André Saidenberg

Rotavirus detection and isolation from chickens with or without symptoms

Rotaviruses have been identified as one of the main etiological agents of diarrhea and enteritis in mammals, including humans, and in avian species. Few studies have been published about enteric viruses in Brazilian poultry, including those related to rotavirus infection. Such studies demonstrate significant occurrence and the importance of enteric viruses in poultry presenting intestinal problems. Enteric viruses are the primary cause of injuries to the gut, allowing other agents, especially bacteria, to attach, to penetrate, and to replicate in the enteric tissue, leading to further damage. The aim of the present study was to detect rotavirus in the intestinal contents of layers and broilers by polyacrylamide gel electrophoresis (PAGE) and virus isolation in MA-104 cell culture. A total of 45.3% of all samples were positive to rotavirus; rotavirus frequencies were 48.7% in samples from flocks with diarrhea, 46.4% in flocks with delayed growth, and 30% in asymptomatic flocks. It was possible to isolate rotavirus in MA-104 cells from the nine rotavirus-positive randomly chosen samples. These results indicate that rotavirus may have an important role in pathogenesis of enteric disease.

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.

Molecular detection of enteropathogenic Escherichia coli in asymptomatic captive psittacines

Psittaciformes are one of the most endangered groups of birds, and several Brazilian species are classified between vulnerable and critically endangered. It is thus necessary to identify agents that cause infections in captive wild animals and to assess the risks posed thereof and to design interventions to minimize the possibility of disease outbreaks, leading to the conservation of endangered species. The purpose of this study was to identify enteropathogenic Escherichia coli (EPEC) cloacal isolates from asymptomatic psittacines in captivity and evaluate the distribution of the EPEC pathotype. Cloacal swabs were obtained from 46 asymptomatic birds, and resulting isolates were tested by polymerase chain reaction (PCR) for the presence of the attaching and effacing gene (eae) and bundle-forming pilus structural gene (bfpA) of EPEC. Samples from several species were tested, and three samples were found to be positive for the eae and bfpA genes and characterized as typical EPEC. This is the first report of this pathotype in asymptomatic psittacines. Although certain E. coli strains are more pathogenic than others, various factors should be considered when determining the potential of E. coli isolates to cause disease in captive psittacines. Birds that are positive for the EPEC (typical) strain could be zoonotic sources of infection, and may have acquired these strains through contact with humans or domestic animals. These findings may also be valuable for the long-term management of endangered species ex situ as one EPEC sample was isolated from a Red-tailed Amazon (Amazona brasiliensis).

Serogroups and virulence genes of Escherichia coli isolated from psittacine birds

Escherichia coli isolates from 24 sick psittacine birds were serogrouped and investigated for the presence of genes encoding the following virulence factors: attaching and effacing (eae), enteropathogenic E. coli EAF plasmid (EAF), pili associated with pyelonephritis (pap), S fimbriae (sfa), afimbrial adhesin (afa), capsule K1 (neu), curli (crl, csgA), temperature-sensitive hemagglutinin (tsh), enteroaggregative heat-stable enterotoxin-1 (astA), heat-stable enterotoxin -1 heat labile (LT) and heat stable (STa and STb) enterotoxins, Shiga-like toxins (stx1 and stx2), cytotoxic necrotizing factor 1 (cnf1), haemolysin (hly), aerobactin production (iuc) and serum resistance (iss). The results showed that the isolates belonged to 12 serogroups: O7; O15; O21; O23; O54; O64; O76; O84; O88; O128; O152 and O166. The virulence genes found were: crl in all isolates, pap in 10 isolates, iss in seven isolates, csgA in five isolates, iuc and tsh in three isolates and eae in two isolates. The combination of virulence genes revealed 11 different genotypic patterns. All strains were negative for genes encoding for EAF, EAEC, K1, sfa, afa, hly, cnf, LT, STa, STb, stx1 and stx2. Our findings showed that some E. coli isolated from psittacine birds present the same virulence factors as avian pathogenic E. coli (APEC), uropathogenic E. coli (UPEC) and Enteropathogenic E. coli (EPEC) pathotypes.

Serratia marcescens Infection in a Swallow-tailed Hummingbird

A swallow-tailed hummingbird (Eupetomena macroura) was presented with a history of prostration and inability to fly. After a 2-day hospitalization, the bird died and necropsy findings included diffuse hyperemia of the small intestine serosal and mucosal surfaces and the presence of a small quantity of clear ascitic fluid in the coelomic cavity. Intestinal contents and cardiac blood were collected for microbiologic exams yielding pure cultures of a pigmented strain of Serratia marcescens. This strain was susceptible to gentamicin, enrofloxacin, streptomycin, trimethoprim, and sulfamethoxazole and had intermediate susceptibility to chloramphenicol and resistance to cephalotin. The source of the infection could not be ascertained, but possible contamination of hummingbird feeders could be involved, because the infection seemed to originate from the digestive tract.

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.

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

Abstract:u2003 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.

Pandemic extra-intestinal pathogenic Escherichia coli (ExPEC) clonal group O6-B2-ST73 as a cause of avian colibacillosis in Brazil

Extra-intestinal pathogenic Escherichia coli (ExPEC) represent an emerging pathogen, with pandemic strains increasingly involved in cases of urinary tract infections (UTIs), bacteremia, and meningitis. In addition to affecting humans, the avian pathotype of ExPEC, avian pathogenic E. coli (APEC), causes severe economic losses to the poultry industry. Several studies have revealed overlapping characteristics between APEC and human ExPEC, leading to the hypothesis of a zoonotic potential of poultry strains. However, the description of certain important pandemic clones, such as Sequence Type 73 (ST73), has not been reported in food sources. We characterized 27 temporally matched APEC strains from diverse poultry farms in Brazil belonging to the O6 serogroup because this serogroup is frequently described as a causal factor in UTI and septicemia in humans in Brazil and worldwide. The isolates were genotypically characterized by identifying ExPEC virulence factors, phylogenetically tested by phylogrouping and multilocus sequence type (MLST) analysis, and compared to determine their similarity employing the pulsed field gel electrophoresis (PFGE) technique. The strains harbored a large number of virulence determinants that are commonly described in uropathogenic E. coli (UPEC) and sepsis associated E. coli (SEPEC) strains and, to a lesser extent in neonatal meningitis associated E. coli (NMEC), such as pap (85%), sfa (100%), usp (100%), cnf1 (22%), kpsMTII (66%), hlyA (52%), and ibeA (4%). These isolates also yielded a low prevalence of some genes that are frequently described in APEC, such as iss (37%), tsh, ompT, and hlyF (8% each), and cvi/cva (0%). All strains were classified as part of the B2 phylogroup and sequence type 73 (ST73), with a cluster of 25 strains showing a clonal profile by PFGE. These results further suggest the zoonotic potential of some APEC clonal lineages and their possible role in the epidemiology of human ExPEC, in addition to providing the first description of the O6-B2-ST73 clonal group in poultry.

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.

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

Abstract An atypical enteropathogenic Escherichia coli was isolated from an asymptomatic nestling of the endangered Lears 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.