Alessandro de Sá Guimarães

High seroprevalence of caseous lymphadenitis in Brazilian goat herds revealed by Corynebacterium pseudotuberculosis secreted proteins-based ELISA

We conducted a seroepidemiological survey to determine the prevalence of caseous lymphadenitis (CLA) in goat herds in Minas Gerais state, Brazil. Serum samples were collected from goats (n=676) from 108 rural properties in 2001, covering most of the sub-regions of this ca. 586,500 square kilometer state. Antibodies against Corynebacterium pseudotuberculosis secreted proteins were detected by an indirect enzyme-linked immunosorbent assay (ELISA). Most of the animals (78.9%) tested positive for CLA; 98% of flocks presented at least one seropositive animal. Goats managed under an extensive production system had a significantly higher seroprevalence of CLA than those in intensive and semi-intensive operations. The age distribution of the animals in the flocks affected the prevalence of this disease; however, goat breed did not. We found seropositivity against C. pseudotuberculosis to be highly prevalent in these Brazilian goat herds; consequently, appropriate management practices for the control of CLA should be implemented.

Molecular characterization of Corynebacterium pseudotuberculosis isolates using ERIC-PCR

Caseous lymphadenitis is an infectious sheep and goats disease caused by Corynebacterium pseudotuberculosis and characterized by abscesses in superficial and visceral lymph nodes. C. pseudotuberculosis strains isolated from these hosts have been shown to be very difficult to type by the existing methods. The aim of this study is evaluating the potential of the Enterobacterial Repetitive Intergenic Consensus (ERIC-PCR) as a tool for molecular typing of C. pseudotuberculosis strains isolated in sheep. One hundred and twenty seven isolates of C. pseudotuberculosis were isolated from lesions suspected to have had caseous lymphadenitis collected from sheep at the slaughterhouse. Animals were from 24 flocks in 13 municipalities of the Minas Gerais State, Brazil. Species identification of the isolates was performed by routine biochemical tests and mPCR. Fingerprint was performed by RAPD using ERIC-1R, ERIC-2 and ERIC-1R+ERIC-2 primers. Seventeen different genotypes were generated by ERIC 1-PCR, 21 genotypes by ERIC 2-PCR and 21 genotypes by ERIC 1+2-PCR. Hunter-Gaston Discrimination Index (HGDI) found for ERIC 1, ERIC 2, ERIC 1+2 PCR were 0.69, 0.87, and 0.84, respectively. For most herds evaluated observed at most three different genotypes among isolates from animals of these property, in all ERIC-PCR assays. However a few flocks observed between four and nine genotypes per flock. The W Kendall value found for correlation among the three techniques of ERIC-PCR was 0.91 (P<5.0 x 10(-6)). The results show that ERIC-PCR has good discriminatory power and advantages over other DNA-based typing methods, making it a useful tool to discriminate C. pseudotuberculosis isolates.

Seroprevalence and risk factors of caprine toxoplasmosis in Minas Gerais, Brazil

The objective of this work was to carry out a study on caprine toxoplasmosis in the state of Minas Gerais, Brazil. To determine the prevalence of toxoplasmosis in goats in Minas Gerais, 767 sera from goats were tested by ELISA (enzyme-linked immunosorbent assay) and IFAT (indirect fluorescence antibody test). The prevalence of antibodies to Toxoplasma gondii was 43.0% and 46.0% by ELISA and IFAT, respectively. It was observed that 26.8% of the goats show low-avidity IgG to T. gondii. These results suggest the presence of animals in recent phase of toxoplasmosis in Minas Gerais. The risk factors for toxoplasmosis in goats were: age over 36 months (OR=1.21; IC 95% 1.02-1.44), use of pen (OR=1.83; IC 95%1.01-3.31) and pure breed animals (OR=2.49; IC 95% 1.11-5.59).

Evaluation of ERIC-PCR as Genotyping Method for Corynebacterium pseudotuberculosis Isolates

The aim of this study was to evaluate the Enterobacterial Repetitive Intergenic Consensus (ERIC-PCR) as a tool for molecular typing of C. pseudotuberculosis isolates from eight different hosts in twelve countries. Ninety-nine C. pseudotuberculosis field strains, one type strain (ATCC 19410T) and one vaccine strain (1002) were fingerprinted using the ERIC-1R and ERIC-2 primers, and the ERIC-1R+ERIC-2 primer pair. Twenty-nine different genotypes were generated by ERIC 1-PCR, 28 by ERIC 2-PCR and 35 by ERIC 1+2-PCR. The discriminatory index calculated for ERIC 1, ERIC 2, and ERIC 1+2-PCR was 0.89, 0.86, and 0.92, respectively. Epidemiological concordance was established for all ERIC-PCR assays. ERIC 1+2-PCR was defined as the best method based on suitability of the amplification patterns and discriminatory index. Minimal spanning tree for ERIC 1+2-PCR revealed three major clonal complexes and clustering around nitrate-positive (biovar Equi) and nitrate-negative (biovar Ovis) strains. Therefore, ERIC 1+2-PCR proved to be the best technique evaluated in this study for genotyping C. pseudotuberculosis strains, due to its usefulness for molecular epidemiology investigations.

Species-level identification of staphylococci isolated from bovine mastitis in Brazil using partial 16S rRNA sequencing

Staphylococci isolated from bovine milk and not classified as Staphylococcus aureus represent a heterogeneous group of microorganisms that are frequently associated with bovine mastitis. The identification of these microorganisms is important, although it is difficult and relatively costly. Genotypic methods add precision in the identification of Staphylococcus species. In the present study, partial 16S rRNA sequencing was used for the species identification of coagulase-positive and coagulase-negative staphylococci isolated from bovine mastitis. Two hundred and two (95%) of the 213 isolates were successfully identified at the species level. The assigning of an isolate to a particular species was based on ≥99% identity with 16S rRNA sequences deposited in GenBank. The identified isolates belonged to 13 different Staphylococcus species; Staphylococcus chromogenes, S. aureus and Staphylococcus epidermidis were the most frequently identified species. Eight isolates could not be assigned to a single species, as the obtained sequences showed 99% or 100% similarity to sequences from two or three different Staphylococcus species. The relatedness of these isolates with the other isolates and reference strains was visualized using a cladogram. In conclusion, 16S rRNA sequencing was an objective and accurate method for the proper identification of Staphylococcus species isolated from bovine mastitis. Additional target genes could be used in non-conclusive cases for the species-level identification of these microorganisms.

Management practices to control gastrointestinal parasites in dairy and beef goats in Minas Gerais; Brazil

Parasitic infection is recognized worldwide as a limiting factor in the production of goats, and various control methods are used to reduce economic losses, often without considering the epidemiology of the parasites. This has led to the development of highly tolerant parasite populations and the presence of chemical residues in the beef and milk. The objective of this study was to determine the level of knowledge of goat farmers about parasitic diseases and to correlate this with the epidemiology of endoparasites and parasite control practices in goat farms in the state of Minas Gerais, Brazil. The analysis was based on a questionnaire applied by trained veterinarians. The sample was homogeneous throughout the state, covering 18.4% (157/853) of municipalities. Eighty-four dairy goat farms in 81 municipalities and 200 properties with beef goats in 76 municipalities were evaluated. The herd size per goat farm ranged from 4 to 57 (average 24) for beef herds and from 2 to 308 (average 63) for dairy farms. The majority of the beef herd production was extensive and semi-extensive (98.5%), while the dairy herds were maintained under intensive farming (98.8%). The mixed production of goats and sheep was reported by 36.5% of beef goat farmers and by 20.2% of dairy goat farmers. Among the beef goats farms on which the technological level was determined, 2.0% were categorized as having high technological level, 34.5% as medium, and 63.5% as low. Of the 84 dairy farms, 30% operated at a high, 47% at a medium, and 23% at a low technological level. The adoption of practices to reduce parasitism, such as the quarantine of animals, treatment of newly arrived animals, regular cleaning of the floor, and technical assistance, was significantly higher on dairy farms than on beef farms. Although 85.7% of dairy farmers and 83% of beef farmers medicate their animals, the treatments were performed without technical criteria, and deworming intervals ranged from 30 to 120 days or more. The average interval between treatments was significantly longer in dairy goat herds (4.8 months) than in the beef herds (3.6 months). The most commonly used drugs were macrocyclic lactones (37.7% in dairy and 39.5% in beef herds) and benzimidazoles (48.9% in dairy and 31.5% in beef herds). Goat production in Minas Gerais is still in its infancy, and even though using a control program associated with other health practices, producers still rely heavily on chemicals to get satisfactory results.

High sero-prevalence of caseous lymphadenitis identified in slaughterhouse samples as a consequence of deficiencies in sheep farm management in the state of Minas Gerais, Brazil

BackgroundCaseous lymphadenitis (CLA), caused by Corynebacterium pseudotuberculosis, is one of the most important diseases of sheep and goats, causing considerable economic losses for herd owners.ResultsWe assessed the seroprevalence of infection with C. pseudotuberculosis in 805 sheep from 23 sheep farms that supply slaughterhouses in the state of Minas Gerais; we also analyzed management practices that could be associated with CLA occurrence, used on these and nearby farms that also supplied animals to the slaughterhouse (n = 60). The serum samples for assaying CLA infection were taken at the slaughterhouse. Frequency of infection with C. pseudotuberculosis was estimated at 43.7%, and farm frequency was estimated at 100%. Management practices were analyzed through a questionnaire. All farmers (60/60) had extensive/semi-extensive rearing system; 70.0% (42/60) identified sheep individually; 11.7% (7/60) had periodical technical assistance; 41.7% (25/60) disinfected the facilities; 86.7% (52/60) used barbed wire fences and did not implement adequate CLA control measures; only 11.7% (7/60) of breeders reported vaccination against C. pseudotuberculosis; 13.3% (8/60) took note of animals with clinical signs of CLA; 1.7% (1/60) opened and sanitized abscesses, and isolated the infected animals; 10.0% (6/60) knew the zoonotic potential of this disease and 1.7% (1/60) of the farmers culled animals in case of recurrence of abscesses.ConclusionsIt can be concluded that C. pseudotuberculosis infection is widely spread in sheep flocks in Minas Gerais state in Brazil and that there is a lack of good management measures and vaccination, allowing transmission of this infectious agent throughout the production network.

Seroprevalence for Neospora caninum in goats of Minas Gerais state, Brazil

This study was aimed at determining the prevalence of anti-Neospora caninum antibodies in serum samples from 667 goats, collected from 90 herds in 90 municipalities in eight of the twelve mesoregions of Minas Gerais State. The presence of anti-N. caninum antibodies was determined by IFAT with a cut-off value of 1:50. The true herd-level seroprevalence in the entire study area was 75.2% (CI 95%: 59.5-90.9%). The true individual-level seroprevalence in the entire study area was 10.7% (CI 95%: 8.3-13.4%) with titers ranging from 50 (50.5%) to 3200 (1.7%). The serological evidence from this study indicated that N. caninum infection is widely distributed among goat-rearing herds in Minas Gerais and further studies are needed in order to evaluate the impact of this parasite as a cause of reproductive disorders.

Molecular characterization of Corynebacterium pseudotuberculosis isolated from goats using ERIC-PCR.

Corynebacterium pseudotuberculosis, the infectious agent of caseous lymphadenitis (CLA), is responsible for substantial economic losses in goat and sheep production. Molecular characterization of C. pseudotuberculosis isolates by enterobacterial repetitive intergenic consensus (ERIC)-PCR has shown promising results in genotyping strains isolated from sheep with CLA. We evaluated the genetic diversity of C. pseudotuberculosis isolates collected from the Sertão region of the Pernambuco (PE) State, Brazil, and investigated the potential of ERIC-PCR as a tool for the molecular typing of strains of C. pseudotuberculosis isolated from goats. Thirty-two C. pseudotuberculosis strains isolated from goats in the municipalities of Floresta and Ibimirim, PE, C. pseudotuberculosis type strain ATCC 19410, the 1002 vaccine strain, and a field isolate of Rhodococcus equi were fingerprinted using the primers ERIC-1R and ERIC-2 and the primer pair ERIC- 1R+ERIC-2. Using 100% similarity as the cutoff, 8, 10, and 7 genotypes were obtained with ERIC-1-PCR, ERIC-2-PCR, and ERIC-1+2-PCR, respectively. The Hunter-Gaston discriminatory index calculated for the ERIC-1-PCR was 0.75. The index for the ERIC-2-PCR was 0.88, and the index for the ERIC-1+2-PCR was 0.79. Among goat isolates of C. pseudotuberculosis, three, two and four genotypes (found by ERIC-1-PCR, ERIC-2-PCR, and ERIC-1+2-PCR, respectively) had been previously described among sheep isolates from Minas Gerais State, Brazil. These results showed that ERIC-PCR has good discriminatory power and typeability, making it a useful tool for discrimination among C. pseudotuberculosis isolates from goats.

Presence of mecA-positive multidrug-resistant Staphylococcus epidermidis in bovine milk samples in Brazil

Bacteria of the genus Staphylococcus are one of the major pathogens causing bovine mastitis. In recent decades, resistance of this genus to oxacillin (methicillin) has been a matter of concern due to the possibility of reducing the effectiveness of mastitis treatments and the transfer of resistance determinants to other bacteria. Oxacillin resistance was studied in 170 staphylococci from bovine milk samples, including 79 Staphylococcus aureus and 91 coagulase-negative staphylococci (CNS). The susceptibility profile of 10 antimicrobial agents used in veterinary practice was determined by the Etest method. In addition to the Etest, the phenotypic characterization of oxacillin resistance was tested using the cefoxitin disk diffusion test. All isolates were screened by PCR to detect the mecA gene in 2 different regions of the gene. The isolates with an oxacillin minimum inhibitory concentration ≥0.5 µg/mL or resistant to cefoxitin were identified by sequencing a 536-bp fragment of the 16S rRNA gene. This group of isolates was also evaluated for the presence of blaZ and mecC genes. Molecular analysis of the mecA gene was carried out by typing of the staphylococcal cassette chromosome mec (SCCmec). The relatedness of the mecA-positive isolates was evaluated by macrorestriction of chromosomal DNA followed by pulsed-field gel electrophoresis. With the exception of penicillin and oxacillin, 86% of the isolates showed susceptibility to cephalothin, gentamicin, erythromycin, sulfonamide, trimethoprim-sulfamethoxazole, and tetracycline. All S. aureus isolates were susceptible to oxacillin, whereas 47% (n=43) of the CNS isolates were resistant. The CNS isolates showed a higher resistance to cephalothin, erythromycin, tetracycline, and gentamicin in comparison with S. aureus. The mecA gene was only detected in 10 CNS isolates, identified as Staphylococcus epidermidis, and classified into 3 pulsotypes (A, B, and C) and 4 subtypes (A1, B1, B2, and B3). Among the isolates with an oxacillin resistance phenotype, 12 were positive for the blaZ gene, and 9 of them were mecA-positive. Two of the oxacillin-resistant isolates amplified the mecA homolog gene of Staphylococcus sciuri and none amplified mecC. Three SCCmec types, I, IV, and V, were found. Our results suggest that Staphylococcus epidermidis can be a reservoir for mecA for other Staphylococcus species. Studies investigating the molecular and phenotypic profile of antimicrobial resistance in staphylococcal species should be performed for controlling the spread of resistance and the selection of appropriate therapeutic measures.