Purification and properties of cholesterol oxidase and choline phosphohydrolase from Rhodococcus equi.

Cholesterol oxidase (CO) and choline phosphohydrolase (CPH) exoenzymes were isolated from culture supernatants of Rhodococcus equi ATCC 33701 and their hemolytic and cytotoxic activities examined. The purifications involved differential ammonium sulphate precipitation, ion exchange and gel filtration chromatography. A purification of 32.8-fold and a yield of 0.3% of CO were determined by synergistic hemolysis of sheep red blood cells (SRBC) presensitized with Staphylococcus aureus beta toxin. The enzymatic activity of CO was also demonstrated by oxidation of aqueous cholesterol suspensions. The activity of CO was reversibly inhibited by concentration. A purification of 412.4-fold and a yield of 1.7% of CPH were determined by hydrolysis of p-nitrophenyphosphorylcholine. Purity of both exoenzymes was confirmed by immunoblotting. On sodium dodecyl sulphate polyacrylamide gel electrophoresis, the CO had a molecular mass (Mr) of 60 kd and the CPH a Mr of 65 kd. Choline phosphohydrolase did not hydrolyse sphingomyelin. Sphingomyelinase C (SMC) activity was however demonstrated in concentrated culture supernatants. This dissociation of SMC from CPH activity indicates that R. equi produces two distinct phospholipase C exoenzymes, a CPH and a SMC. Both CO and CPH combined, or individually, did not lyse native SRBC even with subsequent chilling of the cells at 4 degrees C ("hot-cold" treatment). Purified CO lysed beta toxin-sensitized SRBC. The CPH showed only minor hemolytic activity against such sensitized SRBC even at high concentrations. Combination of CO and CPH in lysis of beta toxin sensitized SRBC showed only minor additive rather than synergistic effects.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sporadic, severe bronchointerstitial pneumonia of foals

Bronchointerstitial pneumonia was diagnosed postmortem in 19 foals in a 10 year retrospective study of submissions to a diagnostic center in Ontario. Mean age at death was 2.0 ± 0.05 (SEM) mo (range five days to four months). Fourteen of 19 were aged from 1.5 to 2.5 mo. Clinically, the disease was generally characterized by sudden onset of fever and increasingly severe dyspnea which developed into respiratory distress before death. Mean length of illness was 7.0 ± 0.33 days (range 1-21 days). The disease appeared to affect only individual foals on 19 different farms.

At postmortem, lungs were typically diffusely red, wet, firm, and failed to collapse. The major lesion recognized histologically was epithelial necrosis of alveoli and terminal bronchioles. Alveolar lumens contained large epithelioid cells, which were probably macrophages, and multinucleated syncytial cells were present in 16 of the 19 lungs. Inflammatory cells were sparse. Intraalveolar fibrin was prominent in all lungs. Bacteriological examination revealed no significant pathogen in 12 animals, but Rhodococcus equi was isolated from seven foals, associated in two animals with extensive abscesses. Viruses were not recovered from eight foals examined.

On the basis of the similarity and severity of lesions, the sporadic nature of the disease, and the similar age at onset which appears to coincide with declining maternally-derived immunoglobulins, we speculate that this disease may be the result of a viral infection.

     

Is canine leptospirosis underdiagnosed in southern Ontario? A case report and serological survey

An eight-year-old city-dwelling Cairn Terrier was presented to a veterinary hospital in acute renal failure with evidence of hepatic insufficiency. The dog was treated symptomatically over three days, during which time vomiting was largely controlled, but it became jaundiced as hepatic insufficiency worsened. Leptospira pomona was demonstrated in large numbers by immunofluorescent staining of urinary sediment. It was isolated and its identity confirmed as L. pomona genotype kennewicki. The source of the infection was thought to be raccoons.

Sera from 474 blood samples submitted for diagnostic purposes to two clinical pathology laboratories in southern Ontario were examined with the microscopic agglutination test for antibodies to selected leptospiral serovars. Of the sera tested, 39.2% reacted at titers ≥1:100 with one or more serovars, the majority of all sera (26.2%) reacting at low titers to canicola or icterohaemorrhagiae, or both. These reactions likely resulted from vaccination. A smaller proportion reacted to other serovars tested: autumnalis (3.8%), bratislava (8.2%), grippotyphosa (1.9%), hardjo (3.0%), and pomona (3.2%). Among dogs reacting to these latter serovars (other than bratislava), many had broadly cross-reacting and relatively high titers. One dog with a titer of 1:800 to pomona had had a disease typical of leptospirosis two years previously. Three other dogs with high titers to autumnalis, bratislava, or mixed serovars had clinical histories compatible with leptospirosis.

We suggest that leptospiral bacterins for dogs in Ontario be broadened to include at least serovars autumnalis and pomona.

     

Relatedness of Streptococcus canis from canine streptococcal toxic shock syndrome and necrotizing fasciitis.

The emergence of streptococcal toxic shock syndrome (STSS) and necrotizing fasciitis (NF) in dogs caused by Streptococcus canis has been reported by our laboratory. Since clonal expansion is thought to be partially responsible for the spread of invasive strains of Streptococcus pyogenes in humans, the relatedness of 15 isolates of S. canis from canine STSS and/or NF was examined using pulsed field gel electrophoresis and biotyping; production of proteases and of a CAMP-like reaction were also examined. Only 2 of the 15 STSS and/or NF isolates were clonally related, suggesting that the emergence of canine STSS/NF is not the result of clonal expansion of one or more highly virulent strains of S. canis. All of the isolates produced proteases and demonstrated a CAMP-like reaction, which appear to be additional characteristics of S. canis.     

Antibody to equi factor(s) in the diagnosis of Corynebacterium equi pneumonia of foals.

Antibody to equi factor(s) in cases of Corynebacterium equi pneumonia in foals was detected using C. pseudotuberculosis exotoxin sensitized calf red blood cells. The test was standardized using antitoxin produced in rabbits by injection of equi factor(s). All sera from ten foals with culture-diagnosed C. equi pneumonia had antibodies to equi factor(s) (titre range 8-256, mean 74.0) and nine sera from 11 foals with suspected C. equi pneumonia also showed antibodies (titre range 4-512, mean 136.4). Two of five pneumonia foals with transtracheal aspirate cultures not yielding C. equi had such antibodies. Fifty-eight of 59 control horse sera had no antibodies; the one positive serum came from a foal on a farm where C. equi pneumonia was endemic. By contrast only five of 15 foals with experimentally-induced C. equi pneumonia had antibodies to equi factor(s), probably because the acute nature of the disease produced did not mimic the chronic course of the natural disease. Antibody to equi factor(s) can be used in the diagnosis of naturally-occurring corynebacterial pneumonia in foals.     

Pasteurella multocida infection in the domestic rabbit: immunization with a streptomycin-dependent mutant.

Fourteen Pasteurella multocida-free rabbits were inoculated intranasally with a streptomycin-dependent mutant of P. multocida serotype 12:A. Vaccinations with approximately 10(8) colony forming units were done on days 0, 14 and 28. Two weeks later the animals were separated into groups, which included 12 rabbits divided into two control groups of six unvaccinated Pasteurella-free animals. Seven vaccinated rabbits were challenged intranasally with the homologous virulent parent strain and the other seven vaccinates were challenged with a virulent strain of serotype 3:A. Rabbits were necropsied two weeks later. The vaccinated group challenged with the parent strain showed a more rapid nasal clearance of the organism than the vaccinated group challenged with the heterologous strain. However, the number of positive cultures of P. multocida recovered from tissues post-challenge were similar in vaccinated and control animals. In a significant number of animals, vaccination with serotype 12:A induced detectable antibody production to somatic antigens of both 12:A and heterologous strain 3:A.     

Experimental Studies on the Pathogenesis of Corynebacterium equi Infection in Foals

Four month-old foals were infected orally with 75 mL of a suspension of 5.0 × 10⁸ Corynebacterium equi per mL. Two foals were killed after ten days and had scanty number of C. equi in the caeco-colic lymph nodes. No C. equi were recovered from the other two foals, killed 20 days after infection. No gross pathological change was detected in these four foals, although mild microscopic lesions were seen in the ileum of one foal. Results of lymphocyte blastogenesis using peripheral blood lymphocytes and C. equi antigens showed, however, that lymphocytes became sensitized to C. equi following this challenge.

In a second experiment four month-old foals were given orally the same dose of organisms but on five consecutive days. Two foals were killed ten days after infection and showed mild histological changes in the large bowel mucosa and C. equi could be recovered from all intestinal lymph nodes cultured. In one of these foals moderate numbers of C. equi were present in the bronchial lymph node. Of the other two foals, one died after 22 days with severe ulcerative enterocolitis and intestinal lymphadenitis. Only one small pulmonary abscess was detected despite large numbers of C. equi in the lungs. The other foal developed similar intestinal changes and was euthanized 25 days after infection. No C. equi were detected in the lungs or bronchial lymph node. Lymphocyte blastogenesis in these animals showed a rapid rise in response to C. equi antigens.

These studies suggest that C. equi pneumonia in foals does not always arise from an intestinal infection, that minor intestinal infection causes a cellular immune response and that massive exposure of the bowel over a sustained period is necessary to induce intestinal lesions.