A. Gunnarsson

Clostridium difficile: prevalence in horses and environment, and antimicrobial susceptibility

REASONS FOR PERFORMING STUDY Clostridium difficile has been associated with acute colitis in mature horses. OBJECTIVES To survey C. difficile colonisation of the alimentary tract with age, occurrence of diarrhoea and history of antibiotic therapy; and to study the occurrence and survival of C. difficile in the environment and antimicrobial susceptibility of isolated strains. METHODS A total of 777 horses of different breeds, age and sex were studied. Further, 598 soil samples and 434 indoor surface samples were examined. Antimicrobial susceptibility of 52 strains was investigated by Etest for 10 antibiotics. RESULTS In horses that developed acute colitis during antibiotic treatment, 18 of 43 (42%) were positive to C. difficile culture and 12 of these (28%) were positive in the cytotoxin B test. Furthermore, C. difficile was isolated from a small number of diarrhoeic mature horses (4 of 72 [6%]) with no history of antibiotic treatment, but not from 273 healthy mature horses examined or 65 horses with colic. An interesting new finding was that, in normal healthy foals age < 14 days, C. difficile was isolated from 1/3 of foals (16 of 56 [29%]). All older foals (170) except one were negative. Seven of 16 (44%) nondiarrhoeic foals treated with erythromycin or gentamicin in combination with rifampicin were also excretors of C. difficile. On studfarms, 14 of 132 (11%) outdoor soil samples were positive for C. difficile in culture, whereas only 2 of 220 (1%) soil samples from farms with mature horses were positive for C. difficile (P = < 0.001). By PCR, it was demonstrated that strains from the environment and healthy foals can serve as a potential reservoir of toxigenic C. difficile. The experimental study conducted here found that C. difficile survived in nature and indoors for at least 4 years in inoculated equine faeces. The susceptibility of 52 strains was investigated for 10 antibiotics and all were susceptible to metronidazole (MIC < or = 4 mg/l) and vancomycin (MIC < or = 2 mg/l). CONCLUSIONS C. difficile is associated with acute colitis in mature horses, following antibiotic treatment. Furthermore, C. difficile was isolated from 1 in 3 normal healthy foals age < 14 days. POTENTIAL RELEVANCE Strains from healthy foals and the environment can serve as a potential reservoir of toxigenic C. difficile.

Molecular typing of isolates of Clostridium perfringens from healthy and diseased poultry

The bacterium Clostridium perfringens can cause both clinical and subclinical disease in poultry. To study the pathogenesis and epidemiology of disease caused by C. perfringens, methods for typing its various strains need to be evaluated. C. perfringens isolates from healthy and diseased poultry from different parts of Sweden were analysed by polymerase chain reaction (PCR) in order to establish the presence of alpha-, beta-, beta2-, epsilon -, iota- and enterotoxin genes. In order to subtype C. perfringens isolates, the two methods amplified fragment length polymorphism (AFLP) and pulsed field gel electrophoresis (PFGE) were compared on 21 C. perfringens isolates from 10 different farms. In a second study, 32 isolates of C. perfringens type A from three broilers from a healthy flock reared without ionophorous anticoccidials were subtyped by PFGE. All 53 isolates analysed with PCR belonged to the toxin type A of C. perfringens, with the gene coding for alpha-toxin production. Two isolates possessed the beta2-gene as well, but none had the other toxin genes. Both AFLP and PFGE differentiated 21 strains into 10 different subtypes. This differentiation correlated closely with the origins of the isolates. Unique subtypes were isolated from seven farms. Only isolates from birds of one farm demonstrated more than one subtype of C. perfringens. The subtyping of the isolates from a healthy flock showed that each bird carried two to three different subtypes and two different subtypes were found in the same kind of tissue sample in four cases. Three of the four different subtypes found in this study were new, compared with the first study. AFLP and PFGE were found to be equally suitable for subtyping of C. perfringens isolates. The wide variation in subtypes in the healthy broilers could be the result of the antibiotic-free rearing of these birds.

Identification of the causative agent of granulocytic ehrlichiosis in Swedish dogs and horses by direct solid phase sequencing of PCR products from the 16S rRNA gene

Seven Swedish isolates of Ehrlichia species from the blood of four dogs and three horses with clinical granulocytic ehrlichiosis, were identified by direct solid phase DNA sequencing of polymerase chain reaction (PCR) products from the 16S rRNA gene. The amplified DNA fragments were produced with primers complementary to the universal regions, U1, U2, U5 and U8 of the 16S rRNA molecule. Identical sequences were obtained from all seven isolates. This nucleotide sequence was similar to the sequences deposited in GenBank for Ehrlichia phagocytophila and E equi. The sequence of the Swedish ehrlichiae differed in two nucleotide positions from the E phagocytophila sequence and in three positions from the E equi sequence, and it is tentatively proposed that it is a subspecies of one of these two. The alignment of the sequence of the Swedish isolates with a recently deposited sequence from human cases of ehrlichiosis in the USA revealed 100 per cent identity in a segment of about 1400 bp.

Detection of granulocytic Ehrlichia species DNA by PCR in persistently infected dogs.

Three female beagle dogs inoculated with granulocytic Ehrlichia species were monitored for four to six months to determine whether there was evidence that the organisms persisted. The dogs were inoculated intravenously with blood containing an Ehrlichia species closely related to Ehrlichia equi and Ehrdichia phagocytophila, and identical to the human granulocytic ehrlichiosis agent with respect to its 16S rRNA gene sequence. The clinical signs were evaluated, and blood samples were collected for haematology, serum biochemistry and serology. Ehrlichial inclusions in the blood were monitored by microscopy, and ehrlichial DNA was detected by the polymerase chain reaction (PcR). Two of the dogs were injected with prednisolone on days 54 to 56 and days 152 to 154 after infection, and the other was injected with prednisolone on days 95 to 97 after infection. The dogs were euthanased and examined postmortem. Ehrlichial inclusions were demonstrated in the neutrophils and seroconversion occurred shortly after inoculation. Two of the dogs developed acute disease with rectal temperatures above 39.°C, after which no further clinical signs were observed. The administration of corticosteroids seemed to facilitate the detection of ehrlichial inclusions. Ehrlichial DNA was detected intermittently by PCR in blood samples from two of the dogs throughout the study. Persistent infection was demonstrated up to five-and-a-half months after inoculation.

Emended descriptions of indole negative and indole positive isolates of Brachyspira (Serpulina) hyodysenteriae.

Two type/reference strains of Brachyspira (B.) hyodysenteriae, 14 Belgian and German indole negative, and 14 Belgian, German and Swedish indole positive field isolates of strongly beta-haemolytic intestinal spirochaetes were compared by pulsed-field gel electrophoresis (PFGE) patterns, biochemical reaction patterns, 16S rDNA sequences and MIC determinations of six antibacterial substances. Three tests for indole production, including a spot indole test, were compared with congruent results. All field isolates were classified as B. hyodysenteriae due to a high genetic and phenotypic similarity with the type strains. The Belgian and German indole negative isolates had identical and unique PFGE patterns for the tested restriction enzymes MluI and SalI, as well as identical 16S rDNA sequences, and they could not be differentiated by any of the methods used. Seven unique PFGE patterns were achieved from the 14 indole positive field isolates. The patterns were identical and unique for epidemiologically related isolates. Type/reference strains and isolates without known relation to other tested isolates showed unique banding patterns. The MICs of tylosin, tiamulin, erythromycin, clindamycin, carbadox and virginiamycin were determined in broth for all isolates. In contrast to Belgian and German isolates, the majority of the Swedish field isolates were susceptible to tylosin, erythromycin and clindamycin. Probable pathways of infection for some of the Swedish isolates were determined. The PFGE patterns of epidemic clones of B. hyodysenteriae remained stable for a period of up to 8 years. In vivo development of resistance to macrolide and lincosamide antibiotics due to use of tylosin was clearly indicated for two epidemic clones.

Early manifestations of granulocytic ehrlichiosis in dogs inoculated experimentally with a Swedish Ehrlichia species isolate

Seven beagles were inoculated experimentally with a Swedish canine Ehrlichia species isolate to study its pathogenicity. With respect to the 16S rRNA gene sequence, the isolate was identical to the human granulocytic ehrlichiosis (HGE) agent and closely related to both Ehrlichia equi and E phagocytophila. After an incubation period of four to 11 days, the most prominent clinical signs were high fever for two to five days and depression. AU the dogs developed profound thrombocytopenia, moderate leukopenia and a strong serological antibody response. Ehrlichial inclusions were detected in blood neutrophils from four to 14 days after inoculation for four to eight days. Ehrlichial DNA could be detected by polymerase chain reaction during the parasitaemic stage and a few days before and after microscopic inclusions were visible. Postmortem, the dogs showed reactive splenic hyperplasia and non-specific mononuclear reactive hepatitis.

Susceptibility to pleuromutilins in Brachyspira (Serpulina) hyodysenteriae.

Abstract The pleuromutilins are the only antimicrobial agents with sufficient minimum inhibitory concentration (MIC) values left to treat swine dysentery in Sweden. Other antimicrobials are either not approved for use against swine dysentery or only partly active against Brachyspira hyodysenteriae. To date, in Sweden two pleuromutilins, tiamulin and valnemulin, are authorized for use in pigs. This study includes a comparison between MICs of tiamulin and valnemulin for Swedish field isolates of B. hyodysenteriae, as determined by broth dilution. For different isolates the MIC of tiamulin was between 0 and 8 times higher than that of valnemulin. No resistance to pleuromutilins was recorded (tiamulin MIC range 0.031–2 μg/ml, valnemulin MIC range ≤0.016–1 μg/ml). In vitro development of tiamulin resistance was also studied. Two B. hyodysenteriae and two B. pilosicoli strains became resistant to tiamulin following reiterated passages on agar containing tiamulin in increasing concentrations. The resistance emerged slowly and three of the strains that went through more than 60 passages increased their tiamulin MICs from 0.031–0.25 to more than 128 μg/ml. The tiamulin MIC for one B. hyodysenteriaestrain that went through 29 passages increased from 0.0125 to 4 μg/ml. One B. pilosicoli strain developed cross-resistance to valnemulin; the MIC increased from 0.25 to more than 64 μg/ml. The valnemulin MIC for one B. hyodysenteriae strain increased from 0.031 μg/ml to 32 μg/ml. Valnemulin MIC was not determined for the B. hyodysenteriae strain that only went through 29 passages. The valnemulin MIC of the other B. pilosicoli strain increased from 0.031 to 4 μg/ml.

Leptospira seroprevalence and associations between seropositivity, clinical disease and host factors in horses.

BackgroundA 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.MethodsSera 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%).ResultsThe 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.ConclusionThere 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.

Prevalence of verotoxin-producing Escherichia coli (VTEC) 0157 in Swedish dairy herds

A prevalence study of verotoxin-producing Escherichia coli O157 (VTEC O157) was performed in 371 randomly selected dairy herds distributed throughout Sweden. Faecal and manure samples were collected and analysed by immunomagnetic separation and culturing. Data were recorded for each herd regarding herd size, age of sampled animals and whether, in addition to cattle, the farm kept other animals. VTEC O157 was isolated from 33 (8.9%) of the 371 investigated herds. The prevalence was higher (23.3%) in Halland county than in the rest of Sweden (P > 0.01). Halland was also the county in Sweden that during the study period had the highest incidence of human VTEC O157 cases. VTEC O157 was not detected on any farm in northern Sweden. Identified risk factors, in the multivariate analyses, for herds being VTEC O157 positive were herd size, geographical localization, presence of pigs on the farm and median age of sampled animals.

Sero-prevalence of granulocytic Ehrlichia spp. and Borrelia burgdorferi sensu lato in Swedish dogs 1991-94

In Sweden, 2 tick-borne zoonotic diseases, granulocytic ehrlichiosis and borreliosis, are frequently diagnosed in dogs, using serological assays. The aims of this study were to determine the sero-prevalences of antibodies to Ehrlichia spp. and Borrelia burgdorferi sensu lato during 1991-94 in dogs, not clinically suspected to be infected with either of the 2 agents. Samples (n=611) were selected from a serum bank using a systematic sampling strategy, stratified across the 4-y period. The stored sera had originally been submitted in order to verify or rule out infection with Sarcoptes scabiei. The overall sero-prevalence for Ehrlichia spp. was 17.7% and for Borrelia burgdorferi sensu lato 3.9% (n=588). Only a few dogs in the northern part of Sweden were sero-positive for Ehrlichia spp. and none were positive for Borrelia burgdorferi sensu lato. An increased sero-prevalence of Ehrlichia spp. was seen during the years studied. The sero-prevalence of Ehrlichia spp. varied with season. Sero-positivity to both agents increased with age. Both diseases are considered zoonotic, and the increase in sero-prevalence of Ehrlichia spp. over the years may reflect the degree of infection in ticks and may have implications for human health.In Sweden, 2 tick-borne zoonotic diseases, granulocytic ehrlichiosis and borreliosis, are frequently diagnosed in dogs, using serological assays. The aims of this study were to determine the sero-prevalences of antibodies to Ehrlichia spp. and Borrelia burgdorferi sensu lato during 1991-94 in dogs, not clinically suspected to be infected with either of the 2 agents. Samples (n = 611) were selected from a serum bank using a systematic sampling strategy, stratified across the 4-y period. The stored sera had originally been submitted in order to verify or rule out infection with Sarcoptes scabiei. The overall sero-prevalence for Ehrlichia spp. was 17.7% and for Borrelia burgdorferi sensu lato 3.9% (n = 588). Only a few dogs in the northern part of Sweden were sero-positive for Ehrlichia spp. and none were positive for Borrelia burgdorferi sensu lato. An increased sero-prevalence of Ehrlichia spp. was seen during the years studied. The sero-prevalence of Ehrlichia spp. varied with season. Sero-positivity to both agents increased with age. Both diseases are considered zoonotic, and the increase in sero-prevalence of Ehrlichia spp. over the years may reflect the degree of infection in ticks and may have implications for human health.