Molecular Characterization by LSSP‐PCR and DNA Sequencing of a Pathogenic Isolate of Leptospira interrogans from Brazil

We report the initial characterization of a leptospiral isolate, Leptospira interrogans, serogroup Sejroe, serovar Hardjo, genotype Hardjoprajitno, strain Norma, and its relatedness with L. interrogans, serogroup Sejroe, serovar Hardjo, genotype Hardjoprajitno, strain Hardjo and Leptospira borgpetersenii, serogroup Sejroe, serovar Hardjo, genotype Hardjobovis, strain Sponselee. The Norma strain singled out during a leptospirosis outbreak in cattle immunized with antigens from the reference strain Hardjoprajitno (OMS). By applying a microscopic agglutination serological test (MAT) to cattle (n = 2966) with symptoms of leptospirosis between 2003 and 2007, more than 50% of sera were found positive for one of the following serotypes: Hardjoprajitno (31–21%), Hardjo Norma (46–40%), Hardjo hardjobovis (18–10%), Mini (8–4%) and Wolffi (7–4%). In immunization trials using six isolates plus Norma isolate, the remission of MAT in these isolates was observed following 6 months of the initial vaccination. To provide molecular ground for the high MAT Norma frequency found in these isolates, a DNA polymorphic analysis was conducted by comparing the Norma isolate with reference strains Hardjoprajitno and Sponselee. The polymorphic analysis in secY showed five base changes in Norma relative to Hardjoprajitno strain, corresponding to 98% identity, while Sponselee displayed 49 polymorphic sites relative to the Hardjoprajitno strain, representing 80% identity. The alignment of secY translated sequences shows no differences between Hardjoprajitno and Norma, and eight polymorphisms between genotype hardjoprajttno and strain Sponselee. Three‐dimensional modelling located these variations within the loop region connecting helices 7 and 8 from secY which is less conserved. DNA sequencing of 23S ribosomal conserved fragment revealed a single polymorphism between Hardjoprajitno and Norma, and 13 polymorphisms between strains Sponselee, Hardjoprajitno and Norma. The differences between Hardjo and Norma were confirmed by low stringency single‐specific primer polymerase chain reaction (LSSP‐PCR) signature experiments with the primer G2, using as template the 285 bp fragment initially amplified with G1/G2 primers.     

The devil is in the details—Host disease and co‐infections are associated with zoonotic pathogen carriage in Norway rats (Rattus norvegicus)

Traditionally, zoonotic pathogen ecology studies in wildlife have focused on the interplay among hosts, their demographic characteristics and their pathogens. But pathogen ecology is also influenced by factors that traverse the hierarchical scale of biological organization, ranging from within‐host factors at the molecular, cellular and organ levels, all the way to the host population within a larger environment. The influence of host disease and co‐infections on zoonotic pathogen carriage in hosts is important because these factors may be key to a more holistic understanding of pathogen ecology in wildlife hosts, which are a major source of emerging infectious diseases in humans. Using wild Norway rats (Rattus norvegicus) as a model species, the purpose of this study was to investigate how host disease and co‐infections impact the carriage of zoonotic pathogens. Following a systematic trap and removal study, we tested the rats for the presence of two potentially zoonotic bacterial pathogens (Bartonella tribocorum and Leptospira interrogans) and assessed them for host disease not attributable to these bacteria (i.e., nematode parasites, and macroscopic and microscopic lesions). We fitted multilevel multivariable logistic regression models with pathogen status as the outcome, lesions and parasites as predictor variables and city block as a random effect. Rats had significantly increased odds of being infected with B. tribocorum if they had a concurrent nematode infection in one or more organ systems. Rats with bite wounds, any macroscopic lesion, cardiomyopathy or tracheitis had significantly increased odds of being infected with L. interrogans. These results suggest that host disease may have an important role in the ecology and epidemiology of rat‐associated zoonotic pathogens. Our multiscale approach to assessing complex intrahost factors in relation to zoonotic pathogen carriage may be applicable to future studies in rats and other wildlife hosts.     

Leptospira detection in flood‐prone environment of Jakarta,Indonesia

The study about Leptospira, particularly pathogenic strain, was conducted in the flood‐prone area in the Special Capital Region of Jakarta. The aim of this study was to discover and identify the serovars of pathogenic Leptospira in the environment, which might infect human during flooding. Seventy‐three samples, consisted of 36 samples of environmental water and 37 samples of soil, were collected from 5 districts of Jakarta. Their pH was measured, and the samples were then cultured in a modified Korthof's medium with 5‐fluorouracil (5‐FU) addition. Polymerase chain reaction, targeted on 23S rDNA (rrl), FlaB and LipL32 genes, was performed to identify Leptospira genus and differentiate the pathogenic. Identification of the pathogenic Leptospira was done utilising DNA sequencing. Seven samples showed amplification of rrl‐gene. flaB and lipl32‐PCR assay indicated one positive amplification band, each. Confirmation of flaB and lipl32 amplicons by DNA sequencing and BLAST analysis showed flaB amplicon (G1B; GenBank accession number MK006031.1 ) had 94% similarity with L. licerasiae ( LC005426.1 ), while lipl32 amplicon was not identified as lipl32 of Leptospira. Based on those results, one intermediate pathogenic and six saprophytic Leptospira were obtained from the environment in Jakarta.     

Conjunctival habronemiasis in a square‐lipped rhinoceros (Ceratotherium simum)

A captive female square‐lipped rhinoceros born in 1993 had been showing intermittent signs of bilateral conjunctivitis and conjunctival proliferation since 1998. Periodic improvement was noted, especially in winter, but overall the condition had deteriorated over the years. Treatment with various topical, intralesional, and systemic antibiotics and glucocorticosteroids was largely ineffective, as were repeated dewormings. No primary cause for these lesions was found in biopsies taken in 2000 and 2006, although a severe infiltrate of numerous eosinophils was observed in the latter. As the condition worsened, secondary corneal changes were noted, and eventually vision was lost due to proliferative conjunctival tissue. Aggressive resection of the proliferating tissue in 2013 restored vision and submitted biopsies yielded a diagnosis of severe allergic conjunctivitis, eosinophilic granuloma, and habronematid (Habronema or Draschia) larval infection. As no other rhinoceros in the herd was affected, including two calves born to the patient who were in close contact with their mother, it was concluded the presentation was most likely due to a hypersensitivity reaction to the dead or dying larvae. Fly repellent is now regularly applied around the eye of this rhinoceros, and a protective face mask has been fitted. Ongoing periodic relapses are treated with oral ivermectin, topical antibiotics, and steroids.     

Canine spinal meningiomas and nerve sheath tumours in 34 dogs (2008‐2016): Distribution and long‐term outcome based upon histopathology and treatment modality

The purpose of this retrospective, multicentre case series was to describe the outcome following surgery and/or radiation of spinal meningiomas and nerve sheath tumours (NSTs) based upon treatment modality, with a specific aim to evaluate the survival times and time to recurrence following treatment for each histopathological diagnosis. Our hypothesis was that the addition of radiation therapy modalities to treatment will yield longer time to recurrence of clinical signs and survival time. Thirty‐four dogs met the inclusion criteria of histopathologically diagnosed extramedullary spinal meningioma or NST. Sixteen extramedullary spinal meningiomas and 18 NSTs were diagnosed. A diagnosis of meningioma was associated with a significantly longer survival time compared with NSTs, with median survival times (MST) of 508 days (95% confidence interval [CI]: 66‐881) vs 187 days (95% CI: 76‐433; P = .02). Dogs (seven) treated with stereotactic radiation therapy (SRT) for recurrence after surgery alone or SRT alone as their initial treatment gained an additional 125 to 346 days survival time.     

Meat Juice Serology and Improved Food Chain Information as Control Tools for Pork‐Related Public Health Hazards

The seroprevalence of Salmonella spp., pathogenic Yersinia spp., Toxoplasma gondii and Trichinella spp. was studied in 1353 finishing pigs from 259 farms that were allocated according to farm types: large fattening farms (≥1000 pig places), small fattening farms (< 1000 pig places) and farrow‐to‐finish farms. The antibodies were analysed with commercial ELISA kits in meat juice samples that were collected at Finnish slaughterhouses. Salmonella antibodies were rare (3% of pigs, 14% of farms) when the cut‐off optical density (OD) value 0.2 was used. Antibodies to pathogenic Yersinia spp. and T. gondii were detected in 57% of pigs and 85% of farms (OD ≥0.3) and in 3% of pigs and 9% of farms (OD ≥0.15), respectively. No antibodies to Trichinella spp. were detected (OD ≥0.3). The European Food Safety Authority (EFSA) considers Salmonella spp., Yersinia enterocolitica, T. gondii and Trichinella spp. as the most relevant biological hazards in the context of meat inspection of pigs. The seroprevalence of these important zoonotic pathogens was low in Finland, except that of Yersinia. The seroprevalence of Toxoplasma was significantly higher in pigs originating from small‐scale fattening farms (P < 0.05). Strong positive correlation was observed at the animal level between Salmonella and Yersinia seropositivity and between Salmonella and Toxoplasma seropositivity (P < 0.05). We suggest that these results reflect the level and importance of biosecurity measures applied on the farms. Meat juice serology at slaughter is a useful tool for targeting measures to control these pathogens. The information obtained from analyses should be used as part of the food chain information (FCI).     

Phenotypic Resistance to Disinfectants and Antibiotics in Methicillin‐Resistant Staphylococcus aureus Strains Isolated from Pigs

The aim of this research was to study the phenotypic resistances to disinfectants and antibiotics in strains of methicillin‐resistant Staphylococcus aureus (MRSA) obtained from Canary black pigs. Analyses were performed on 54 strains of MRSA, isolated in Canary black pigs from the province of Tenerife (Spain); all of them carried the mecA gene. The strains were isolated by means of nasal swab samples of healthy pigs, collected under veterinarian supervision. Bactericidal activity of antiseptics and disinfectants was tested by means of the dilution–neutralization method. Susceptibility to the disinfectants glutaraldehyde, peracetic acid and silver nitrate was assessed, as well as to the antiseptics chlorhexidine, benzalkonium chloride and povidone iodine. Susceptibility to a wide array of antibiotics representing the main groups was determined by means of the disc diffusion method. All the strains demonstrated susceptibility to the disinfectants tested at the recommended concentration, and even to dilutions equal to or lesser than 1/16. The most effective antiseptic and disinfectant were, respectively, chlorhexidine and silver nitrate. With regard to the antibiotics, the strains proved to be multiresistant. All presented phenotypic resistance to the β‐lactam antibiotics ampicillin, penicillin and cefoxitin, as well as to numerous aminoglycosides, tetracycline and trimethoprim–sulfamethoxazole. It was also observed that 61.1% of the strains were carriers of plasmids. Our results underline that in the strains such as MRSA, which show multiple resistances to antibiotics, the antiseptics and disinfectants show great efficacy. Moreover, as other authors also suggest, for the treatment and prevention of infections caused by MRSA, the use of β‐lactam and aminoglycoside antibiotics may be less effective.     

Isolation of a Campylobacter lanienae‐like Bacterium from Laboratory Chinchillas (Chinchilla laniger)

Routine necropsies of 27 asymptomatic juvenile chinchillas revealed a high prevalence of gastric ulcers with microscopic lymphoplasmacytic gastroenteritis and typhlocolitis. Polymerase chain reaction (PCR) analysis using Campylobacter genus‐specific partial 16S rRNA primers revealed the presence of Campylobacter spp. DNA in the faeces of 12 of 27 animals (44.4%). Species‐specific partial 16S rRNA PCR and sequencing confirmed that these animals were colonized with Campylobacter lanienae, a gram‐negative, microaerophilic bacterium that was first identified on routine faecal screening of slaughterhouse employees and subsequently isolated from faeces of livestock. Campylobacter lanienae was isolated from the faeces of six PCR‐positive animals and identified with species‐specific PCR and full 16S rRNA sequencing. Phylogenetic analysis showed that these isolates clustered with C. lanienae strain NCTC 13004. PCR analysis of DNA extracted from gastrointestinal tissues revealed the presence of C. lanienae DNA in the caecum and colon of these chinchillas. Gastrointestinal lesions were scored and compared between C. lanienae‐positive and C. lanienae‐negative animals. There was no correlation between colonization status and lesion severity in the stomach, liver, duodenum, or colon. Possible routes of C. lanienae infection in chinchillas could include waterborne transmission and faecal–oral transmission from wild mice and rats or livestock. Based on these findings, the authors conclude that C. lanienae colonizes the lower bowel of chinchillas in the absence of clinical disease. This is the first report of C. lanienae in any rodent species. Campylobacter lanienae isolates from different mammalian species demonstrate heterogeneity by 16S rRNA sequence comparison. Analysis using rpoB suggests that isolates and clones currently identified as C. lanienae may represent multiple species or subspecies.     

Aortic rupture causing cardiac tamponade in a 24‐day‐old Friesian colt with concurrent colonic Chlamydiosis and Balantidiasis

A 24‐day‐old Friesian colt died suddenly and a physical examination the morning the foal died showed no abnormalities and serum IgG levels >8.0 g/l. Necropsy examination revealed haemopericardium and a 2 cm transverse tear at the root of the aorta. The foal was also found to have Chlamydophila spp. in the epithelium and Balantidium coli on the mucosal surface of the large colon. An aortic rupture is a novel finding in a foal, colonic Chlamydiosis has not been previously reported in horses and Balantidium coli has not been reported in equids in North America.     

The occurrence of Salmonella,extended‐spectrum β‐lactamase producing Escherichia coli and carbapenem resistant non‐fermenting Gram‐negative bacteria in a backyard poultry flock environment

Increase in the number of small‐scale backyard poultry flocks in the USA has substantially increased human‐to‐live poultry contact, leading to increased public health risks of the transmission of multi‐drug resistant (MDR) zoonotic and food‐borne bacteria. The objective of this study was to detect the occurrence of Salmonella and MDR Gram‐negative bacteria (GNB) in the backyard poultry flock environment. A total of 34 backyard poultry flocks in Washington State (WA) were sampled. From each flock, one composite coop sample and three drag swabs from nest floor, waterer‐feeder, and a random site with visible faecal smearing, respectively, were collected. The samples were processed for isolation of Salmonella and other fermenting and non‐fermenting GNB under ceftiofur selection. Each isolate was identified to species level using MALDI‐TOFF and tested for resistance against 16 antibiotics belonging to eight antibiotic classes. Salmonella serovar 1,4,[5],12:i:‐ was isolated from one (3%) out of 34 flocks. Additionally, a total of 133 ceftiofur resistant (Cef^R) GNB including Escherichia coli (53), Acinetobacter spp. (45), Pseudomonas spp. (22), Achromobacter spp. (8), Bordetella trematum (1), Hafnia alvei (1), Ochrobactrum intermedium (1), Raoultella ornithinolytica (1), and Stenotrophomonas maltophilia (1) were isolated. Of these, 110 (82%) isolates displayed MDR. Each flock was found positive for the presence of one or more Cef^R GNB. Several MDR E. coli (n = 15) were identified as extended‐spectrum β‐lactamase (ESBL) positive. Carbapenem resistance was detected in non‐fermenting GNB including Acinetobacter spp. (n = 20), Pseudomonas spp. (n = 11) and Stenotrophomonas maltophila (n = 1). ESBL positive E. coli and carbapenem resistant non‐fermenting GNB are widespread in the backyard poultry flock environment in WA State. These GNB are known to cause opportunistic infections, especially in immunocompromised hosts. Better understanding of the ecology and epidemiology of these GNB in the backyard poultry flock settings is needed to identify potential risks of transmission to people in proximity.     

Comparatively examining of the apelin‐13 levels in the Capoeta trutta (Heckel, 1843) and Cyprinus carpio (Linnaeus, 1758)

Apelin is a recently discovered peptide produced by several tissues in the various vertebrates and fish. Apelin has been suggested to have role in regulation of many diverse physiological functions including food intake, energy homoeostasis, immunity, osmoregulation and reproduction. In this study, apelin‐13 levels in the blood serum of Cyprinus carpio and Capoetta trutta were determined. Then the results were compared between two species and sexes of each species. Apelin‐13 level was analysed using the enzyme‐linked immunoassay (ELISA) kit (Rat apelin‐13 ELISA kit, catalog no: CSB‐E14367r). Apelin‐13 level in the blood serum of C. trutta was significantly higher than those of the C. carpio (p < 0.05). However, its levels were observed to be no significant difference (p > 0.05) that compared to between sexes of each species. There was a significant negative correlation (r = ?0.829, p = 0.0001) between the apelin‐13 level and body weight of C. carpio. However, no significant correlation (r = ?0.022, p = 0.924) between the apelin‐13 level and weight of C. trutta observed.     

Hepatitis E virus in Common voles (Microtus arvalis) from an urban environment,Hungary: Discovery of a Cricetidae‐specific genotype of Orthohepevirus C

Hepatitis E virus is a major causative agent of acute hepatitis worldwide. Despite its zoonotic potential, there is limited information about the natural chain of hepevirus infection in wildlife, and the potential reservoir species. In this study, we performed a HEV survey by heminested RT‐PCR on rodent samples from an urban environment (in the city of Pécs, Hungary) and investigated the prevalence of the virus among these native rodent species (Apodemus agrarius, Apodemus flavicollis, Apodemus sylvaticus, Microtus arvalis and Myodes glareolus). HEV was detected exclusively in Common voles (M. arvalis), in 10.2% of screened voles, and 3.2% of all investigated samples from all species. Based on the phylogenetic analysis, our strain showed the closest homology with European Orthohepevirus C strains detected previously in faecal samples of birds of prey and Red fox, supporting the possibility of the dietary origin of these strains. In addition, our samples showed close phylogenetic relation with a South American strain detected in Necromys lasiurus (Cricetidae), but separated clearly from other Muridae‐associated strains, suggesting the presence of a Cricetidae‐specific genotype in Europe and South‐America. Based on these results, we hypothesize the reservoir role of M. arvalis rodents for the European Cricetidae‐specific Orthohepevirus C genotype.     

Diversity of Plasmids and Antimicrobial Resistance Genes in Multidrug‐Resistant Escherichia coli Isolated from Healthy Companion Animals

The presence and transfer of antimicrobial resistance genes from commensal bacteria in companion animals to more pathogenic bacteria may contribute to dissemination of antimicrobial resistance. The purpose of this study was to determine antimicrobial resistance gene content and the presence of genetic elements in antimicrobial resistant Escherichia coli from healthy companion animals. In our previous study, from May to August, 2007, healthy companion animals (155 dogs and 121 cats) from three veterinary clinics in the Athens, GA, USA area were sampled and multidrug‐resistant E. coli (n = 36; MDR, resistance to ≥2 antimicrobial classes) were obtained. Of the 25 different plasmid replicon types tested by PCR, at least one plasmid replicon type was detected in 94% (34/36) of the MDR E. coli; four isolates contained as many as five different plasmid replicons. Nine replicon types (FIA, FIB, FII, I2, A/C, U, P, I1 and HI2) were identified with FIB, FII, I2 as the most common pattern. The presence of class I integrons (intI) was detected in 61% (22/36) of the isolates with eight isolates containing aminoglycoside‐ and/or trimethoprim‐resistance genes in the variable cassette region of intI. Microarray analysis of a subset of the MDR E. coli (n = 9) identified the presence of genes conferring resistance to aminoglycosides (aac, aad, aph and strA/B), β‐lactams (ampC, cmy, tem and vim), chloramphenicol (cat), sulfonamides (sulI and sulII), tetracycline [tet(A), tet(B), tet(C), tet(D) and regulator, tetR] and trimethoprim (dfrA). Antimicrobial resistance to eight antimicrobials (ampicillin, cefoxitin, ceftiofur, amoxicillin/clavulanic acid, streptomycin, gentamicin, sulfisoxazole and trimethoprim‐sulfamethoxazole) and five plasmid replicons (FIA, FIB, FII, I1 and I2) were transferred via conjugation. The presence of antimicrobial resistance genes, intI and transferable plasmid replicons indicate that E. coli from companion animals may play an important role in the dissemination of antimicrobial resistance, particularly to human hosts during contact.