Comparative Genomics of 42 Arcanobacterium phocae Strains.

For the last 13 years, the fur industry in Europe has suffered from epidemic spouts of a severe necrotizing pyoderma. It affects all species currently farmed for fur and causes animal welfare problems and significant losses to the farmers. The causative agent of this disease was identified as Arcanobacterium phocae. Previously, this bacterium has been isolated from seals and other marine mammals, apparently causing wound and lung infections. Attempts at antibiotic treatment have been unsuccessful and the current advice on preventing the disease is to cull all animals with clinical signs. This poses an urgent question regarding possible vaccine development, as well as the need for further understanding of the pathogenicity of this organism. This study compared the whole genomes of 42 A. phocae strains isolated from seals, blue foxes, finnraccoons, mink and otter. The sequences were created using the Illumina technology and annotations were done using the RAST pipeline. A phylogenetic analysis identified a clear separation between the seal strains and the fur-animal-derived isolates, but also indicated that the bacterium readily adapts to new environments and host species with reasonable diversity. A pan- and core-genome was created and analyzed for proteins. A further analysis identified several virulence factors as well as multiple putative and secreted proteins of special interest for vaccine development.

Comparison of Streptococcus halichoeri isolates from canine and fur animal infections: biochemical patterns, molecular characteristics and genetic relatedness.

BACKGROUND: Streptococcus halichoeri infections have been reported in grey seals, a European badger, a Stellar sea lion and humans, but its presence in companion and fur animals is unknown. Since 2010, S. halichoeri-like bacteria (SHL) have been isolated from fur animals and dogs in Finland. Our aim was to retrospectively investigate laboratory records for SHL from canine and fur animal infections, characterize the isolates and compare their genetic relatedness in relation to three reference strains: CCUG 48324T, originating from a grey seal, and strains 67100 and 61265, originally isolated from humans. RESULTS: A total of 138 and 36 SHLs from canine and fur animal infections, respectively, were identified in the laboratory records. SHL was commonly associated with skin infections, but rarely as the only species. A set of 49 canine and 23 fur animal SHLs were further characterized. MALDI-TOF confirmed them as being S. halichoeri. The growth characteristics were consistent with the original findings, but isolates were catalase positive. In total, 17 distinct API 20 Strep patterns were recorded among all 75 isolates tested, of which pattern 5563100 was the most common (n = 30). Antimicrobial resistance to erythromycin and clindamycin was common in canine isolates, but rare in fur animal isolates. Three clusters were observed by PFGE, and 16S rRNA sequencing revealed 98.1-100% similarities with the human strains and 98.1-99.5% with the seal strain. A phylogenetic tree of concatenated 16S rRNA and rpoB revealed closely related isolates with two clades. Fifteen canine isolates were identical to the human strains based on concatenated 16S rRNA and rpoB sequencing. CONCLUSIONS: Streptococcus halichoeri appears to be quite a common bacterial species in the skin of dogs and fur animals. The clinical significance of S. halichoeri is uncertain, as it was rarely isolated as a monoculture. No apparent temporal or spatial clustering was detected, but isolates from different sources were genetically very similar. Because many canine isolates were genetically similar to the human reference strains, transmission between dogs and humans may be possible. WGS sequencing of strains from different sources is needed to further investigate the epidemiology and virulence of S. halichoeri.

Streptococcus halichoeri: Comparative Genomics of an Emerging Pathogen.

Streptococcus halichoeri is an emerging pathogen with a variety of host species and zoonotic potential. It has been isolated from grey seals and other marine mammals as well as from human infections. Beginning in 2010, two concurrent epidemics were identified in Finland, in fur animals and domestic dogs, respectively. The fur animals suffered from a new disease fur animal epidemic necrotic pyoderma (FENP) and the dogs presented with ear infections with poor treatment response. S. halichoeri was isolated in both studies, albeit among other pathogens, indicating a possible role in the disease etiologies. The aim was to find a possible common origin of the fur animal and dog isolates and study the virulence factors to assess pathogenic potential. Isolates from seal, human, dogs, and fur animals were obtained for comparison. The whole genomes were sequenced from 20 different strains using the Illumina MiSeq platform and annotated using an automatic annotation pipeline RAST. The core and pangenomes were formed by comparing the genomes against each other in an all-against-all comparison. A phylogenetic tree was constructed using the genes of the core genome. Virulence factors were assessed using the Virulence Factor Database (VFDB) concentrating on the previously confirmed streptococcal factors. A core genome was formed which encompassed approximately half of the genes in Streptococcus halichoeri. The resulting core was nearly saturated and would not change significantly by adding more genomes. The remaining genes formed the pangenome which was highly variable and would still evolve after additional genomes. The results highlight the great adaptability of this bacterium possibly explaining the ease at which it switches hosts and environments. Virulence factors were also analyzed and were found primarily in the core genome. They represented many classes and functions, but the largest single category was adhesins which again supports the marine origin of this species.

Population Genetics and Characterization of Campylobacter jejuni Isolates from Western Jackdaws and Game Birds in Finland.

Poultry are considered a major reservoir and source of human campylobacteriosis, but the roles of environmental reservoirs, including wild birds, have not been assessed in depth. In this study, we isolated and characterized Campylobacter jejuni from western jackdaws (n = 91, 43%), mallard ducks (n = 82, 76%), and pheasants (n = 9, 9%). Most of the western jackdaw and mallard duck C. jejuni isolates represented multilocus sequence typing (MLST) sequence types (STs) that diverged from those previously isolated from human patients and various animal species, whereas all pheasant isolates represented ST-19, a common ST among human patients and other hosts worldwide. Whole-genome MLST revealed that mallard duck ST-2314 and pheasant ST-19 isolates represented bacterial clones that were genetically highly similar to human isolates detected previously. Further analyses revealed that in addition to a divergent ClonalFrame genealogy, certain genomic characteristics of the western jackdaw C. jejuni isolates, e.g., a novel cdtABC gene cluster and the type VI secretion system (T6SS), may affect their host specificity and virulence. Game birds may thus pose a risk for acquiring campylobacteriosis; therefore, hygienic measures during slaughter and meat handling warrant special attention.IMPORTANCE The roles of environmental reservoirs, including wild birds, in the molecular epidemiology of Campylobacter jejuni have not been assessed in depth. Our results showed that game birds may pose a risk for acquiring campylobacteriosis, because they had C. jejuni genomotypes highly similar to human isolates detected previously. Therefore, hygienic measures during slaughter and meat handling warrant special attention. On the contrary, a unique phylogeny was revealed for the western jackdaw isolates, and certain genomic characteristics identified among these isolates are hypothesized to affect their host specificity and virulence. Comparative genomics within sequence types (STs), using whole-genome multilocus sequence typing (wgMLST), and phylogenomics are efficient methods to analyze the genomic relationships of C. jejuni isolates.

Etiology of acute respiratory disease in fattening pigs in Finland.

BACKGROUND: The objective of our study was to clinically and etiologically investigate acute outbreaks of respiratory disease in Finland. Our study also aimed to evaluate the clinical use of various methods in diagnosing respiratory infections under field conditions and to describe the antimicrobial resistance profile of the main bacterial pathogen(s) found during the study. METHODS: A total of 20 case herds having finishing pigs showing acute respiratory symptoms and eight control herds showing no clinical signs suggesting of respiratory problems were enrolled in the study. Researchers visited each herd twice, examining and bleeding 20 pigs per herd. In addition, nasal swab samples were taken from 20 pigs and three pigs per case herd were necropsied during the first visit. Serology was used to detect Actinobacillus pleuropneumoniae (APP), swine influenza virus (SIV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine respiratory coronavirus (PRCV) and Mycoplasma hyopneumoniae antibodies. Polymerase chain reaction (PCR) was used to investigate the presence of porcine circovirus type 2 (PCV2) in serum and SIV in the nasal and lung samples. Pathology and bacteriology, including antimicrobial resistance determination, were performed on lung samples obtained from the field necropsies. RESULTS: According to the pathology and bacteriology of the lung samples, APP and Ascaris suum were the main causes of respiratory outbreaks in 14 and three herds respectively, while the clinical signs in three other herds had a miscellaneous etiology. SIV, APP and PCV2 caused concurrent infections in certain herds but they were detected serologically or with PCR also in control herds, suggesting possible subclinical infections. APP was isolated from 16 (80%) case herds. Marked resistance was observed against tetracycline for APP, some resistance was detected against trimethoprim/sulfamethoxazole, ampicillin and penicillin, and no resistance against florfenicol, enrofloxacin, tulathromycin or tiamulin was found. Serology, even from paired serum samples, gave inconclusive results for acute APP infection diagnosis. CONCLUSIONS: APP was the most common cause for acute respiratory outbreaks in our study. SIV, A. suum, PCV2 and certain opportunistic bacteria were also detected during the outbreaks; however, viral pathogens appeared less important than bacteria. Necropsies supplemented with microbiology were the most efficient diagnostic methods in characterizing the studied outbreaks.

Experimental Infection of Mink Enforces the Role of Arcanobacterium phocae as Causative Agent of Fur Animal Epidemic Necrotic Pyoderma (FENP).

Fur Animal Epidemic Necrotic Pyoderma (FENP) is a severe, often lethal infectious disease affecting all three fur animal species: mink (Neovision vision), foxes (Vulpes lagopus) and finnraccoons (Nyctereutes procyonoides). Previous studies showed an association between Arcanobacterium phocae and FENP. An experimental infection was conducted to confirm the ability of A. phocae to infect mink either alone or concurrently with a novel Streptococcus sp. found together with A. phocae in many cases of FENP. Different inoculation methods were tested to study possible routes of transmission. Typical signs, and gross- and histopathological findings for FENP were detected when naïve mink were infected with the tissue extract of mink with FENP, using a subcutaneous/ intradermal infection route. Edema, hemorrhage, necrosis and pus formation were detected in the infection site. A pure culture preparation of A. phocae alone or concurrently with the novel Streptococcus sp. caused severe acute signs of lethargy, apathy and anorexia and even mortality. The histopathological findings were similar to those found in naturally occurring cases of FENP. In contrast, the perorally infected mink presented no clinical signs nor any gross- or histopathological lesions. This study showed that A. phocae is able to cause FENP. The study also indicated that predisposing factors such as the environment, the general condition of the animals, temperature and skin trauma contribute to the development of the disease.

Isolation of Clostridium limosum from an outbreak of metritis in farmed mink.

BACKGROUND: An outbreak of sudden death of pregnant farmed mink in Finland occurred during the busiest whelping period in the spring of 2013. The affected farms were all located in western Finland in a rather narrow geographic area, Ostrobothnia. Dead mink from 22 farms were submitted for laboratory diagnostics to the Finnish Food Safety Authority (Evira). The carcasses were necropsied and tissue specimens were prepared for histology. Samples of internal organs and peritoneal fluid were cultured bacteriologically. RESULTS: Major pathological findings included hemorrhagic vaginal discharge, severely inflamed uteri with luminal hemorrhagic exudate and dead fetuses. Dead fetuses were present in the peritoneal cavity and associated severe peritonitis occurring as sequela of uterine rupture were found in most minks. Histological findings included hemorrhages, neutrophil infiltrations, degenerative inflammatory cells, edema, fibrin and rod-shaped bacteria on all layers of the uterine wall. In most samples abundant and pure anaerobic bacterial growth of Clostridium limosum was found. CONCLUSIONS: This is the first report of C. limosum associated metritis in farmed mink. Disease was only observed in pregnant females and the uterus was the primary site of infection. The source of infection and the route of transmission remained unclear, but feed borne transmission was suspected.

Characterization of a new epidemic necrotic pyoderma in fur animals and its association with Arcanobacterium phocae infection.

A new type of pyoderma was detected in Finnish fur animals in 2007. The disease continues to spread within and between farms, with severe and potentially fatal symptoms. It compromises animal welfare and causes considerable economic losses to farmers. A case-control study was performed in 2010-2011 to describe the entity and to identify the causative agent. Altogether 99 fur animals were necropsied followed by pathological and microbiological examination. The data indicated that the disease clinically manifests in mink (Neovison vison) by necrotic dermatitis of the feet and facial skin. In finnraccoons (Nyctereutes procyonoides), it causes painful abscesses in the paws. Foxes (Vulpes lagopus) are affected by severe conjunctivitis and the infection rapidly spreads to the eyelids and facial skin. A common finding at necropsy was necrotic pyoderma. Microbiological analysis revealed the presence of a number of potential causative agents, including a novel Streptococcus sp. The common finding from all diseased animals of all species was Arcanobacterium phocae. This bacterium has previously been isolated from marine mammals with skin lesions but this is the first report of A. phocae isolated in fur animals with pyoderma. The results obtained from this study implicate A. phocae as a potential causative pathogen of fur animal epidemic necrotic pyoderma (FENP) and support observations that the epidemic may have originated in a species-shift of the causative agent from marine mammals. The variable disease pattern and the presence of other infectious agents (in particular the novel Streptococcus sp.) suggest a multifactorial etiology for FENP, and further studies are needed to determine the environmental, immunological and infectious factors contributing to the disease.

Prevalence of and risk factors associated with viral and bacterial pathogens in farmed European wild boar.

The aim of this study was to estimate in farmed European wild boars the prevalence of and risk factors associated with a range of common porcine viral and bacterial infections, namely, porcine parvovirus (PPV), porcine circovirus type 2 (PCV2), swine influenza virus (SIV), Aujeszky's disease virus (ADV), classical swine fever virus (CSFV), swine vesicular disease virus (SVDV), coronavirus causing transmissible gastroenteritis (TGEV), porcine reproductive and respiratory syndrome virus (PRRSV), Mycoplasma hyopneumoniae, Lawsonia intracellularis, Brucella spp., and Leptospira spp. A sampling frame was compiled based on a national record of wild boar farmers, and 32 farms were surveyed. Serological screening was carried out on 303 samples from animals slaughtered between 2005 and 2008, and random-effect logistic regression models were developed for pathogens with a 'non-zero' prevalence. The apparent animal prevalence for PPV, PCV2, and L. intracellularis was 46.5% (95% confidence interval [CI] 41-52%), 51.1% (95% CI 45-57%) and 59.2% (95% CI 54-65%), respectively. Apparent farm seroprevalence rates for PPV, PCV2 and Lawsonia intracellularis were 56.3% (95% CI, 39-73%), 21.9% (95% CI, 8-36%) and 78.1% (95% CI, 64-92%), respectively. No antibodies were detected against SIV, ADV, CSFV, SVDV, TGEV, PRSSV, Leptospira spp., Brucella spp., or M. hyopneumoniae. Increasing herd size, proximity to dense populations of domestic swine and later sampling times within the survey period were found to be risk factors. Overall, the seroprevalence of these pathogens in farmed wild boar was similar to that in the farmed domestic pig population in Finland. However, it is possible that the rearing of wild boars in fenced estates may predispose them to particular infections, as reflected in higher antibody titres.