Comparison of Multiple-Locus Variable-Number Tandem Repeat Analysis Profiles of Enteropathogenic Yersinia spp. Obtained from Humans, Domestic Pigs, Wild Boars, Rodents, Pork and Dog Food.

The enteropathogenic Yersinia genus is commonly detected in wildlife including wild boars. Difficulties in its cultivation may hamper subsequent epidemiological studies and outbreak investigations. Multiple-locus variable-number tandem repeat analysis (MLVA) of Yersinia (Y.) enterocolitica and Y. pseudotuberculosis has proven useful in source attribution and epidemiological studies but has hitherto relied on the analysis of isolates. In the present study, MLVA profiles generated from 254 isolates of Y. enterocolitica indicated similarities between human, pig and rodent isolates. Further, MLVA analyses of 13 Y. pseudotuberculosis pure-cultured isolates were compared to MLVA analyses performed directly on the 14 PCR-positive enrichment broths from which the isolates originated, which showed matching MLVA profiles. This indicates that MLVA analysis performed directly on enrichment broths could be a useful method for molecular epidemiological investigations. In addition, 10 out of 32 samples of wild boar minced meat obtained from private hunters and from approved wild-game-handling establishments were PCR-positive for the presence of Y. enterocolitica and may indicate a risk for public health.

Genome characteristics related to the virulence of Streptococcus suis in Swedish pigs.

The impact of S. suis on Swedish pig production has increased in recent years, and characterization of the strains present in the pig population is needed to aid in surveillance and prevention. Therefore, the aim of this study was to identify and characterize differences in the genomes between Swedish S. suis isolates associated with disease and isolates from healthy animals. Isolates categorized as being pathogenic (n = 100) or non-pathogenic (n = 117) were whole-genome sequenced, serotyped in silico, and sequence-typed using traditional MLST and core-genome MLST, and a genome-wide association study was performed to identify virulence-associated genes. In decreasing order, serotypes 2, 1, and 7 were the most common in the pathogenic group, and serotypes 15 and 12 were the most common in the non-pathogenic group. Among the commonly disease-associated sequence types, ST28 and ST25 were identified, whereas ST1 was scarcely found. The majority of isolates belonged to novel sequence types, revealing differences between Swedish isolates and those reported from other countries. The genomes of the pathogenic isolates were on average smaller and less heterogenic as compared to those of the non-pathogenic isolates. Although a majority of the previously published virulence-associated genes included in the study were found in the genomes of both pathogenic and non-pathogenic isolates, several new, significantly virulence-associated genes were identified.

Whole-Genome Sequencing Evaluation of MALDI-TOF MS as a Species Identification Tool for Streptococcus suis.

Streptococcus suis is an important bacterial pathogen in pigs that may also cause zoonotic disease in humans. The aim of the study was to evaluate matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) identification of S. suis case isolates from diseased pigs and tonsil isolates from healthy pigs and wild boar using sequence analysis methods. Isolates (n = 348) that had been classified as S. suis by MALDI-TOF MS were whole-genome sequenced and investigated using analyses of (i) the 16S rRNA gene, (ii) the recN gene, and (iii) whole-genome average nucleotide identity (ANI). Analysis of the 16S rRNA gene indicated that 82.8% (288 out of 348) of the isolates were S. suis, while recN gene analysis indicated that 75.6% (263 out of 348) were S. suis. ANI analysis classified 44.3% (154 out of 348) as S. suis. In total, 44% (153 out of 348) of the investigated isolates were classified as S. suis by all of the species identification methods employed. The mean MALDI-TOF MS score was significantly higher for the S. suis case isolates than for the tonsil isolates; however, the difference is of limited practical use. The results show that species confirmation beyond MALDI-TOF MS is needed for S. suis isolates. Since the resolution of 16S rRNA gene analysis is too low for Streptococcus spp., ANI analysis with a slightly lowered cutoff of 94% may be used instead of, or in addition to, recN gene analysis. Supplementation of the MALDI-TOF MS reference library with mass spectra from S. orisratti, S. parasuis, S. ruminantium, and additional S. suis serotypes should be considered in order to produce more accurate classifications.

Streptococcus suis in Swedish grower pigs: occurrence, serotypes, and antimicrobial susceptibility.

BACKGROUND: Streptococcus suis is a major cause of meningitis, arthritis, and pneumonia in pigs worldwide, and an emerging pathogen in humans. In Sweden, S. suis has previously received little attention but has in recent years become increasingly recognized as affecting the pig production. The aim of the present study was to investigate the occurrence, serotypes and antimicrobial susceptibility of S. suis in Swedish grower pigs from herds with and without reported S. suis associated disease, as well as possible associations between S. suis associated disease and selected environmental and production factors. Swab samples were taken from the tonsils of clinically healthy 8-13-week-old grower pigs from ten case herds and ten control herds. Isolates were cultured, identified using MALDI-TOF MS, and serotyped using latex agglutination. The antimicrobial susceptibility of 188 isolates was tested using broth microdilution. Production data was gathered and environmental parameters were measured on the farms. RESULTS: Streptococcus suis was isolated from 95% of the sampled pigs in both the case and the control herds. Serotypes 3, 4, 5, 7, 9, 10, 11, 15, 16, and 17-34 were detected, although a majority of the isolates (81.5%) were non-typeable. There was less diversity among the serotypes isolated from the case herds than among those from the control herds; four and nine different serotypes, respectively. Isolates resistant to penicillin (3.8%) were reported for the first time in Sweden. Tetracycline resistance was common (88.4%). No association was noted between the production and the environmental factors investigated, and the carriership of S. suis. CONCLUSIONS: The carriership of S. suis was found to be higher in clinically healthy Swedish pigs than previously estimated, and for the first time, the presence of Swedish isolates resistant to penicillin was reported. Many of the most commonly disease-associated serotypes, e.g. serotypes 2, 9, 3, and 7, were detected in healthy grower pigs although further studies are needed to investigate the virulence of these isolates.

Effectiveness of alternative measures to reduce antimicrobial usage in pig production in four European countries.

BACKGROUND: The reduction of antimicrobial usage (AMU) is in the focus in modern pig production. The objective of this study was to assess the effectiveness of alternatives to reduce AMU at herd level. In a prospective study, 68 farrow-to-finish pig herds located in Belgium, France, Germany and Sweden were recruited on a voluntary basis to implement tailor-made intervention plans to reduce their AMU. Alternative measures included improvement of biosecurity (n = 29 herds), vaccination (n = 30), changes of feeding schemes or drinking water quality (n = 45), improved pig health and welfare care (n = 21) as well as changes in stable climate and zootechnical measures (n = 14). Herds were followed for 1 year after implementation of measures. Annual antimicrobial expenditures or treatment records, as well as disease incidence scores were collected and compared to those of the year before intervention. AMU was measured as the treatment incidence and calculated by age category, antimicrobial class and administration route. RESULTS: Compliance with the intervention plans was high (median 93%). AMU was significantly reduced following the implementation of alternative measures: in the median herd of the four countries, pigs were treated before intervention 25% of their expected lifespan (200 days from birth to slaughter) and after intervention 16%. AMU of suckling and weaned pigs were significantly reduced by 37 and 54%, respectively. The usage of polymyxins and tetracyclines was significantly reduced by 69 and 49%, respectively. AMU via feed and water, as well as parenteral AMU were significantly reduced by 46 and 36%, respectively. Herds with a higher AMU level before intervention achieved a bigger reduction. The majority of disease incidence were similar before and after intervention, with a few exceptions of disorders related to the gastro-intestinal tract in suckling pigs (decreased) and in breeding pigs (increased). CONCLUSION: Following tailor-made implementation of alternative measures, a substantial reduction of AMU in pig production was achievable without jeopardizing animal health. The AMU reduction in the youngest age categories (suckling and weaned pigs) and the reduction of group treatments via feed and water was in line with the recent European Guidelines on the prudent use of antimicrobials in veterinary medicine.

Isolates from Colonic Spirochetosis in Humans Show High Genomic Divergence and Potential Pathogenic Features but Are Not Detected Using Standard Primers for the Human Microbiota.

Colonic spirochetosis, diagnosed based on the striking appearance in histological sections, still has an obscure clinical relevance, and only a few bacterial isolates from this condition have been characterized to date. In a randomized, population-based study in Stockholm, Sweden, 745 healthy individuals underwent colonoscopy with biopsy sampling. Of these individuals, 17 (2.3%) had colonic spirochetosis, which was associated with eosinophilic infiltration and a 3-fold-increased risk for irritable bowel syndrome (IBS). We aimed to culture the bacteria and perform whole-genome sequencing of the isolates from this unique representative population sample. From 14 out of 17 individuals with spirochetosis we successfully isolated, cultured, and performed whole-genome sequencing of in total 17 isolates, including the Brachyspira aalborgi type strain, 513A. Also, 16S analysis of the mucosa-associated microbiota was performed in the cases and nonspirochetosis controls. We found one isolate to be of the species Brachyspira pilosicoli; all remaining isolates were of the species Brachyspira aalborgi Besides displaying extensive genetic heterogeneity, the isolates harbored several mucin-degrading enzymes and other virulence-associated genes that could confer a pathogenic potential in the human colon. We also showed that 16S amplicon sequencing using standard primers for human microbiota studies failed to detect Brachyspira due to primer incompatibility.IMPORTANCE This is the first report of whole-genome analysis of clinical isolates from individuals with colonic spirochetosis. This characterization provides new opportunities in understanding the physiology and potentials of these bacteria that densely colonize the gut in the individuals infected. The observation that standard 16S amplicon primers fail to detect colonic spirochetosis may have major implications for studies searching for associations between members of the microbiota and clinical conditions such as irritable bowel syndrome (IBS) and should be taken into consideration in project design and interpretation of gastrointestinal tract microbiota in population-based and clinical settings.

Detection of Leptospira in Urban Swedish Rats: Pest Control Interventions as a Promising Source of Rats Used for Surveillance.

Rat carcasses obtained from pest control interventions can potentially be used for an efficient surveillance of zoonotic diseases such as leptospirosis. To evaluate the performance of different laboratory methods for detection of pathogenic Leptospira spp., heart and kidney samples from wild Norway rats were analyzed by microscopic agglutination test (MAT, the gold standard), a commercial IgG enzyme-linked immunosorbent assay, and by an optimized quantitative PCR (secY qPCR, followed by sequencing). We found secY qPCR to be as sensitive as MAT for screening of Leptospira infection in pest control rats and selected secY qPCR for a larger screening of rats from urban and rural areas in central and southern Sweden. We identified secY qPCR positive rats from the cities Stockholm, Gothenburg, and Malmö, which were further confirmed by sequencing.

The occurrence of Treponema spp. in gingival plaque from dogs with varying degree of periodontal disease.

Periodontal disease is common in dogs and is initiated by gingival plaque composed of several hundred bacterial species. Some of these species have specifically been pointed out as potential periodontal pathogens, such as Treponema spp. Treponema spp. are difficult to culture and therefore the majority have been detected by culture-independent methods, such as PCR (Polymerase Chain Reaction). This leaves many Treponema spp. uncharacterized and unnamed. In this study, treponemes were investigated in gingival plaque from dogs with varying degree of periodontal disease with the aim to describe their occurrence and diversity in dogs. The methods used were culture, phase-contrast microscopy, PCR targeting the 16SrRNA-tRNAIle intergenic spacer region (ISR2), sequencing of the ISR2 and phylogenetic analysis. Treponema spp. were detected in samples from 10 out of 11 dogs and isolates were obtained from six dogs. Both healthy and periodontitis affected dogs were Treponema positive. Phylogenetic analysis, based on ISR2 sequences, revealed a large diversity of treponemes in the study population that were found to be distributed mainly in two groups, corresponding to the human oral treponeme phylogroups II (Treponema denticola) and IV (Treponema maltophilum) genetic groups. They were generally more distantly related to other treponemes in these groups. Treponemes from dogs with periodontitis and dogs with mild gingivitis without periodontitis did not differ in any obvious way. The results indicate that several phylotypes of oral treponemes are common in dogs regardless of periodontal status.

Distribution of enteropathogenic Yersinia spp. and Salmonella spp. in the Swedish wild boar population, and assessment of risk factors that may affect their prevalence.

BACKGROUND: Pure Eurasian wild boars and/or hybrids with domestic pigs are present in the wild on most continents. These wild pigs have been demonstrated to carry a large number of zoonotic and epizootic pathogens such as Salmonella spp., Yersinia enterocolitica and Y. pseudotuberculosis. Wild boar populations throughout Europe are growing and more and more wild boar meat is being consumed, the majority within the homes of hunters without having passed a veterinary inspection. The aim of this study was to investigate if factors such as population density, level of artificial feeding, time since establishment of a given population, and the handling of animal by-products from slaughtered animals could influence the presence of these pathogens in the wild boar. RESULTS: In total, 90 wild boars from 30 different populations in Sweden were sampled and analysed using a protocol combining pre-cultivation and PCR-detection. The results showed that 27% of the sampled wild boars were positive for Salmonella spp., 31% were positive for Y. enterocolitica and 22% were positive for Y. pseudotuberculosis. In 80% of the sampled populations, at least one wild boar was positive for one of these enteropathogens and in total, 60% of the animals carried at least one of the investigated enteropathogens. The presumptive risk factors were analysed using a case-control approach, however, no significant associations were found. CONCLUSION: Human enteropathogens are commonly carried by wild boars, mainly in the tonsils, and can thus constitute a risk for contamination of the carcass and meat during slaughter. Based on the present results, the effect of reducing population densities and number of artificial feeding places might be limited.

Experimental inoculation of Treponema pedis T A4 failed to induce ear necrosis in pigs.

Ear necrosis is a syndrome affecting pigs shortly after weaning and is regarded as an animal welfare issue. The etiology is unknown but Treponema spp., predominantly Treponema pedis, are commonly detected in the lesions. Oral treponemes have been suggested as source of infection, transferred by biting and licking behavior. In this study, five pigs were intradermally inoculated with Treponema pedis strain T A4 with the aim of investigating if this strain would induce ear lesions. Three pigs served as controls. The inoculation was repeated after 29 days, and the study continued for 56 days. Serum samples were collected throughout the study and analyzed by ELISA for IgG antibodies towards T. pedis T A4 lysate. Skin biopsies were taken from the inoculation area at the end of the study. Gingival samples were collected and cultivated for treponemes, for comparison to the inoculation strain and to follow colonisation. The challenged pigs did not develop any clinical signs of infection and no spirochetes were detected in sections from skin biopsies. The number of Treponema-positive gingival samples increased during the study. In the challenge group, IgG towards the bacterial lysate peaked 7 days after each inoculation and decreased rapidly hereafter. In the control group a weak IgG response was observed after the second inoculation, possibly caused by the oral treponemes.

Profile of pig farms combining high performance and low antimicrobial usage within four European countries.

Pig farmers are strongly encouraged to reduce their antimicrobial usage because of the rising threat from antimicrobial resistance. However, such efforts should not compromise the herd health status and performance. This study aimed to describe the profile of so-called 'top-farms' that managed to combine both high technical performance and low antimicrobial usage. A cross-sectional study was conducted among 227 farrow-to-finish farms in Belgium, France, Germany and Sweden. Among them, 44 farms were allocated to the top-farms group and were compared with the 'regular' farms group in terms of farm characteristics, biosecurity and health status. Top-farms had fewer gastrointestinal symptoms in suckling pigs and fewer respiratory symptoms in fatteners, which could partly explain their reduced need for antimicrobials and higher performance. They also had higher biosecurity and were located in sparsely populated pig areas. However, 14 farms of the top-farms group were located in densely populated pig areas, but still managed to have low usage and high technical performance; they had higher internal biosecurity and more extensive vaccination against respiratory pathogens. These results illustrate that it is possible to control infectious diseases using other approaches than high antimicrobial usage, even in farms with challenging environmental and health conditions.

Evaluation of the relationship between the biosecurity status, production parameters, herd characteristics and antimicrobial usage in farrow-to-finish pig production in four EU countries.

BACKGROUND: High antimicrobial usage and the threat of antimicrobial resistance highlighted the need for reduced antimicrobial usage in pig production. Prevention of disease however, is necessary to obtain a reduced need for antimicrobial treatment. This study aimed at assessing possible associations between the biosecurity level, antimicrobial usage and farm and production characteristics in order to advice on best practices for a low antimicrobial usage and maximum animal health and production. A cross-sectional study was conducted in 227 farrow-to-finish pig herds in Belgium, France, Germany and Sweden between December 2012 and December 2013. Associations between biosecurity status, antimicrobial usage, and production parameters were evaluated with multivariable general linear models, according to an assumed causal pathway. RESULTS: The results showed that higher antimicrobial usage in sows tended to be associated with higher antimicrobial usage from birth until slaughter (p = 0.06). The antimicrobial usage from birth until slaughter was positively associated with the number of pathogens vaccinated against (p < 0.01). A shorter farrowing rhythm (p < 0.01) and a younger weaning age (p = 0.06) tended to be also associated with a higher antimicrobial usage from birth until slaughter whereas a better external biosecurity (p < 0.01) was related with a lower antimicrobial usage from birth until slaughter. CONCLUSION: Management practices such as weaning age and biosecurity measures may be important factors indirectly impacting on antimicrobial usage. We therefore promote a holistic approach when assessing the potential to reduce the need for antimicrobial treatments.

Antimicrobial use in Swedish farrow-to-finish pig herds is related to farmer characteristics.

BACKGROUND: Antimicrobial resistance is an increasing problem and reducing AM use is critical in limiting its severity. The underlying causes of antimicrobial use at pig farm level must be understood to select effective reduction measures. We previously showed that antimicrobial use on Swedish pig farms is comparatively low but varies between farms, although few farms are high users. In the present survey of a convenience sample of 60 farrow-to-finish herds in Sweden, we investigated farmers' attitudes to antimicrobials and the influence of information provided by veterinarians about antimicrobial resistance. Farm characteristics were also recorded. We had previously quantified antimicrobial use for different age categories of pigs during one year, as well as external and internal biosecurity. Risk factors based on hypothetical causal associations between these and calculated treatment incidence (TI) for the different age categories were assessed here in a linear regression model. RESULTS: There were no significant associations between biosecurity and TI for any pig age category. Increasing farmer age was associated with higher TI for suckling piglets and fatteners. For suckling piglets, the age group with the highest frequency of treatment, TI was also significantly associated with farmer and education of the staff, where female farmers, and university educated staff was associated with a higher TI. Larger farms were associated with a higher TI in fatteners. CONCLUSIONS: In the investigated Swedish pig farms, factors that influenced antimicrobial usage were more related to characteristics of the individual farmer and his/her staff than to biosecurity level, other management factors or farmers' attitudes to antimicrobials.

Antimicrobial usage in 60 Swedish farrow-to-finish pig herds.

The aim of this study was to quantify antimicrobial consumption in Swedish farrow-to-finish pig herds. Sixty herds with 100 sows or more producing more than 500 fatteners per year participated in a study where data on antimicrobial consumption over a period of one year were collected. Data on antimicrobial use were collected by substance, administration route and per age category. Antimicrobial use was measured as defined daily doses and expressed as treatment incidence (TI) per 1000 pig-days at risk. The TIs for growing pigs varied between herds, from 1.6 to 116.0 with a median of 14.3. The highest TI was recorded for suckling piglets with a median of 54.7 (range; 1.6-367.9), while the median TIs for weaners, fatteners and adults were 6.2, 2.8 and 8.4, respectively (range; 0.0-260.5; 0.0-64.9; 0.0-45.0, respectively). The within herd TIs for the different age categories were not correlated. Individual treatment, mainly consisting of injectables, was the most common form of application except for weaners for which a majority (54.8%) of the treatments were group treatments. Benzylpenicillin was the most commonly applied substance except for weaners for which oral formulations of tylosin were most common. For fatteners, group treatments constituted 8.4% of the total TI. Group treatments with oral colistin were applied to suckling piglets in five herds. Group treatments were not applied to adult pigs. The TI for weaners was significantly lower for specific pathogen-free herds. The results show that the overall antimicrobial use in Swedish farrow-to-finish pig herds varied to a great extent, and the between-herd variation indicates that there is room for improvement of pig health. Targeting suckling piglets may be most beneficial, but further studies are required to identify specific focus areas which may reduce the need for antimicrobials in this particular age group.

Biosecurity level and health management practices in 60 Swedish farrow-to-finish herds.

BACKGROUND: Biosecurity measures are important tools to maintain animal health in pig herds. Within the MINAPIG project, whose overall aim is to evaluate strategies to raise pigs with less antimicrobial use, biosecurity was evaluated in medium to large farrow-to-finish pig herds in Sweden. In 60 farrow-to-finish herds with more than 100 sows, the biosecurity level was evaluated using a previously developed protocol (BioCheck). In a detailed questionnaire, internal and external biosecurity was scored in six subcategories each. An overall score for biosecurity was also provided. Information regarding production parameters as well as gender and educational level of personnel working with the pigs was also collected. Descriptive statistics were used to examine the recorded data. RESULTS: The median scores for external and internal biosecurity were 68 and 59, respectively, where 0 indicates total absence of biosecurity and 100 means maximal possible biosecurity. The subcategories for external and internal biosecurity that had the highest scores were "Purchase of animals" (external) and "Nursery unit"/"Fattening unit" (internal), while "Feed, water and equipment supplies" (external) and "Measures between compartments and equipment"/"Cleaning and disinfection" (internal) received the lowest scores. A female caretaker in the farrowing unit, a farmer with fewer years of experience and more educated personnel were positively associated with higher scores for some of the external and internal subcategories. In herds with <190 sows, fattening pigs were mixed between batches significantly more often than in larger herds. CONCLUSIONS: The herds in this study had a high level of external biosecurity, as well as good internal biosecurity. Strong biosecurity related to the purchase of animals, protocols for visitors, the use of all-in, all-out systems, and sanitary period between batches. Still, there is room for improvement in preventing both the introduction of disease to herds (external) and the spread of infections within herds (internal). Systems for animal transport can be improved and with respect to internal biosecurity, there is especially room for improvement regarding hygiene measures in and between compartments, as well as the staff's working procedures between different groups of pigs.

Alternatives to the use of antimicrobial agents in pig production: A multi-country expert-ranking of perceived effectiveness, feasibility and return on investment.

Nineteen alternatives to antimicrobial agents were ranked on perceived effectiveness, feasibility and return on investment (ROI) from 0 (not effective, not feasible, no ROI) to 10 (fully effective, completely feasible, maximum ROI) by 111 pig health experts from Belgium, Denmark, France, Germany, Sweden and Switzerland. The top 5 measures in terms of perceived effectiveness were (1) improved internal biosecurity, (2) improved external biosecurity, (3) improved climate/environmental conditions, (4) high health/Specific Pathogen Free/disease eradication and (5) increased vaccination. The top 5 measures in terms of perceived feasibility were (1) increased vaccination, (2) increased use of anti-inflammatory products, (3) improved water quality, (4) feed quality/optimization and (5) use of zinc/metals. The top 5 measures in terms of perceived ROI were (1) improved internal biosecurity, (2) zinc/metals, (3) diagnostics/action plan, (4) feed quality/optimization and (5) climate/environmental improvements. Univariate linear regression showed that veterinary practitioners rank internal biosecurity, vaccination, use of zinc/metals, feed quality optimization and climate/environmental on average highest, while researchers and professors focused more on increased use of diagnostics and action plans. Financial incentives/penalties ranked low in all countries. Belgian respondents ranked feed quality significantly lower compared to the German respondents while reduction of stocking density was ranked higher in Belgium compared to Denmark. Categorical Principal Component Analysis applied to the average ranking supported the finding that veterinary practitioners had a preference for more practical, common and already known alternatives. The results showed that improvements in biosecurity, increased use of vaccination, use of zinc/metals, feed quality improvement and regular diagnostic testing combined with a clear action plan were perceived to be the most promising alternatives to antimicrobials in industrial pig production based on combined effectiveness, feasibility and ROI.

Rodents on pig and chicken farms - a potential threat to human and animal health.

Rodents can cause major problems through spreading various diseases to animals and humans. The two main species of rodents most commonly found on farms around the world are the house mouse (Mus musculus) and the brown rat (Rattus norvegicus). Both species are omnivorous and can breed year-round under favourable conditions. This review describes the occurrence of pathogens in rodents on specialist pig and chicken farms, which are usually closed units with a high level of bio-security. However, wild rodents may be difficult to exclude completely, even from these sites, and can pose a risk of introducing and spreading pathogens. This article reviews current knowledge regarding rodents as a hazard for spreading disease on farms. Most literature available regards zoonotic pathogens, while the literature regarding pathogens that cause disease in farm animals is more limited.

Phenotypic and molecular characterization of Brachyspira spp. isolated from wild rodents.

The occurrence of intestinal spirochaetes of genus Brachyspira in wild rodents was studied by cultivating 209 caecal samples. Spirochaetal cultures were obtained from 83% of rats and 33% of house mice. Biochemical characterization and six different species-specific PCR methods were applied to 101 of 118 isolates and a selection of 34 brachyspiras were further studied by sequencing of the 16S rRNA gene. The results showed that isolates representing all the established biochemical phenotypes could be cultured from the rodents, including the porcine pathogens Brachyspira hyodysenteriae and Brachyspira pilosicoli. Phylogenetic studies indicated that rodents carry Brachyspira spp. that are closely related to porcine and avian isolates, as well as variants previously not described. One group of hippurate-negative rat isolates were shown to possess the 16S rRNA gene hexa(T) nucleotide segment, previously described only in B. pilosicoli and 'Brachyspira corvi', and phylogenetically they formed a sister lineage of the B. pilosicoli cluster. Furthermore, a large number of the rodents were colonized by slowly growing, non- or weakly haemolytic spirochaetes. Most of these brachyspiras were isolated at 37°C and phylogenetically they formed two separate clusters. Sequence analysis of their 16S rRNA genes indicated that the new variants of Brachyspira spp. may constitute novel species of the genus Brachyspira.

From mouse to moose: multilocus genotyping of Giardia isolates from various animal species.

Giardia intestinalis is a protozoan parasite that consists of seven genetically distinct assemblages (A to G). Assemblage A and B parasites have been detected in a wide range of animals including humans, while the other assemblages (C to G) appear to have a narrower host range. However, the knowledge about zoonotic transmission of G. intestinalis is limited. To address this question, 114 Giardia isolates from various animals in Sweden including pets, livestock, wildlife and captive non-human primates were investigated by a sequence-based analysis of three genes (beta-giardin, glutamate dehydrogenase and triose phosphate isomerase). Assemblage A infections were detected in nine ruminants, five cats and one dog, while three sheep were infected with both assemblages A and E. Multilocus genotypes (MLGs) were defined for assemblage A, and three of these MLGs have previously been detected in Giardia isolates from humans. The newly described sub-assemblage AIII, until now reported mainly in wild hoofed animals, was found in one cat isolate. Assemblage B occurred in three monkeys, one guinea pig and one rabbit. The rabbit isolate exhibited sequences at all three loci previously detected in human isolates. The non-zoonotic assemblages C, D, E, F or G were found in the remaining 83 G. intestinalis isolates, which were successfully amplified and genotyped, generating a wide variety of both novel and known sub-genotypes. Double peaks in chromatograms were seen in assemblage B, C, D and E isolates but were never observed in assemblage A, F and G isolates, which can reflect differences in allelic sequence divergence. No evidence of genetic exchange between assemblages was detected. The study shows that multilocus genotyping of G. intestinalis is a highly discriminatory and useful tool in the determination of zoonotic sub-groups within assemblage A, but less valuable for subtyping assemblages B, C, D and E due to the high frequency of double peaks in the chromatograms. The obtained data also suggest that zoonotic transmission of assemblages A and B might occur to a limited extent in Sweden.

Isolation of spirochetes of genus Treponema from pigs with ear necrosis.

Various ear lesions, often caused by ear biting, are common in pigs. Some herds have a high frequency of ear necrosis, a syndrome characterized by necrotic lesions along the rim of the pinna, often bilateral and sometimes resulting in loss of the entire ear. In samples from such lesions spirochetes have been observed microscopically but never isolated or identified. In this study two herds with periodic outbreaks of ear necrosis among weaners were investigated. Samples were collected from ear lesions and from the gingiva of the pigs. Spirochetes were observed in silver stained histological sections and by phase contrast microscope in scrapings from the necrotic lesions. From an ear lesion a pure spirochete isolate was obtained and identified as a yet unnamed species of genus Treponema, closely related to spirochetes found in digital dermatitis in cattle. From the oral samples two pure isolates were obtained. One of these isolates was identified as the same species as in the ear lesion and one as Treponema socranskii. Species identification was based on 16S rRNA gene sequences.