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Isolation of Pathogenic Strains of Haemophilus Somnus from the Female Bovine Reproductive Tract

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Isolation of Pathogenic Strains of Haemophilus Somnus from the Female Bovine Reproductive Tract Isolation of Pathogenic Strains of Haemophilus somnus from the Female Bovine Reproductive Tract Jacek M. Kwiecien and Peter B. Little ABSTRACT pathogenic strains of H. somnus in the (43/o) provoquerent une maladie neu- genital tract of apparently normal rologique suraigue fatale tandis que The prevalence of Haemophilus cows as well as of those with inflam- huit n'etaient pas pathogenes. somnus in the genital tract of slaugh- matory disease. Une etude pathologique compara- tered and live cows in southern Ontario tive des isolats pathogenes et non- was investigated. The vagina and pathogenes a demontri que les pre- uterus of slaughtered cows were RESUME miers causent une meningo-encephalite swabbed separately. Live cows were suppurative fatale tandis que les non- examined and sampled in two field On a etudie la prevalence d'Haemo- pathogenes ne provoquent aucune surveys: Centre A and Centre B. In the philus somnus dans le tractus genital lesion ou provoquent une legere lepto- former, aspirated mucus secretions de vaches abattues et de vaches vivantes. meningite leucocytaire. and in the latter, specimens obtained Chez les vaches abattues, le vagin et Les resultats qui ressortent de cette by guarded swabbing were examined l'uterus furent ecouvillonnes separe- etude indiquent qu'il y a des souches bacteriologically. Haemophilus somnus ment. L'examen et l'ecouvillon chez les pathogenes d'H. somnus dans le trac- was isolated from 28 genital tracts of vaches vivantes furent menes au cours tus genital de vaches apparemment 461 slaughtered (6.1%1), and seven of de deux etudes cliniques: etude A et normales aussi bien que chez celies 199 live (3.5%) cows during the cen- etude B. Dans le premier cas, des secre presentant une maladie inflammatoire. tre B survey. The isolates were tions et du mucus etaient aspires; dans (Traduit par Dr Patrick Guay) recovered from both normal and le second cas, les specimens etaient diseased reproductive tracts. Fourteen obtenus au moyen d'un ecouvilion strains isolated from genital organs protege avant d'etre evalues au plan INTRODUCTION were examined for pathogenicity bacteriologique. in vivo to test the occurrence of patho- Haemophilus somnus fut isole de Haemophilus somnus is an impor- genic isolates. In the initial stage of the 28 tractus genitaux de 461 specimens tant pathogen in cattle causing peracute in vivo study on pathogenicity, each of d'abattoir (6.1/o) et de sept des fatal septicemia and thrombotic men- the fourteen isolates was examined on 199 specimens provenant d'animaux ingoencephalitis (TME) (1), pneumonia one calf using an intracisternal inocu- vivants (3.5%) dans l'etude B. Les (2,3), arthritis (2), myocardial absces- lation. Subsequently, one pathogenic isolats furent obtenus de tractus geni- sation (4), mastitis (5-7), and abortion and one nonpathogenic strain were taux normaux et anormaux. La patho- (3,8). The frequent isolation of inoculated into five calves each to sta- genicite in vivo de 14 souches isolees H. somnus from the genital tract (9) tistically confirm their pathogenic des organes genitaux fut examinee and semen (10,11-13) of bulls is not potential. Of 14 genital isolates of pour evaluer la frequence des isolats accompanied by overt disease (9). H. somnus examined in an intracister- pathogenes. Cows harbor this organism in the nal calf assay, six (43/o) caused a fatal Au debut de l'etude in vivo sur la genital tract at a lower frequency peracute neurological disease, while pathogenicite, chacun des 14 isolats fut (10-27%o) but prevalence may vary eight were nonpathogenic. A compara- examine chez un veau apres une ino- depending on the geographical area tive pathological study of pathogenic culation intracisternale. Par la suite, surveyed (3,14-17). Haemophilus and nonpathogenic isolates showed une souche pathogene et une souche somnus may cause inflammatory that the former caused a severe fatal non pathogene furent inoculees female genital disease (8,17) or persist suppurative meningoencephalitis chez cinq veaux pour confirmer la in the genital tract for a considerable whereas the latter caused no lesions pathogenicite. time as a commensal (3,17). No work whatsoever or a mild leukocytic lepto- Des 14 isolats genitaux d'H. somnus has been carried out to determine the meningitis. The salient data obtained etudies par inoculation dans la citerne pathogenicity of isolates from the in this study indicate that there are cerebello-medullaire chez le veau, six genital tract of the cow. In limited Department of Pathology, Ontario Veterinary College, University of Guelph, Guelph, Ontario NIG 2W1. Reprint requests to Dr. P.B. Little. The work was supported by the Ontario Ministry of Agriculture and Food and the Canadian Association of Animal Breeders. Submitted July 2, 1991. Can J Vet Res 1992; 56: 127-134 127 in vivo studies, seminal and preputial this work. Colonies of H. somnus were The mucus specimens collected in strains were determined to be non- identified morphologically (24,25), and survey A were transported at ambient pathogenic in an intracisternal (I/C) the isolation confirmed by an indirect temperature to the laboratory within calf assay (18,19), a method which is fluorescent antibody test (IFAT) and 12 h and cultured on the media used regarded as the reliable way to dif- immunodiffusion test (IDT). in the abattoir survey and also on ferentiate pathogenic from nonpatho- In the IFAT the colonies were McConkey agar to aid in further genic strains of H. somnus. Fatal suspended in PBS, mounted on a glass bacteriological evaluation of the peracute meningitis, however, occurred microscope slide, air-dried and fixed specimens. in young calves inoculated I/C with with acetone for 10 min. Anti- The swab specimens from survey B TME and pneumonic isolates (18,19) H. somnus bovine hyperimmune serum were cultured on BHI-agar, McConkey which correlates with their ability to 704 against reference strain 43826 agar, and a modified Columbia agar cause septicemia and lesions typical of (20,26) was applied for 20 min and as a selective medium. The medium TME after intravenous inoculation incubated at room temperature in a differed from the selective medium (20,21). This paper addresses the humidified chamber. After washing used in the abattoir survey and in important epidemiological issue of the with PBS, a rabbit anti-704 immune survey A in that it was devoid of neo- pathogenicity of strains of H. somnus serum conjugated with fluorescein iso- mycin and the concentration of sodium isolated from normal and diseased thiocyanate was applied for 20 min and azide was lowered to 2 jig/mL (from female bovine genital tracts. then the slide washed with PBS. The 50 fig/mL) (23). The isolation of reference strain 43826 of H. somnus H. somnus was performed using was prepared in the same manner and methods described for the abattoir sur- MATERUILS AND METHODS used as a positive control. The slides vey. Organisms other than H. somnus were examined using a Leitz Wetzlar® were identified using standard methods ABATTOIR SURVEY Orthoplan fluorescent microscope in the Clinical Microbiology Labora- Reproductive tracts of dairy and beef (Walter A. Carveth Ltd., Toronto, tory, Veterinary Teaching Hospital, cows were opportunistically sampled at Ontario) and were considered positive Ontario Veterinary College. a local abattoir (J.M. Schneider's Inc., when there was an "apple" green Nine strains of H. somnus isolated Kitchener, Ontario). The vagina was fluorescence associated with the from the slaughtered cows and five cut away from the rectum, and bacterial cell wall. The IDT was per- strains isolated from live cows were together with the cervix and uterus formed using H. somnus reference cultivated in the yolk sac of five to examined for gross mucosal lesions. strain 43826 and the antigens tested seven day old embryonating eggs and A sterile swab (6 inch absorbent buds, were prepared by sonication of bacte- stored at - 70°C (20). Seven of those Johnson and Johnson Ltd., Toronto, ria (18). strains were recovered from inflam- Ontario) was inserted to sample the matory uterine exudate, one from the mucosa of the anterior vagina and FIELD SURVEY cervix of a live cow with vulvitis and posterior cervix. The serosa of the Numerous dairy herds in southern the remaining six isolates were uterus was rinsed with 70%o alcohol, Ontario were visited with theriogenolo- recovered from grossly normal cervix the wall incised, and a separate swab gists from centre A (United Breeders or vagina. inserted into the exposed uterine Inc., Guelph, Ontario) and centre B cavity. The swabs were placed sepa- (Western Ontario Breeders Inc., STUDY OF STRAIN PATHOGENICITY rately into sterile tubes containing Woodstock, Ontario). The cows were Fourteen genital strains of 1 mL of phosphate buffered saline selected for sampling according to the H. somnus were selected on the basis (PBS), pH 7.2, with 0.1 % porcine breeding history which indicated a of whether the genital tract had inflam- gelatin (Sigma Chemical Company, reproductive problem. A genital matory lesions. The isolates were tested St. Louis, Missouri) as a transport examination per rectum was per- for pathogenicity by intracisternal medium. formed by the theriogenologist during inoculation into young Holstein bull The specimens, maintained at the visit. calves (#1-14), whereby each strain of ambient temperature, were plated on Cows in survey A (Centre A) were H. somnus was examined separately in solid media within 3 h. The vaginal sampled by introduction of a regular one animal. Two strains, 87121531 and and uterine swabs were cultured on insemination catheter into the uterus 88159, were later inoculated into five two kinds of media: (I) a Columbia and/or cervix and aspiration of some calves (#15-24) to confirm their agar selective medium, developed for mucus or mucopurulent material into pathogenicity.
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