Leptospira Status in Sweden during the Past Century, Neglected and Re-Emerging?

We compiled data on notified cases of leptospirosis in animals and humans in Sweden. Published studies on leptospirosis in humans and animals from the beginning of the 20th century onwards are summarized. During the Second World War, hundreds of leptospirosis cases in humans were reported in Sweden, but since then, there have been only a few severe cases. Surveillance of leptospirosis in domestic animals demonstrates that the pathogen is still occurring. The occurrence of Leptospira in humans and animals in the other Nordic countries resembles that in Sweden. Leptospirosis is an underdiagnosed and underreported disease globally, both in animals and humans, partly due to the lack of simple, rapid diagnostic tools but possibly also due to the lack of awareness among physicians, veterinarians and nurses. Traditionally, leptospirosis has been mostly diagnosed by serology, but development of molecular methodshas improved the capability for correct diagnosis. As of today, leptospirosis is regarded as a relatively uncommon disease in the Nordic countries, but in some other countries, it is considered a neglected zoonosis or a (re-)emerging disease that may become more common in the future. Possible factors that could contribute to an increase in incidence are discussed in this review. Active surveillance of humans and domestic and wild animals and stringent rodent control in society and animal farms are of outmost importance for prevention.

Detection of Campylobacter species in different types of samples from dairy farms.

BACKGROUND: Livestock, domestic pets and wildlife can be intestinal carriers of thermotolerant Campylobacter species. These reservoirs can in turn contaminate the environment and food products, thus creating pathways to campylobacteriosis in human beings. The purposes of this study were to investigate sampling strategies applied for surveillance of Campylobacter on dairy cattle farms and to identify the presence and species of Campylobacter in different age groups. METHODS: Boot sock and faecal samples were collected from five dairy herds from three age groups-cows, heifers and calves younger than 12 months-and from milk filters. RESULTS: Campylobacter species were isolated in 152 of 250 samples, of which 93 isolates were identified as C jejuni, 51 as C hyointestinalis, two as C lari and one as C coli, whereas five isolates could not be identified to species level. Campylobacter species were isolated from 86 of 110 faecal samples, 60 of 97 sock samples and six of 43 milk filter samples. CONCLUSION: Faecal samples were the optimal sample type for detection of Campylobacter on dairy farms. However, taking multiple types of samples could be recommended in order to optimise the recovery rate and variety of Campylobacter species detected when investigating the presence of Campylobacter on dairy farms.

Characterization of Campylobacter spp. isolated from wild birds in the Antarctic and Sub-Antarctic.

A lack of knowledge of naturally occurring pathogens is limiting our ability to use the Antarctic to study the impact human-mediated introduction of infectious microorganisms have on this relatively uncontaminated environment. As no large-scale coordinated effort to remedy this lack of knowledge has taken place, we rely on smaller targeted efforts to both study present microorganisms and monitor the environment for introductions. In one such effort, we isolated Campylobacter species from fecal samples collected from wild birds in the Antarctic Peninsula and the sub-Antarctic island of South Georgia. Indeed, in South Georgia, we found Campylobacter lari and the closely related Campylobacter peloridis, but also distantly related human-associated multilocus sequence types of Campylobacter jejuni. In contrast, in the Antarctic Peninsula, we found C. lari and two closely related species, Campylobacter subantarcticus and Campylobacter volucris, but no signs of human introduction. In fact, our finding of human-associated sequence types of C. jejuni in South Georgia, but not in the Antarctic Peninsula, suggests that efforts to limit the spread of infectious microorganisms to the Antarctic have so far been successful in preventing the introduction of C. jejuni. However, we do not know how it came to South Georgia and whether the same mode of introduction could spread it from there to the Antarctic Peninsula.

Foodborne pathogens in unpasteurized milk in Sweden.

Raw milk may be a risk for public health if it is contaminated with zoonotic pathogens. To study the prevalence in unpasteurized milk from Swedish farms, bovine and small ruminant dairy farms were sampled. Since the sampling method and transport conditions may influence the outcome of analyses, efforts were made to optimize the methodology. Culturing of bacteria was done from in-line milk filters collected from the milk pipe at the point where it enters the milk bulk tank at the farms and this way of sampling was compared to sampling bulk tank milk (BTM) directly. Analysing milk filters were found to be superior to analysing BTM directly. Conditions for transport of milk filter samples were further improved by the addition of Cary Blair transport medium, which significantly increased the number of positive samples for pathogenic bacteria. The isolation of several foodborne pathogens from milk filters was demonstrated. The prevalence of samples with Staphylococcus aureus was 71% and 64%, and Listeria spp. 21% and 29% from dairy cow and goat/sheep farms, respectively. Campylobacter jejuni, Yersinia enterocolitica and verotoxigenic Escherichia coli (VTEC) O157 were detected in 9%, 2% and 2% of samples from bovine milk, respectively. We conclude that the choice of sampling method and sample handling influence the results of bacterial culturing. From the results of this study, we strongly recommend to sample in-line milk filters instead of BTM directly and to use Cary Blair medium during transport, especially if the samples are to be analysed for Campylobacter spp. and/or Listeria spp. The findings also show that unpasteurized milk from Swedish farms occasionally contain bacteria with zoonotic potential.

Outbreak of Campylobacteriosis Following a Dairy Farm Visit: Confirmation by Genotyping.

In April-May 2014, an outbreak of campylobacteriosis occurred after a preschool visit to a dairy farm in the South Western part of Sweden. During the visit, a meal, including unpasteurized milk, was served. A retrospective cohort study using a web-based questionnaire was performed among the participants (n = 30) of the farm visit. A total of 24 of the 30 (80%) cohort members completed the questionnaire. Eleven cases were identified, and Campylobacter jejuni was isolated from eight of them. Seven of the cases were 2- to 7-year-old children. We found the highest attack rates among those who usually drink milk (45%) and those who consumed unpasteurized milk during the farm visit (42%). No cases were unexposed (risk ratio incalculable). As result of the farm investigation, Campylobacter was isolated from cattle on the farm. Genotyping with pulsed-field gel electrophoresis and whole genome sequencing confirmed that human and cattle isolates of C. jejuni belonged to one cluster. Thus, cattle on the farm are considered the source of infection, and the most likely vehicle of transmission was contaminated unpasteurized milk. We recommend consumption of heat-treated milk only and increased awareness of the risk of consuming unpasteurized milk.

Lipooligosaccharide locus classes and putative virulence genes among chicken and human Campylobacter jejuni isolates.

BACKGROUND: Campylobacter cause morbidity and considerable economic loss due to hospitalization and post infectious sequelae such as reactive arthritis, Guillain Barré- and Miller Fischer syndromes. Such sequelae have been linked to C. jejuni harboring sialic acid structures in their lipooligosaccharide (LOS) layer of the cell wall. Poultry is an important source of human Campylobacter infections but little is known about the prevalence of sialylated C. jejuni isolates and the extent of transmission of such isolates to humans. RESULTS: Genotypes of C. jejuni isolates from enteritis patients were compared with those of broiler chicken with pulsed-field gel electrophoresis (PFGE), to study the patterns of LOS biosynthesis genes and other virulence associated genes and to what extent these occur among Campylobacter genotypes found both in humans and chickens. Chicken and human isolates generally had similar distributions of the putative virulence genes and LOS locus classes studied. However, there were significant differences regarding LOS locus class of PFGE types that were overlapping between chicken and human isolates and those that were distinct to each source. CONCLUSIONS: The study highlights the prevalence of virulence associated genes among Campylobacter isolates from humans and chickens and suggests possible patterns of transmission between the two species.

Epidemiology of Campylobacter jejuni infections in Sweden, November 2011-October 2012: is the severity of infection associated with C. jejuni sequence type?

BACKGROUND: Campylobacter jejuni is among the most frequent causes of bacterial gastroenteritis in Europe. Over 8,000 C. jejuni multilocus sequence typing sequence types (STs) have been described; ST-21 and ST-45 have been identified as the most frequent types in all human studies so far. In contrast to other STs, ST-22 has been associated with the Guillain-Barré syndrome and ST-677 was recently linked to severe systemic infections in Finland. We investigated risk factors associated with hospitalisation in individuals with C. jejuni infections acquired in Sweden. METHODS: A total of 1,075 individuals with domestically acquired C. jejuni infection diagnosed between November 2011 and October 2012 in Sweden were included in this retrospective cohort study. Typing data for the isolates as well as clinical data including hospitalisation dates and diagnosis codes for individuals with C. jejuni infection were obtained. Factors associated with hospitalisation and length of hospitalisation were investigated by multivariable analysis. RESULTS: A total of 289 individuals were hospitalised due to C. jejuni infection (26.8%); those with co-morbidities were over 14 times more likely to become hospitalised than those without (odds ratio [OR]: 14.39, 95% confidence interval [CI]: 6.84-30.26). Those with underlying co-morbidities were also hospitalised longer than those without (4.22 days vs. 2.86 days), although this was not statistically significant. C. jejuni ST-257 (OR: 2.38; CI: 1.08-5.23), but not ST-22 or ST-677, was significantly associated with hospitalisation. CONCLUSION: ST-677 was not associated with increased hospitalisation or a longer hospital stay in our study whilst ST-257 was. However, individuals with C. jejuni infections were generally more frequently hospitalised than previously demonstrated; this requires further consideration including possible targeted interventions.

Genetic diversity and host associations in Campylobacter jejuni from human cases and broilers in 2000 and 2008.

Campylobacter jejuni is an important food-borne pathogen, with a global distribution. It can colonize numerous host species, including both domestic and wild animals, but is particularly associated with birds (poultry and wild birds). For human campylobacteriosis, poultry products are deemed the most significant risk factor for acquiring infection. We conducted a genotyping and host attribution study of a large representative collection of C. jejuni isolated from humans and broilers in Sweden in the years 2000 and 2008. In total 673 broiler and human isolates from 10 different abattoirs and 6 different hospitals were genotyped with multilocus sequence typing. Source attribution analyses confirmed the strong linkage between broiler C. jejuni and domestic human cases, but also indicated a significant association to genotypes more commonly found in wild birds. Genotype distributions did not change dramatically between the two study years, suggesting a stable population of infecting bacteria.

Prevalence of thermophilic Campylobacter species in Swedish dogs and characterization of C. jejuni isolates.

BACKGROUND: The aims of this study were to investigate the prevalence of Campylobacter species in Swedish dogs, to identify the species of the Campylobacter isolates and to genotype the C. jejuni isolates. Young and healthy dogs were targeted and the sampling was performed at 11 veterinary clinics throughout Sweden from October 2011 to October 2012. Faecal swab samples were collected and sent to the laboratory at the National Veterinary Institute (SVA) for isolation of Campylobacter, speciation and genotyping. RESULTS: Campylobacter spp. were isolated from 67 of the 180 sampled dogs which yields an overall prevalence of 37%. The most prevalent species of Campylobacter among the participating dogs was C. upsaliensis with 52 of the 67 identified isolates. A lower prevalence was observed for C. jejuni with seven identified isolates and one isolate was identified as C. helveticus. Multi-locus sequence typing (MLST) was carried out on the seven C. jejuni isolates and all sequence types that were found are also commonly found in humans. The dogs were divided into three age groups; 1) under 12 months, 2) 12 to 23 months and 3) 24 months and older. The highest prevalence was found in the two younger age groups. Dogs shedding C. jejuni were between 3-12 months of age while dogs shedding C. upsaliensis were found in all ages. CONCLUSIONS: The present investigation finds that Campylobacter spp. known to cause campylobacteriosis in humans are present in Swedish dogs. The results suggest an age predisposition where dogs under 2 years of age are more likely to shed Campylobacter spp. than older dogs. The most commonly isolated species was C. upsaliensis followed by C. jejuni, which was only detected in dogs up to 12 months of age. All C. jejuni isolates identified in the present study were of the same MLST types that have previously been described both in humans and in animals. The awareness of the Campylobacter risk of healthy young dogs may be an important way to reduce the transmission from dogs to infants, young children and immunocompromised adults.

Monitoring Campylobacter in the poultry production chain­from culture to genes and beyond.

Improved monitoring tools are important for the control of Campylobacter bacteria in poultry production. Standardized reference culture methods issued by national and international standardization organizations are time-consuming, cumbersome and not amenable to automation for screening of large numbers of samples. The ultimate goal for rapid monitoring of Campylobacter is to prevent contaminated meat from entering the food market. Currently, real-time PCR is fulfilling abovementioned criteria to a certain extent. Further development of real-time PCR, microarray PCR, miniaturized biosensors, chromatographic techniques and DNA sequencing can improve our monitoring capacity at a lower cost. Combined with innovative sampling and sample treatment, these techniques could become realistic options for on-farm and liquid-sample monitoring at slaughterhouses.

A prospective follow-up study on transmission of Campylobacter from poultry to abattoir workers.

Contact with poultry or poultry meat is a well-known risk factor for campylobacteriosis, but prospective studies on transmission of Campylobacter from chickens to humans during slaughter are scarce. In this study, we monitored transmission of Campylobacter from slaughtered chicken to originally culture-negative abattoir workers during the peak season of colonized chicken and human Campylobacter infection. Stool samples were obtained from 28 abattoir workers together with data on health status once a month between June and September 2010, with a follow-up sample collected in February 2011. Campylobacter-positive individuals and chicken flocks were identified by culture, and isolates were further characterized using molecular techniques. Campylobacter was isolated from seven asymptomatic individuals. Four of them had been newly employed and had not reported any previous Campylobacter infection. Four human isolates had matching genetic fingerprints with isolates from recently slaughtered chickens. Our results further support the role of chicken as the source of human Campylobacter infection but suggest that asymptomatic Campylobacter infection may occur even in individuals with only limited earlier exposure to Campylobacter.

The workshop on animal botulism in Europe.

A workshop on animal botulism was held in Uppsala, Sweden, in June 2012. Its purpose was to explore the current status of the disease in Europe by gathering the European experts in animal botulism and to raise awareness of the disease among veterinarians and others involved in biopreparedness. Animal botulism is underreported and underdiagnosed, but an increasing number of reports, as well as the information gathered from this workshop, show that it is an emerging problem in Europe. The workshop was divided into 4 sessions: animal botulism in Europe, the bacteria behind the disease, detection and diagnostics, and European collaboration and surveillance. An electronic survey was conducted before the workshop to identify the 3 most needed discussion points, which were: prevention, preparedness and outbreak response; detection and diagnostics; and European collaboration and surveillance. The main conclusions drawn from these discussions were that there is an urgent need to replace the mouse bioassay for botulinum toxin detection with an in vitro test and that there is a need for a European network to function as a reference laboratory, which could also organize a European supply of botulinum antitoxin and vaccines. The foundation of such a network was discussed, and the proposals are presented here along with the outcome of discussions and a summary of the workshop itself.

Management of animal botulism outbreaks: from clinical suspicion to practical countermeasures to prevent or minimize outbreaks.

Botulism is a severe neuroparalytic disease that affects humans, all warm-blooded animals, and some fishes. The disease is caused by exposure to toxins produced by Clostridium botulinum and other botulinum toxin-producing clostridia. Botulism in animals represents a severe environmental and economic concern because of its high mortality rate. Moreover, meat or other products from affected animals entering the food chain may result in a public health problem. To this end, early diagnosis is crucial to define and apply appropriate veterinary public health measures. Clinical diagnosis is based on clinical findings eliminating other causes of neuromuscular disorders and on the absence of internal lesions observed during postmortem examination. Since clinical signs alone are often insufficient to make a definitive diagnosis, laboratory confirmation is required. Botulinum antitoxin administration and supportive therapies are used to treat sick animals. Once the diagnosis has been made, euthanasia is frequently advisable. Vaccine administration is subject to health authorities' permission, and it is restricted to a small number of animal species. Several measures can be adopted to prevent or minimize outbreaks. In this article we outline all phases of management of animal botulism outbreaks occurring in wet wild birds, poultry, cattle, horses, and fur farm animals.

Evaluation of commercial dysferlin antibodies on canine, mouse and human skeletal muscle.

Immunostaining of muscle biopsy cryosections is a powerful tool for identifying protein deficiencies. For dysferlin, a protein associated with limb-girdle muscular dystrophy and Miyoshi myopathy, weak immunostaining of normal muscle has been a problem in reliably identifying dysferlin deficiency in human patients or dystrophic animals. Here we use skeletal muscle cryosections from dog, mouse and human to test several dysferlin antibodies under different conditions of fixation, and without fixation. NCL-Hamlet antibody (mouse monoclonal), following fixation in acetone/methanol, provided the strongest and most reliable staining in sections of human muscle as well as of dog and mouse muscle. Unlike animal tissue, unfixed human muscle also gave strong and reliable staining. NCL-Hamlet 2 gave good staining in all species. Epitomics (rabbit monoclonal) antibody gave good staining of all muscles, and did not stain muscle of dysferlin-deficient mice. However, it strongly stained muscle sarcolemma of patients with dysferlin deficiency, making the antibody less useful. Abcam antibody gave weak staining, and Santa Cruz antibodies did not immunostain muscle dysferlin in any species tested. NCL-Hamlet antibody was optimal for immunoblotting in all species. Use of select antibodies for immunostaining and immunoblotting, and optimization of immunostaining methods, should increase the sensitivity of detecting dysferlin deficiency in skeletal muscle.

Within-flock variations of Campylobacter loads in caeca and on carcasses from broilers.

The proportions of broiler caeca and carcass rinse samples positive for Campylobacter spp. within broiler flocks were determined. Twenty intact caeca per flock from 27 flocks were analyzed individually. Rinse samples were obtained from 5 to 25 carcasses from 27 other flocks. In total, 540 caecum samples and 445 carcass rinse samples were analyzed. The proportion of positive caeca within flocks ranged from 10 to 100%, and the proportion of positive carcasses ranged from 85 to 100%. The highest and lowest numbers of Campylobacter spp. found in positive caecum samples were 8.6 and 1.7log cfu/g caecal contents, respectively. The number of Campylobacter spp. in the caeca from individual broilers within a flock varied by up to 6.3log cfu/g caecal contents. The highest number found on one carcass was 4.2log cfu/ml carcass rinse sample, and the within-flock variation in Campylobacter spp. was up to 3.2log cfu/ml rinse sample. There was thus great variation in the load of Campylobacter spp. in individual caecum and carcass samples obtained from each positive broiler flock. This large variation in the numbers of Campylobacter spp. carried by individual birds should be considered when only one or a few samples are collected from a flock and the results are used for risk assessments.

Multilocus sequence typing of Campylobacter jejuni from broilers.

Campylobacter jejuni isolates from a national Swedish Campylobacter monitoring in broilers were characterized by multilocus sequencing typing (MLST) in order to study the genetic diversity of this bacterial population. Isolates were initially characterized by pulsed-field gel electrophoresis (PFGE). One hundred were chosen for MLST genotyping. PFGE identified 69 distinct types compared to 44 different sequence types (STs) identified with MLST. Eighteen STs had not been described previously, while the remaining 26 STs were assigned to previously known clonal complexes. The majority of isolates were of genotypes noted in broilers and in humans in earlier studies. However, three clonal complexes, ST-206 complex, ST-677 complex and ST-1034 complex, previously associated with wild bird and environmental samples, were among the genotypes found. This study shows that most of the Swedish broiler isolates were of genotypes noted as common in broilers. However, it also highlights the potential influence of environmental sources on the broiler C. jejuni genotypes.

Muscular dystrophy associated with alpha-dystroglycan deficiency in Sphynx and Devon Rex cats.

Recent studies have identified a number of forms of muscular dystrophy, termed dystroglycanopathies, which are associated with loss of natively glycosylated alpha-dystroglycan. Here we identify a new animal model for this class of disorders in Sphynx and Devon Rex cats. Affected cats displayed a slowly progressive myopathy with clinical and histologic hallmarks of muscular dystrophy including skeletal muscle weakness with no involvement of peripheral nerves or CNS. Skeletal muscles had myopathic features and reduced expression of alpha-dystroglycan, while beta-dystroglycan, sarcoglycans, and dystrophin were expressed at normal levels. In the Sphynx cat, analysis of laminin and lectin binding capacity demonstrated no loss in overall glycosylation or ligand binding for the alpha-dystroglycan protein, only a loss of protein expression. A reduction in laminin-alpha2 expression in the basal lamina surrounding skeletal myofibers was also observed. Sequence analysis of translated regions of the feline dystroglycan gene (DAG1) in affected cats did not identify a causative mutation, and levels of DAG1 mRNA determined by real-time QRT-PCR did not differ significantly from normal controls. Reduction in the levels of glycosylated alpha-dystroglycan by immunoblot was also identified in an affected Devon Rex cat. These data suggest that muscular dystrophy in Sphynx and Devon Rex cats results from a deficiency in alpha-dystroglycan protein expression, and as such may represent a new type of dystroglycanopathy where expression, but not glycosylation, is affected.

Identification of nine sequence types of the 16S rRNA genes of Campylobacter jejuni subsp. jejuni isolated from broilers.

BACKGROUND: Campylobacter is the most commonly reported bacterial cause of enteritis in humans in the EU Member States and other industrialized countries. One significant source of infection is broilers and consumption of undercooked broiler meat. Campylobacter jejuni is the Campylobacter sp. predominantly found in infected humans and colonized broilers. Sequence analysis of the 16S rRNA gene is very useful for identification of bacteria to genus and species level. The objectives in this study were to determine the degree of intraspecific variation in the 16S rRNA genes of C. jejuni and C. coli and to determine whether the 16S rRNA sequence types correlated with genotypes generated by PFGE analysis of SmaI restricted genomic DNA of the strains. METHODS: The 16S rRNA genes of 45 strains of C. jejuni and two C. coli strains isolated from broilers were sequenced and compared with 16S rRNA sequences retrieved from the Ribosomal Database Project or GenBank. The strains were also genotyped by PFGE after digestion with SmaI. RESULTS: Sequence analyses of the 16S rRNA genes revealed nine sequence types of the Campylobacter strains and the similarities between the different sequence types were in the range 99.6-99.9%. The number of nucleotide substitutions varied between one and six among the nine 16S rRNA sequence types. One of the nine 16S rRNA sequence profiles was common to 12 of the strains from our study and two of these were identified as Campylobacter coli by PCR/REA. The other 10 strains were identified as Campylobacter jejuni. Five of the nine sequence types were also found among the Campylobacter sequences deposited in GenBank. The three 16S rRNA genes in the analysed strains were identical within each individual strain for all 47 strains. CONCLUSION: C. jejuni and C. coli seem to lack polymorphisms in their 16S rRNA gene, but phylogenetic analysis based on 16S rRNA sequences was not always sufficient for differentiation between C. jejuni and C. coli. The strains were grouped in two major clusters according to 16S rRNA, one cluster with only C. jejuni and the other with both C. jejuni and C. coli. Genotyping of the 47 strains by PFGE after digestion with SmaI resulted in 22 subtypes. A potential correlation was found between the SmaI profiles and the 16S rRNA sequences, as a certain SmaI type only appeared in one of the two major phylogenetic groups.

Non-pathogenic protein aggregates in skeletal muscle in MLF1 transgenic mice.

Protein aggregate formation in muscle is thought to be pathogenic and associated with clinical weakness. Over-expression of either wild type or a mutant form of myeloid leukemia factor 1 (MLF1) in transgenic mouse skeletal muscle and in cultured cells resulted in aggregate formation. Aggregates were detected in MLF1 transgenic mice at 6 weeks of age, and increased in size with age. However, histological examination of skeletal muscles of MLF1 transgenic mice revealed no pathological changes other than the aggregates, and RotaRod testing did not detect functional deficits. MLF1 has recently been identified as a protein that could neutralize the toxicity of intracellular protein aggregates in a Drosophila model of Huntington's disease (HD). We also demonstrate that MLF1 interacts with MRJ, a heat shock protein, which can independently neutralize the toxicity of intracellular protein aggregates in the Drosophila HD model. Our data suggest that over-expression of MLF1 has no significant impact on skeletal muscle function in mice; that progressive formation of protein aggregates in muscle are not necessarily pathogenic; and that MLF1 and MRJ may function together to ameliorate the toxic effects of polyglutamine or mutant proteins in myodegenerative diseases such as inclusion body myositis and oculopharyngeal muscular dystrophy, as well as neurodegenerative disease.