Epitope mapping of Campylobacter jejuni flagellar capping protein (FliD) by chicken (Gallus gallus domesticus) sera

Campylobacter jejuni, a Gram-negative rod, is a zoonotic pathogen associated with human acute bacterial gastroenteritis worldwide. The flagellum, composed of more than 35 proteins, is responsible for colonization of C. jejuni in the host gastrointestinal tract as well as inducing protective antibodies against the homologous serotype. In our previous study, we demonstrated that the flagellar capping protein (FliD) is an immunodominant protein that reacted strongly to sera from field chickens. In this communication, we mapped linear immunoreactive epitopes on FliD using a set of 158 synthetic peptides of 15-mer overlapping with 11 amino acid residues on peptide microarrays with sera from field chickens. The results from peptide microarrays showed (1) no cross-reactivity of the immobilized peptides with the secondary anti-chicken antibody in the control incubation, and (2) heterogeneous patterns of sera reacting to the immobilized peptides. The peptides that reacted to more than three chicken sera and had higher averages of fluorescence units were selected for further validation by the peptide ELISA. The results showed peptides 24, 91 and 92 had relatively high reactivity and less variation among 64 individual serum samples, indicating these peptides represented the shared immunodominant epitopes on the C. jejuni FliD protein. These peptides were also recognized by sera from chickens immunized with the purified recombinant FliD protein. The findings of the specific shared linear immunodominant epitopes on FliD in this study provide a rationale for further evaluation to determine their utility as epitope vaccines covering multiple serotypes for chicken immunization, and subsequently, for providing safer poultry products for human consumption.     

Characterization of the Campylobacter jejuni Population in the Barnacle Geese Reservoir

Campylobacter spp. are the most common cause of bacterial gastroenteritis worldwide and have been isolated from a wide number of different hosts and environmental sources. Waterfowl is considered a natural reservoir for this zoonotic bacterium and may act as a potential infection source for human campylobacteriosis. In this study, faecal samples from 924 barnacle geese were tested for the presence of C. jejuni and C. coli. The resulting C. jejuni and C. coli populations were characterized by multilocus sequence typing (MLST), structure analysis by BAPS and phylogenetic analysis based on full genome sequences. The prevalences of C. jejuni in barnacle geese faeces were 11.5% and 23.1% in 2011 and 2012, respectively, and only 0.2% of the samples were positive for C. coli in both years. Furthermore, a possible adaption of the clonal complexes (CCs) ST‐702 and ST‐1034 to the barnacle geese reservoir was found, as these two CCs represented the majority of the typed isolates and were repeatedly isolated from different flocks at several time‐points. Further core genome phylogenetic analysis using ClonalFrame revealed a formation of a distinct monophyletic lineage by these two CCs, suggesting a certain degree of clonality of the C. jejuni population adapted to barnacle geese. Therefore, although STs also commonly found in humans patients (e.g. ST‐45) were among the barnacle geese C. jejuni isolates, this reservoir is probably an infrequent source for human campylobacteriosis.     

Multilocus Sequence Types of Environmental Campylobacter jejuni Isolates and their Similarities to those of Human,Poultry and Bovine C. jejuni Isolates

In this study, we investigated the multilocus sequence type (MLST) diversity and population genetics of Campylobacter jejuni isolates collected from the natural waters (n = 57), wild birds (n = 37) and zoo animals (n = 19) in southern Finland, the Helsinki area and the Helsinki Zoo, respectively. On average, we found C. jejuni in 20%, 10.4% or 11.5% of the samples collected from natural waters, wild birds and zoo animals, respectively. High ST diversity was detected in all three sources and 41.2% of the STs were novel, but the multi‐host adapted ST‐45 was the most common ST detected. The MLST data, supplemented with C. jejuni isolates from domestically acquired human infections (n = 454), poultry (n = 208) and bovines (n = 120), were utilized in a population structure study. The results indicate four groups of strains with varying ecological associations, demonstrating presence of genetically distinct lineages within each of the studied sources. We discovered that the greatest ST overlap occurs between human isolates and isolates from natural waters and poultry, which suggests that the latter two are the most important sources of C. jejuni among domestically acquired infections in Finland.     

High‐Level Ciprofloxacin‐Resistant Campylobacter jejuni Isolates Circulating in Humans and Animals in Incheon,Republic of Korea

Campylobacter jejuni is one of the major causative pathogens of outbreaks or sporadic cases of diarrhoeal diseases worldwide. In this study, we compared the phenotypic and genetic characteristics of C. jejuni isolates of human and food‐producing animal origins in Korea and examined the genetic relatedness between these two groups of isolates. Regardless of isolation source, all C. jejuni isolates harboured four virulence genes, cadF, cdtB, ciaB and racR, whereas the wlaN and virB11 genes were more frequently observed in human isolates. Antimicrobial susceptibility testing showed that the majority of C. jejuni isolates displayed high‐level resistance to fluoroquinolone (95.2%) or tetracycline (76.2%) antibiotics, and 12.4% of isolates exhibited multidrug resistance (more than three classes of antibiotics tested). Pulsed‐field gel electrophoresis (PFGE) of all Campylobacter isolates revealed 51 different SmaI‐PFGE patterns and six major clusters containing both human and animal isolates. These results indicate that genetically diverse strains of C. jejuni with antimicrobial drug‐resistance and virulence properties have prevailed in Incheon. Nevertheless, some particular populations continue to circulate within the community, providing the evidence for an epidemiological link of C. jejuni infections between humans and food‐producing animals. Therefore, the continued monitoring and surveillance of C. jejuni isolates of human and food‐producing animal origins are required for public health and food safety.     

Distribution and Genotypic Characterization of Campylobacter jejuni Isolated from Poultry in Split and Dalmatia County,Croatia

Consumption of poultry contaminated with Campylobacter jejuni has been recognized worldwide as the leading cause of campylobacteriosis. Therefore, the aim of our study was to investigate the prevalence and genotype diversity of Campylobacter jejuni in poultry meat intended for consumption in Split and Dalmatia County, which is the second biggest County in Croatia. Furthermore, we also wanted to discover possibly stable clones of C. jejuni appearing in different samples and periods of time, which would indicate their ability to persist in or adapt to poultry. In the period from March 2008 until June 2010, 834 samples of poultry from various sources were examined using a surface swab technique. Isolation of C. jejuni was performed by Preston broth and Karmali agar. Identification of the isolates was carried out using biochemical tests. C. jejuni was found in 84 of 574 chicken samples (14.6%) and in nine of 260 samples of turkey (3.5%). Pulse‐field gel electrophoresis (PFGE) was used to analyse 61 obtained isolates using SmaI and KpnI. Of 22 different macrorestriction profiles (MRP) that were found, five were detected in poultry from both different locations and periods of time. Samples from 11 locations were found to be contaminated with more than two different genotypes of C. jejuni. Interestingly, the same MRP were found both in poultry declared to be of domestic origin and in the poultry imported from abroad. The prevalence of C. jejuni in poultry samples was in accordance with previously reported results. Genotypic analysis indicated that the population of C. jejuni in Split and Dalmatia County was diverse and that multiple strains of C. jejuni could be found in the same poultry samples. Furthermore, the same genotypes were identified from the samples obtained from different locations and periods of time, which could support the theory of a global existence of certain MRP that are able to persist in or adapt to poultry.     

Urban foci of murine typhus involving cat fleas (Ctenocephalides felis felis) collected from opossums in Mexico City

Murine typhus, a neglected rickettsiosis caused by Rickettsia typhi, is a common disease in several Latin‐American countries. The sylvatic life cycle of R. typhi encompasses the presence of several wild mammals, particularly opossums of the genus Didelphis and their associated fleas. Due to the colonization of wild environments by human populations, the increase in contact with opossum fleas has generated the presence of urban outbreaks of typhus. For this reason, the aim of our study was to identify the presence and diversity of Rickettsia sp. in fleas collected from opossums of an urban reserve in Mexico City. Opossums were captured from February to September 2017. For the detection of Rickettsia DNA, fragments of 800 bp of the citrate synthase (gltA) and the outer membrane protein B (ompB) were amplified. A total of 141 fleas (111 ♀, 30 ♂) of a single species (Ctenocephalides felis felis) were recovered from 31 Didelphis virginiana. Rickettsia DNA was detected in 17.7% (25/141) of the analysed fleas, recovered from seven infested opossums. The Maximum likelihood of sequences exhibited an identity of 99%–100% with sequences of R. typhi from southern United States. This work represents the first record of R. typhi in fleas from opossums in Mexico.     

Epidemiology of Campylobacter jejuni in raccoons (Procyon lotor) on swine farms and in conservation areas in southern Ontario

Campylobacter is a leading cause of foodborne illness in humans worldwide. Sources of infection are often difficult to identify, and are, generally, poorly understood. Recent work suggests that wildlife may represent a source of Campylobacter for human infections. Using a repeated cross‐sectional study design, raccoons were trapped on five swine farms and five conservation areas in southern Ontario from 2011 to 2013. Our objectives were to: (a) assess the impact of seasonal, climatic, location, annual and raccoon demographic factors on the occurrence of Campylobacter jejuni in these animals; and (b) identify clusters of C. jejuni in space, time and space‐time using spatial scan statistics. Multi‐level multivariable logistic regression was used to examine the odds of isolating C. jejuni, with site and animal modelled as random intercepts. The following independent variables were examined: raccoon age and sex, year, location type, season, temperature and rainfall. A total of 1,096 samples were obtained from 627 raccoons; 46.3% were positive for C. jejuni. The following interactions and their main effects were significant (p < .05) and retained in the final model: season × temperature, year × rainfall, year × temperature. Based on the results from our multivariable model and spatial scan statistics, climatic variables (i.e. rainfall, temperature and season) were associated with the carriage of C. jejuni by raccoons, but the effects were not consistent, and varied by location and year. Although raccoons may pose a zoonotic risk due to their carriage of Campylobacter, further work is required to characterize the transmission and movement of this microorganism within the ecosystem.     

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.     

Distribution and Diversity of Salmonella Strains in Shipments of Hatchling Poultry,United States, 2013

Multistate outbreaks of salmonellosis associated with live poultry contact have been occurring with increasing frequency. In 2013, multistate outbreaks of salmonellosis were traced back to exposure to live poultry, some of which were purchased at a national chain of farm stores (Farm store chain Y). This study was conducted at 36 stores of Farm store chain Y and was concurrent with the timing of exposure for the human outbreaks of salmonellosis in 2013. We used environmental swabs of arriving shipment boxes of hatchling poultry and shipment tracking information to examine the distribution, diversity and anti‐microbial resistance of non‐typhoidal Salmonella (NTS) across farm stores and hatcheries. Isolates recovered from shipment boxes underwent serotyping, anti‐microbial resistance (AMR) testing and pulsed‐field gel electrophoresis (PFGE). Postal service tracking codes from the shipment boxes were used to determine the hatchery of origin. The PFGE patterns were compared with the PFGE patterns of NTS causing outbreaks of salmonellosis in 2013. A total of 219 hatchling boxes from 36 stores in 13 states were swabbed between 15 March 2013 and 18 April 2013. NTS were recovered from 59 (27%) of 219 hatchling boxes. Recovery was not significantly associated with species of hatchlings, number of birds in the shipment box, or the presence of dead, injured or sick birds. Four of the 23 PFGE patterns and 23 of 50 isolates were indistinguishable from strains causing human outbreaks in 2013. For serotypes associated with human illnesses, PFGE patterns most frequently recovered from shipment boxes were also more frequent causes of human illness. Boxes positive for the same PFGE pattern most frequently originated from the same mail‐order hatchery. Only one of 59 isolates was resistant to anti‐microbials used to treat Salmonella infections in people. This study provides critical information to address recurrent human outbreaks of salmonellosis associated with mail‐order hatchling poultry.     

First detection of Hepatitis E virus (Orthohepevirus C) in wild brown rats (Rattus norvegicus) from Great Britain

In the United Kingdom, there has been an increase in the number of hepatitis E virus (HEV) infections in people annually since 2010. Most of these are thought to be indigenously acquired Orthohepevirus A genotype 3 (HEV G3), which has been linked to pork production and consumption. However, the dominant subgroup circulating in British pigs differs from that which is found in people; therefore, an alternative, potentially zoonotic, source is suspected as a possible cause of these infections. Rodents, brown rats (Rattus norvegicus) in particular, have been shown to carry HEV, both the swine HEV G3 genotype and Orthohepevirus C, genotype C1 (rat HEV). To investigate the prevalence of HEV in British rodents, liver tissue was taken from 307 rodents collected from pig farms (n = 12) and other locations (n = 10). The RNA from these samples was extracted and tested using a pan‐HEV nested RT‐PCR. Limited histopathology was also performed. In this study, 8/61 (13%, 95% CI, 5–21) of brown rat livers were positive for HEV RNA. Sequencing of amplicons demonstrated all infections to be rat HEV with 87%–92% nucleotide identity to other rat HEV sequences circulating within Europe and China (224 nt ORF‐1). Lesions and necrosis were observed histologically in 2/3 samples examined. No rat HEV RNA was detected in any other species, and no HEV G3 RNA was detected in any rodent in this study. This is the first reported detection of rat HEV in Great Britain. A human case of rat HEV infection has recently been reported in Asia, suggesting that rat HEV could pose a risk to public health.     

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.     

Changes in the prevalence of Salmonella serovars associated swine production and correlations of avian,bovine and swine‐associated serovars with human‐associated serovars in the United States (1997–2015)

As Salmonella enterica is an important pathogen of food animals, surveillance programmes for S. enterica serovars have existed for many years in the United States. Surveillance programmes serve many purposes, one of which is to evaluate alterations in the prevalence of serovars that may signal changes in the ecology of the target organism. The primary aim of this study was to evaluate changes in the proportion of S. enterica serovars isolated from swine over a near 20‐year observation period (1997–2015) using four longitudinal data sets from different food animal species. The secondary aim was to evaluate correlations between changes in S. enterica serovars frequently recovered from food animals and changes in S. enterica serovars associated with disease in humans. We found decreasing proportions of S. enterica serovar Typhimurium, serovar Derby and serovar Heidelberg and increasing proportions of S. enterica serovar 4,[5],12:i:‐, serovar Infantis and serovar Johannesburg in swine over time. We also found positive correlations for the yearly changes in S. enterica serovar 4,[5],12:i:‐, serovar Anatum and serovar Johannesburg between swine and human data; in S. enterica Worthington between avian and human data; and in S. enterica serovar 4,[5],12:i:‐ between bovine and human data. We found negative correlations for the yearly changes in S. enterica serovar 4,[5],12:i:‐ and serovar Johannesburg between avian and human data.     

Autochthonous Chagas disease in the southern United States: A case report of suspected residential and military exposures

Chagas disease is a parasitic infection that can result in a progressive dilated cardiomyopathy. Here, we present the epidemiologic details of a suspected locally acquired transmission case originating from the southern United States. This is the first published report of Chagas disease in a young, healthy United States veteran with repeat triatomine exposures in Arizona. Military personnel and Arizona residents should be aware of their Chagas disease transmission risks.     

Epidemiology of Salmonella on the Paws and in the Faeces of Free‐Ranging Raccoons (Procyon Lotor) in Southern Ontario,Canada

Raccoons are common in urban and rural environments and can carry a wide range of bacteria, including Salmonella, that can negatively affect human and livestock health. Although previous studies have reported that raccoons shed a variety of Salmonella serovars in their faeces, it is unknown whether Salmonella is carried on raccoon paws. Our objective was to compare the prevalence of Salmonella on the paws and in the faeces of raccoons in south‐western Ontario. Raccoons were sampled in a repeat cross‐sectional study on five swine farms and five conservation areas from May to October 2012. A total of 416 paired faecal and paw samples were collected from 285 individual raccoons. Salmonella was detected in 18% (75/416; 95% CI, 14–22%) and 27% (111/416; 95% CI, 22–31%) of paw and faecal samples, respectively. Salmonella was detected only on paws in 8% (35/416; 95% CI, 5.9–11.5%), only in faeces in 17% (71/416; 95% CI, 13.6–21.0%) and on both paws and in faeces in 10% (40/416; 95% CI, 7.0–12.9%) of raccoon captures. Multilevel logistic regression models were used to examine associations between the presence of Salmonella and age (adult, juvenile), sex (male, female), location type (swine farm, conservation area), sample type (faeces, paw) and season (May–July and August–October). Random intercepts were included to account for clustering by individual animal and location. Significant differences, that varied by sample type and season, were noted in the prevalence of Salmonella carriage between sexes. Raccoons can carry Salmonella serovars known to infect humans and livestock on their paws and/or in their faeces and therefore have the potential to mechanically and biologically disseminate Salmonella among livestock facilities and human recreational areas.     

Randomized control trial to test the effect of a feed additive on Campylobacter contamination in commercial broiler flocks up to slaughter

A randomized controlled trial (RCT) was carried to evaluate the effect of a feed additive on Campylobacter contamination of broilers reared in commercial conditions. Twenty‐four broiler flocks naturally contaminated with Campylobacter were enrolled in the RCT: 12 were assigned to a control group (C) fed with a conventional finishing feed from 4 weeks of age to slaughter (around 35 days), and the other group of 12 flocks (S) was fed with a finishing feed supplemented with 250 ppm of a patented feed additive (an ion‐exchanged clay compound) previously proven to reduce Campylobacter contamination in broiler caeca under experimental conditions. Enumeration of Campylobacter colonies in caeca (8 per flock) was carried out following ISO standards before feed distribution and at slaughter. Before treatment, the caecal Campylobacter load tended to be lower in C flocks (7.1 ± 1.9 log CFU/g, CI_95% [6.6–7.5]) than in S flocks (7.7 ± 1.0 log UFC/g, CI_95% [7.5–7.9]) (p = .05). At slaughter, the bacterial load was similar in the S (7.7 ± 1.0 log CFU/g, CI_95% [7.5–7.9]) and C groups (7.5 ± 1.2 log CFU/g, CI_95% [7.2–7.8]) (p = .73). Therefore, the feed additive had no significant effect on the caecal Campylobacter load at slaughter under the tested conditions. The logistical constraints inherent in field trials and the natural variability of Campylobacter contamination in naturally infected broiler flocks make it difficult to reproduce experimental results in in situ farm conditions. RCT testing of an intervention strategy in commercial situation is therefore a key step in evaluating pre‐harvest interventions against food‐borne pathogens.     

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.