Epidemiology of Haemophilus somnus in young rams.

Epidemiology of Haemophilus somnus in Young Rams V. Wayne Lees, Alan H. Meek and

ABSTRACT

The prevalence of Haemophilus somnus in the prepuce of young rams was examined. Of 473 rams entering

Record of Performance (ROP) stations at 50 days of age, 43 (9.1%) were positive. Average daily gain was not affected by Haemophilus status, but was influenced by breed of ram. Suffolks were predicted to gain 0.515 kg daily compared to 0.427 kg for a group combining all other breeds. Using logistic regression to identify risk factors for individual H. somnus infection, rams in 1989 were 0.382 times as likely to be infected as rams in 1988, and Suffolks were 0.314 times as likely to be infected as the other breeds group, but these factors were not significant at the flock level. Of 80 eligible flocks of origin, 22 (27.5%) were classified as infected with H. somnus, based on rams submitted to the ROP station. Infected flocks contributed 133 rams, 43 (32.3%) of which were positive. There was no association between H. somnus status and lambing percent or the percent of abortions and stillbirths, but there was a statistically significant association with the percent of ewes which failed to lamb. In the model developed, 6% of the bred ewes in noninfected flocks failed to lamb, compared to a rate of 12% in infected flocks. These results suggest H. somnus may influence ewe fertility earlier, rather than later in gestation. Purchasing replacement animals and having cattle on the farm were risk factors for Haemophilus infection in the flock. Where replacements had been purchased within the previous year, the risk of flock infection rose 8.5 times, and on farms having cattle as well as sheep, the risk

Soren Rosendal

13.2 times. It is possible interspecies transmission may play a role in the epidemiology of Haemophilus infections, but further studies are required for validation. rose

RESUME Cette etude visait a evaluer la prevalence de la bacterie Haemophilus somnus dans le pretpuce de jeunes beliers. Sur 473 beliers arrivant en station d'epreuve (ROP) a l'age de 50 jours, 43 (9,1%) etaient porteurs. Le gain moyen journalier ne sembla pas etre affecte par la presence d'Haemophilus, bien qu'il l'eut ete par la race des beliers. Les animaux de race Suffolk auraient en effet gagne 0,515 kg par jour, comparativement a 0,427 kg pour un groupe compose de plusieurs autres races. En utilisant la methode de regression logistique pour identifier de fason statistique les facteurs de risque pour les infections individuelles a H. somnus, les beliers avaient 0,382 fois plus de chances d'etre infectes en 1990 qu'en 1988, et les sujets de race Suffolk avaient 0,314 fois plus de chances d'etre infectes que les sujets d'autres races. Toutefois ces facteurs n'etaient pas statistiquement significatifs a l'echelle du troupeau. Si on se fie aux beliers soumis a la station d'epreuve des 80 troupeaux eligibles, 22 (27,5%) furent classifies comme infectes par H. somnus. Les troupeaux infectes ont fourni 133 beliers a la station, dont 43 (32,3%) etaient positifs. On n'observa aucun rapport entre le statut de positivite a H. somnus et le pourcentage de mises bas, d'avortements ou de naissances prematurees, bien qu'il y ait eu une association statistiquement significa-

tive avec le pourcentage de brebis qui n'ont pas agnele. Dans ce modele, 6% des brebis saillies issues de troupeaux non infectes n'ont pas mis bas, comparativement a 12% dans des troupeaux infectes. Ces resultats portent a croire qu'H. somnus pourrait influencer davantage la fertilite des brebis tot apries la saillie plutot qu'en fin de gestation. Le fait d'acheter des sujets de ramplacement tout en ayant des bovins sur la ferme, apparut comme un facteur de risque supplementaire pour une infection a Haemophilus dans un troupeau. La ou on avait achete des sujets de remplacement au cours des annees pr&cedentes, le risque d'infection du troupeau augmenta de 8,5 fois, et sur des fermes ou on gardait egalement des bovins en plus des moutons, le risque augmenta de 13,2 fois. Il apparait donc possible qu'un phenomene de transmission inter-espieces puisse jouer un rBle important dans l'epidemiologie des infections a Haemophilus: il faudrait toutefois d'autres etudes pour valider cette hypothese. (Traduit par Dr. Andre Cecyre).

INTRODUCTION

Haemophilus somnus, Haemophilus agni and Histophilus ovis have been recognized as important pathogens in domestic ruminants (1-9). Although they have been described under various names, these organisms are very similar and may be identical (2, 10-13). Haemophilus somnus causes a variety of syndromes in cattle including infectious thromboembolic meningoencephalitis (ITEME), pneumonia, abortion, myocarditis and arthritis, but is also a normal inhabit-

Agriculture Canada, Animal Diseases Research Institute, P.O. Box 640, Lethbridge, Alberta TIJ 3Z4 (Lees) and Department of Population Medicine (Meek), Department of Veterinary Microbiology and Immunology (Rosendal), Ontario Veterinary College, University of Guelph, Guelph, Ontario NIG 2W1. Submitted October 23, 1989.

Can J Vet Res 1990; 54: 331-336

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ant of the prepuce and vagina in healthy animals. Previous studies indicate that 60-77% of bulls and steers, and 8-10% of normal cows carry the organism in the genital tract

(14-16).

Similarly, H. agni and H. ovis have been associated with ovine septicemia, pneumonia, mastitis and epididymitis, but have been isolated from the prepuce of normal rams (8, 9, 17-21). On one sheep experimental station in Idaho, H. ovis was isolated from up to 87.5% of 20 week old rams and 8% of yearlings, but not from rams less than 12 weeks of age (9). In another report, a variety of bacteria, including Haemophilus spp., were cultured from 48 Montana rams with clinical epididymitis (17). Septicemia caused by H. ovis was first described in lambs from the Ottawa valley by Mitchell in 1925 (18). Since then, there have been relatively few reports regarding the role of Haemophilus spp. in producing disease in sheep (8, 19-21). The purpose of this investigation was to determine the prevalence of H. somnus in an opportunistic sample of young Record of Performance (ROP) rams and their flocks of origin, to examine the association of infection with production, reproduction, morbidity and mortality, and to identify risk factors for infection of rams and their flock of origin. MATERIALS AND METHODS The study was conducted from March 1988 to May 1989 at three ROP ram test stations, two in Ontario and one in Alberta. BACTERIOLOGY

negative coccobacilli which produced positive indole and negative catalase reactions (23). An agar gel immunodiffusion technique using serum from a hyperimmunized calf confirmed the identity of the organism (24). INDIVIDUAL RAMS

Every ram accepted into the station on selected entry dates was sampled and included in the study population. Rams entering the ROP test stations were approximately 50 days old (range 35-65 days) and weighed 15-45 kg. After passing a physical examination, rams were individually identified and vaccinated against clostridial diseases. Throughout the 50-day feeding period, weight gain and backfat measurements were recorded, and at the end of the trial, the summary average daily gain (ADG) was computed.

questionnaire were mailed to each eligible contributor asking them to supply production data for their flock, such as the number and breeds of sheep kept, the number of ewes bred and those which lambed, the number and sex distribution of liveborn lambs, the number of abortions and stillborn lambs, the number and ages of rams used, the number, age, sex and source of replacement animals purchased in the previous year, and the number and breed of cattle on the farm. Questionnaire data were coded and stored in a computerized database (Paradox 2.0, Ansa Software, Belmont, California) before being statistically analyzed. STATISTICAL METHODS

The definitions of all independent and dependent variables for individual rams and their flock of origin are listed in Table I. Variables were eligible for initial testing in the model if they were CONTRIBUTING FARMS unconditionally associated with the Farms were included in the study if dependent variable at the 15% signifiat least two rams in a single year were cance level (p < 0.15). Then a condicultured on entry to the ROP station. tional selection was performed so that A letter of introduction and a all variables included in the final

TABLE I. Deflnitions of variables Independent variables AVRAMAGEb = the mean age, in years, of all rams on the farm BOTREPLb = 0 if no replacement animals were purchased in the previous year = 1 if replacements were purchased CATTLEb = 0 if no cattle were present on the farm = I if cattle were present on the farm EWEPRAMb = number of ewes bred per ram used FLOKSIZEb = number of ewes bred NUMFLOKSb = the number of separate flocks kept on farm (1, 2 or 3) ONTESTWTC = initial weight (kg) of rams at beginning of test period PROVINCEb c = 0 for Ontario = I for Alberta SUFFOLKb'C (individual rams) = 0 for all non-Suffolk rams =1 for Suffolk rams (farm level) =0 if no Suffolk flocks were kept =1 if at least one flock of Suffolks was kept YEARb c = 0 for 1988 = I for 1989

A cotton-tipped culture swab (22) of the preputial cavity and a serum sample were collected from each ram when it entered and when it left the Dependent variables station. Within 3 h, swabs were ADGaC = daily weight gain in kg over the 50 day feeding period streaked onto bovine brain heart ENTRYHEMaC = 0theformean a negative preputial culture for H. somnus on entry to the ROP station infusion blood yeast extract (BHI= 1 if H. somnus was isolated BYE) plates which were incubated in FARMHEMab = 0 if H. somnus was not isolated from any ram submitted by that contributor I if H. somnus was isolated from at least one ram 5% CO2 at 370C for 48 h. A presump- FLTOLAMBb == the percentage of ewes bred which failed to lamb tive identification of H. somnus was LAMBING%b = the flock average of the number of live lambs born per ewe bred x 100 made on the basis of morphology and LAMBMORTb = the percentage of total lambs born which were stillborn or aborted biochemical reactions, i.e. small, aAlso used as an independent variable circular, slow growing, CO2 depend- bFlock level of organization ent, butter-colored colonies of gram- clndividual ram level of organization

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model were significant at the 5% level (p < 0.05). Statistical analyses of individual ROP ram and farm questionnaire data were performed using two computer statistical analysis programs. SAS 6.03 (SAS Institute, Cary, North Carolina) was used to compute Mantel-Haenszel summary odds ratios and least squares regressions for continuous outcomes, while MULTLR (25) was used for logistic regression models involving binary outcomes. Risk factors for being classified as H. somnus positive were examined for individual rams and for their flock of origin using logistic regression. For individual rams (ENTRYHEM), the independent variables offered for selection in the model were YEAR, PROVINCE, SUFFOLK and ONTESTWT. For flocks of origin (FARMHEM), the independent variables offered were AVRAMAGE, BOTREPL, CATTLE, EWEPRAM, FLOKSIZE, NUMFLOKS, PROVINCE, SUFFOLK and YEAR. Least squares regression was performed in a stepwise manner to identify significant variables for continuous outcomes such as average daily gain (ADG) at the individual level, and lambing percent (LAMBING%), percent of stillbirths and abortions (LAMBMORT) and percent of bred ewes failing to lamb (FLTOLAMB) at the flock level. When individual average daily gain (ADG) was modelled, the independent variables offered for selection were YEAR, SUFFOLK, PROVINCE and ENTRYHEM. When the three continuous flock outcomes (LAMBING%, LAMBMORT, FLTOLAMB) were modelled, the variables offered were FARMHEM, YEAR, SUFFOLK, PROVINCE, AVRAMAGE, BOTREPL, CATTLE, EWEPRAM, FLOKSIZE and NUMFLOKS.

RESULTS

positive preputial isolations from 14 of these animals. This difference in isolation rate was statistically significant (logit x2 = 12.687, p < 0.001). In Ontario, there was no significant difference between the proportion of positive animals sampled at the two stations (20 positive/ 178 at one station and three positive/47 at the other), therefore the results were combined. There was no significant difference in the distribution of positive rams between the two provinces (X2 = 0.665, p = 0.415). In Ontario, 23 of 225 rams, and in Alberta, 20 of 248 rams were positive. Eleven breeds of sheep were represented in the survey. Suffolks represented 58% (273/473) of the total, Dorsets 19% (92/473) and a variety of other breeds made up the remaining 23% (108/473). Since there was no significant difference in the proportion of infected rams among Dorsets (16/ 92) and the group of other breeds (14/ 108), both groups were combined and labelled as Other Breeds in the analysis. There was a significant difference in the prevalence of infected sheep between years (X2 = 10.238, p = 0.001), as well as a significant difference in the proportion of Suffolks (x2 = 8.143, p = 0.004) between 1988 (118/231) and 1989 (155/ 242), therefore the effect of breed controlling for year was examined (Table II). The Breslow-Day chi-square test for homogeneity between strata was not significant at the 5% level (X2 = 3.521, p = 0.061), therefore a Mantel-Haenszel summary odds ratio was computed. Based on a 95% confidence interval (CI) of 0.156 to 0.599, individual Suffolk rams were 0.3 times as likely to be infected with preputial H. somnus as rams of Other Breeds. A similar result was obtained using logistic regression to model risk

FLOCKS OF ORIGIN

On the basis of a positive culture from at least one ram submitted to the ROP station, 22 (27.5%) of the 80 eligible flocks of origin were classified as infected with H. somnus. Positive rams were not uniformly distributed, but appeared to cluster within certain flocks. Among the rams submitted from the 22 infected flocks of origin, 32% (43/133) were positive. Of the 80 eligible contributors, 56 returned the questionnaire, for a response rate of 70%. Of the 56 responding contributors, 15 (26.8%) had infected flocks, while of the 24 nonresponding contributors, seven (29.2%) had flocks which were infected. There was no significant difference in the prevalence of flock infection between the two response groups (X2 = 0.01, p > 0.25). The characteristics of Haemophilus negative and Haemophilus positive

TABLE II. Distribution ofH. somnus positive and negative ROP rams (n = 473) by breed controlling for year

INDIVIDUAL RAMS

The prevalence of preputial H. somnus in all ROP rams cultured was 9.1% (43/473). In a subset of 66 of these rams, swabs were also obtained from the conjunctival sac and nasal passages. No Haemophilus spp. were isolated from either location despite

factors for the outcome ENTRYHEM (Table III). Rams in 1989 were 0.382 times as likely to be infected as rams in 1988, and Suffolk rams were 0.314 times as likely to be infected as rams of Other Breeds. Average daily gain was not significantly affected by Haemophilus status, but was affected by breed. Given the regression model, ADG for Suffolk rams was predicted to be 0.515 kg, compared to 0.427 kg for Other Breeds (Table III). Over the 50-day feeding period, there were no statistically significant differences in crude morbidity and mortality rates between rams which were negative for H. somnus and those which were positive. Illness was seen in 15 (3.5%) of the 430 negative animals and in three (6.7%) of the 43 positive ones, while mortality occurred in five (1.2%) of the negative rams and in none of the positive ones.

H. somnus culture Pos

Neg 1988

Other breeds Suffolk

88 112

25 6

x2 = 14.424

p < 0.0001

Other breeds Suffolk missing = I

81 148

5 7

x2 = 0.197

p = 0.657

1989

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earlier, rather than later in gestation. Haemophilus somnus has been asso(Continuous) Outcome = ADG with infertility in cows (16,26) ciated ADG = 0.427 + 0.088 SUFFOLK and has been shown to be detrimental IV lodel R2 Coefficient SE Variable p to bovine embryos in vitro (27-29). INTERCEPT 0.427 0.005 0.0001 Given the known pathogenic role of 0.28 0.088 0.007 0.0001 SUFFOLK H. somnus in bovine reproductive = (Binary) Outcome ENTRYHEM this study indicates a similar disease, ENTRYHEM = e*(-1.393 -1.159 SUFFOLK -0.964 YEAR) effect may be occuring in sheep. Variable Coefficient SE p OR 95% CI No significant flock predictors were -1.393 0.224 0.0001 INTERCEPT found for the rate of stillbirths and -1.159 0.350 0.0009 0.314 0.158, 0.623 SUFFOLK -0.964 0.359 0.0072 0.382 YEAR 0.189, 0.770 abortions (LAMBMORT). Lambing percent (LAMBING%) was affected only by geographic location, not by H. flocks which responded are summar- times if rep lacements had been somnus status of the flock. Although ized in Table IV. No significant purchased in the previous year, and we would have expected lambing differences were seen in the distribu- 8.5 times if ca ttle were present on the percent to be 6% lower in infected tion of infected and noninfected flocks farm. flocks, this small difference may have regarding PROVINCE, YEAR or been masked by other more influential BREED. factors represented by PROVINCE. DIISCUSSION The regression models for predictProvincial differences in lambing ing continuous outcomes at the flock percent may be related to differences Confusion exists in the literature in management and flock size, as the level are presented in Table V. Haemophilus status (FARMHEM) concerning tihe relationship of H. median flock in Ontario was 30 ewes, was found to be a significant predictor somnus, H. agrni and H. oviS, and the compared to 90 in Alberta. As of the percentage of ewes which failed exact taxonomic position of these indicated by a relatively low coeffito lamb (FLTOLAMB). In nonin- three organisnns is a question of some cient of determination (R2 = 0.08), fected flocks, 6.022% of the bred ewes debate. Becauise these organisms are inclusion of other, as yet unidentified, failed to lamb, compared to 12.012% very similar, some workers have variables,are required to improve the in infected flocks. When LAMBING% suggested they should be considered as model's predictive value. Buying replacement animals in the was examined, PROVINCE was the a group (1 1- 13, 23). In this study, only significant variable found. When organisms wiere identified as H. previous year and having cattle were the percentage of stillbirths and somnus on thte basis of morphology, significant risk factors for flock i and aar reatios ad abortions (LAMBMORT) was exam- biochemical rreactions agar gel with H. somnus. Flocks elinfection immunodiffusion. no were variables ined, independent containing replacement sheep which In our study, flock infection with H. were bought in the previous year were significant at the 5% level. According to the model, the lambing percentage somnus was a significant predictor of 13 times more at risk of being infected for Ontario farms was predicted to be the percentage of ewes which failed to than flocks which did not. As with 150%, compared to 166% for Alberta lamb. In the FLTOLAMB model, most infectious diseases, movement of noninfected flocks experienced a 6% animals commonly disseminates the farms. The risk factors for being classified failure rate, whereas infected flocks organism from one location to the as H. somnus-infected flock were had a 12% failure rate. As FLTO- next, and maintaining a closed flock examined using logistic regression, LAMB was influenced by Haemophi- may be one method to control, or at and two variables were found to be lus status, whereas LAMBING% and least minimize the risk of Haemophistatistically significant, BOTREPL LAMBMORT were not, the data lus spp. infection. If cattle were also and CATTLE (Table V). The odds of suggest any effect H. somnus may present on the farm, the risk of flock being classified as infected rose 13.2 have on ewe fertility likely occurs infection increased 8.5 times. Although interspecies transmission has not been widely investigated, these TABLE IV. Distribution of H. somnus positive and negative flocks (n = 56) by various factors findings suggest having cattle and sheep on the same farm significantly H. somnus status the risk of infection, but increases Pos Neg further experimental and field studies By province are required to investigate the mode of p = 0.955 27 10 x2 = 0.003 Ontario of H. somnus both transmission 5 14 Alberta within and between species. Year of By year entry and breed were risk factors for 5 p = 0.770 x2 = 0.086 12 1988 infection with H. somnus for individ10 29 1989 ual rams, but these factors were not By breed significant at the flock level. This 5 p = 0.955 14 x2 = 0.003 Other breeds 10 27 Suffolk suggests flock infection is similarly TABLE III. Regression models for individual ram data

334

TABLE V. Regression models for contributing farm data (Continuous) Outcome LAMBING% LAMBING% = 150.435 + 15.716 PROVINCE Variable Coefficient SE p Model R2 INTERCEPT 150.435 4.130 0.0001 PROVINCE 5.716 7.090 0.031 0.08 (Continuous) Outcome FLTOLAMB FLTOLAMB = 6.022 + 5.990 FARMHEM Variable Coefficient SE p Model R2 INTERCEPT 6.022 1.251 0.0001 FARMHEM 5.990 2.479 0.0192 0.099 (Binary) Outcome = FARMHEM FARMHEM = e*(-4.096 + 2.143 CATTLE + 2.582 BOTREPL) Variable Coefficient SE p OR INTERCEPT -4.096 1.230 0.0009 CATTLE 2.143 0.730 0.0033 8.5 BOTREPL 2.582 1.164 0.0264 13.2

distributed among all breeds of sheep and over a wide geographic area. The prevalence of infected flocks reported in this study (27.5%) is probably conservative due to potential misclassification. Although the culture technique would not produce false positive results (i.e. the positive predictive value approaches 100%), false negative results are possible (the negative predictive value is unknown), therefore some infected rams, and some flocks, may have been misclassified as noninfected. Haemophilus somnus infection seemed to cluster within some flocks, therefore the prevalence among young rams on infected farms is probably much higher than the prevalence found in the general population of ROP rams. Keeping in mind the nonrandom sampling technique used, this study suggests approximately 33% (43/133) of rams from positive farms may be infected, but further field studies are needed to verify this estimate. The rate of genital infection with Haemophilus spp. found in individual rams was lower than that found in bulls and steers (14,15), but was comparable to that of normal cows (16). Part of this difference may relate to the age difference in the animals sampled. From a study undertaken in a single flock at an Idaho sheep experimental station, Walker and LeaMaster (9) linked the prevalence of Histophilus ovis to the age of ram, suggesting the organism was a transitory component of the genital flora. They did not identify the organism in any rams less than 12 weeks of age, but

95% CI -

2.0, 35.6 1.3, 129.4

found 87.5% of rams were infected at 20 weeks. By the time they were yearlings, the prevalence had declined to 8%. While prevalence may be age related, without further field work it would be difficult to interpret the significance of their findings in regard to the general sheep population. In another report, McGillivery et al (13) used restriction endonuclease analysis to identify the source of H. ovis epididymitis in unmated rams. They suggested neonatal rams were infected as they passed through the contaminated birth canal of their dams. The results of our study support the view that infection occurs early in life. We found H. somnus can be recovered from the prepuce of approximately 10% of rams as young as seven weeks of age, however further work is required to pinpoint both the source and time of infection for young rams. It appears that infection with H. somnus is not detrimental to ROP growth performance, as average daily gain was not influenced by infection, after accounting for other factors. Breed differences did exist, but factors such as geographic location, and year of entry were not significant. Given the peracute, rather than chronic, nature of septicemic Haemophilus infection, one would not expect the growth performance of positive rams to be affected unless clinical disease was detected. The differences in crude morbidity and mortality rates between infected and noninfected rams were nonsignificant. This is not surprising given the low level of clinical disease in ROP stations, where the level of care is

generally very good. At these low rates, a much larger study population would have been required to demonstrate any statistically significant differences in morbidity and mortality. In conclusion, we found flock infection with H. somnus was associated with an increase in the percentage of bred ewes which failed to lamb. Buying replacement animals and having cattle on the same farm significantly increased the risk of H. somnus infection in the flock. It is clear from a number of questions raised that more field and experimental work is needed to clarify the role of H. somnus in ovine disease.

ACKNOWLEDGMENTS Thanks are extended to the many participants without whose support this project would not have been possible. The ROP advisors, station managers and staff, and contributors are commended for their cooperation and patience. Excellent technical assistance was provided by staff at ADRI (Lethbridge) and the Department of Veterinary Microbiology and Immunology, OVC, Guelph, and financial support was gratefully received from Agriculture Canada and the Ontario Ministry of Agriculture and Food.

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