BackgroundLocomotor 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.ResultsFifteen 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.ConclusionsOur 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12917-016-0762-0) contains supplementary material, which is available to authorized users.
A recent (November 2010) outbreak of infectious laryngotracheitis (ILT) in a multi-age laying hen facility in Minas Gerais state, Brazil, is described. Previous ILT outbreak in laying hens was only notified in São Paulo state, Brazil, in 2002. In the outbreak described here, the affected population was approximately eight million hens, with flock sizes ranging from 100,000 to 2,900,000 chickens. The average mortality ranged from 1 to 6%, and morbidity was around 90% (most of the twenty seven farms of the area were positive for ILT virus). Three multi-age laying farms from one company were selected for this report. Clinical signs included prostration, dyspnea, conjunctivitis, occasional swelling of the paranasal sinuses and bloody mucous nasal discharge. Severely affected chickens presented with dyspnea, gasping and became cyanotic before death. At necropsy, these chickens had fibrinous exudate blocking the larynx and the lumen of cranial part of the trachea. In addition, conjunctivitis with intense hyperemia, edema and sinuses with caseous exudate were present. On histopathology, there were marked necrosis and desquamation of respiratory ephitelium and conjunctiva with numerous syncytial cells formation and fibrinous exudate. Moderate to marked non suppurative (especially lymphocytes and plasma cells) infiltration in the lamina propria also was observed. Sixteen out of 20 examined chickens, eosinophilic intranuclear inclusion bodies were observed in the syncytial cells. The DNA extracted from larynx and trachea produced positive PCR results for ILT virus (ILTV) DNA using formalin-fixed, paraffin embedded (FFPE) samples. Amplicons from a small region of ICP4 gene were submitted to sequencing and showed 100% identity with ILTV EU104910.1 (USA strain), 99% with ILTV JN596963.1 (Australian strain) and 91% with ILTV JN580316.1 (Gallid herpesvirus 1 CEO vaccine strain) and JN580315.1 (Gallid herpesvirus 1 TCO vaccine strain).
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.
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.
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.
Pesq. Vet. Bras. 33(9) The goal of this study was to characterize the epidemiological situation and the factors involved in the prevalence of babesiosis and anaplasmosis in cattle in the dairy basin of Parnaíba, Piauí, Brazil. The study was conducted in 22 farms, and collected blood samples from 202 cattle to study serological, molecular and determination of the packed cell volume (PCV). On the farms were applied surveys involving epidemiological aspects. Seroprevalence rates were: Babesia bigemina 52.5%, B. bovis 68.8%, and Anaplasma marginale 89.1%. Of the samples analyzed, 73.3% were reactive for Babesia spp. and A. marginale, showing co-infection. In PCR, B. bigemina and B. bovis were positive in 52.0% and 33.2% respectively, and A. marginale in 76.2%. Of these, 51.5% amplified DNA of Babesia spp. and A. marginale. The semi-intensive management predominated in 68.0% of the farms studied. The clinical history of babesiosis and anaplasmosis, was reported from 73% of the farms. There was no significant difference (p>0.05) between age groups and for the PCV of positive compared with negative animals. The study indicates that in this region is enzootic instability for babesiosis and enzootic stability for anaplasmosis, reinforcing the fact that in Brazil there are areas of enzootic instability, even in tropical regions of the country. The PCR technique was a valuable tool for the diagnosis of these diseases and may be used to characterize a geographic region. ões tropicais do país. A técnica de PCR demonstrou ser uma ferramenta valiosa para o diagnóstico destas doenças e pode ser utilizada para caracterizar uma região geográfica.
KeywordsAvian infectious laryngotracheitis, conventional PCR, histopathology, immunohistochemistry, laying hen.Submitted: November/2013 Approved: June/2014 ABSTRACT Seventy-eight chickens from a very high poultry density (approximately eight million) region and twelve backyard chickens from neighboring areas were analyzed by histopathology and additional techniques for the presence of the infectious laryngotracheitis virus. The virus distribution was determined in different tissues using immunohistochemistry (IHC) and polymerase chain reaction (PCR). The disease was histopathologically diagnosed in 41.0% (32/78) of the commercial layers. Lesions were mainly characterized by syncytial cells with eosinophilic intranuclear inclusion body formed from the hyperplastic epithelium of the upper respiratory tract, primary and secondary bronchi, and conjunctiva. IHC showed 70% (21/30) positive signal in the larynx/trachea and, 53.8% (14/26) in the lungs, either in epithelial cells or syncytia. In the turbinates and paranasal sinuses, 29.6% (8/27) of samples showed positive signal. PCR detected the following gallid herpesvirus 1-positive percentages: conjunctiva 63.2% (31/49), lungs 57.6% (30/52), turbinates and paranasal sinuses 56% (28/50), and larynx/trachea 50% (39/78). IHC showed to be a useful additional tool for definitive ILT diagnosis, especially during the subacute phase of the disease when syncytial cells with intranuclear inclusion bodies are no longer observed. PCR using specific primers from ICP4 gene, generating a product of 237 base pairs, was sensitive for ILT diagnosis, and very useful for rapid detection of GaHV-1 in chickens. Fixed tissues allowing histopatological examination and detection of GaHV-1 by PCR, are a good option in areas where farms are located several hundred kilometers away from a diagnostic center, reducing problems with conservation of fresh samples and the risk of virus spread.
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