Expression of genes encoding resistance in Staphylococcus spp. isolated from bovine subclinical mastitis in Brazil.

INTRODUCTION: Staphylococci are the most important agents associated with bovine mastitis. This study aimed at characterizing resistance factors to antimicrobials in Staphylococcus spp. isolated from the milk of cows with subclinical mastitis. METHODOLOGY: In vitro resistance of 243 Staphylococcus spp. isolates to antimicrobials commonly used in clinical practice was evaluated. The detection and expression of genes encoding resistance mecA (gene encoding penicillin binding protein 2a) mecALGA251 (mecA homologue), blaZ (gene encoding penicillin resistance), femA and femB (genes encoding essential factors - A and B - for the expression of methicillin resistance) and aacA-aphD (gene encoding for a bifunctional enzyme that confers resistance to gentamicin) using PCR and RT-PCR was investigated. RESULTS: One or more genes encoding resistance to different antimicrobials were detected in 184 Staphylococcus spp. SAMPLES: The femA and femB genes were the most frequent. Regarding the variables' detection (N = number of strains) and expression (% of strains), the following results were obtained: blaZ (N = 40 - 82.5%), femA (N = 147 - 47.6%), aacAaphD (N = 30 - 43.3%), femB (N = 138 - 29.7%), mecA (N = 33 - 27.3%), mecALGA251 (N = 01 - 0.0%). There was a higher occurrence of phenotypic resistant strains for amoxicillin, ampicillin and penicillin in isolates positive for detection and/or expression of blaZ gene when compared with the other genes. CONCLUSIONS: The present study provides new information on genotypic traits of Staphylococcus isolates from bovine subclinical mastitis especially regarding the evaluation of expression of genes associated with antimicrobial resistance in Staphylococcus spp. using molecular tools.

An Atypical Enteropathogenic Escherichia coli Isolate with Possible Human and Domestic Animal Origin from a Free-Living Lear's Macaw (Anodorhynchus leari).

An atypical enteropathogenic Escherichia coli was isolated from an asymptomatic nestling of the endangered Lear's Macaw (Anodorhynchus leari). Phylogenetically, it was identical to bovine and human strains and highly similar to other human and domestic animal isolates. We discuss potential routes of transmission and risks to conservation of this species.

HEALTH-SCREENING PROTOCOLS FOR VINACEOUS AMAZONS (AMAZONA VINACEA) IN A REINTRODUCTION PROJECT.

Reintroduction is a growing field in the conservation of endangered species. The vinaceous Amazon parrot (Amazona vinacea) is extinct in several areas, and a project to release confiscated individuals to their former range is currently underway. The objective of this study was to evaluate and improve the selection and treatment of individual release candidates by detecting possible pathogen carriers using samples taken before and during release. As part of prerelease health protocols, samples were obtained from 29 parrots on three different occasions while in captivity and once after their release. Samples were screened for paramyxovirus type 1, avian influenza, poxvirus, coronavirus, psittacine herpesvirus 1, Chlamydia psittaci , enteropathogenic Escherichia coli (EPEC), Salmonella spp., and endoparasites. The majority of samples returned negative results, with the exception of two individuals that tested positive for C. psittaci in the first sampling and for Ascaridia spp. in the second pooled sampling. Treatments for C. psittaci and endoparasites were administered prior to release, and negative results were obtained in subsequent exams. The number of positive results for E. coli (non-EPEC) decreased during the rehabilitation period. Adequate quarantine procedures and health examinations greatly minimize disease risks. The protocols employed in this study resulted in acceptable health status in accordance with current environmental legislation in Brazil. Additionally, protocols allowed informed decisions to release candidates, minimized risks, and favored the selection of healthy individuals, thereby contributing to the recovery of this species. It is important to determine appropriate minimum health-screening protocols when advanced diagnostics may not be available or high costs make the tests prohibitive in countries where confiscations occur. We hypothesize that a minimum panel of tests of pooled samples can serve as an alternative approach that minimizes costs and overall workload and supports projects intended to restore and promote flagship species and hamper their illegal trade.

Detection of bacteria and fungi and assessment of the molecular aspects and resistance of Escherichia coli isolated from confiscated passerines intended for reintroduction programs.

Many native bird species are currently considered rare in Brazil because they have been indiscriminately collected by animal traffickers and commercialized, leading to dwindling numbers in their natural habitats. Confiscated animals are at times destined for reintroduction programs that must ensure these animals do not pose a risk to native populations. Healthy or sick wild passerines may carry a great diversity of microorganisms. Therefore, knowledge of the sanitary status of confiscated animals destined for reintroduction is critical to assess whether these animals act as microorganism carriers and to investigate the epidemiology of transmissible diseases, a crucial aspect for animal and human health preservation. This study examined the occurrence of aerobic and facultative anaerobic bacteria and fungi in cloacal swabs collected from wild confiscated passerines intended for reintroduction programs. In vitro susceptibility tests of the most frequent isolates as well as studies of the molecular aspects of Escherichia coli isolates were also performed. There was microorganism growth in 62.5% of 253 samples. The microorganisms that were most frequently isolated were Staphylococcus spp. (15.0%), Micrococcus spp. (11.5%), E. coli (10.7%) and Klebsiella spp. (10.7%). Fifteen bacteria genera and seven fungi genera were isolated. Multidrug-resistance to antimicrobials was observed in Staphylococcus spp., Micrococcus spp., E. coli and Klebsiella spp. isolates. The high occurrence of Enterobacteria observed is possibly related to the sanitary conditions in which confiscated animals are usually kept. One E. coli sample (out of 27 isolates) was positive for the S-fimbrial adhesion encoding gene (sfa). Considering the low occurrence of genes that encode virulence factors, confiscated passerines may represent a low risk for the potential transmission of EPEC, APEC, UPEC and NMEC isolates to other animals or humans. The potential risk of intra- or inter-specific transmission of multidrug-resistant isolates and the introduction of these microorganisms into the environment must be considered, although there are still therapeutic alternatives for treatment of these animals among the antimicrobials which were tested. The stress and poor hygiene conditions imposed on animals during trafficking may have caused their contamination by multidrug-resistant agents transmitted by humans or by the precarious environment to which they were subjected. Risks related to the dissemination of Salmonella spp., Cryptococcus spp. and Candida spp. are low when reintroduction programs are considered.

Occurrence of genes coding for MSCRAMM and biofilm-associated protein Bap in Staphylococcus spp. isolated from bovine subclinical mastitis and relationship with somatic cell counts.

This study aimed to elucidate aspects of the epidemiology of bovine subclinical mastitis through the assessment of genes encoding MSCRAMM (microbial surface components recognizing adhesive matrix molecules - a group of adhesins) and protein Bap (implicated in biofilm formation), in coagulase-positive (CPS) and coagulase-negative (CNS) Staphylococcus isolated from subclinical mastitis. Milk samples were collected for microbiological exams, somatic cell count (SCC) and a survey of the genes coding for MSCRAMM (cna, eno, ebpS, fnbA, fnbB and fib) and biofilm-associated protein Bap (bap) in 106 Staphylococcus spp. isolates using PCR. The frequencies of occurrence of eno (82.1%), fnbA (72.6%), fib (71.7%) and bap (56.6%) were higher (P < 0.0001) compared with the other assessed genes (cna, ebpS and fnbB). The higher frequency of occurrence (P < 0.005) of the bap gene in CNS compared with CPS suggests that in these species biofilm formation is an important mechanism for the persistence of the infection. The medians of the SCCs in the samples where eno, fnbA, fib and bap genes were detected were higher compared with Staphylococcus without the assessed genes (P < 0.05) and negative samples (P < 0.01), which indicated that the presence of these MSCRAMM may be related to a higher intensity of the inflammatory process.

Virulence factors genes of Staphylococcus spp. isolated from caprine subclinical mastitis.

The aim of this study was to investigate genes involved in adhesion expression, biofilm formation, and enterotoxin production in isolates of Staphylococcus spp. from goats with subclinical mastitis and associate these results with the staphylococcal species. One hundred and twenty-four isolates were identified and polymerase chain reaction (PCR) was performed to detect the following genes: cna, ebpS, eno, fib, fnbA, fnbB, bap, sea, seb, sec, sed and see. The most commonly Staphylococcus species included S. epidermidis, S. lugdunensis, S. chromogenes, S. capitis ss capitis and S. intermedius. With the exception of fnbB, the genes were detected in different frequencies of occurrence in 86.3% of the Staphylococcus spp. isolates. Eno (73.2%) and bap (94.8%) were more frequently detected in coagulase-negative staphylococci (CNS); ebpS (76%), fib (90.9%) and fnbA (87%) were the most frequent genes in coagulase-positive staphylococci (CPS). Regarding enterotoxins, genes sed (28.2%) and see (24.2%) had a higher frequency of occurrence; sec gene was more frequently detected in CPS (58.8%). There was no association between the presence of the genes and the Staphylococcus species. Different virulence factors genes can be detected in caprine subclinical mastitis caused by CNS and CPS. The knowledge of the occurrence of these virulence factors is important for the development of effective control and prevention measures of subclinical mastitis caused by CNS and CPS in goats.

Occurrence of Chlamydophila felis in a cattery in Osasco city, São Paulo state / Ocorrência de Chlamydophila felis em um gatil na região de Osasco, São Paulo

Chlamydophila felis is associated with upper respiratory tract infections. In the present study, 31 cats from a noncommercial shelter located in Osasco, SP, Brazil, were examined. The cats presented with clinical manifestations, which were classified from grade 1 to 4, with 4 indicating severe manifestations. In total, 16.13% of the cats presented with grade 1 severity of clinical manifestations, 25.81% presented with grade 2, 38.71% presented with grade 3, and 19.35% presented with grade 4. PCR was used to detect C. felis in samples taken from the oral mucosa and ocular conjunctiva of both eyes using sterile, dry cotton swabs. Overall, 58% of the samples were positive for C. felis. Of these animals, none showed clinical manifestations that were classified as grade 1, whereas 5.56% of cats were classified as grade 2, 61.11% were classified as grade 3, and 33.33% were classified as grade 4. The median clinical manifestation intensity score for the first group was 3 and ranged from 2 to 4. In the second group not positive for C. felis, 38.45% of the animals (5/13) presented with manifestations classified as grade 1, 53.85% (7/13) were classified as grade 2, 7.69% (1/13) were classified as grade 3, and no animals were classified as grade 4. The median clinical manifestation intensity score for the second group was 2 and ranged from 1 to 3. In this study, there was a high occurrence of C. felis in animals with clinical manifestations.

A Chlamydophila felis está associada à infecção de trato respiratório superior. No presente estudo, foram utilizados 31 felinos de um gatil não comercial, localizado em Osasco/SP. Os gatos apresentavam manifestações clínicas, classificadas de 1 a 4, sendo 4 atribuído àqueles que apresentavam pior manifestação clínica. Foi observada a intensidade de manifestação clínica grau 1 em 16,13% dos gatos, a 2 em 25,81%, a 3 em 38,71% e a 4 em 19,35%. A detecção de C. felis foi realizada por técnica de PCR em amostras obtidas com suabes de algodão, seco e estéril, de mucosa oral e de conjuntiva ocular de ambos os olhos. Verificou-se que 58% das amostras para C. felis foram positivas, entre esses animais, nenhum apresentou manifestação clínica classificada como grau 1, o grau 2 foi observado em 5,56% dos gatos, 61,11% para o 3 e 33,33% dos animais apresentava a intensidade 4. Verificou-se que para o primeiro grupo a mediana dos escores de intensidade das manifestações clínicas observadas foi de 3, variando de 2 a 4. No segundo grupo, foi observado 38,45% (5/13) dos animais para a intensidade 1, 53,85% (7/13) para a 2 e 7,69% (1/13) para a 3, nenhum animal deste grupo apresentou o grau 4. Verificou-se para o segundo grupo, a mediana dos escores de intensidade das manifestações clínicas observadas foi de 2, variando de 1 a 3. Neste trabalho foi observada uma elevada ocorrência de C. felis em animais com manifestação clínica.