Genetic diversity and antimicrobial resistance profile of Enterococcus faecalis isolated from broilers with vertebral osteomyelitis in Southeast Brazil.

Vertebral osteomyelitis (VO) is a worldwide emerging disease that affects broilers. Recently, the isolation of Enterococcus faecalis in cases of the disease has been described. This study aimed at determining the genetic diversity and antimicrobial resistance profile of 12 E. faecalis strains isolated from broilers with VO. Strains were isolated from nine flocks from six farms in a high-density poultry production area in Southeast Brazil and were evaluated using multilocus sequence typing and phylogenetic analysis. Antimicrobial susceptibility tests and PCR were performed to detect antimicrobial resistance genes. E. faecalis isolates belonged to different sequence types (ST), six of which (ST49, ST100, ST116, ST202, ST249, and ST300) have been previously described. Strains ST708 and ST709 were newly identified in this study. Strain ST49 was most frequently isolated (50% of the flocks) from the analysed VO cases. No phylogenetic or phylogeographic relationship was found among the strains. The VO isolated E. faecalis strains showed highest resistance to aminoglycosides, mainly gentamicin (40%), but were highly susceptible to vancomycin (10%). Aminoglycoside resistance genes were detected in seven E. faecalis strains, and AAC6'-APH2″ genes were most frequently detected. The results showed that E. faecalis strains isolated from recently reported VO cases were highly diverse genetically. The diversity of genotypes in circulation in the analysed flocks, without apparent relationship among them, raises questions on aetiopathogenesis of the disease in broilers and evolutionary aspects of E. faecalis.

Diversity of Escherichia coli strains involved in vertebral osteomyelitis and arthritis in broilers in Brazil.

BACKGROUND: Locomotor disorders and infections by Escherichia coli represent major concerns to the poultry industry worldwide. Avian pathogenic E. coli (APEC) is associated with extraintestinal infections leading to respiratory or systemic disease known as colibacillosis. The most common lesions seen in cases of colibacillosis are perihepatitis, airsacculitis, pericarditis, peritonitis/salpingitis and arthritis. These diseases are responsible for significant economic losses in the poultry industry worldwide. E. coli has been recently isolated from vertebral osteomyelitis cases in Brazil and there are no data on molecular and phenotypic characteristics of E. coli strains isolated from lesions in the locomotor system of broilers. This raised the question whether specific E. coli strains could be responsible for bone lesions in broilers. The aim of this study was to assess these characteristics of E. coli strains isolated from broilers presenting vertebral osteomyelitis and arthritis in Brazil. RESULTS: Fifteen E. coli strains from bone lesions were submitted to APEC diagnosis and setting of ECOR phylogenic group, O serogroup, flagella type, virulence genes content, genetic patterns by Pulsed Field Gel Electrophoresis (PFGE) and Multilocus Sequence Typing (MLST). In addition, bacterial isolates were further characterized through a lethality test, serum resistance test and antibiotic resistance profile. E. coli strains harbored different genetic pattern as assessed by PFGE, regardless of flock origin and lesion site. The strains belonged to seven sequence types (STs) previously described (ST117, ST101, ST131, ST 371 and ST3107) or newly described in this study (ST5766 and ST5856). ECOR group D (66.7 %) was the most frequently detected. The strains belonged to diverse serogroups (O88, O25, O12, and O45), some of worldwide importance. The antibiotic resistance profile confirmed strains' diversity and revealed a high proportion of multidrug-resistant strains (73 %), mainly to quinolones and beta-lactams, including third generation cephalosporin. The percentage of resistance to tetracycline was moderate (33 %) but always associated with multidrug resistance. CONCLUSIONS: Our results demonstrated that vertebral osteomyelitis and arthritis in broilers can be associated with highly diverse E. coli based on molecular and phenotypic characteristics. There was no specific virulence patterns of the E. coli strains associated with vertebral osteomyelitis or arthritis. Also, E. coli strains were frequently multidrug resistant and belonged to STs commonly shared by APEC and human ExPEC strains.

Vertebral osteomyelitis associated with single and mixed bacterial infection in broilers.

Vertebral osteomyelitis (VO) is a worldwide emerging disease that affects broilers. The objective of this study was to determine the frequency and aetiology of VO in broilers in a highly productive broiler region. For this, 608 broilers with locomotory problems were analysed from 18 farms. Clinical signs were recorded, necropsy was performed and samples were collected from vertebral bodies with gross changes for molecular and histopathological analysis and for bacterial isolation. From broilers with locomotory changes, 5.1% (31/608) had VO and, of these, 93.5% were 40 days old or older and 89.7% were males. The birds with VO presented varying degrees of limited mobility and this was related to the level of compression to the spinal cord. Bacterial species of the genus Enterococcus (DNA detected in 53.6%) were the aetiological agents involved in most VO cases. Enterococcus faecalis was detected most frequently (35.7%), but Enterococcus hirae was also present in some lesions (7.1%). Escherichia coli was detected in 35.7% of vertebral lesions and co-infection with E. faecalis was confirmed in 7.1% cases. Staphylococcus aureus was involved in 14.3% of the cases, being 7.1% in co-infection with Enterococcus spp. or E. hirae. Our study has indicated that, in Brazil, VO in broilers may not be caused by a single infectious agent and has a lower frequency than recently reported in other countries. This study suggests that there are geographical differences between Brazil and other countries concerning the frequency and aetiology of VO.

Comparative B-mode and Doppler renal ultrasonography with histopathological findings in dogs positive for canine visceral leishmaniasis.

Morphological parameters and renal flow were assessed and measured in six clinically healthy dogs with negative serological and parasitological examinations for canine visceral leishmaniasis (CVL) and six dogs with CVL-positive serological and parasitological tests and asymptomatic condition of the disease. Kidney length and diameter were measured and compared with the aorta/kidney ratio. Alterations in flow were measured using the flow velocity (cm/s) and resistance index (RI). Renal function was assessed by serum biochemical examination (urea, creatine, total proteins, albumin, and globulin) and urinalysis. CVL-positive animals presented heterogeneous kidney echotexture, reduced vascular flow, hypoperfusion, lesser visualization of the arcuate arteries, high RI values (1.06 ± 0.28 for the right kidney and 1.10 ± 0.30 for the left kidney), reduced diastolic flow, and high systolic peak. They also presented positive creatine/RI correlation and between the renal artery vascular flow and kidney diameter. Histopathological analysis of CVL-positive animals was compatible and confirmed hemodynamic changes observed by Doppler ultrasound. The findings in the present study showed that Doppler ultrasonography is a valid method to assess and detect alterations in flow in renal pathological processes with kidney vascular damage, as in the case of leishmaniasis, and can help in the diagnosis of animals with this infection. Microsc. Res. Tech. 79:637-645, 2016. © 2016 Wiley Periodicals, Inc.

Molecular characterization of infectious laryngotracheitis virus in naturally infected egg layer chickens in a multi-age flock in Brazil.

The virus responsible for an outbreak of infectious laryngotracheitis (ILT) in a multi-age flock of egg layer chickens under quarantine in Brazil was characterized. Layer chickens from this area with circulating gallid herpesvirus 1 (GaHV 1) were evaluated using histopathology and molecular characterization techniques based on sequences of infected-cell polypeptide 4 (ICP4) and thymidine kinase (TK) genes. The infected chickens that were analyzed were PCR-positive for GaHV-1 in the trachea and negative in most trigeminal ganglia. The lack of ILT lesions in the conjunctiva and respiratory tissues, combined with detection of viral DNA in the trachea, was found to be associated with latent infection. The sequences from five farms obtained in the present study were identical, and there were no deletions within the 272- to 283-bp region of the ICP4 gene, as observed in the sequences of vaccine strains (CEO and TCO). The lack of a deletion in the ICP4 fragment analyzed in this study indicates that the chickens were infected with a field virus. The absence of the T252M mutation in a fragment of the TK gene, in addition to the low mortality rate observed, suggests that the outbreak in the state of Minas Gerais was not caused by a highly virulent strain but rather by a field virus of lower virulence. In addition, using phylogenetic reconstructions, it was found that this field strain was grouped together in a separate branch, apart from the previously characterized Brazilian strains. The introduction of vectored vaccines apparently has been effective in reducing clinical disease and lesions, and preventing new outbreaks of disease.

Collared pecary (Tayassu tajacu) as a new model of renal ischemic injury induced by clamping the renal artery.

PURPOSE: The use of the collared peccary as an experimental model for ischemic nephropathy. METHODS: A total of 12 collared peccary (Tayassu tajacu) was used and ischemic nephropathy was induced in six of these animals that constituted the experimental group (G1) while the other six formed the control group (G2). Ischemic nephropathy was induced surgically by partial occlusion of the left renal artery. The disease course was assessed by hematological tests, serum chemistry, urinalysis, ultrasound (US) and doppler ultrasound function of the renal artery before induction, and at five, 10, 15 and 20 days after surgery. Twenty days after the occlusion, unilateral nephrectomy and histopathological examination were performed to assess renal morphology. RESULTS: Statistical analysis by Fischer's test showed a significant difference (p<0.05) between the control group and the experimental group. The histopathological examination showed glomerular, tubular and interstitial lesions. In the experimental group, 83.3% (5 /6) showed moderate renal lesions and only 16.7% (1/6) were classified with no lesions. The ultrasound examination of the right kidney presented statistical difference between day 5 and day 10 post occlusion. CONCLUSION: The collared peccary as a good experimental model for ischemic renal disease, because it could be manipulated during the research time without death, with health conditions that permit any subsequent procedure for disease therapy.

Collared Pecary (tayassu tajacu) as a new model of renal ischemic injury induced by clamping the renal artery

PURPOSE: The use of the collared peccary as an experimental model for ischemic nephropathy. METHODS: A total of 12 collared peccary (Tayassu tajacu) was used and ischemic nephropathy was induced in six of these animals that constituted the experimental group (G1) while the other six formed the control group (G2). Ischemic nephropathy was induced surgically by partial occlusion of the left renal artery. The disease course was assessed by hematological tests, serum chemistry, urinalysis, ultrasound (US) and doppler ultrasound function of the renal artery before induction, and at five, 10, 15 and 20 days after surgery. Twenty days after the occlusion, unilateral nephrectomy and histopathological examination were performed to assess renal morphology. RESULTS: Statistical analysis by Fischer's test showed a significant difference (p<0.05) between the control group and the experimental group. The histopathological examination showed glomerular, tubular and interstitial lesions. In the experimental group, 83.3% (5 /6) showed moderate renal lesions and only 16.7% (1/6) were classified with no lesions. The ultrasound examination of the right kidney presented statistical difference between day 5 and day 10 post occlusion. CONCLUSION: The collared peccary as a good experimental model for ischemic renal disease, because it could be manipulated during the research time without death, with health conditions that permit any subsequent procedure for disease therapy. .

Splendore-Hoeppli phenomenon in a cat with osteomyelitis caused by Streptococcus species.

A 9-month-old male neutered mixed-breed cat had a history of chronic lameness of the right hind limb, which was non-responsive to antibiotic treatment. Hematologic analysis revealed marked neutrophilia and mild monocytosis. Radiography revealed extensive loss of cortical bone, and replacement with irregular and disorganized bone. There was loss of the normal cortico-medullary distinction, and the medullary cavity had an irregular radiodensity suggestive of osteomyelitis. Surgical curettage and antibiotics did not improve the clinical condition, and amputation was performed. Grossly, the skin over the right tibia was ulcerated with a viscous and granular exudate. At histopathology, there was marked diffuse pyogranulomatous dermatitis, myositis, periostitis and osteomyelitis associated with Splendore-Hoeppli phenomenon. In addition, there was marrow osteoproliferation and multifocal cortical loss, reabsorption, fibroplasia and endosteal bone formation. Gram staining revealed myriad slightly elongated Gram-positive bacteria, arranged in pairs or single chains, confirmed by polymerase chain reaction as Streptococcus species.