streptomycin (9.5%), followed by tetracycline (3.5%), clindamycin (9.3%), and erythromycin (2.8%). The tsst-1 gene was detected in 36.8% of isolates and pvl was not observed. One hundred and thirty-six (47.7%) isolates possessed agr type II, followed by types III (20%) and I (8.1%), with type IV not being detected. We observed that the same spa type could result in different PFGE profiles, so the exclusive use of spa type sequences can lead to incorrect interpretations regarding the spread of clones in an epidemiological context.

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)

In vitro algicidal effect of guanidine on Prototheca zopfii genotype 2 strains isolated from clinical and subclinical bovine mastitis.

Prototheca species have increasingly been reported to be opportunistic pathogens that cause mastitis in dairy herds, and it poses an emergent problem because at present, there are no effective therapies for the treatment of protothecal mastitis. This study investigated the in vitro algicidal effect of guanidine on 75 Prototheca zopfii genotype 2 strains isolated from 75 cases of clinical and subclinical bovine mastitis. All strains were susceptible to guanidine in vitro with minimal algaecide concentrations ranging from 0·001 to 0·035%. Guanidine is known to have a high microbicidal effect and is considered to be a new generation microbicidal compound. It is not toxic to human mucous membranes and conjunctivas at low concentrations and has been used as a disinfectant in swimming pools and as an antiseptic for human wounds. The algicidal action of guanidine at low concentrations indicates that it could be an alternative disinfectant or antiseptic for cleaning of the dairy environment and milking equipment, in pre- and postdipping solutions, in the chemical dry therapy of bovine teats and even in the intramammary therapy of P. zopfii infections. This is the first report of the in vitro algicidal effect of guanidine on P. zopfii strains of animal origin. SIGNIFICANCE AND IMPACT OF THE STUDY: Prototheca zopfii genotype 2 is an opportunistic pathogen of bovine mastitis. To date, no effective therapies against protothecal mastitis have been developed. The in vitro algicidal effect of guanidine on 75 P. zopfii genotype 2 strains isolated from cows revealed that all of the isolates were susceptible to the compound at low concentrations, which indicates that guanidine may be used as an antiseptic/disinfectant for dairy milking equipment, in pre- and postdipping solutions, and as a chemical dry therapy or an intramammary therapy. This study describes the in vitro algicidal effect of guanidine on P. zopfii for the first time.

Short communication: Outbreak of methicillin-resistant Staphylococcus aureus (MRSA)-associated mastitis in a closed dairy herd.

Cows are probably the main source of contamination of raw milk with Staphylococcus aureus. Mammary glands with subclinical mastitis can shed large numbers of Staph. aureus in milk. Because of the risk of this pathogen to human health as well as animal health, the aim of this paper was to describe an outbreak of mastitis caused by methicillin-resistant Staph. aureus (MRSA), oxacillin-susceptible mecA-positive Staph. aureus (OS-MRSA), and methicillin-susceptible Staph. aureus (MSSA) on a dairy farm. Milk samples were obtained from all quarters, showing an elevated somatic cell count by the California Mastitis Test. The isolates were identified by phenotypic and genotypic methods. Staphylococcus spp. were isolated from 53% (61/115) of the milk samples, with 60 isolates identified as Staph. aureus (98.4%) and 1 isolate identified as Staphylococcus epidermidis (1.6%). The presence of the mecA gene was verified in 48.3% of Staph. aureus isolates. Of the Staph. aureus isolates, 23.3% were MRSA and 25.0% were OS-MRSA. The total of mastitis cases infected with MRSA was 12.2%. The detection of this large percentage of mastitis cases caused by MRSA and OS-MRSA is of great concern for the animals' health, because ß-lactams are still the most important antimicrobials used to treat mastitis. In addition, Staph. aureus isolates causing bovine mastitis represent a public health risk.

Methicillin-resistant Staphylococcus aureus of lineage ST398 as cause of mastitis in cows.

UNLABELLED: The objective of this study was to analyse the prevalence and molecular characteristics of methicillin-resistant Staphylococcus aureus (MRSA) in milk of cows with mastitis. The California mastitis test (CMT) was used to detect the presence of mastitis in all 100 cows of a farm in Brazil. The CMT was positive in milk of 115 mammary quarters from 36 cows (36%). MRSA isolates were recovered from 4 of these 36 cows with mastitis (11%), and they were further characterized (one MRSA/sample). The four MRSA isolates were typed as t011-ST398-agr1-SCCmecV and presented two different pulsed-field-gel-electrophoresis-ApaI patterns. These four MRSA isolates showed resistance to tetracycline, streptomycin and ciprofloxacin, carried the mecA, blaZ, tet(K), and tet(M) resistance genes, and presented the S84L and S80F amino acid substitutions in GyrA and GrlA proteins, respectively. Two ST398 isolates exhibited resistance to gentamicin and tobramycin [with aac(6)-aph(2") and ant(4)-Ia genes] and one isolate resistance to clindamycin [with lnu(B) and lsa(E) genes]; this latter isolate also carried the spectinomycin/streptomycin resistance genes spw and aadE. MRSA of lineage ST398 is worldwide spread, normally multidrug resistant and may be responsible for bovine mastitis. To our knowledge, this is the first detection of MRSA-ST398 in Brazil. SIGNIFICANCE AND IMPACT OF THE STUDY: Few studies on the epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) from bovine isolates have been performed in Brazil. MRSA of lineage ST398 is worldwide spread and associated with farm animals. Multidrug-resistant MRSA-ST398 isolates were recovered in 11% of mastitic cows from a single farm, with one isolate carrying the unusual lsa(E), spw and aadE genes. To our knowledge, this is the first detection of MRSA-ST398 isolates in milk samples of cows with mastitis in Brazil.

Sensitivity and specificity of serological tests, histopathology and immunohistochemistry for detection of Toxoplasma gondii infection in domestic chickens.

Since free-range chickens are important for the epidemiology of toxoplasmosis, this study evaluated the sensitivity and specificity of different laboratory techniques for the diagnosis of Toxoplasma gondii in these animals. Serum samples from 135 adult domestic chickens were tested for anti-T. gondii antibodies by the indirect fluorescent antibody test (IFAT), enzyme-linked immunosorbent assay (ELISA), modified agglutination test (MAT), and indirect hemagglutination test (IHAT). Tissue samples from all animals were analyzed by histopathology, immunohistochemistry and mouse bioassay (gold standard). Fifty-four chickens were positive for T. gondii in the bioassay. The sensitivity and specificity of the different tests were, respectively, 85% and 56% for ELISA; 80% and 52% for IFAT; 76% and 68% for MAT; 61% and 80% for IHAT; 7% and 98% for immunohistochemistry, and 6% and 98% for histopathology. The MAT was the most effective method for the diagnosis of T. gondii infection in chickens, followed by ELISA. Histopathology and immunohistochemistry are useful tools for the diagnosis of T. gondii infection in chickens due to their specificity.

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.

Diversity of Staphylococcus species and prevalence of enterotoxin genes isolated from milk of healthy cows and cows with subclinical mastitis.

The objectives of this study were to determine the occurrence and diversity of Staphylococcus spp. in milk from healthy cows and cows with subclinical mastitis in Brazil and to examine the profile of enterotoxin genes and some enterotoxins produced by Staphylococcus spp. A total of 280 individual mammary quarter milk samples from 70 healthy cows and 292 samples from 73 cows with subclinical mastitis were collected from 11 farms in the state of São Paulo, Brazil. Staphylococcus spp. were recovered from 63 (22.5%) samples from healthy cows and from 80 samples (27.4%) from cows with mastitis. The presence of Staphylococcus aureus was significantly different between these 2 groups and was more prevalent in the cows with mastitis. The presence of Staphylococcus saprophyticus was also significantly different between these 2 groups, but this organism was more prevalent in healthy cows. No statistically significant differences were observed in the numbers of other staphylococci in milk samples from the 2 groups. The sea gene was the most prevalent enterotoxin gene in both groups. Eight of 15 (53.3%) Staph. aureus carried this gene and all produced the SEA toxin. In the coagulase-negative staphylococci (CNS) group, 61 of 128 (47.5%) had the same gene and just 1 (1.6%) Staphylococcus epidermidis strain produced the enterotoxin in vitro. Because CNS were isolated from both groups of cows and most CNS contained enterotoxin genes but did not produce toxins, the role of CNS in mastitis should be carefully defined.

Molecular characterization and clonal diversity of methicillin-susceptible Staphylococcus aureus in milk of cows with mastitis in Brazil.

Mastitis is an important disease for the dairy industry worldwide, causing economic losses and reducing milk quality and production. Staphylococcus aureus is a worldwide agent of this intramammary infection, which also causes foodborne diseases. The objective of this study was to determine the frequency of methicillin-susceptible Staphylococcus aureus (MSSA) isolates in milk of mastitis cows in Brazil and to analyze the genetic lineages and the content of antimicrobial resistance genes and virulence factors among these isolates. Fifty-six MSSA isolates were recovered from 1,484 milk samples (positive for the California mastitis test) of 518 cows from 11 different farms in Brazil (representing 51% of total Staph. aureus obtained), and they were further characterized. Methicillin-susceptible Staphylococcus aureus were isolated from 3.7% of California mastitis test-positive tested milk samples and from 6.2% of tested mastitic cows. Methicillin-susceptible Staphylococcus aureus isolates were characterized by spa typing, agr typing, and multilocus sequence typing, and resistance and virulence traits were investigated by PCR. Seven spa types were identified among MSSA (% of isolates): t127 (44.6), t605 (37.5), t002, t1784, t2066 (1.8), and 2 new ones: t10856 (10.7) and t10852 (1.8). Five distinct sequence types (ST) were detected (% of isolates): ST1 (46.4), ST126 (37.5), ST133 (10.7), ST5 (3.6), and a novel ST registered as ST2493 (1.8). Resistances were detected for streptomycin, chloramphenicol, and tetracycline. One strain contained the chloramphenicol resistance gene (fexA; included within transposon Tn558) and 3 strains contained the tetracycline resistance gene [tet(K)]. Methicillin-susceptible Staphylococcus aureus strains were susceptible to most of the antibiotics studied and lacked the virulence genes of Panton-Valentine leukocidin (lukF/S-PV), toxic shock syndrome toxin 1 (tst), exfoliative toxin A (eta), and exfoliative toxin B (etb), as well as the genes of the immune evasion cluster. Methicillin-susceptible Staphylococcus aureus isolates were detected in a relatively low proportion of cows with mastitis (6.2%) and recovered isolates presented high diversity of genetic lineages, with CC1 and CC126 the predominant clonal complexes, and CC133 also being detected. Larger epidemiological studies with molecular characterization of isolates are required to deepen the knowledge on the circulating genetic lineages among the cow population with mastitis.

Detection of cross infections by Leishmania spp. and Trypanosoma spp. in dogs using indirect immunoenzyme assay, indirect fluorescent antibody test and polymerase chain reaction.

The aim of this study was to detect cross infections by Leishmania spp. and Trypanosoma spp. using enzyme-linked immunosorbent assay (ELISA), indirect fluorescent antibody test (IFAT) and polymerase chain reaction (PCR). Thus, 408 blood samples were collected from dogs domiciled in Araçatuba Municipality, São Paulo State, Brazil; the dogs were of both sexes, of several breeds and aged 6 months. For Leishmania spp., 14.95% (61 out of 408) of dogs were reactive using IFAT. Positivity was 20.10% (82 out of 408) using ELISA and 29.66% (121 out of 408) using PCR, with significant differences for the sex and age of these animals (p < 0.05). For Trypanosoma spp., antibody occurrence using ELISA was 10.54% (43 out of 408), while PCR indicated 2.45% (10 out of 408) positive dogs. Using IFAT, 10.29% (42 out of 408) of animals were considered positive and only sex showed a significant difference (p < 0.05). In this study, 10.54% (43 out of 408) of animals were seropositive according to ELISA for Trypanosoma spp., of which 79.07% (34 out of 43) showed positive results in the molecular diagnosis for Leishmania spp., while of the 10.29% (42 out of 408) positive dogs according to IFAT, 95.24 % (40 out of 42) had confirmed infection by this parasite. The obtained results demonstrate evidence of cross infections by both protozoa in the animals analysed in this study.