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. diff i c i l e in the environment and antimicro b i a l 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. R e s u l t s : In horses that developed acute colitis during antibiotic t reatment, 18 of 43 (42%) were positive to C. diff i c i l e c u l t u re and 12 of these (28%) were positive in the cytotoxin B test. F u rt h e r m o re, C. diff i c i l e was isolated from a small number o f d i a r rhoeic 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. diff i c i l e w a s isolated from 1/3 of foals (16 of 56 [29%]). All older foals (170) except one were negative. Seven of 16 (44%) nondiarrh o e i c foals treated with ery t h romycin or gentamicin in combination with rifampicin were also excretors of C. diff i c i l e. On studfarms, 14 of 132 (11%) outdoorsoil 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 re s e r v o i r of toxigenic C. diff i c i l e. T h e 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 4 mg/l) and vancomycin (MIC 2 mg/l). C o n c l u s i o n s : C. difficile is associated with acute colitis in mature horses, following antibiotic treatment. Furt h e r m o re, C. diff i c i l e was isolated from 1 in 3 normal healthy foals age <14 days. Potential relevance: Strains from healthy foals and the e n v i ronment can serve as a potential re s e r v o i r of toxigenic C. diff i c i l e .

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Summary Clostridium (C.) difficile, or its cytoxin, was demonstrated in faecal samples from 10 of 25 (40%) mature horses investigated with acute colitis treated primarily with antibiotics for disorders other than diarrhoea. C. difficile was not found in faecal samples from 140 horses without signs of enteric disorders, from 21 nondiarrhoeic horses treated with antibiotics, nor from 22 horses with colitis untreated with antibiotics. Except for C. difficile neither Salmonella nor any other investigated intestinal pathogen was isolated in any of the diarrhoeic horses. The findings strongly support some earlier reports that C. difficile is associated with acute colitis in mature horses treated with antibiotics. Of the 10 horses, 4 proved positive for C. difficile both in culture and in the cytotoxin test, 4 in culture only and 2 only in the cytotoxin test. Eight of 10 horses positive for C. difficile were or had recently been hospitalised, indicating that C. difficile may be a nosocomial infection in horses. All horses positive for C. difficile were treated with β‐lactam antibiotics. The authors are grateful to A. Hellander‐Edman, B. Green and J. Skidell for fruitful cooperation and for providing specimens and to G. Holmström, G. Sigstam and H. Ljung for excellent technical assistance. This research was supported by ATG (the Swedish Horserace Totalizator Board) and the AGRIA Foundation for Research.

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In Sweden, mares sometimes develop acute, often fatal, colitis when their foals are treated orally with erythromycin and rifampicin for Rhodococcus (R.) equi infection. Clostridium (C.) dificile, or its cytotoxin, was demonstrated in faecal samples from 5 of 11 (45%) mares with diarrhoea. By contrast C. di&ile was not found in the faecal flora of 12 healthy mares with foals treated for R. equi infection or in 56 healthy mares with healthy untreated foals. No other enteric pathogen was isolated from any diarrhoeic mare. Of 7 investigated treated foals, 4 had a high (1651.0, 1468.3, 273.0 and 88.8 pglg) faecal concentration of erythromycin. The dams of those 4 foals developed acute colitis, whereas the dams of 3 foals with a lower (26.3, 4.6 and 3.7 pglg) faecal erythromycin concentration remained healthy, indicating that there might have been an accidental intake of erythromycin by mares. The foals treated with antibiotics were regarded as asymptomatic carriers and potential reservoirs, as C. dificile was found in 7 of 16 foals investigated, while 56 untreated foals proved negative. The isolated C. difJicile strains proved resistant to both erythromycin (MIC >256 mg/l) and rifampicin (MIC >32 mg/l), a fact that may have favoured the growth of C. difJicile in the foal intestine. All mares found positive for C. dimile were, or had recently been, hospitalised together with their foals, indicating that C. dificile may be a nosocomial infection, in horses. The results emphasise that routine testing for C. di&ile and its cytotoxin is recommended when acute colitis occurs in mares when their foals are treated with erythromycin and rifampicin. Preventive measures in order to avoid accidental ingestion of erythromycin by mares from the treatment of their foals are suggested.

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. All 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.

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 Ehrlichia 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 39.0 degrees 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.

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