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. the isolation of H. somnus (22) and the catheter. One to three mL of the A total of 24 Holstein bull calves adapted in our laboratory through aspirate was immediately placed into three to eight weeks old were kept in substitution of Columbia agar (Oxoid) sterile tubes containing 1 mL of the isolation facilities, fed with antibiotic with Columbia agar (Difco) (23), and transport medium. Cows examined in free-rations and examined clinically for (II) a brain heart infusion agar (BHI- survey B (Centre B) were sampled one week prior to the challenge. The agar) used as a nonselective medium using guarded sterile swabs (Kalayjian Animal Care Committee guidelines (18). Plates were incubated for 24 h at Industries Inc., Long Beach, Califor- were followed during all experiments. 37°C in an atmosphere containing 5q7o nia), which were exposed in the uterus The inoculum of each strain examined CO2. These conditions of incubation or the posterior cervix and immediately in vivo was prepared as described pre- were used for all bacterial cultures in placed into the transport medium. viously (20). The prepared H. somnus

128 suspension was estimated to contain and handled as described previously RESULTS 105 colony-forming units (CFU)/mL and examined for presence of homol- and 1 mL of this suspension was used ogous IgG2 anti-H. somnus antibodies ABATTOIR SURVEY as an intracisternal inoculum within prior to the challenge. The titers were A total of 461 reproductive tracts 1 h after preparation. The actual num- measured using a dot-blot immuno- from 405 dairy and 56 beef cows were ber of bacterial CFU inoculated was assay described previously (27). examined. Haemophilus somnus was counted in residual inoculum immedi- The first group of five animals isolated from 28 cows (6.1%) compris- ately after the animal inoculation (20). (#15-19) were inoculated with strain ing 24 isolates from dairy (5.97o) and The calves were sedated with an 88159 shown in the preliminary trial to four from beef (7.1 070) cows. Twenty- intramuscular dose of xylazine be pathogenic and the second group of four (85.7!7o) strains were isolated (Rompun®, Bayvet Division, Chemagro five (#20-24) with strain 87121531 from grossly healthy genital organs Ltd., Etobicoke, Ontario) 0.25-0.5 which was considered nonpathogenic. and four strains (14.3%) from diseased mg/kg body weight and then by an Histological lesions observed in two organs of two cows with pyometra and intravenous dose of chloral hydrate- sections, the thalamus and the two with endometritis (Table I). MgSO4 solution until profound nar- cerebellum/rostral medulla of the Haemophilus somnus colonies cultured cosis occurred (20,21). The inoculation calves, were assessed using 13 criteria on Columbia agar (22) were small, technique into the cisterna magna has in order to compare the same type of nonhemolyzing and grey. Numbers of been described (20). Approximately lesions among the examined calves. colonies were markedly higher on BHI- 1 mL of cerebrospinal fluid (CSF) was The 13 histological criteria were as fol- agar than on Columbia agar. Twenty- withdrawn and 1 mL of the inoculum lows: in meninges, infiltration by intact one specimens were overgrown by con- containing H. somnus was injected. leukocytes (i) and infiltration by taminants including Escherichia coli in All calves were observed during necrotic leukocytes (ii); in choroid 12 plates, Proteus sp. in seven, Actino- recovery from anesthesia and then plexus, severity of the interstitial mycespyogenes in one, and Pasteurella repeatedly examined clinically until inflammation (iii); in pyencephalus, multocida in one. Of 28 isolated strains euthanasia was performed. The calves numbers of intact leukocytes (iv) and of H. somnus, 27 were positively iden- with a severe acute neurological disease numbers of necrotic leukocytes (v); tified on the IFAT and 12 on the IDT. were euthanized when in lateral vasculitis in the brain parenchyma, recumbency near extremis, while tran- perivascular leukocytosis (vi), fibrinoid FIELD SURVEY siently mildly affected animals were vascular necrosis (vii), and perivascular Survey A killed three to four days after the chal- protein-rich fluid exudation or hemor- A total of 154 dairy cows were lenge. Withdrawal of some CSF from rhage (viii). The remaining criteria examined and sampled in 30 herds. the cisterna magna through the spinal included; vasculitis in the meninges, One strain of H. somnus was isolated needle was attempted immediately perivascular leukocytosis (ix), flbrinoid in a mixed culture with other organisms after euthanasia. A routine total post- vascular necrosis (x), and perivascular from an asymptomatic repeat breeder. mortem examination was performed. protein-rich fluid exudation or hemor- Of the examined animals four cows The brain and other tissues were rhage (xi); neutrophilia in the neuropil had a purulent metritis, 60 were repeat removed for histopathology and a CSF (xii), and microglial reaction in the breeders, 30 were recently calved and sample and sections of the brain, lung, neuropil (xiii). The lesions in each cat- had a prolonged mucopurulent post- liver, kidney and spleen were taken for egory were scored according to severity: parturient discharge requiring antimi- bacteriological examination and 0, 1, 2 and 3. The total scores for the crobial treatment. Five cows aborted cultured on BHI-agar. Tissues for section of cerebellum/medulla and within a few weeks prior to the sam- histopathology were fixed by immer- thalamus from five calves in the first pling and required treatment, and sion in 10%7o buffered formalin, and the group were compared with the scores 55 cows which were apparently normal sections of basal ganglia, parietal cor- for both these sections for the second were examined and sampled in the tex, thalamus, midbrain, cerebellum group. An analysis of variance postparturient interval. In total, ten and rostral medulla, and caudal (ANOVA) procedure from SAS Inc. bacterial species, including H. somnus, medulla, as well as sections of the (28) was used to determine statistically were identified from the 126 cows. other organs were processed routinely significant differences. A large number of specimens con- for histological examination. The examination of the microscopic slides was performed with no knowledge of the severity of the clinical disease TABLE I. Isolation site of H. somnus from the genital tract of slaughtered cows and findings on the postmortem examination. Condition of the genital tract Two genital isolates, 88159 and Isolation site Normal Diseaseda Total (¾) 87121531, were selected from the Vagina only 14 0 14 (50) Uterus only 5 1 6 (21.4) group of 14 to confirm in vivo the Both sites 3 8 reproducibility of their pathogenic 5 (28.6) potential. Both strains were recovered Total (%) 24 (85.7) 4 (14.3) 28 (100) from the normal vagina of slaughtered aInflammatory lesions were associated in two animals with endometritis and two with pyometra cows. Ten calves were kept in isolation as diagnosed on gross examination

129 tained contaminants. The most com- PATHOGENICITY STUDIES which were variably scattered on the monly isolated bacteria were E. coli, The number of bacterial CFU in the serosal surfaces of organs of the 69 times, a-streptococci, 57, Bacillus inocula of the 14 I/C studies varied thoracic and abdominal cavity. sp., 30 and A. pyogenes, (22). from 0.11-15 x 105. This moderate Of the eight calves which did not variation of numbers of bacteria used develop fatal neurological disease after Survey B in the challenge did not appear to the I/C challenge, six had no gross A total of 197 dairy and two beef influence the mortality of calves. All pathological lesions. One animal (#8) cows were examined and sampled in 85 challenged animals recovered well had a minor amount of slightly cloudy different herds. Seven (3.52%) strains from anesthesia within 30-60 min after CSF and cloudy yellow basal lepto- of H. somnus were isolated from seven the inoculation. meninges, whereas the other (#9) had dairy cows of which four were repeat a mild fibrinous exudate in the men- breeders, two had a prolonged post- Clinical signs inges over the dorsal aspect of the parturient discharge and one had Six calves (#1-6) developed severe medulla oblongata. aborted four days prior to the sam- neurological disease, became anorexic, pling. All strains of H. somnus were progressively depressed, ataxic and Bacteriology isolated either in a mixed or pure cul- recumbent within 5 h to 10 h after the On bacteriological examination of ture on BHI-agar and four strains on challenge and developed progressively the CSF, large numbers of H. somnus the KD-Columbia agar, when it was severe opisthotonus and rigidity of the were found in all calves with fatal employed. Colonies of H. somnus limbs. These signs were followed by neurological disease. Basilar sections cultured on the KD-Columbia agar hypothermia and lateral recumbency of the rostral brain collected from were similar in size and morphology with severe tonic-clonic convulsions of these calves yielded large to moderate to colonies grown in parallel on the limbs prior to euthanasia, 16-29 h numbers of H. somnus from four BHI-agar. after the challenge. The six genital calves (#1,3,5,6) and no bacterial Overall, 13 cows were diagnosed as isolates of H. somnus inoculated I/C growth from two calves (#2,4). having metritis, 141 were repeat in these six calves were designated Haemophilus somnus colonies were breeders, 31 had a prolonged inflam- pathogenic strains. always isolated from the CNS in pure matory postparturient discharge which In the remaining group of eight culture. There was no significant bac- required treatment, and 14 cows had animals (#7-14) the intracisternal terial growth from the remaining aborted in the past few weeks. From inoculation of H. somnus had only organs cultured. 99 (49.75%) cows which were positive mild effects, characterized by mild to Of the eight nonfatally affected on bacterial culture, a-streptococci moderate anorexia, hyperthermia, animals, the CSF and brain tissue from were isolated most frequently, at 39 depression, mild ataxia and variable all were negative on bacterial culture, times, and were followed in number by opisthotonus all of which were tran- except for low numbers of H. somnus E. coli, 24, A. pyogenes, 22, Bacillis sient. All eight calves were healthy colonies in the CSF of one calf (#14). sp., 19, diphtheroids, 9, nonhemolyz- when euthanized three to four days ing staphylococci, 8, and H. somnus, after the I/C challenge. The genital Histopathology 7. Bacterial contamination of the spec- strains of H. somnus inoculated I/C in On histological examination five imens was not a problem in this part these eight calves were designated non- severely affected calves (#1-5) had pro- of the field study. pathogenic strains. nounced fibrinopurulent meningitis The presence of moderate to high composed of a massive infiltration of numbers of E. coli and ca-streptococci Necropsy intact and necrotic neutrophils and and the presence of Bacillus sp. colo- The six calves with prominent neu- mononuclear cells sometimes accom- nies on the nonselective medium was rological disease had severe lesions in panied by a localized leukocytic infil- considered bacterial contamination. the central nervous system (CNS). All tration of the subjacent brain paren- The proportion of samples containing of these animals had a moderate chyma (Fig. 1). Large bacterial colonies potential contaminants to the total amount of fibrinopurulent exudate in were frequent in the leptomeninges of number of samples for the repeat the atlantooccipital joint and a large the brain stem. Meningeal and paren- breeder category was calculated in volume of cloudy CSF containing chymal blood vessels adjacent to the both field surveys and the values com- strands of fibrin. Cloudy yellow col- meninges and ependyma had fibrinoid pared using a Chi square test (29). The ored meninges covered the flattened necrosis of the tunica adventitia and repeat breeder cows sampled in sur- cerebral gyri and there was a cone tunica media, accompanied by intra- vey A had a significantly higher shaped posterior cerebellar herniation vascular leukocytosis and swollen or incidence of bacterial contamination in into the foramen magnum. There was exfoliated endothelium (Fig. 2). Peri- mucus samples than those sampled extensive fibrin deposition in the men- vascular exudation of a protein rich using guarded swabbing in survey B inges of the lower brain stem and fluid and hemorrhage surrounded (p < 0.05). Although the difference in rostral cervical spinal cord. Both some damaged meningeal and paren- the proportion of contamination lateral and third ventricles were mildly chymal blood vessels. In all cerebral between the total number of cows in to moderately distended with cloudy ventricles, there was severe purulent both field studies appeared obvious, it CSF. All six animals had marked inflammation of the choroid plexus was not found to be significant pulmonary congestion, edema and and large amounts of fibrinous statistically. petechial and ecchymotic hemorrhages pyencephalus containing scattered

130 plexus. This animal had a severe leptomeningitis adjacent to the pituitary with visible bacterial colonies. Animal #7 had a moderate leptomeningitis of the brain stem and moderate vasculitis in the meninges, adjacent parenchyma and subependymal neuropil. Inflam- mation of the choroid plexus and pyencephalus was composed of intact leukocytes occasionally surrounding small bacterial colonies. This animal also had a fibrinopurulent pituitary adenitis. The thalamic section of calf #13 and the cerebellomedullary section of calf #14 were not examined microscopic- ally. Total histological lesion scores in the thalamic and cerebellomedullary sections of the 14 calves are presented graphically in Fig. 4. Fig. 1. Section of the rostral medulla oblongata from calf #2 inoculated I/C with a pathogenic strain of H. somnus 8711337 and euthanized 21 h after challenge, showing severe fibrinopurulent Statistical study meningitis and localized invasion of the adjacent brain parenchyma by inflammatory cellular exudate. Ten calves with no significant IgG2 There is leukocytic infitration of the Virchow-Robin space of the adjacent parenchymal blood vessels. anti-H. somnus antibody titers were H&E. Bar 300 iLm. inoculated with two strains, five animals per strain. Group 1 (#15-19) were each inoculated with 2.5 x 105 CFU of strain 88159, which had been shown to be pathogenic. These all recovered well from the general anesthesia but subsequently all suc- cumbed to a severe neurological disease and were euthanized when in lateral i-17" recumbency, and near death within 12 to 21 h after the challenge. All had a severe fibrinopurulent meningitis and 'o 4. the cultured CSF samples yielded 9 heavy pure growth of H. somnus. The brain sections from these animals yielded large to low numbers of A; H. somnus, while the remaining organs sampled were bacteriologically negative. Histologically severe fibrinopurulent meningitis with a large pro- fe " _ portion of necrotic leukocytes was A.-§-r associated with scattered bacterial col- Fig. 2. Midbrain section from calf #1 inoculated I/C with a pathogenic strain of H. somnus 87102721 and euthanized 16 h after challenge, showing severe fibrinopurulent meningitis. A large bacterial onies. The lesions of vasculitis, inflam- colony (arrow) is adjacent to the wall of a blood vessel with mural fibrinoid necrosis, loss of mation of the choroid plexus and endothelium and intravascular leukocytosis with fibrin deposition. Also, massive mixed leukocytic pituitary adenitis were similar to those infiltration of the perivascular space is composed of neutrophils and mononudear cells, large numbers in severely affected calves examined of which are necrotic. H&E. Bar 40 1sm. previously. bacterial colonies and much leukocytic the remaining five severely affected Group 2 (#20-24) were inoculated necrosis. A pituitary adenitis was due calves. with 1.2 x 106 CFU of H. somnus to neutrophilic infiltration of the pars Of eight calves with mild and non- strain 87121531 each and showed only nervosa and intermedia and adjacent fatal disease, two animals, (#11,12) transient clinical signs of anorexia, meningitis. The remaining fatally had no histological lesions. Two calves apathy and elevation in body temper- affected calf (#6) had similar CNS (#13,14) had a mild diffuse leptomen- ature. No animal in this group had any lesions, but the inflammatory infiltrate ingitis (Fig. 3) and calf #9 additionally abnormal findings on gross necropsy was predominantly composed of intact had mild leukocytic cuffing of some of examination when killed three days leukocytes. The vascular lesions were the parenchymal blood vessels and postinoculation. Numbers of H. somnus also less severe in this animal than in mild inflammation of the choroid colonies ranging from three to 20 were

131 so

so-

mm ._ _

Fig. 3. Section of caudal medulla oblongata from calf #7 inoculated I/C with a nonpathogenic Fig. 4. Scoring of severity of histological lesions strain of H. somnus 87121531 and euthanized 95 h after the challenge, showing mild subacute men- observed in the section of cerebellum and ingitis with low numbers of mononuclear leukocytes scattered in the leptomeninges. H&E. Bar 14-medulla and the section of thalamus following 100 Am. intracisternal inoculation of 14 genital strains of H. somnus into 14 calves. solid bar-cerebellum and medulla, crossed bar isolated from the CSF of three animals ductive tract. No association between - thalamus, (#20,21 and 24), whereas the CSF from the pathogenicity of 14 isolates and the 148710622* -only the section of cerebellum and the two remaining calves, (#22 and 23), clinical status of the animal at the time medulla were examined and scored, 87101435t-only the section of thalamus was and the remaining organs of all animals of isolation could be made, since of six examined and scored. were negative on culture. Histo- pathogenic strains three were recovered pathological examination of the CNS from grossly normal genital tracts and revealed mild generalized leukocytic three from diseased genital tracts. leptomeningitis and mild leukocytic Similarly, of eight nonpathogenic cuffing of the parenchymal vasculature strains, four were recovered from nor- adjacent to the meninges or ependyma. mal and four from diseased reproduc- There was mild inflammation of the tive tracts. choroid plexus and calf #24 had a The I/C calf assay reliably differen- fibropurulent coagulum in the fourth tiated one pathogenic strain, which ventricle with intact and necrotic caused a rapidly progressing fatal Is 16 1 @ 9 s a leukocytes. All calves of this group had neurological disease and severe fibri- severe suppurative pituitary adenitis nopurulent meningoencephalitis in six and adjacent meningitis, and calf #22 calves, from one nonpathogenic isolate had scattered bacterial colonies in the which caused only a mild transient meningeal exudate. disease and mild to moderate, histolog- The scores of histological lesions for ical lesions. The remaining 12 isolates the section of cerebellum/medulla and were tested in one animal each, and the section of thalamus from five therefore can only presumptively be calves from the first group were signif- called "pathogenic" (five strains) or pawnic nq icantly higher than the scores for the "nonpathogenic" (seven strains). Fig. 5. Scoring of histological lesions in the corresponding sections from five calves Histological lesions produced in the thalamic region and in the region of the cere- from the second group (p < 0.0001 for CNS by these 14 strains indicated the bellum and medulla of ten calves, following of an or none intracisternal inoculation of a pathogenic, 88159 both sections). Total scores of the absence all phenomenon or nonpathogenic, 87121531 strain of H. somnus. histological lesions in both sections regarding pathogenicity of genital The scores for calves inoculated with the from both groups of calves are pre- isolates of H. somnus. In addition to pathogenic strain were significantly higher than sented graphically in Fig. 5. a well defined group of isolates pro- those for calves inoculated with the nonpathoge- mild or no and a nic strain, p < 0.0001. ducing damage, Solid bar-cerebellum and medulla, crossed bar DISCUSSION group of strains producing severe - thalamus. lesions, there were also strains which This study demonstrated the fell into an intermediate group. One a similar moderate degree. Although prevalence of pathogenic strains of pathogenic and two nonpathogenic histological lesions indicated a substan- 'H. somnus in the female bovine repro- isolates produced lesions in the CNS of tial inflammatory response to both

132 "nonpathogens", the mild course of reproducibly differentiate pathogenic genital mucosa, rarely causing a local clinical disease caused by these isolates from nonpathogenic isolates. The inflammatory response. showed this term to be useful. pathogenicity of an H. somnus isolate It should now be determined Differences among individual in the I/C test corresponds to its poten- whether there are pathogenic strains of animals used for the I/C testing of tial to cause septicemia and lesions of H. somnus in the genital tract of the isolates in the initial stage of the study TME after intravenous inoculation of bull and in semen samples. Humphrey might have had an influence on their calves as shown by Stephens et al (18) and Groom (19) tested a limited susceptibility to infection. Haemophilus (20,21). In the present study the ability number of male genital isolates in the somnus antibody titers were not of genital isolates of H. somnus to I/C calf assay and found them to be examined in those 14 calves, but all of cause disease syndromes other than nonpathogenic, but the testing of a them were procured from one herd meningoencephalitis, i.e. arthritis, large number of strains is required to with no history of H. somnus-related myocardial abscessation, pneumonia clarify this issue. The development of disease and were raised in isolation and inflammation of the genital tract, an in vitro model for the reliable prior to the experiments. Calves used was not investigated. The other in vivo testing of large numbers of genital in the statistically valid test originated models used for testing the pathogenic- strains of H. somnus is an important from the same herd and had no signif- ity of strains of H. somnus, i.e. a calf requirement to replace the I/C calf icant IgG2 anti-H. somnus antibody subcutaneous chamber inoculation assay which is costly and raises serious titers. Measurement of the IgG2 class (19) and a pulmonary assay (31) have humane concerns. Such a putative of antibody has been proposed as the been used to a limited extent. Also in vitro test would be an important step most reliable method to assess serolog- testing of a limited number of strains for further studies on the epidemiology ically the status of prior infection with has been done by the infusion of the of the H. somnus disease complex and H. somnus in cattle (30). bacterial suspension into the nongravid the screening of exported live cattle The prevalence data obtained in live uterus or cervix, and infection of the and semen samples for pathogenic cows examined in field survey B pregnant uterus (reviewed in 32). isolates. The identification of impor- (3.52%), was lower than that obtained Pathogenic strains tested in the intra- tant factors of bacterial virulence may in the abattoir (p < 0.1). Comparison venous septicemic challenge produced also be accelerated by the use of such of both data cannot be made in this disease in 60-70% of animals and a test, and the findings could lead to study however, because in contrast to needed large numbers of calves for the development of more effective the centre B study, the origin of validation (20,21). The I/C calf assay vaccines. slaughtered cows was not determined. was used in the present study because The bacteriological results from of its confirmed clearcut reliability to both field surveys indicated that sur- determine pathogenicity of H. somnus ACKNOWLEDGMENTS vey B provided reliable data on the isolates and the relatively simple inocu- prevalence of H. somnus in live cows. lation and examination procedure, The authors thank Dr. N. Shain, A guarded swabbing technique com- contrary to most of the other in vivo United Breeders Inc., and Drs. E. bined with KD-Columbia agar as a assays. Empringham and G. McKay, Western selective medium (survey B), was Since pathogenic strains of H. som- Ontario Breeders, Inc., for their con- found to be superior to the technique nus were found among genital isolates tribution in the field studies. of aspiration of genital mucus (sur- from the cow this provides an impor- vey A) for successful isolation of tant link in understanding the epide- H. somnus. Guarded swabs had little miology of the TME syndrome. The REFERENCES contamination and the KD-Columbia female bovine reproductive tract is a agar was not suppressive for H. somnus source of not only nonpathogenic but 1. KENNEDY PC, BIBERSTEIN EL, but inhibited gram-positive organisms, also pathogenic strains of H. somnus. HOWARTH JA, FRAZIER LM, As DUNGWORTH DL. Infectious meningo- particularly a-streptococci, the most suggested before, orovaginal con- encephalitis in cattle caused by a important contaminant in survey B. tact between cattle may lead to tran- Haemophilus-like organism. Am J Vet Res The sampling of the anterior vagina sient infection of the nasal passages 1960; 21: 403-409. and cervix was elected in the abattoir and upper respiratory tract (25) and in 2. PANCIERA RJ, DAHLGREN RR, and survey B to avoid fecal contamina- suitable circumstances be followed by RINKER HB. Observations on septicemia of cattle caused by a Haemophilus-like tion. Although the rate of isolation of pneumonia and/or bacteremia with organism. Pathol Vet 1968; 5: 212-226. H. somnus can be higher in the subsequent septicemia and TME. This 3. CORBEIL LB, WIDDERS PR, GOGO- vestibular glands than in the anterior work supports observations made by LEWSKI RP, ARTHUR J, INZANA TJ, vagina and cervix (14,16), sampling of other workers using the I/C calf assay WARD ACS. Haemophilus somnus: Bovine reproductive and respiratory disease. Can vestibular glands in live animals is dif- (18,19) that pathogenicity of H. somnus Vet J 1986; 27: 90-94. ficult and prone to fecal contamination. for the CNS is determined by individ- 4. GUICHON PT, PRITCHARD J, JIM GK. The I/C calf assay has been used ual strains. Haemophilus somnus myocarditis in a previously by Humphrey (18) and The comparison of the prevalence of feedlot steer. Can Vet J 1988; 29: 1012-1013. Groom (19) to test virulence of isolates H. somnus in cows with genital disease 5. ARMSTRONG KR, OSBORNE AD, JANZEN ED. Haemophilus somnus mastitis of H. somnus including those recovered and in healthy cows indicates, similarly in a dairy cow. Can Vet J 1986; 27: 211-212. from the male genital tract. Both to other studies (32), that H. somnus 6. HIGGINS R, MARTIN JR, LAROUCHE authors were able to reliably and is a commensal well adapted to the Y, GOYETTE G. Mastitis caused by

133 Haemophilus somnus in a dairy cow. Can 15. WEISSER W, ALBERT K. Haemophilus 24. GARCIA-DELGADO GA, LITTLE PB, Vet J 1987; 28: 117-119. somnus und Corynebacterium pyogenes BARNUM DA. A comparison of various 7. GREER D, McCONNEL W, BALL H. Vorkommen im Vaginalsecret fruchtbar- Haemophilus somnus strains. Can J Comp Isolation of Haemophilus somnus from keitsgestorter Rinder in Nordwurttemberg. Med 1977; 41: 380-388. bovine milk. Vet Rec 1989; 125: 381-382. Tierarztl Umsch 1987; 42: 596-600. 25. HUMPHREY JD, STEPHENS LR. 8. KLAVANO GG. Observations of 16. NOWACKI W, MOLENDA J, S1 FANIAK 'Haemophilus somnus': A review. Vet Bull Haemophilus somnus infection as an agent T, CHELMONSKA A, NIKOLAJCZUK 1983; 53: 987-1004. producing reproductive disease: Infertility M. Izolacja Haemophilus somnus z dr6g 26. STEPHENS LR. Studies on infectious and abortion. Proc Soc Theriogenology rodnych kr6w. Medycyna Weterynaryjna thromboembolic meningoencephalitis in cat- 1980: 139-149. 1988; 44: 36-39. tle. PhD thesis, University of Guelph, 1982: 9. HUMPHREY JD, LIT'LE PB, STEPHENS 17. STEPHENS LR, SLEE KJ, POULTON P, 193. LR, BARNUM DA, DOIG PA, THORSEN LARCOMBE M, KOSIOR E. Investigation 27. YARNALL M, CORBEIL LB. Antibody J. Prevalence and distribution of Haemo- of purulent vaginal discharge in cows with response to Haemophilus somnus Fc recep- philus somnus in the male bovine reproduc- particular reference to Haemophilus somnus. tor. J Clin Microbiol 1989; 27: 111-117. tive tract. Am J Vet Res 1982; 43: 791-795. Aust Vet J 1986; 63: 182-185. 28. SAS INSTITUTE INC. SASO User's Guide: 10. HUMPHREY JD, LITTLE PB, BARNUM 18. HUMPHREY JD. 'Haemophilus somnus': Statistics. 5th ed. Cary, North Carolina: SAS DA, DOIG PA, STEPHENS LR, THOR- Colonization of the bovine reproductive Institute Inc., 1985. SEN J. Occurrence of Haemophilus somnus tract. PhD thesis, University of Guelph, 29. COCHRANE GW, COX GM. Experimen- in bovine semen and in the prepuce of bulls 1982: 298. tal design. New York, Chichester, Brisbane, and steers. Can J Comp Med 1982; 46: 19. GROOM SC. Haemophilus somnus: Studies Toronto, Singapore: John Wiley and Sons, 215-217. of virulence and pathogenesis. PhD thesis, 1957. 11. JANZEN ED, CATES WF, BARTH University of Guelph, 1990: 193. 30. WIDDERS PR, PAISLEY LG, GOGO- A, NECHALA L, PAWLYSHYN V, 20. STEPHENS LR, LITLE PB, WILKIE LEWSKI RP, EVERMANN JF, SMITH SAUNDERS JR, OSBORNE AD. Pre- BN, BARNUM DA. Humoral immunity in JW, CORBEIL LB. Experimental abortion valence of Haemophilus somnus in the experimental thromboembolic meningo- and the systemic immune response to semen of bulls in Saskatchewan. Can Vet J encephalitis in cattle caused by Haemophilus 'Haemophilus somnus' in cattle. Infect 1981; 22: 361-362. somnus. Am J Vet Res 1981; 42: 468-473. Immun 1986; 54: 555-560. 12. KROGH HV, PEDERSEN KB, BLOM E. 21. STlEPHENS LR, LFrILE PB, HUMPHREY 31. GROOM SC, LHTLE PB, ROSENDAL S. Haemophilus somnus in semen from Danish JD, WILKIE BN, BARNUM DA. Vaccina- Virulence differences among three strains of bulls. Vet Rec 1983; 112: 460. tion of cattle against experimentally induced Haemophilus somnus following intratracheal 13. STEFANIAK T, MOLENDA J, KROLIN- thromboembolic meningoencephalitis with inoculation of calves. Can J Vet Res 1988; SKI J, CHELMONSKA A. Wstepne badania a Haemophilus somnus bacterin. Am J Vet 52: 349-354. nad zakazeniem narzadu rozrodczego Res 1982; 43: 1339-1342. 32. KWIECIEN JM, LITTLE PB. Haemophilus buhaj6w, wywolanych paleczka Haemo- 22. SLEE KJ, STEPHENS LR. Selective somnus and reproductive disease in the cow: philus somnus. Medycyna Weterynaryjna medium for isolation of Haemophilus A review. Can Vet J 1991; 32: 595-601. 1987; 43: 621-623. somnus from cattle and sheep. Vet Rec 1985; 14. MILLER RB, BARNUM DA, McENTEE 116: 215-217. KE. Haemophilus somnus in the reproduc- 23. KWIECIEN JM, LITTLE PB. Failure of a tive tracts of slaughtered cows: location and selective medium to isolate Haemophilus frequency of isolations and lesions. Vet somnus strains. Aust Vet J 1989; 66: Pathol 1983; 20: 515-521. 159-160.

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