Aloysio de Mello Figueiredo Cerqueira

Shigatoxigenic Escherichia coli serogroups O157, O111 and O113 in feces, water and milk samples from dairy farms

The objective of this study was to determine the prevalence of Shigatoxigenic Escherichia coli (STEC) and STEC serogroups O157, O111 and O113 in feces, water and milk sampled in dairy farms in Jaboticabal, SP, Brazil. Feces (n=454), water (n=54) and milk samples (n=30) were collected from 10 herds and assessed for the presence of the virulence genes stx1, stx2 and eae by polymerase chain reaction (PCR). All stx and eae positive samples were submitted to a second PCR reaction targeting the sequences rfb O157, rfb O111 and rfb O113. High prevalence of stx was detected (59.9%) in fecal samples, whereas the prevalence of sequences rfb O157, rfb O111 and rfb O113 was 18.9%, 3.3% and 30.4%, respectively. All sequences were detected more frequently in calves and heifers. Sequences stx2 and eae were prevalent in the fecal samples. stx sequences were detected in 1.9% and 3.3% of water and milk samples, respectively. Low prevalence of E. coli O113 was observed in water samples, whereas no E. coli O157 or E. coli O111 was detected. Furthermore, none of the serogroups were identified in milk samples. STEC was identified in all herds (100%), and serogroups O157, O111 and O113 were observed in 40%, 50% and 90% of the herds, respectively. In conclusion, the high STEC prevalence detected in dairy herds evidences that bovine feces might play an important role as a contamination source in the region of Jaboticabal, Brazil.

Detection and molecular characterization of piroplasms species from naturally infected dogs in southeast Brazil

Rangelia vitalii is a protozoon described from dogs in the south and southeast regions of Brazil. It is phylogenetically related to Babesia spp. that infects dogs, but data on this enigmatic parasite is still limited. The aim of this work was to detect piroplasm species in dogs in the state of Rio de Janeiro, Brazil, by 18S rRNA gene-based PCR assay, restriction fragment length polymorphism (RFLP) and sequence analyses. Of 103 dogs examined, seven (6.8%) were positive for Babesia spp. by PCR. The amplified products were digested by restriction enzymes to differentiate the Babesia species, and one sample was identified as Babesia vogeli. The pattern observed for the other six amplification products did not match with pattern described for large Babesia infecting dogs. Sequencing analysis confirmed these six samples as R. vitalii, with high homologies (99-100%) with a sequence from south Brazil. This study confirms the presence of Babesia vogeli and Rangelia vitalii circulate in domestic dogs in Teresópolis, Rio de Janeiro, Brazil.

Characterization of atypical Enteropathogenic Escherichia coli (aEPEC) isolated from dogs.

Enteropathogenic Escherichia coli (EPEC), an important human pathogen has the ability to form attaching and effacing lesions on the intestinal epithelium and has been isolated from a wide range of species. Two EPEC subgroups are recognized: typical (tEPEC) and atypical (aEPEC) strains, differing by the presence of EAF plasmid and bundle-forming pilus (BFP) in typical strains and their absence in atypical strains. This study searched the presence of EPEC strains in 101 fecal samples of diarrheic (n=65) and non-diarrheic (n=36) dogs from two cities in Rio de Janeiro State, Brazil. The isolates were evaluated for the presence of eae, tir, espA, espB and bfpA genes, EAF plasmid, and for the insertion site of the LEE locus. Cell-adherence assays, fluorescent actin staining (FAS) test, hemolysin production and serotyping were also performed. Twenty eight aEPEC isolates were recovered from 48 eae-positive fecal samples, 24 from diarrheic animals and 4 from non-diarrheic ones. PCR showed that most isolates was positive for β or γ intimin, and for β or α subtypes of tir, espA and espB. Six isolates showed a selC insertion of locus LEE. Only two isolates from the same diarrheic animal harbored the bfpA gene, and none presented the EAF plasmid. Most isolates was FAS-positive and showed a localized adherence-like (LAL) in a 6h HeLa cell-adherence assay. A wide diversity of serotypes was detected including O4:H16 and O51:H40, previously described in human disease. The phenotypic and genotypic markers of aEPEC isolated from diarrheic and non-diarrheic dogs were similar to those found in isolates recovered from human disease.

Characterization of epidemic clones of Listeria monocytogenes serotype 4b isolated from humans and meat products in Brazil

INTRODUCTION Listeria monocytogenes is an important foodborne pathogen and the 4b serotype is responsible for many cases of human listeriosis reported in Brazil. Several listeriosis outbreaks worldwide have involved a small number of well-defined clonal groups, designated as epidemic clones (ECs). METHODOLOGY We studied 71 strains of serotype 4b, including 25 isolates from human cases of listeriosis and 46 from meat-based foods, collected in Brazil between 1977 and 2010. The presence of ECs (I and II) markers and virulence genes (inlA, inlB, ilnC, inlJ and actA) were evaluated by PCR assay. The genetic relationship of ECs-positive strains was assessed by pulsed field gel electrophoresis. RESULTS ECI and ECII markers were found both in human and food strains, with 19.7% positive for the ECI marker and 40.8% for ECII. Most strains (97.2%) were positive for the virulence genes that were studied. Nevertheless, the actA gene amplicons showed two distinct sizes, with all ECI positive strains exhibiting a 105bp deletion. Pulsed field gel electrophoresis (PFGE) analysis allowed the recognition of highly related strains, particularly from two outbreaks of neonatal listeriosis in São Paulo State occurred in 1992 and 1997, both ECII-positive; and two ECI strains from a human case (1982) and from bovine meat (2009). CONCLUSIONS The presence of ECs among clinical samples and beef isolates of serotype 4b from some regions of Brazil highlights the need for rigorous control of production procedures. Furthermore, the association of ECII with two nosocomial outbreaks suggests its ability to spread in these settings.

Genetic diversity of Ehrlichia canis strains from naturally infected dogs in Rio de Janeiro, Brazil.

The aim of this study was to characterize Ehrlichia canis strains from naturally infected dogs in Rio de Janeiro, Brazil. In addition, all the clinical and hematological findings observed in these dogs were reported. PCR targeting the 16S rRNA gene was used for diagnostic purposes, and the TRP19 and TRP36 genes were sequenced to evaluate the genetic diversity. Fifteen samples were positive for E. canis. The polymerase chain reaction for the TRP19 gene resulted in 11 amplicons (11/15), which were cloned into the pGEM-T easy vector for sequencing. The complete sequence of TRP19 gene was compared to those in the GenBank, revealing high identicalness. Phylogenetic analysis on the TRP36 gene sequences demonstrated two distinct strains from two dogs, named 56C and 70C. The 56C strain was grouped with the strain Cuiaba 16, which is a hybrid strain formed by Brazilian and US genogroups; and the 70C strain was grouped with other strains of the US genogroup, thus suggesting that there are at least two genogroups of E. canis in Rio de Janeiro (US and Brazilian). Those animals, in which the 70C and 56C strains were isolated, showed distinct clinical and hematological manifestations of the disease. The appearance of different genotypes may express new phenotypes, thus resulting in different forms of presentation of the disease and making its diagnosis more complex.

Microbiological Findings in Tracheal Wash From Mule Foals With and Without Clinical Evidence of Respiratory Disease

Abstract Respiratory diseases are common in horses; however, there is a lack of information in the literature on respiratory disease affecting mule foals. The objective of this study was to investigate the presence of aerobic bacteria in tracheal wash samples from 20 mule foals up to 6 months of age, with and without clinical evidence of respiratory disease. Samples were collected via endoscopy in two separate occasions and sent for cytology, microbial culture, and PCR for detection of Rhodococcus equi. Based on clinical evidence, 32.5% (13/40) of the samples were obtained from mule foals displaying signs of a respiratory condition, whereas 50% (20/40) of the samples showed cytologic evidence of respiratory tract infection. One hundred percent of samples provided positive cultures with Escherichia coli (45%), Enterococcus (37.5%), and coagulase‐negative Staphylococcus (30%) being the most common bacterial genera isolated. R. equi was not identified in any of the samples. The correlation between isolated bacterial agents and the presence of respiratory infection was not statistically significant. The microorganisms found in the samples may be naturally present in the soil, feces, and environment in which the animals live, presenting a risk of opportunistic respiratory infection. HighlightsAerobic bacteria was investigated in endoscopically obtained tracheal wash of mule foals up to 6 months of age with and without clinical evidence of respiratory disease.Bacterial growth of at least one type of bacteria was obtained in 100% of endoscopically obtained traqueal wash samples from mule foals, and at least two bacteria genera were identified in 87% of the samples.Escherichia coli, Enterococcus, and coagulase‐negative Staphylococcus were the most commonly isolated bacteria found in 45%, 37.5%, and 30.0% of samples, respectively.When frequency of bacteria genera in mule foals with or without cytologic evidence of infection was submitted to statistical analysis, no significant association was detected.