seven herds with nocardial mastitis were matched with control herds based on ..... Epidemiologic und diagnostik der Nocardia asteroides mastitis des rindes.
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An investigation of risk factors for nocardial mastitis in central Alberta dairy herds Gerald W. Ollis, Matthew Schoonderwoerd, Casey Schipper
Abstract A case-control study was undertaken during the summer of 1989 in central Alberta dairy herds to identify independent predictors of nocardial mastitis. Thirtyseven herds with nocardial mastitis were matched with control herds based on herd size, milk production, and enrolment in Alberta Dairy Herd Improvement Services. Control herds were considered free of nocardial mastitis based on negative cultures of four weekly bulk tank milk samples and one composite milk sample collected during the same period from each lactating cow in the herd. A detailed questionnaire on herd management was completed during farm visits. The use of blanket dry cow therapy was not found to be a risk factor for nocardial mastitis. Dry cow therapy with intramammary products containing neomycin and the use of multidose vials of dry cow medications were the only predisposing factors identified as being significantly associated with nocardial mastitis in central Alberta dairy herds. Use of neomycin as a dry cow therapy increased the odds of nocardial mastitis occurring in these dairy herds by 169 times.
R6sume ivaluation des facteurs susceptibles de causer uns mammite a Nocardla chez les bovins laltiers, au centre de I'Alberta Une etude de cas fut entreprise chez des troupeaux
laitiers, au centre de l'Alberta, durant l'ete de 1989, afin d'identifier les differents facteurs influencant la mammite A Nocardia. Trente-sept troupeaux prisentant de la mammite a Nocardia furent jumeles A des troupeaux temoins selon leur grosseur, leur production laitiere et leur participation au Service d'amelioration des troupeaux laitiers d'Alberta. Les troupeaux temoins ont ete classifies absents de mammite A Nocardia selon les resultats negatifs de cultures bacteriennes effectuees a partir de quatre echantillons de lait par semaine, preleves dans la citerne et d'un echantillon preleve a partir de lait compose provenant de chacune des vaches du troupeau. Tous les echantillons ont dte prtleves durant la meme periode. Un questionnaire detalle sur la regie du troupeau fut compl6te a chaque visite sur la ferme. L'emploi de la therapie de couverture pour
Animal Health Division, Alberta Agriculture, O.S. Longman Building, 6909 - 116 Street, P.O. Box 8070, Edmonton, Alberta T6H 4P2. Can Vet J Volume 32, April 1991
vache tarie ne fut pas considere comme un facteur predisposant A la mammite a Nocardia. La therapie pour vache tarie avec utilisation de produits intramammaires contenant de la neomycine et l'emploi de ffoles a doses multiples pour medication pour vache tarie furent les seuls facteurs pr6disposant identiflis comme ayant une association significative avec la mammite A Nocardia dans les troupeaux laitiers du centre de l'Alberta. L'utilisation de la niomycine comme th6rapie pour vache tarie augmente de 169 fois les chances d'apparition de mammite a Nocardia chez ces troupeaux (Traduit par Dr ThOrse Lanthier) laitiers. Can Vet J 1991; 32: 227-231
Introduction M astitis caused by Nocardia spp. is usually sporadic in occurrence, affecting only one or two animals in a herd (1). Nocardiae are aerobic soil saprophytes (2) that can become pathogens when introduced into the mammary gland (3). This type of mastitis has usually been associated with repeated intramammary antibiotic therapy or unsanitary practices during intramammary infusion, and herd outbreaks have been traced back to a common source (4,5). Predisposing factors which have been identified include contaminated home-mixed intramammary antibiotics, use of multidose vials, repeated use of a single infusion cannula with only momentary immersion in alcohol between quarters, and failure to disinfect the ends of teats prior to udder infusion (6). In Alberta, we have experienced an epizootic of nocardial mastitis which started in early 1988. Over 200 infected herds have been detected, with up to 20% of the cows in some herds infected in one or more quarters. During 1989, a total of 420 infected cows in 160 herds (there are 1,450 dairy herds in Alberta) were identified following the culture of milk samples from infected quarters. This outbreak is part of a crossCanada epizootic which started in the fall of 1987 (7). Similar disease outbreaks occurred in France between 1981 and 1985 (8) and in Switzerland in 1989 (9). An increase in the number of cases of nocardial mastitis in Brazil has been reported as well (10). In response to the concerns raised by this Canadian epizootic, the Ontario Ministry of Agriculture and Food hosted a national nocardial mastitis strategy 227
Table 1. Comparison of somatic cell count, standard plate count, herd size, and production in case and control herds (Alberta Nocardia study, 1989)
Parameter
BSCC 1988C (000's) SPC 1988d (000's) Herd size (No. lactating) Milk production (BCA)
Case, herd n=37 Mean ± SD 197 ± 147 6.2 ± 4.3 66.5 ± 35.5 156.1 + 20.1
Control herd n=37 Mean ± SD
220 8.1 67.8 156.1
± ± ± ±
125 6.9 38.0 19.1
p valueb
0.52 0.14 0.87 0.99
aDifferences in values between groups of herds were not significant bValues of greater than 0.05 are considered nonsignificant CBSCC = bulk tank somatic cell count dSPC = standard plate count meeting on February 15, 1989 (11). Numerous participants from across Canada shared their experiences and observations on nocardial mastitis. A common concern was that present lactational or dry cow therapy practices needed to be re-evaluated, and this meeting resulted in a set of recommendations for farms with Nocardia-infected cows (11). Initial observations in Alberta supported the idea that the disease occurred most frequently in herds with low bulk tank somatic cell counts and which practiced blanket dry cow therapy (usually neomycin). Multidose vials as well as individual syringes had been used and only cows in their second or subsequent lactation were observed to be affected. During the summer of 1989, a case-control study was conducted in central Alberta dairy herds; it included about 501o of Alberta's dairy herds. The objective of this study was to identify independent predictors of nocardial mastitis.
Materials and methods Thirty-seven case herds were selected based on their proximity to Edmonton, enrolment in Alberta Dairy Herd Improvement Services (ADHIS), and willingness by the dairyman to cooperate. A herd was designated Nocardia-positive (case herd) if one or more cows were found to have at least one quarter infected with Nocardia spp. These herds were identified from routine diagnostic submissions of milk samples to the provincial veterinary laboratory in Edmonton. The case herds were matched closely with 37 control herds selected from a list of dairy herds in the same geographical area. The control herds were also enrolled in ADHIS and were matched with the case herds on herd size and level of milk production recorded during May 1989. Control herds had no history of nocardial mastitis. Milk samples from every lactating cow in each control herd, as well as four weekly bulk tank milk samples, were negative for Nocardia spp. The individual cow milk samples were taken from the milk metering equipment by a technician during one of the regularly scheduled ADHIS test days. These samples were chilled and submitted within 18 hours to the provincial veterinary bacteriology laboratory in Edmonton. All bulk tank milk samples were part of the provincial milk quality monitoring program and 228
were
obtained from the Central Milk Testing Labora-
tory. The four bulk tank milk samples for each control herd were submitted for bacteriology at weekly intervals commencing one week prior to the week when
the individual cow samples were obtained. Nine herds, initially selected as control herds, were replaced due to the isolation of Nocardia spp. from one or more of their bulk tank milk samples and/or individual cow samples. Nine new control herds of similar size and production level were selected from the list of eligible control herds. One swab (0.02 mL) from each individual cow milk sample was inoculated on half of a plate of blood agar (5% citrated ovine blood, Columbia agar base) with gentamicin (25 mg/L). A 0.15 mL aliquot of each bulk tank milk sample was spread, using a swab, over an entire plate of the same medium. The presence of gentamicin reduced the overgrowth of environmental bacteria to a great extent and allowed Nocardia spp. to be more easily identified. During the farm visit, the technician interviewed the dairyman and completed a detailed questionnaire. The questions covered information related to herd size, source of herd additions, type of housing and bedding materials, general management and nutrition, as well as mastitis management and milking techniques. Specific milking techniques of interest included udder hygiene prior to milking, type of milking equipment, and whether or not teat dips were used. The type of teat dip used and how it was applied were noted. Intramammary antibiotics used during lactation or at drying off were recorded. Also, the infusion techniques employed were reviewed. Average bulk tank somatic cell counts and standard plate counts for each of the herds were obtained for 1988 from Dairy Production Branch records.
Data analysis Descriptive statistics were performed using Epi Info ver. 5, an epidemiology statistics system for microcomputers (12). Least square means of continuous variables were calculated by analysis of variance. The Chi-square analysis was performed on categorical variables to explore simple associations between a number of presumed risk factors for Nocardia infection. Can Vet J Volume 32, April 1991
Table 2. Distribution of herd size and BCA for milk of case and control herds (Alberta Nocardia study, 1989) Case 37
Control n = 37
Herd size < 50 50-100 101-200
16 15 6
17 14 6
Herd BCA 100-125 126-150 151-175 176-200
3 9 16 9
4 9 17 7
Variable
n =
appeared to have a sparing effect on infection with Nocardia spp. (OR 0.28, p 0.02). As regards other selected general herd management procedures, both groups had used similar techniques and no significant differences between case and control herds were observed (Table 3). Unconditional analyses, controlling for the matching variable based on herd size and production, generated the same unconditional odds ratios as the matched analyses listed in Table 3. =
=
Conditional associations The use of multidose vials was a confounder of the neomycin Nocardia association because it was causally associated with Nocardia infection (OR = 8.82, 0.00) and noncausally associated with neomycin p 5.13, p 0.00). The housing factor was use (OR noncausally associated with neomycin use (OR 0.24, p 0.00) as well as with Nocardia infection (OR 0.28, p 0.02). Neither blanket dry cow therapy (OR 1.75, p 0.29) nor animal purchases (OR 2.23, p 0.11) were associated with neomycin use. Hence, the latter two variables did not qualify as confounding variables to be entered into the final logistic regression model. Except for the use of multidose vials, all the above independent input variables which had shown significant unconditional associations with herd nocardial mastitis were rendered insignificant after neomycin was entered into the logistic regression equation. The variables entered into the final logistic regression model, for predicting the likelihood for a dairy herd becoming infected with Nocardia spp., are listed in Table 4. Adjusting neomycin use for the practice of using multidose vials of dry cow therapy increased the unconditional odds of a herd becoming infected from 144 times to 169 times. If neomycin was administered from multidose vials only, the odds of becoming infected with Nocardia spp. were increased to 2,298 times (e5'9 + 2.66); see Table 4. The addition of the housing variable to the logistic model did not improve the goodness of fit. There were no significant interactions. =
=
=
=
Multivariate logistic regression was used to obtain estimates of adjusted odds ratios and their 95% confidence limits with the assistance of MULTR, a microcomputer program (13). The goodness of fit of the final logistic regression model, fitted to the herd data, was determined by the Likelihood Ratio Test (14).
Results The herds ranged in size, from 50 to 200 cows (mean 67 ± SD 37). Breed class average (BCA) for milk of herds in the study was 156 ± SD 20. The effectiveness of the matching procedure was confirmed in that there were no statistically significant (p < 0.05) differences between case and control herds in terms of herd size, milk production, and average bulk tank somatic cell counts and average standard plate counts for 1988 (Table 1). Similarly, the number of herds belonging to each of three herd size categories and four BCA categories was not different (Table 2).
=
=
=
=
=
=
Simple associations Five variables were unconditionally associated with Nocardia infection. They included purchasing replacement animals, blanket dry cow therapy, the use of multidose vials, the use of neomycin as a dry cow antibiotic, and housing dry cows with the lactating herd for the first few days of the dry period. More case than control herds purchased replacement stock (48.6/o vs. Discussion 21.6%), which increased the likelihood of infection Our objective was to identify independent predictors with Nocardia spp. by 3.43 times. Dry cow therapy of nocardial mastitis by means of a case-control study. in a blanket application occurred in 83.8% of the case To qualify as a valid case-control study, there had to herds but in only 58.80o of control herds. This prac- be confidence that our control herds were truly free tice raised the odds of nocardial mastitis by a factor of nocardial mastitis. In a previous Canadian study, of 5.46. The use of products in multidose vials for dry the control status was determined by asking the herd owner if Nocardia spp. had been identified in the herd cow therapy was observed in 54.1%o of case and 11.8 % of control herds. This practice increased the odds of (15). However, previous work in Edmonton revealed a given dairy herd becoming Nocardia positive by a that many dairy herds were infected, but the owners factor of 8.82. The most remarkably significant dif- were not aware of it (16). As well, the subsequent bulk ference between the two groups was that 35 of the 37 tank monitoring program in Alberta has detected 198 (94.60/o) case herds had used a dry cow therapy prod- dairy herds infected with Nocardia spp. over a uct containing neomycin while only four of the 37 12 month period. The owners of these herds were (10.8%7o) control herds had done so. This increased the unaware of the presence of this pathogen. As a result of the bacteriology done on the control odds of a herd becoming infected with Nocardia spp. 144 times. Unconditionally, housing dry cows with the herds in our study, nine (24.30%o) of the herds selected lactating herd for the first few days of the dry period as controls had to be replaced because of the isolation Can Vet J Volume 32, April 1991
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Table 3. Frequency and odds ratios of management variables in case and control herds (Alberta Nocardia study, 1989)
Variable
Replacements purchased Blanket dry cow therapy Used multidose vials only Neomycin dry cow product Abrupt drying-off Housing first few daysa Housing last two weeksa Dry cows with heifers Bedding for dry cows Calving outside Teat dip before milking Teat dip after milking Udder wash Lead feeding
Control herds n = 37 Yes No 8 29 20 14 4 30 4 33 21 16 15 22 22 15 34 3 20 17 20 17 1 36 4 33 31 6 25 12
Case herds n = 37 Yes No 18 19 31 6 20 17 35 2 16 21 6 31 19 18 28 9 11 26 18 19 0 37 33 4 32 5 24 14
Odds ratios 3.43 5.46 14.16 144.38 0.58 0.28 0.72 0.27 0.50 0.81 N/A 1.00 0.81 1.13
aDry cows housed with the lactating herd at the start or end of the dry Table 4. Maximum likelihood estimates of logistic parameters relating neomycin use, multidose vials and a housing variable to the risk of Nocardia spp. Infection in Alberta dairy herds (Alberta Nocardia study, 1989) Variable ORb 95% CLC p value ba 3.75 Intercept 5.19 169.0 18.0 1778.0 0.000 Neomycin use Multidose vial 2.66 14.2 1.2 163.0 0.032 Housing variabled 0.25 1.3 0.1 11.5 0.825 aEstimated logistic regression coefficient bAdjusted Odds Ratio (OR = exp b) C95% Confidence limits for the Odds Ratio (95% CL = expb ± X %* Seb) dHousing dry cows with the lactating herd for the first few days of the dry period
of Nocardia spp. from bulk tank milk and/or individual cow milk samples. Further confirmation of control status was gained by culturing a composite milk sample from each lactating animal in the herd. Only one of the control herds in our study has subsequently been detected as infected with Nocardia spp. by the bulk tank monitoring program in Alberta. This herd was one of the four control herds which had used neomycin containing dry cow products prior to our study. One cow was eventually confirmed to be infected with Nocardia spp. in one quarter. The initial impression in Alberta was that herds with low somatic cell counts were at higher risk for nocardial mastitis. We did not match our case and control herds for this factor but found there was no significant difference between the case and control herds regarding bulk tank somatic cell count data. The results of this study indicate that it is not the practice of blanket dry cow therapy that predisposes a herd to developing a problem with nocardial mastitis. This is in contrast to a previous case-control study by Stark and Anderson (15), in which they concluded that 230
Significance of difference, p value 0.01 0.02 0.00 0.00 0.24 0.02 0.48 0.11 0.15 0.64 0.31 1.00 0.74 0.81 period
blanket dry cow therapy and a specific dry cow product were associated with the diagnosis of nocardial mastitis in a dairy herd. Blanket dry cow therapy is an important tool in controlling contagious forms of mastitis in some herds. To discontinue this management procedure could have detrimental effects in some herds as far as mastitis control is concerned. Our study indicated that producers would be wiser to evaluate which dry cow products they are using, rather than question the use of blanket dry cow therapy per se. After adjusting for the use of multidose vials, the odds ratio in our study for using either of two products containing neomycin was 169. In the study by Stark and Anderson, the probability of a herd experiencing nocardial mastitis was increased by 6.7 times if a product containing neomycin and hydrocortisone was used as a dry cow medication. The discrepancy between these two Canadian case-control studies exists because our control herds were much more likely to be free of nocardial mastitis during the time of our study. The reasons for neomycin dry cow medications being a risk factor for nocardial mastitis are unclear at this time. Nocardia spp. have been reported to be resistant in vitro to neomycin (6). The Nocardia spp. associated with the current cross-Canada epizootic are resistant to neomycin (J. Lynch, personal communication, 1989). It is possible that nocardiae could be introduced accidentally into the udder during intramammary infusion and proliferate if neomycin is used as a dry cow medication. This does not seem likely because the cross-Canada epizootic of nocardial mastitis occurred suddenly after neomycin had been used in Canada as a dry cow medication for about twenty years. We could not detect any obvious differences in the udder infusion techniques used in control or infected herds. All producers from the case herds indicated that no changes had been made in the manner in which intramammary infusions were carried out. As far as we are aware, in Alberta there were only three products containing neomycin which were marketed exclusively as dry cow medications. Only the Can Vet J Volume 32, April 1991
use of these three products has been associated with nocardial mastitis in Alberta but only two of the three products were used by any of the herds in this study. The most popular product was marketed primarily in multidose vials while another was available only in individual syringes. When we controlled the use of multidose vials in our analysis for the use of neomycin, multidose vials remained a risk factor for nocardial mastitis. Contamination of the multidose vial due to poor hygiene during use is possible, but, unfortunately, we have been unsuccessful in growing Nocardia spp. from several suspect vials. The reason why multidose vials remain a risk factor for nocardial mastitis is not clear at this time and further research is needed to clarify this question. Various management variables pertaining to the dry period, parturition, or the immediate postpartum period were not predisposing factors for nocardial mastitis in our study. One would expect that leaving the recently dried off animal with the lactating herd for several days would predispose that animal to developing a new udder infection. Unconditionally, a sparing effect on nocardial mastitis was observed in our study but this effect disappeared when the confounding variables were controlled. The failure to provide bedding for dry stock does not appear to be an important risk factor for nocardial mastitis. Seventy percent of the case herds provided bedding to the dry stock while only 54% of the control herds did the same. In summary, we found that dairy herds in central Alberta which used dry cow products containing neomycin were 169 times more likely to become Nocardia problem herds than those in which neomycin was not used.
Acknowledgments We express our gratitude to the following people for their assistance: Dianne Mallas, Dave Domes (Dairy Production Branch), Andy Van Biert and staff (Alberta Dairy Herd Improvement Services), Kim Whitehead and staff (Central Milk Testing Laboratory), Suzanne Gibson, Lorna McFadzen, Julie cvJ Popowicz, and Dr. E.L. Franco.
References 1. Blood DC, Radostits OM. Veterinary Medicine. 7th ed. London:
Bailliere Tindall, 1989: 531-532. 2. Lechevalier HA. Nocardioforms. In: Sneath PHA, Mair NS, Sharpe ME, Holt JG, eds. Bergey's Manual of Systematic Bacteriology. Vol 2. Baltimore: Williams and Wilkins, 1986: 1458-1471. 3. Pier AC, Willers EH, Mejia MJ. Nocardia asteroides as a mammary pathogen of cattle. II. The sources of nocardial infection and experimental reproduction of the disease. Am J Vet Res 1961; 22: 698-703. 4. Barnum DA, Fuller DS. A report on the isolation of two species of Nocardia from bovine mastitis. Annu Meet Northeast Mastitis Council, St. Hyacinthe, Quebec, Canada, 1956.
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5. Sears PM. Nocardia mastitis. Bovine Practitioner 1983; 18: 4-6. 6. Pier AC, Gray DM, Fossatti MY. Nocardia asteroides - a newly recognized pathogen of the mastitis complex. Am J Vet Res 1958; 19: 319-331. 7. Dohoo I. Nocardia spp. mastitis in Canada. Can Vet J 1989; 30: 969. 8. Pellerin JL, Bodin G, Rai-el-Balhaa G, Asseman E. La nocardiose mammaire bovine. Revue Med Vet 1987; 138: 999-1005. 9. Penseyres JH, Meyer B, Battig U, Wegmann P, Breer C. Epidemiologic und diagnostik der Nocardia asteroides mastitis des rindes. Proc Int Conf Mastitis, Austria, 1989: 55-60. 10. Pianta C. Mammary infections with Nocardia asteroides. Bull Inst Pesq Vet Desiderio Finamor 1987; 10: 17-21. 11. Schoonderwoerd M, Lynch JA. A report on the bovine nocardial mastitis meeting. Can Vet J 1989; 30: 555-558. 12. Dean AD, Dean JA, Burton AH, Dicker RC. Epi Info, Version 5: a word processing, database, and statistics program for epidemiology on micro-computers. USD Incorporated, Stone Mountain, Georgia, 1990. 13. Campos-Filho N, Franco EL. Epidemiologic programs for computers and calculators. A microcomputer program for multiple logistic regression by unconditional and conditional maximum likelihood methods. Am J Epidemiol 1989; 129: 439-444. 14. Schlesselman JJ. Case-control Studies, Design, Conduct, Analysis. New York: Oxford University Press, 1982: 257-259. 15. Stark DA, Anderson NG. A case-control study of Nocardia mastitis in Ontario dairy herds. Can Vet J 1990; 31: 197-201. 16. Schoonderwoerd M, McFadzen LL, Manninen KL, Ollis GW. Culturing of bulk tank milk for the presence of Nocardia spp. Can Vet J 1990; 31: 453-454.
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