Susceptibility of Escherichia coli from bovine mastitis to new antimicrobial drugs.
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Biomedical subjects
Publications and source records attributed to J F Prescott.
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Outbreaks of colitis, where Campylobacter jejuni and Campylobacter coli were the only pathogens isolated occurred in weanling mink (Mustella vision) on two commercial mink ranches in Ontario. Lesions were restricted to the proximal colon and were characterized by multiple 1 mm focal or 1 mm linear erosions/ulcers in the region 2 cm distal to the ileal-colonic junction. Histological changes included thickening of the colonic mucosa, inflammatory cell infiltrate in the lamina propria and submucosa, cellular debris and inflammatory exudate within cryptal lumens and multiple areas of mucosal erosion/ulceration. Four C. jejuni negative mink were challenged with 5.1 X 10(9) colony forming units of C. jejuni by oral inoculation. Three of four experimentally infected mink developed diarrhea by day 4 postinfection with lesions grossly and microscopically similar to mink in the naturally occurring outbreak. Examination of lesions by transmission electron microscope failed to show evidence of C. jejuni invasion of intestinal epithelium. Feeding uncooked slaughterhouse chicken offal was the likely source of C. jejuni in the naturally occurring outbreaks.
Polymorphonuclear leukocytes from adult horses were separated from whole blood, using a 2-step Percoll gradient, and were tested for bactericidal function against Corynebacterium equi. Staphylococcus aureus, an organism against which equine neutrophils have proved efficacy, was a positive control. The percentage of uptake after a 15-minute preincubation of the neutrophils and bacteria in the presence of normal horse serum was also calculated. The results indicated that equine neutrophils effectively phagocytosed and killed C equi and S aureus. The percentage of uptake for S aureus (95% +/- 3%) was greater than that for C equi (85% +/- 6%) (P less than 0.001), but the bactericidal efficacy was equivalent. More than 90% of the ingested or attached bacteria were destroyed during the 3-hour incubation period (mean percentage of C equi killed = 96 +/- 2%; mean percentage of S aureus killed = 91 +/- 8%). These results indicated that a failure of bacterial killing by neutrophils is unlikely to be important in the pathogenesis of C equi pneumonia in the horse.
Combined antimicrobial drug treatment was recommended for foals with Corynebacterium equi pneumonia. The preferred combination is orally administered erythromycin estolate (25 mg/kg of body weight, QID) plus rifampin (10 mg/kg, BID). Erythromycin estolate also can be combined for synergistic effect with sodium benzyl penicillin given IV (100,000 IU/kg, QID) or with ampicillin given IV (11 to 15 mg/kg, QID). A third choice is sodium benzyl penicillin IV with gentamicin IM (2.2 mg/kg, TID) or with kanamycin IM (10 mg/kg, QID). Gentamicin should be combined with penicillin G or ampicillin and not used for longer than one week without monitoring for nephrotoxicosis. Rifampin should be used only in combination with erythromycin or penicillin. Erythromycin or rifampin and gentamicin give antagonistic interactions in vitro. Chloramphenicol or trimethoprim-sulfamethoxazole may be effective if given in high doses but are not preferred drugs. Treatment response should be monitored clinically and radiographically and treatment should be continued for 2 weeks after the foal is clinically and radiographically normal.
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The production of M-protein antigen of Streptococcus equi was studied during in vitro growth in equine blood and in various media. Of 11 S equi strains studied, seven which had initially possessed 0.04 mg or less M-protein per 10 mg of streptococcal cell extract showed an increase in M-protein content after successive culture in heparinised horse blood. Maximum proliferation occurred in Todd-Hewitt (TH) medium with added 0.2 per cent w/v glucose when compared with TH medium alone or TH medium with 2 per cent w/v sucrose, starch, neopeptone or normal horse serum. The M-protein of these strains did not change after the addition of either neopeptone or normal horse serum to TH medium but declined with the addition of sugars. In experiments involving phagocytosis of S equi by equine polymorphs, the percentage of polymorphs which engulfed cocci was higher with a capsule-deficient strain (69.0 +/- 11.6 per cent) than with five typical encapsulated strains (21.1 +/- 7.0 per cent to 30.9 +/- 13.3 per cent). Phagocytosis of five typical strains was greater after growth in a trypsin-containing medium than in medium devoid of trypsin. Trypsin-grown cells took longer to kill mice than did normal cells. It was concluded that M-protein was one of the factors involved in the virulence of S equi.
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.
Bronchoalveolar lavage was performed 5 times, sequentially, on 3 healthy foals while each foal was 6 to 63 days of age. Phagocytosis and bactericidal assays were performed on recovered alveolar macrophages. Corynebacterium equi and alveolar macrophages at a ratio of 10:1 were incubated for 1 hour in medium containing 1% heat-inactivated rabbit anti-C equi serum. After incubation, greater than 90% of the alveolar macrophages contained at least 1 ingested bacterium and each alveolar macrophage contained 9.4 +/- 1.0 bacteria (mean +/- SE). After alveolar macrophages and C equi were incubated for 1 hour in medium containing heat-inactivated pooled normal horse serum, approximately 24% of the alveolar macrophages contained at least 1 bacterium and each alveolar macrophage contained 0.8 +/- 0.7 bacteria. From 6 to 61 days of age, each foal had significantly (P less than 0.05) decreased phagocytic activity by alveolar macrophages, but a significant change in killing of C equi by alveolar macrophages was not found in the foals from 21 to 61 days of age. After incubating alveolar macrophages and C equi for 4 hours in vitro, approximately 75% of ingested C equi remained viable.
The minimal inhibitory concentrations of penicillin G, ampicillin, gentamicin, erythromycin and rifampicin were determined for nine strains of Corynebacterium equi. The effect of combinations of any two of these antibiotics on the killing of these strains was determined at antibiotic concentrations achievable in horses using recommended drug dosages (ampicillin 4.0 microgram/ml, gentamicin 1.0 microgram/ml using recommended drug dosages (ampicillin 4.0 microgram/ml, gentamicin 1.0 microgram/ml and erythromycin 0.25 microgram/ml). Penicillin G was used at 4.0 microgram/ml and rifampicin at 0.063 microgram/ml. The combinations of gentamicin with erythromycin or rifampicin gave antagonistic effects on killing compared to either drug alone. Combinations of erythromycin with rifampicin or penicillin showed synergistic effects, as did penicillin--gentamicin. All other combinations, and a triple combination of penicillin--rifampicin--erythromycin, showed additive effects only.
Campylobacter jejuni were isolated in large numbers from the majority of birds sampled in colonic swabs from 28 of 60 flocks at slaughter. By contrast only small numbers of birds from 11 of the same 60 flocks yielded Salmonella enteritidis serotypes. Three C. jejuni isolates from each flock were serotyped on the basis of their heat-stable antigens, using antisera prepared against 16 serotypes common in Campylobacter diarrhea in man. The majority (72 of 83) of the chicken isolates could be serotyped.
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.
Corynebacterium equi was cultured from manure or soil on five horse-breeding farms in Ontario at monthly intervals on three occasions during the summer of 1982. The organism was widespread. Contamination by C. equi of the loafing paddock and pasture areas was significantly greater in a farm established 30 years than in two established for four and six years and there was a significant correlation between the C. equi burden in stables, paddocks and pastures and the length of use of the five farms for horses. In all farms, numbers of C. equi in pasture soil exceeded numbers in fresh manure, suggesting that environmental multiplication of the organism might occur. A farm with an endemic C. equi pneumonia problem differed significantly from the other four farms, where disease was not endemic, in the larger number of C. equi isolated in the stable area. By contrast the farm with a C. equi pasture soil burden significantly heavier than on all other farms had no deaths due to C. equi pneumonia. There was a correlation (r = 0.78, p = 0.061) between the number of cases of C. equi pneumonia on the farms and numbers of C. equi in the area of the stables, but not on the paddocks or pastures. About two-thirds of randomly chosen isolates from the farms belonged to the three capsular serotypes most commonly found in pneumonic foals.
In a survey for the somatic and capsular serotypes of Pasteurella multocida present in domestic rabbits in Canada, but mainly in Ontario, samples were obtained from research facilities, commercial rabbitries and from abattoir and necropsy specimens. Sources of isolates were upper respiratory tract infections, localized bronchopneumonias , acute fibrinous pneumonias, abscesses and otitis media. Of 59 isolates obtained, 47.0% were type 12:A, 30.5% 3:D and 12.0% were 3:A. Less common types were 12(4):A, 12:D, 4(12):A and 3:untypable. Somatic group 3 was most commonly isolated from acute pneumonic disease, while serogroup 12:A was most commonly found in upper respiratory tract infections and in localized chronic bronchopneumonia. Two serotypes of P. multocida were isolated from four pneumonic lungs collected from abattoir specimens. Most isolates were susceptible to the commonly used antibiotics.
The pharmacokinetic parameters of erythromycin in foals were determined following intravenous administration of 5.0 mg/kg to animals aged 1, 3, 5 and 7 weeks. The distribution of the drug was described by a two-compartment open model, and no significant differences were observed between coefficients on which the parameters were based. Pharmacokinetic values were also determined for four mares given 5.0 mg/kg intravenously and for six 10-12-week-old foals given 20.0 mg/kg intravenously. The half-life of erythromycin for all groups of animals (foals less than 7 weeks, mares, foals 10-12 weeks) was 1.0-1.1 h; the apparent volume of distribution was between 2.3 and 7.2 l/kg, and the clearance of the drug from the body was between 1.9 and 5.0 mg/kg/h. No drug could be detected in the serum following oral administration of 5.0 mg/kg erythromycin estolate; detectable levels were found for 5 h in mares given 12.5 mg/kg, and for 8 h in foals given 20.0 mg/kg orally. Peak levels in foals given the drug orally were 0.42 micrograms/ml at 120 min after administration. Foals given 10.0 mg/kg of erythromycin base intramuscularly had serum concentrations detectable 12 h later; the peak level achieved was 1.44 micrograms/ml serum 90 min after administration and concentrations exceeded 0.25 micrograms/ml for 6 h. In the mares the milk concentrations were approximately twice those in serum. Recommendations were made for drug dosage to be used in the treatment of Corynebacterium equi pneumonia of foals.
A total of 191 Campylobacter jejuni and 125 Campylobacter coli were isolated from the intestinal content of 398 chickens, 421 cattle, and 203 pigs. All 108 chicken isolates and 73 of 80 cattle isolates were C. jejuni, but 115 of the 118 pig isolates were C. coli. A total of 84% of the C. jejuni and 64% of the C. coli isolates were typed on the basis of thermostable antigens with 20 antisera prepared against frequently occurring serotypes in Campylobacter enteritis in man (15 C. jejuni, 6 C. coli serotypes). A total of 96% of the chicken isolates and 67% of the cattle isolates belonged to 11 C. jejuni serotypes that occur most frequently in human cases of enteritis (serotypes 1, 2, 3, 4, 5, 13/16, 18, 21, 23, 31, and 36). Serotype 8, a relatively common human isolate, was not recovered. The C. coli isolates from pigs belonged to serotypes uncommon among human isolates.
Six foals were inoculated intrabronchially with a suspension of Corynebacterium equi. Six weeks before this challenge, three foals were vaccinated with a C. equi bacterin. Three foals were unvaccinated controls. All foals developed a severe bronchopneumonia in the inoculated lung, indicating that vaccination was not protective. Three foals (two vaccinated, one control) were killed eight to nine days after infection. One control died on day 9 with lesions of disseminated intravascular coagulation. The remaining two foals (one vaccinated, one control) were killed on day 17. C. equi was cultured in large numbers from affected lung and bronchial lymph nodes, and in smaller numbers from unaffected lung, spleen, and liver in all foals. In the 8- to 9-day-old lung lesions, the alveoli were filled with macrophages, neutrophils, and multinucleate giant cells and most contained numerous C. equi. The few foci of alveolar necrosis were associated with groups of bacteria-laden macrophages undergoing degeneration. In the lesions of 17-day duration, there was extensive parenchymal destruction with little fibrous tissue reaction. Lesions common to both groups included hyperplastic bronchiolitis, pulmonary edema, and perivascular lymphocytic cuffs and a pyogranulomatous lymphadenitis in bronchial nodes. One vaccinated foal had a microscopic pyogranulomatous colitis. The lesions in the experimentally infected foals are compared with those in naturally infected foals and discussed in terms of likely pathogenetic mechanisms involved in C. equi pneumonia in foals.
The intragastric inoculation of a suspension of Corynebacterium equi on five consecutive days induced severe ulcerative colitis, typhlitis, and lymphadenitis of colonic and cecal nodes in two ponies necropsied three weeks after infection. No gross lesions were observed in two ponies necropsied ten days after infection. A single inoculum of equivalent size failed to induce gross lesions in four ponies killed at ten or 20 days after infection. Microscopic lesions consistent with early C. equi infection of Peyer's patches were seen in two of the ponies killed ten days after infection. Only one small pulmonary abscess occurred in one foal, suggesting that intestinal lesions are not likely the usual precursor of pulmonary disease in naturally infected foals. The gross and microscopic lesions in the experimentally infected ponies were typical of the intestinal form of naturally occurring C. equi associated disease in foals.
One hundred and forty-four lungs obtained postmortem from cattle with pneumonia were cultured for anaerobic bacteria. Forty-five lungs yielded 73 anaerobic isolates belonging to 20 species. The number of isolations of anaerobes from acute fibrinous or suppurative bronchopneumonias (32.5%) was slightly lower than from similar chronic bronchopneumonias (36.5%). Anaerobes were not recovered from 15 lungs showing macroscopic changes not of bacterial origin, nor from 13 healthy lungs. The predominant genera isolated were Bacteroides, Peptococcus, Fusobacterium and Clostridium. The most common species were P. indolicus (15 isolates), B. asaccharolyticus (nine), F. necrophorum (six), C. perfringens (four) and B. fragilis (four). There was a significant correlation between the presence of Corynebacterium pyogenes (p less than 0.001) or Escherichia coli (p less than 0.01) and the presence of anaerobes in the lungs. The isolated anaerobic bacteria were generally susceptible to ampicillin, penicillin G, cefoxitin, cephalothin, clindamycin, chloramphenicol, erythromycin, tetracycline and metronidazole. The B. fragilis and C. perfringens isolates showed multiple antibiotic resistance, and five P. indolicus isolates were resistant to tetracycline.