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An imported dog was confirmed to be positive with canine brucellosis in Sweden in 2010. The whole genome of Brucella canis SVA10 was subjected to phage analysis (WGS-PA) and was assigned to the Asian B. canis cluster. Further analysis indicated that the genome of B. canis SVA10 is smaller compared to genomes of the same species. A 35,781 bp genomic island (GI) was found to be absent in strain SVA10 which was detected by read mapping the paired reads to the genome of B. canis ATCC 23,365T. The lacking genes of genomic island GIFeGSH are mainly coding for iron uptake enzymes and parts of the glutathione pathway. A screening of all available whole genome sequences of Brucella strains confirmed that GIFeGSH is also missing in four more strains of B. canis but present in several strains of B. abortus, B. melitensis, B. suis, B. ovis, B. microti, B. pinnipedialis, and B. ceti. Parts of the GI were present, but scattered in two other B. canis strains. The aim of this study was to find differences in the genomes of Brucella which might explain former described differences in virulence. The analysis was extended to all available Brucella genomes after the detection of a genomic island in strain SVA10.

This study investigated organic laying hen farms for the presence of Erysipelothrix rhusiopathiae in the house environment and from potential carriers (i.e. insects and mice) during ongoing erysipelas outbreaks, and compared the obtained isolates with those from laying hens. The samples were investigated by selective culture followed by species-specific polymerase chain reaction on cultures. E. rhusiopathiae was isolated from the spleen, jejunal contents, manure, dust and swabs from water nipples. Three more samples from the house environment tested positive by polymerase chain reaction compared with selective culture alone. Selected isolates were investigated by pulsed-field gel electrophoresis (PFGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). One farm was represented by isolates from laying hens only, and one of these isolates differed in one PFGE band from the others. Different banding patterns were observed for isolates from laying hens and manure on one farm. On the remaining two farms, the isolates from the house environment and laying hens were identical but differed between farms. Outbreaks reoccurred in the next flock on two of the farms, and different PFGE types were isolated from consecutive flocks. Our results suggest an external source of infection, which would explain the previously reported increased risk of outbreaks in free-range flocks. Contaminated manure and dust may represent sources of transmission. For the isolates, MALDI-TOF MS and biochemical typing results were in agreement but, since the type strain of Erysipelothrix tonsillarum was typed as E. rhusiopathiae using MALDI-TOF MS, further studies into this method are needed.

The Swedish Forum for Biopreparedness Diagnostics (FBD) is a network that fosters collaboration among the 4 agencies with responsibility for the laboratory diagnostics of high-consequence pathogens, covering animal health and feed safety, food safety, public health and biodefense, and security. The aim of the network is to strengthen capabilities and capacities for diagnostics at the national biosafety level-3 (BSL-3) laboratories to improve Sweden's biopreparedness, in line with recommendations from the EU and WHO. Since forming in 2007, the FBD network has contributed to the harmonization of diagnostic methods, equipment, quality assurance protocols, and biosafety practices among the national BSL-3 laboratories. Lessons learned from the network include: (1) conducting joint projects with activities such as method development and validation, ring trials, exercises, and audits has helped to build trust and improve communication among participating agencies; (2) rotating the presidency of the network steering committee has fostered trust and commitment from all agencies involved; and (3) planning for the implementation of project outcomes is important to maintain gained competencies in the agencies over time. Contacts have now been established with national agencies of the other Nordic countries, with an aim to expanding the collaboration, broadening the network, finding synergies in new areas, strengthening the ability to share resources, and consolidating long-term financing in the context of harmonized European biopreparedness., The Swedish Forum for Biopreparedness Diagnostics (FBD) is a network that fosters collaboration among the 4 agencies with responsibility for the laboratory diagnostics of high-consequence pathogens, covering animal health and feed safety, food safety, public health and biodefense, and security.

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.

There have been several outbreaks of botulism among poultry and wild birds in Sweden in recent years. The National Veterinary Institute of Sweden (SVA) has identified botulinum neurotoxin (BoNT)/C1 or the mosaic BoNT/C1D using the mouse bioassay. This is believed to be the first report on the application of the Endopep mass spectrometry (Endopep-MS) method to selected clinical animal (serum and liver) samples and a feed sample that had previously given positive test results with the mouse bioassay. In the mouse bioassay eight of the eleven samples were found to be neutralized by both BoNT/C1 and /D antitoxins; the other three were neutralized only by BoNT/C1 antitoxin, but the mice showed a prolonged survival time when the samples had been treated with /D antitoxin. The Endopep-MS analysis, on the other hand, demonstrated only BoNT/C1 activity for all eleven samples. This suggests that at least eight of the samples were of the chimeric toxin type BoNT/C1D, where the enzymically active site is identical to that of BoNT/C1, while other parts of the protein contain sequences of BoNT/D. This is the first step of a cross-validation between the established mouse bioassay and the Endopep-MS of serotypes BoNT/C1 and /C1D. Endopep-MS is concluded to have potential as an attractive alternative to the mouse bioassay.

Type C botulinum neurotoxin (BoNT/C)-producing Clostridium botulinum causes animal botulism worldwide and has become a serious problem in poultry flocks and waterfowl in Sweden. The objectives of the present study were to isolate, characterize and subtype C. botulinum type C avian isolates in order to increase the knowledge of the genetic diversity. Isolates from 13 birds were identified by 16S rRNA sequencing and BoNT/C gene detection by real-time polymerase chain reaction (PCR). Conventional PCR was used to distinguish a chimeric BoNTC/D gene, often associated with avian botulism, from the BoNT/C gene. The isolates analysed all contained the gene coding for a chimeric toxin type C/D. Two fingerprinting techniques, pulsed-field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA analysis (RAPD), were optimized and used to investigate the epidemiological relatedness among the strains. The isolates were divided into three different pulsotypes based upon their restriction profiles for SmaI and SalI. The RAPD system proved to be as discriminative as PFGE. This study reveals a small genetic diversity among Swedish type C strains, with a high similarity between strains from broilers and herring gulls.

Neonatal porcine diarrhoea of uncertain aetiology has been reported from a number of countries. This study investigated 50 diarrhoeic and 19 healthy piglets from 10 affected Swedish herds. The piglets were blood-sampled for analysis of serum γ-globulin and necropsied, and the intestines were sampled for histopathology and cultured for Escherichia coli, Clostridium perfringens and Clostridium difficile. Escherichia coli isolates (n = 276) were examined by PCR for virulence genes encoding LT, STa, STb, EAST1, VT2e, F4, F5, F6, F18, F41, AIDA-I, intimin, and for the genes aaiC and aggR. Selected isolates were analysed for additional virulence genes by a microarray and subjected to O-typing. Clostridium perfringens isolates (n = 152) were examined by PCR for genes encoding major toxins, enterotoxin and beta2-toxin. There was no difference in serum γ-globulin concentration between diarrhoeic and non-diarrhoeic piglets, and pathological lesions in the intestines were generally mild. Porcine enterotoxigenic Escherichia coli, a common cause of piglet diarrhoea, was only found in two piglets. Further, the virulence gene profiling did not suggest involvement of other diarrhoeogenic pathotypes of Escherichia coli. Growth of Clostridium perfringens did not differ between diarrhoeic and non-diarrhoeic piglets. All isolates were type A, all were negative for enterotoxin, and 151 of 152 isolates were beta2-toxin positive. In pigs ≥ 2 days old, moderate to profuse growth of Clostridium difficile was more common in the controls. In conclusion, it was not possible to relate Escherichia coli, Clostridium perfringens type A and C or Clostridium difficile to neonatal porcine diarrhoea in any of the investigated herds.

Objective—To characterize the bacteria of the genital tract in adult cats; assess the effect of estrus, mating, and administration of progestins on those microorganisms in females; and evaluate whether results of bacteriologic culture of vaginal swabs are affected by cleansing of the vulva prior to sampling or by repeated sampling. Animals—66 female and 29 male cats undergoing routine ovariohysterectomy or castration. Procedure—Specimens were obtained from vaginal and uterine or preputial mucosae with swabs moistened with sterile saline (0.9% NaCl) solution. In 9 cats, vaginal specimens were obtained before and after cleansing of the vulva with ethanol; in 7 female cats, 2 vaginal specimens were obtained in immediate succession. Results—Aerobic bacteria were most commonly isolated from cats' vaginas and prepuces; anaerobic bacteria were isolated frequently from males (41%) but rarely from females (5%). Generally, culture results were not affected by cleansing of the vulva or repeated vaginal sampling. The bacterial population of the vaginas of cats was influenced by stage of the estrous cycle but not by mating or administration of progestins. Bacteria were not isolated from the uterus of any cat. Conclusions and Clinical Relevance—In cats, bacteria of the genital tract in females are predominantly aerobic; in males, aerobic and anaerobic bacteria are found. The bacterial population of the vagina is affected by stage of the estrous cycle. Pure growth of bacteria in culture of genital tract specimens is a normal finding; antimicrobials should only be administered if clinical signs of genital infection are present. (Am J Vet Res 2003;64:963–968)

Avian botulism is a paralytic disease caused by Clostridium botulinum-produced botulinum neurotoxins (BoNTs), most commonly of type C/D. It is a serious disease of waterbirds and poultry flocks in many countries in Europe. The objective of this study was to compare the genetic relatedness of avian C.botulinum strains isolated in Spain with strains isolated in Sweden using pulsed-field gel electrophoresis (PFGE). Fifteen strains were isolated from Spanish waterbirds using an immunomagnetic separation technique. Isolates were characterized by PCR, and all were identified as the genospecies Clostridium novyi sensu lato and eight harboured the gene coding for the BoNT type C/D. PFGE analysis of the strains revealed four highly similar pulsotypes, out of which two contained strains from both countries. It also showed that outbreaks in wild and domestic birds can be caused by the same strains. These results support a clonal spreading of the mosaic C.botulinum type C/D through Europe and give relevant information for future epidemiological studies., This research was supported by/executed in the framework of the EU-project AniBioThreat (Grant Agreement: Home/2009/ISEC/AG/191) with the financial support from the Prevention of and Fight against Crime Programme of the European Union, European Commission – Directorate General Home Affairs. The project was also sponsored by the Spanish Ministry of Environment (Grant OAPN 035/2009). IA was supported by a JAE PRE grant from the Spanish Council of Research (CSIC). The IMS methodology was financed by the Norwegian Agricultural Authority, Animalia, Felleskjøpet fôrutvikling, Fiskå Mølle and Norgesfôr through the Norwegian Research Council (Grant Number 199375/I99).

Background Strangles is a contagious equine-specific disease caused by Streptococcus equi subsp. equi. Unfortunately, detection of S. equi can fail in up to 40% of horses with strangles. Whereas recent molecular biologic methods and sampling techniques have improved recovery of S. equi optimal sampling methods and laboratory analyses remain ill-defined. Objectives To determine the yield of S. equi from horses with acute strangles in confirmed outbreaks by field-sampling methods subjected to culture and biochemical identification, and real-time PCR directly and after culture. Animals Fifty-seven horses of varying breeds and ages from 8 strangles outbreaks. Methods Prospective study. Culture with biochemical identification and real-time PCR directly, and from culture, were performed on nasal swabs, nasopharyngeal swabs, and nasopharyngeal lavages. Results Real-time PCR directly from samples identified the highest number of infected horses, with 45/57 nasal swabs, 41/57 nasopharyngeal swabs, and 48/57 nasopharyngeal lavages S. equi positive. Biochemical identification (highest positives 22/57) was inferior to real-time PCR for S. equi recovery regardless of sampling method. Real-time PCR of nasopharyngeal lavage directly and after culture yielded 52/57 positives whereas direct real-time PCR of nasopharyngeal lavage combined with either nasopharyngeal swabs or nasal swabs yielded 53/57 positives. Three horses were negative on all samples. Conclusions and Clinical Importance Nasopharyngeal lavage analyzed by a combination of real-time PCR directly and after culture or, alternatively, real-time PCR directly on a nasopharyngeal lavage and a nasal/nasopharyngeal swab can identify S. equi in over 90% of acute strangles cases.

Background Treatment and protection of wounds in horses can be challenging; protecting bandages may be difficult to apply on the proximal extremities and the body. Unprotected wounds carry an increased risk of bacterial contamination and subsequent infection which can lead to delayed wound healing. Topical treatment with antimicrobials is one possibility to prevent bacterial colonization or infection, but the frequent use of antimicrobials ultimately leads to development of bacterial resistance which is an increasing concern in both human and veterinary medicine. Methods Standardized wounds were created in 10 Standardbred mares. Three wounds were made in each horse. Two wounds were randomly treated with LHP® or petrolatum and the third wound served as untreated control. All wounds were assessed daily until complete epithelization. Protocol data were recorded on day 2, 6, 11, 16, 21 and 28. Data included clinical scores for inflammation and healing, photoplanimetry for calculating wound areas and swab cytology to assess bacterial colonization and inflammation. Bacterial cultures were obtained on day 2, 6 and 16. Results Mean time to complete healing for LHP® treated wounds was 32 days (95%CI = 26.9-37.7). Mean time to complete healing for petrolatum and untreated control wounds were 41.6 days (95%CI = 36.2-47.0) and 44.0 days (95%CI = 38.6-49.4) respectively. Wound healing occurred significantly faster in LHP® wounds compared to both petrolatum (p = 0.0004) and untreated controls (p < 0.0001). There was no significant difference in time for healing between petrolatum and untreated controls. Total scores for bacteria and neutrophils were significantly (p < 0.0001) lower for LHP® treated wounds compared to petrolatum from day 16 and onwards. Staphylococcus aureus and Streptococcus zooepidemicus were only found in cultures from petrolatum treated wounds and untreated controls. Conclusions Treatment with LHP® reduced bacterial colonization and was associated with earlier complete wound healing. LHP® cream appears to be safe and effective for topical wound treatment or wound protection.

In this study we examined seven queens with normal oestrous cycles and a history of infertility after normal matings. We performed clinical examination, vaginal cytology, evaluation of oestradiol, progesterone and total T4 levels, vaginal bacterial culture, ultrasonography, and serum analyses for detection of antibodies against chlamydia and feline leukaemia virus (FeLV) antigenemia. Ovariohysterectomy (OHE) was recommended for 1/7 queens because of pathological uterine changes detected at ultrasonography and clinical examination. Four out of seven queens were treated with antibiotics and two of these had more litters. One of the queens that were treated was not mated again and one was mated without conceiving and was at a later OHE found to have degenerative uterine changes. No treatment was given and no diagnosis could be established in 2/7 queens. Both of them were later ovariohysterectomised and one showed degenerative uterine changes while the uterus of the other queen could not be obtained for follow-up. In summary, 4/7 cats were diagnosed with uterine pathology and no definitive diagnosis could be established for 3/7 queens.

Reasons for performing study When managing strangles in horses, it is crucial to detect chronic infection with Streptococcus equi (SE), i.e. silent carriers. Objectives Evaluate diagnostics for SE carriers over time in a farm. Study design Longitudinal observational study. Methods Sixty-three Icelandic horses isolated on an island were studied 4 to 26 months after remission of acute strangles, including repeated clinical examination and collection of blood, nasopharyngeal lavage (NPL), and guttural pouch lavage (GPL) samples. Twenty-two horses were treated with penicillin locally and systemically. Serology for S. equi was examined by iELISA [1]. Nasopharyngeal lavage and GPL samples were investigated for S. equi and S. zooepidemicus by real-time PCR [2]. Results Thirty-three per cent were SE carriers after 15 months, despite repeated penicillin treatment. In 16/18 carriers, GPL samples were PCR-positive, but not NPL samples, whereas the opposite was true in 2 horses. Several carriers with persistent aerocystitis were not detected by 3 consecutive NPL samples. Five of 18 carriers were seronegative (27.8%) at 15 months. Following conservative treatment, 7 of these 18 carriers were still carriers at 20 months, 8 were negative, and 3 were lost for sampling. Only GPL samples were positive at this point, and only 1/7 carriers were seropositive. Conclusions To detect chronic carriers of strangles, RT-PCR analysis from both GPL and NPL samples may be necessary. Serological screening at individual level often misses individual carriers in long-term cases. Ethical animal research: The testing was approved by the Swedish Ethical Committee on Animal Experiments and horse owners gave their informed consent for inclusion of animals in the study. Source of funding: The Swedish-Norwegian Foundation for Equine Research. Competing interests: None declared.

Clostridium perfringens is a pathogen of great concern in veterinary medicine, because it causes enteric diseases and different types of toxaemias in domesticated animals. It is important that bacteria in tissue samples, which have been collected in the field, survive and for the classification of C. perfringens into the correct toxin group, it is crucial that plasmid-borne genes are not lost during transportation or in the diagnostic laboratory. The objectives of this study were to investigate the survival of C. perfringens in a simulated transport of field samples and to determine the stability of the plasmid-borne toxin genes cpb1 and etx after storage at room temperature and at 4°C. Stability of the plasmid-borne genes cpb1 and etx of C. perfringens CCUG 2035, and cpb2 from C. perfringens CIP 106526, JF 2255 and 6 field isolates in aerobic atmosphere was also studied. Survival of C. perfringens was similar in all experiments. The cpb1 and etx genes were detected in all isolates from samples stored either at room temperature or at 4°C for 24–44 h. Repeated aerobic treatment of C. perfringens CCUG 2035 and CIP 106526 did not result in the loss of the plasmid-borne genes cpb1, cpb2 or etx. Plasmid-borne genes in C. perfringens were found to be more stable than generally reported. Therefore, C. perfringens toxinotyping by PCR can be performed reliably, as the risk of plasmid loss seems to be a minor problem.

Horses that lack obvious clinical signs of respiratory disease can be silent carries of Streptococcus equi subsp. equi (S. equi) in their guttural pouches. Guttural pouch lavage and repeated nasopharyngeal lavages(1) analyzed by real-time PCR(2) are sensitive methods for analysis for S.equi and are considered to be a standard to identify carrier stage. The carrier screening, identifying and treatment of carriers, in the end of a disease outbreak by sampling guttural pouches is expensive and time consuming. According guidelines carriers that have endoscopic findings of guttural pouch empyema, chondroids or discharging lymph nodules are sampled and treated. The sensitivity of using macroscopic appearance of guttural pouches as an indication of persistent S. equi colonization is poorly studied. The aim of this study was to investigate if there is a correlation between endoscopic findings in guttural pouches and positive qPCR analysis for S.equi. In total 179 endoscopic findings from upper airways from 91 individual horses after a strangles outbreak were compared with positive qPCR results from nasopharyngeal lavage and guttural pouch lavage samples. Nasopharyngeal lavage was performed with 120 ml saline and guttural pouch lavage with 40 ml saline for each pouch and analyzed separately. The nasopharyngeal lavage was performed with 120 ml saline prior endoscopy and endoscopic guttural pouch lavages using 40 ml saline for each pouch and analyzed separately. The endoscopic findings of guttural pouches, including empyema, chondroids or discharging retropharyngeal lymph nodules were recorded and compared to the qPCR analysis for S. equi. Therewas no statistical correlation between endoscopy findings and qPCR results. However, if the first guttural pouch lavage was positive by qPCR for S. equi, it was statistically more likely that the contralateral guttural pouch also was positive, indicating a risk for contamination of the second sample. Horses that lack obvious clinical abnormalities on upper airway endoscopy including the guttural pouches can be silent carriers of S. equi. In conclusion, negative endoscopic findings of the guttural pouches of recovered strangles cases cannot rule out silent carrier status. This study was approved by the Swedish Ethical Committee on Animal Experiments and each horse owner had given their informed consent to participate in the study. Financial support was achieved from The Swedish-Norwegian Foundation for Equine Research and Swedish Research Council Formas.

Background A cross-sectional study was carried out to determine the seroprevalence of different serovars of Leptospira spp. and their association with clinical disease and host factors in Swedish horses. Methods Sera from 2017 horses brought to equine clinics during 1997–98 were investigated. The sera were examined by microscopic agglutination test for the presence of antibodies against the following L. interrogans serovars: Bratislava strain Jez, Icterohaemorrhagiae strain Kantorowicz and Pomona strain Pomona and also L. kirschneri sv Grippotyphosa strain Duyster and L. borgpetersenii sv Sejroe strain M 84. Host factors, disease factors, season, pasture access and outdoor confinement variables were analysed with respect to seropositivity to sv Bratislava and Icterohaemorrhagiae. Multivariable logistic regression was used to model seropositivity to sv Bratislava and Icterohaemorrhagiae (seroprevalence > 8%). Results The seroprevalence, at a cut-off 1:100, were for sv Bratislava (16.6%), Icterohaemorrhagiae (8.3%), Sejroe (1.2%), Pomona (0.5%) and Grippotyphosa (0.4%). In the multivariable analysis, it was demonstrated that seroprevalence increased with age for sv Bratislava and Icterohaemorrhagiae. For sv Bratislava the seasons April – June and October – December and for sv Icterohaemorrhagiae October – December had higher seroprevalences than other seasons. Horses not used for racing had higher levels of seropositivity to sv Bratislava. Furthermore, horses with respiratory problems as well as horses with fatigue had higher levels of seropositivity to sv Bratislava. Ponies and coldbloods, and horses with access to pasture, had lower seroprevalence for sv Icterohaemorrhagiae. Healthy horses had lower seroprevalence for sv Icterohaemorrhagiae, than non-healthy horses. Conclusion There was no significant association between clinical signs and disease and positive titres to sv Bratislava (except for the association between respiratory problems and fatigue and seropositivity to sv Bratislava). The results suggest that horses with increasing age and exposed to factors associated with outdoor life had an increased seroprevalence for sv Bratislava, indicating that horses get infected from outdoor and/or are exposed to shedding from other horses (management dependent). For sv Icterohaemorrhagiae, management possibly plays a role as ponies and coldbloods as well as healthy horses had lower seroprevalence. Overall, the age of the horse should be taken into consideration when evaluating the titre as the average healthy horse has a higher titre than a young horse.