Characterization of multidrug resistant Salmonella recovered from diseased animals

Three hundred and eighty Salmonella isolates recovered from animal diagnostic samples obtained from four state veterinary diagnostic laboratories (AZ, NC, MO, and TN) between 2002 and 2003 were tested for antimicrobial susceptibilities and further characterized for bla(CMY) beta-lactamase genes, class 1 integrons and genetic relatedness using PFGE. Forty-seven serovars were identified, the most common being S. Typhimurium (26%), S. Heidelberg (9%), S, Dublin (8%), S. Newport (8%), S. Derby (7%), and S. Choleraesuis (7%). Three hundred and thirteen (82%) isolates were resistant to at least one antimicrobial, and 265 (70%) to three or more antimicrobials. Resistance was most often observed to tetracycline (78%), followed by streptomycin (73%), sulfamethoxazole (68%), and ampicillin (54%), and to a lesser extent chloramphenicol (37%), kanamycin (37%), amoxicillin-clavulanic acid (20%), and ceftiofur (17%). With regards to animal of origin, swine Salmonella isolates displayed the highest rate of resistance, being resistant to at least one antimicrobial (92%), followed by those recovered from turkey (91%), cattle (77%), chicken (68%), and equine (20%). Serovars commonly showing multidrug resistance (MDR) to > or =9 antimicrobials were S. Uganda (100%), S. Agona (79%), and S. Newport (62%), compared to S. Heidelberg (11%) and S. Typhimurium (7%). Class-1 integrons were detected in 43% of all isolates, and were found to contain aadA, aadB, dhfr, cmlA and sat1 gene cassettes alone or in various combinations. All ceftiofur resistant isolates (n=66) carried the bla(CMY) beta-lactamase gene. A total of 230 PFGE patterns were generated among the 380 isolates tested using XbaI, indicating extensive genetic diversity across recovered Salmonella serovars, however, several MDR clones were repeatedly recovered from different diseased animals.     

Chronological study of Mycoplasma hyopneumoniae infection, seroconversion and associated lung lesions in vaccinated and non-vaccinated pigs

A field trial was conducted to study Mycoplasma hyopneumoniae (Mh) infection dynamics by nested polymerase chain reaction (nPCR) and serology in pigs of a farm affected by enzootic pneumonia (EP). Moreover, correlation of Mh detection at different respiratory tract sites with presence of EP gross and microscopic lung lesions was assessed. These parameters were studied and compared between vaccinated (two doses at 1 and 3 weeks of age versus one dose at 6 weeks of age) and non-vaccinated pigs. Animals were monitored from birth to slaughter by nPCR from nasal swabs and by serology. From 3 to 22 weeks of age, an average of three pigs per treatment and per batch were necropsied (n = 302). The remaining pigs were sent to the slaughter (n = 103). Nasal, bronchial and tonsillar swabs were taken from the necropsied/slaughtered pigs; gross and microscopic EP-suggestive lung lesions were also assessed. Single and double vaccination resulted in earlier seroconversion and higher percentage of Mh seropositive pigs compared to control group. At slaughter, double vaccinated pigs showed lower percentage of EP-compatible gross lung lesions and lower Mh prevalence at upper respiratory tract sites (nasal cavity and tonsil) than control pigs.     

Central nervous system signs in chickens caused by a new avian reovirus strain: a pathogenesis study

The present study describes the pathogenesis of infection of chicks with a new avian reovirus strain, belonging to the so-called enteric reovirus strains (ERS) that is capable of causing central nervous system signs in SPF white leghorns. After intramuscular (IM) or oral inoculation birds were either observed for clinical signs or sacrificed for macroscopic, histological and virological examination for 21 days. Virus isolation was performed on the brain, leg muscle, hock joint, liver and spleen. For the detection of viral antigen the immunohistochemistry (IHC) technique was performed on the caudal part of the cerebrum, spinal cord including spinal ganglia and right N. Ischiadicus. High mortality (79% in 7 days) was seen in birds that were inoculated IM. Survivors were depressed and stayed small until the end of the experiment. One bird had tremor and showed torticollis at 9 days after IM inoculation. Birds that were inoculated orally were depressed from day 4 and stayed small until the end of the experiment. One bird showed a torticollis at 10 days after inoculation. After both IM and oral inoculation ERS was isolated from the brain between 3 and 10 days after inoculation. Other examined organs were positive for virus isolation from day 1 or 5 until day 21. IHC revealed viral antigen positive cells in the Plexus chorioideus (plexus epithelial cells or cells within the underlying connective tissue) and in a spinal ganglion. The results indicate that the pathogenesis of ERS infection in chickens bears some resemblance with that of the mammalian reoviruses serotype 1 in mice.     

Phenotypic and genotypic characterization of Paenibacillus larvae isolates

Paenibacillus larvae is the causative agent of American Foulbrood (AFB), a severe disease of honeybees (Apis melifera). The aim of this work was to develop a strategy for the subtyping and the epidemiological analysis of P. larvae. Phenotypic characterisation, susceptibility to several antibiotics, electrophoresis of whole bacterial proteins, rep-PCR, ribotyping and DGGE were assessed using a collection of P. larvae isolates from different Uruguayan and Argentinean locations. Results indicated that there are two P. larvae genotypes circulating in Uruguay ERIC I-BOX A (worldwide distributed) and ERIC I-BOX C (exclusively detected in Argentina until this study). These results suggest that P. larvae isolates had moved between Argentina and Uruguay, probably through the Uruguay River. Patterns of whole bacterial proteins, DGGE and ribotyping did not improve the P. larvae intraspecific discrimination. Antibiotic susceptibility assays showed that 100% isolates were OTC-sensitive and 22% (belonging to ERIC I-BOX A group) were sulfisoxazole-resistant. This work may contribute to the elucidation of basic aspects related to the epidemiology of AFB in Uruguay and in the region.     

Resistance studies of erythromycin and rifampin for Rhodococcus equi over a 10-year period

This study sought to determine whether an increase in resistance of Rhodococcus equi to the antibiotics rifampin and erythromycin occurred over a 10-year period. This was carried out by the use of E test strips for rifampin and erythromycin to determine the MIC (minimum inhibitory concentration) values of Rhodococcus equi to this combination of antibiotics.The findings of this study indicated that there was an increase in resistance of Rhodococcus equi to rifampin and erythromycin over the 10-year period. The MIC for rifampin increased from 0.081 μg/ml in 1996 to 0.187 μg/ml in 2006 and from 0.258 μg/ml for erythromycin during the years prior to 2000 to 0.583 μg/ml in 2006.This finding suggests that there may be a problem in the treatment of Rhodococcus equi infections in foals in the future, particularly as the number of drugs available for treatment of Rhodococcus equi infection is limited because of the intracellular capabilities of this bacterium. Antibiotics used in its treatment have to be able to penetrate the polysaccharide cell wall of Rhodococcus equi as well as the alveolar macrophages in which the bacterium is capable of surviving.     

The aetiology of generalized alopecia in young calves

The objective of this study was to test for correlations between alopecia and ruminal drinking in young calves. 331 calves up to an age of 31 days were tested for evidence of generalized hair loss daily during their stay in the clinic. Incidence of diarrhoea and the results of ruminal fluid and blood analysis were compared between the groups with and without alopecia. Calves with alopecia showed a significantly higher incidence of diarrhoea and of ruminal acidosis persisting for at least 24 hours. Blood analysis revealed significant differences in degree of acidosis, in concentrations of D-lactate, urea, and creatinine in serum as well as in the activities of glutathione peroxidase, aspartate amino transferase, and creatine kinase. Alopecia in calves is correlated to longer periods of diseases, which are known to be accompanied by the production of D-lactate in the gastrointestinal tract, such as diarrhoea and ruminal drinking. The question, whether alopecia is due to formation of toxic substances or to deficiency of essential substances can not be answered.     

Inhibition of porcine circovirus type 1 and type 2 production in PK-15 cells by small interfering RNAs targeting the Rep gene

Porcine circovirus type 1 (PCV1) and type 2 (PCV2) are two genotypes of porcine circovirus. Both of them are presumed to be widespread in the swine population. Currently, there is no specific treatment for their infections. RNA interference (RNAi) is a sequence-specific RNA degradation mechanism mediated by small interfering RNA (siRNA), which represents a possible therapeutic application for the treatment of viral infections. In this study, three siRNA expression plasmids (pS-RepA, pS-RepB and pS-RepC) were generated to target three different coding regions of the Rep protein (Rep) of PCV. These siRNAs were used to inhibit PCV production in a porcine kidney cell line, PK-15 cells. Our results revealed that Rep gene expression was inhibited by pS-RepA, pS-RepB and pS-RepC to different degrees. Moreover, our study also showed that the production of PCV1 and PCV2 was reduced by these siRNAs. pS-RepC, which targets the middle region of Rep gene, proved to be the most efficient siRNA for inhibition of Rep expression and viral production. Taken together, our data suggest that RNAi could be investigated as a potential treatment for PCV infection.     

Equine influenza vaccine containing older H3N8 strains offers protection against A/eq/South Africa/4/03 (H3N8) strain in a short-term vaccine efficacy study

REASONS FOR PERFORMING STUDY: Surveillance of equine influenza viruses has suggested that strains included in currently licensed vaccines are a poor match for those predominantly circulating in the field. OBJECTIVE: To assess the ability of Duvaxyn IE-T Plus to provide cross protection against the newly evolved South Africa/4/03 (H3N8) strain of equine influenza virus. METHODS: The vaccine efficacy was evaluated by challenge infection with influenza strain A/eq/South Africa/4/03 (H3N8) 2 weeks after a primary course of 2 vaccinations with Duvaxyn IE-T Plus given at a 4-week interval. The outcome of challenge in vaccinated ponies was compared with that in unvaccinated animals. RESULTS: At the time of challenge, all vaccinated ponies had high levels of antibody to Newmarket/1/93, Newmarket/2/93 and South Africa/4/03 strains measured by single radial haemolysis. After challenge infection, there were statistically significantly decreased clinical scores and virus shedding was significantly lower in the vaccinated ponies compared to unvaccinated controls. CONCLUSION: Two doses of Duvaxyn IE-T Plus provides good clinical and virological protection against challenge with a variant virus 2 weeks after the 2 doses of vaccine. POTENTIAL RELEVANCE: When variant strains of equine influenza virus first emerge, booster immunisations with currently available vaccines may limit infection provided sufficiently high antibody levels are achieved, suggesting that vaccination in the face of an outbreak may be beneficial.     

Detection of Mycobacterium avium subsp. paratuberculosis in ovine faeces by direct quantitative PCR has similar or greater sensitivity compared to radiometric culture

The aims of this study were to develop a new real-time quantitative PCR (QPCR) assay based on IS900 for detection and quantification of Mycobacterium avium subsp. paratuberculosis (MAP) DNA in faeces, and to use this to detect infected sheep. Both the C and S strains of MAP were detected by the QPCR assay, and no cross reactions were detected with 51 other species of mycobacteria including 10 which contained IS900-like sequences. One copy of IS900 fragment cloned into plasmid pCR2.1 and 1 fg of MAP genomic DNA were consistently detected, while in spiked faecal samples the detection limit was 10 viable MAP per gram of ovine faeces. A total of 506 individual ovine faecal samples and 27 pooled ovine faecal samples with known culture results were tested. The QPCR assay detected 68 of 69 BACTEC culture positive individual faeces and there was a strong relation between time to detection in culture and DNA quantity measured by QPCR (r= -0.70). In pooled faecal samples, QPCR also agreed with culture (kappa=0.59). MAP DNA was detected from some culture negative faecal samples from sheep exposed to MAP, suggesting that the QPCR has very high analytical sensitivity for MAP in faecal samples and detects non-viable MAP in ovine faeces. None of the faecal samples from 176 sheep that were not exposed to MAP were positive in QPCR. This is the first report of a direct faecal QPCR assay that has similar sensitivity to a gold standard radiometric culture assay.