The effect of subclinical Mycobacterium paratuberculosis infection on days open in Michigan, USA, dairy cows

Preventive Veterinary Medicine 46 (2000) 171±181

The effect of subclinical Mycobacterium paratuberculosis infection on days open in Michigan, USA, dairy cows Y.J. Johnson-Ifearulundua, J.B. Kaneenea,*, D.J. Sprecherb, J.C. Gardinerc, J.W. Lloydd b

a Population Medicine Center, Michigan State University, East Lansing, MI 48823, USA Department of Large Animal Clinical Sciences, Michigan State University, East Lansing, MI 48823, USA c Department of Biostatistics, Michigan State University, East Lansing, MI 48823, USA d Department of Agricultural Economics, Michigan State University, East Lansing, MI 48823, USA

Received 13 April 1999; accepted 17 May 2000

Abstract A prospective cohort study design was used to evaluate the impact of subclinical Mycobacterium paratuberculosis infection on days open in a sample of Michigan dairy herds with a history of cows positive for M. paratuberculosis diagnosed by fecal culture. Participating herds were tested and productivity and reproduction records were monitored for 18 months. All cows 24 months old were tested for M. paratuberculosis infection using the ELISA and radiometric fecal-culture (RFC) techniques. Test-negative cows were re-tested at the conclusion of the monitoring period. Multivariable regression models were used. Using both tests in parallel, the overall sample apparent prevalence for M. paratuberculosis infection was 41.8% (223/533 animals tested). Adjusting for diagnostic sensitivity and specificity, this resulted in a calculated sample true prevalence of 59.9%. ELISA-positive cows (on average) had a 28-day increase in days open when compared to ELISAnegative cows (pˆ0.02). The diagnostic method used to define a case altered the apparent association between paratuberculosis test status and days open. Fecal culture was a less-effective diagnostic tool for use in herds with a high prevalence of infected animals. The increase in days open in the ELISA-positive cows was an indication that perhaps reduced estrus expression or an increased post-partum anestrous period occurred in the subclinically infected ELISA-positive animals. This might have been due to a negative energy balance associated with M. paratuberculosis infection. # 2000 Elsevier Science B.V. All rights reserved. Keywords: Cattle-microbiological disease; Johne's disease; Paratuberculosis; Days open

*

Corresponding author. Tel.: ‡1-517-353-5941; fax: ‡1-517-432-0976. E-mail address: [email protected] (J.B. Kaneene) 0167-5877/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 5 8 7 7 ( 0 0 ) 0 0 1 4 5 - 8

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1. Introduction Few studies have assessed the impact of subclinical Mycobacterium paratuberculosis infection on reproductive outcomes. The biological mechanism for reduced fertility in paratuberculosis-positive cows is based upon the relationship between energy balance and reproduction. Cows that are infected with M. paratuberculosis might be at an increased risk of being in negative energy balance due to reduced intestinal absorption of nutrients. The granulomatous enteritis and mucosal thickening that are characteristic of paratuberculosis result in a malabsorption syndrome with a protein-losing enteropathy (Patterson et al., 1967; Patterson and Berret, 1968; Kreeger, 1991). Negative energy balance can reduce the growth and development of corpora lutea and result in a reduction of serum progesterone (Terhune, 1993; Van DeHaar et al., 1995). In post-partum cows, negative energy balance results in an increased interval to first ovulation, a reduced number of large follicles and reduced growth of pre-ovulatory follicles (Terhune, 1993; Britt, 1994). It has not been determined whether the intestinal lesions in the subclinically infected animal reduce intestinal function sufficiently to cause a negative energy balance. However, it is reasonable to presume that because non-infected cows are prone to a negative energy balance in the early post-partum period through peak lactation, cows that have even a mild reduction in intestinal function due to subclinical enteritis are at an increased risk of severe negative energy balance during the early post-partum to peak lactation period. Early post-partum cows are believed to be energy deficient because nutrients are shifted away from other tissues and organs to the mammary gland in support of milk production. This process is known as `goal-oriented nutrient partitioning.' Hence, milk production takes priority over reproduction (Lucy et al., 1992). Although the first and second ovulatory follicles develop during the pre-parturient dry period at a time when the cow is in positive energy balance, the third, fourth, and fifth ovulatory follicles develop, while the cow is in negative energy balance in the early post-partum period (Lucy et al., 1992). The pre-ovulatory follicles that develop in the face of negative energy balance produce less estradiol, resulting in reduced expression of behavioral estrus, which can lead to reduced estrus detection (Lucy et al., 1992). While the first and second post-partum corpora lutea produce levels of progesterone that are not influenced by the cow's energy balance, the third through fifth corpora lutea are from follicles that are dysfunctional and produce less progesterone (Britt, 1994). Decreased progesterone has been associated with reduced first-service conception risks (Fonseca et al., 1983; Villa-Goday et al., 1988). These impairments in the function of pre-ovulatory follicles and corpora lutea that are associated with negative energy balance can be expected to result in increased days to first service and increased number of days open. Thus, cows subclinically infected with M. paratuberculosis may be at increased risk of having a delayed first post-partum service and subsequent conception. This would reduce the reproductive efficiency of the herd due to increased days open, even though the overall conception rate, however, might be unaffected. Two studies reported an increased risk of premature culling due to infertility among cows with subclinical paratuberculosis (Merkal et al., 1975; Buergelt and Duncan, 1978). However, the extent of the infertility was not quantified, nor was the cause specified.

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Abbas et al. found that calving intervals were on the average 1.73 months greater in M. paratuberculosis-infected cows than negative cows (Abbas et al., 1983). However, that study was not limited to subclinical cases. In contrast, DeLisle and Milestone (1989) and McNab et al. (1991) found no association between subclinical M. paratuberculosis infection and calving interval. The current literature fails to demonstrate an association consistently between M. paratuberculosis test status and reproductive outcomes in the subclinical animal. Previous studies differed in diagnostic technique and study population (culled cows or cows in the current herd). Some defined positive animals on the basis of culture or histopathology (Buergelt and Duncan, 1978; Abbas et al., 1983) and others on the basis of ELISA alone (DeLisle and Milestone, 1989; McNab et al., 1991). Variation of the results across diagnostic techniques may provide an explanation for a failure to find consistent results in the literature regarding the impact of subclinical paratuberculosis on reproduction. Quantifying the economic impact of subclinical paratuberculosis is a vital component of efforts to develop cost-effective Johne's disease control programs. Due to the inconsistency in the literature regarding the impact of subclinical M. paratuberculosis infection on reproductive outcomes, we designed this study. The purpose of our study was to evaluate the impact of subclinical M. paratuberculosis infection on days open; we selected days open because it is a specific reproductive outcome that effects overall reproductive performance. Subclinical M. paratuberculosis-infected cows were compared to their M. paratuberculosis-negative herd-mates on number of days open post-partum. We hypothesized a greater number of days open post-partum on an average among the infected cows. 2. Materials and methods The experimental design for this study was a prospective cohort to assess reproductive outcomes of the cows testing positive for M. paratuberculosis infection compared with herd-mates that tested negative. The prospective nature of the study allowed the determination of M. paratuberculosis-infection status of control animals over time and it ensured that the cases were indeed positive prior to the time for which the productivity was being assessed. To compare our results with those of previous studies, several case definitions (and respective control groups) were determined based upon the results of ELISA and radiometric fecal cultures (RFC) (see Table 1). To evaluate the impact of subclinical infection alone, cases were limited to test-positive animals that were not demonstrating clinical signs of paratuberculosis (chronic or intermittent diarrhea or loss of weight that was unresponsive to anthelmintic or antimicrobial treatment, for a duration of 30 days or more) at the time of testing. Control animals also had to be free of clinical signs of paratuberculosis. Animals that developed clinical paratuberculosis during the study period were eliminated from the data analyses. Because the larger study, of which this was a part, was designed to evaluate the effect of subclinical M. paratuberculosis infection on both reproductive outcomes and on milk

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Table 1 Case definitions and respective control groups for comparison of days open in a prospective cohort study of the effect of M. paratuberculosis test status on reproductive outcomes in paratuberculosis-positive dairy herds in Michigan, USA, monitored from May 1996 to November 1997 Comparison number

Case definition

Control group

ELISA result 1 2 3 4 5 6

Positive Positive Negative Positive Positive ±

and and and or

Fecal result

ELISA result

Positive Negative Positive Positive ± Positive

Negative Negative Negative Negative Negative ±

Fecal result and and and and

Negative Negative Negative Negative ± Negative

production, the number of animals was based on power to detect a significant difference in 305-day mature-equivalent milk. This approach was felt to be valid because there is more variation in milk production than in days open. Given a Type-I error of 5% and Type-II error of 20%, and assuming an intra-herd correlation of 0.04, the objective was to detect a minimum difference in mature-equivalent milk of 1000 lb. The standard deviation of mature-equivalent milk was approximately 3000 lb. Using the method described by Donner, the required sample size was estimated to be 120 infected animals and 120 uninfected (Donner, 1992). To control for the possible loss to follow-up and the conversion of some test-negative animals to positive status, the goal was to obtain 360 negative animals. Participating farms were selected from paratuberculosis-positive Holstein dairy herds enrolled in the Michigan Dairy Herd Improvement Association (DHIA). Participants were referred by private veterinary practitioners and by Michigan Department of Agriculture Veterinary Medical Officers. For this project, a paratuberculosis-positive herd was defined as a herd that had been classified as M. paratuberculosis infected within the past 2 years prior to the study. Infected herd status was based on having at least one animal positive on fecal culture. Participating producers were required to use artificial insemination or hand-mating, so that exact breeding dates could be recorded. Records were maintained by the farm manager and collected by the investigators during quarterly farm visits. For each herd enrolled in the study, all females 24 months old and all primipara females (of any age) were tested. Collection of DHIA milk production data and reproductive records began at the time of initial testing. Herds were monitored for 18 months. Those animals that tested negative with both the ELISA and fecal culture at the initial testing were re-tested at the conclusion of the observation period. Those animals that remained negative were classified as the control animals for comparison to their positive herd-mates. Due to the low sensitivities of the ELISA and fecal culture and the chronic nature of paratuberculosis, any animal that tested positive for M. paratuberculosis infection, with either the ELISA or fecal culture, at any time during the study period was classified as a case.

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The true prevalence for each herd was determined by adjusting the sample apparent prevalence1 for the sensitivity (69%) and specificity (99.7%) of the ELISA and RFC when used in parallel (Collins and Sockett, 1993).2 Blood and feces were collected from the selected animals by the investigators. Ten milliliter of blood were collected from each cow via the middle coccygeal vein using a 20-gauge 1 in. needle and a 10 ml vacutainer tube.3 After clotting, the samples were centrifuged and serum was harvested and stored at ÿ708C in a sterile cryotube. Approximately 20 g of feces were collected from each cow via rectal palpation using a disposable plastic obstetrical glove, placed in a plastic specimen cup, sealed and labeled. Fecal specimens were then mailed via overnight express in styrofoam coolers with cold packs, to Johne's Testing Center at the University of Wisconsin in Madison, WI, for radiometric fecal cultures. Serum samples were tested for antibodies to M. paratuberculosis via the commercial IDEXX ELISA test kit4 using the manufacturer's recommended protocol (Anderson and Seymour, 1991; Collins and Sockett, 1993). Radiometric fecal culturing was conducted at Johne's Testing Center at the University of Wisconsin. The laboratory followed the protocol described in recent studies (Collins et al., 1990). Briefly, BACTEC medium was supplemented with mycobactin J, egg-yolk suspension, vancomycin, amphotericin B, and nalidixic acid in a radiometric broth. Decontaminated fecal specimens were filter concentrated placed in the vial containing the radiometric culture medium. Vials were read weekly on a BACTEC 460. All positive growth vials were subcultured on plate media and M. paratuberculosis isolation was confirmed by IS900 Genetic Probe (see Footnote 4). The sensitivity of fecal culture was enhanced by using the radiometric technique because it has been reported to detect as few as 10 viable organisms per gram of feces in contrast to traditional fecal-culture techniques which are reported to require 100±1000 viable colony-forming units per gram of feces (Whipple and Merkal, 1985; Sanfleban, 1990). General management data for the farm and study-animal identification were collected at the start of the study. The study-animal identification form established the animals' age, lactation number, and current reproductive status. Individual-cow health and reproductive data were collected quarterly during the study period. To collect data on potentially confounding factors such as occurrence of health and disease events other than clinical paratuberculosis, participating farmers were asked to report individual-animal incidence of disease (including clinical mastitis, metritis, lameness, ketosis, hypocalcemia, displaced abomasum, retained placenta, and dystocia) throughout the observation period. Diagnoses made by the producer or the veterinarian regularly serving the herd were recorded during quarterly farm visits. Participating farmers were asked to sign permission forms to provide milk production data directly from DHIA to the investigators quarterly.

1

Sample apparent prevalenceˆnumber of test positive animals/number of animals tested. True prevalenceˆ[apparent prevalence‡(specificityÿ100%)]/[specificity‡(sensitivityÿ100%)]. 3 Corning Glass Works, Corning, NY. 4 IDEXX Laboratories, Portland, ME. 2

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2.1. Data analysis Data were maintained in a computerized database system and statistical analyses were conducted using SAS5. Multivariable regression was used. Four biologically relevant independent variables were included in the multivariable regression model: M. paratuberculosis test status, herd, parity and days in milk. No model selection or reduction was used. Wald's p value was used to test significance of the covariates. Individual-animal M. paratuberculosis test status was the main fixed effect of interest. Because herd members were more likely to be similar with respect to the outcomes of interest, herd was also included in the models as a fixed effect. Cows were tested at the conclusion of the study without regard to their respective stage of lactation, and the stress of peak lactation is believed to increase the probability of bacterial shedding by subclinically infected cows; therefore, days in milk may be associated with M. paratuberculosis test status. It is also believed that as the number of days in milk increases, the risk of a cow being in negative energy balance also increases and then decreases. Negative energy balance has a negative impact upon reproductive efficiency (Terhune, 1993; Britt, 1994; Van DeHaar et al., 1995) and thus can be expected to result in increased days open. Parity and days in milk (at the time of the final DHIA measurement recorded in the study) were included as covariates in the regression models because they affect days open and might confound if not included in the multivariable model. Two herds were dropped from the statistical analyses due to the withdrawal from DHIA (herds 6 and 7). One herd was dropped from the statistical analyses due to unreliable reproductive data (herd 2). Reproductive data for >30% of the study animals from this herd were missing or reported dates were not biologically plausible. The sample of herds used for statistical analysis had apparent and true prevalences of 48.2% (184 positive/382 animals tested) and 69.7% (95% CI: 65.1, 74.3), respectively. In only one of the herds sampled (herd 3), the herd prevalence was low enough to have the target goal of the ratio of 3 negative animals to each positive animal (see Table 2). Thus, cases were frequency matched to controls to adjust for the imbalance in the case to control ratio and to stratify on parity. The resulting sample consisted of 81 negative and 116 positive cows. Individual animals were dropped from the multivariable analyses if there were missing data for the variables of interest. The sample used for multivariable analyses consisted of 84 cases and 40 controls with complete data sets, frequency matched on the parity group. 3. Results A total of 533 animals from 7 herds were enrolled in the study and tested for paratuberculosis. When a case was defined as an animal that was positive on either the RFC or the ELISA, the overall sample apparent prevalence was 41.8% (223 test positive animals/533 animals tested). Adjusting (see Footnote 2) for the 69% sensitivity and 99.7% specificity of the ELISA and RFC (when used in parallel) resulted in a calculated true prevalence of 59.9% (95% CI: 55.7, 64.1). 5

SAS 6.12 (1997), SAS Institute Inc., Cary, NC, USA.

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Table 2 Herd-specific prevalences of M. paratuberculosis infection in Michigan dairy herds monitored from May 1996 to November 1997a Herd number

1 2b 3 4 5

Number of animals tested

Number of tests (‡) for M. paratuberculosis infection

Herd prevalence %

95% CI

47 44 126 77 172

30 14 20 62 86

63.8 31.8 15.9 80.5 50.0

50.0, 77.5 18.0, 45.6 9.5, 22.3 71.6, 89.3 42.5, 57.5

a

Positive status based on the case definition of animals testing positive on either radiometric fecal culture or the ELISA. b Herd 2 was not included in multivariable modeling because breeding dates were not reliable.

A very low reported incidence of potential confounding disease occurrence precluded inclusion of these data in the models (only 2% [11/533] of the study animals had any reported disease event during the study period). See Table 2 for the farm prevalence of test-positive animals from the herds used in the multivariable statistical analyses. Tables 3 and 4 report the univariable descriptions of the two risk factors included in the models (days in milk and parity) by M. paratuberculosis test status, respectively. Tables 5 and 6 report the description and the multivariable analysis of days open by test status. ELISApositive cows had on average a 28-day increase in days open and this difference was significant (pˆ0.02). No other significant differences in days open by M. paratuberculosis test status were found. Table 3 Univariable description of days in milk by M. paratuberculosis test status in paratuberculosis-positive dairy herds monitored from May 1996 to November 1997 in Michigan, USA Case definition

Infection status

ELISA‡ and culture‡

‡ ÿ

17 59

282, 305, 394 49, 161, 293

ELISA‡ and cultureÿ

‡ ÿ

39 59

93, 209, 288 49, 161, 293

ELISAÿ and culture‡

‡ ÿ

51 59

77, 201, 284 49, 161, 293

ELISA‡ or culture‡

‡ ÿ

107 59

96, 242, 295 49, 161, 293

ELISA‡a

‡ ÿ

56 110

126, 271, 305 65, 177, 284

Culture‡a

‡ ÿ

68 98

116, 275, 305 69, 188, 288

a

Significant at p0.05.

Number of cows

Quartiles

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Table 4 Parity by M. paratuberculosis-test status from paratuberculosis-positive herds monitored from May 1996 to November 1997 in Michigan, USA Case definition

Infection status

Parity 1

2

3

4

ELISA‡ and culture‡

‡ ÿ

7 26

5 10

4 14

1 9

ELISA‡ and cultureÿ

‡ ÿ

14 26

9 10

10 14

6 9

ELISAÿ and culture‡

‡ ÿ

15 26

12 10

16 14

8 9

ELISA‡ or culture‡

‡ ÿ

36 26

26 10

30 14

15 9

ELISA‡

‡ ÿ

21 41

14 22

14 30

7 17

Culture‡

‡ ÿ

22 40

17 19

20 24

9 15

4. Discussion The method of defining a case altered the association between days open and M. paratuberculosis test status. When a case was defined as ELISA positive and a control was defined as ELISA negative (without regard to any fecal results), M. paratuberculosis test-positive status was associated with a significant increase in the number of days open Table 5 Univariable analysis of days open by M. paratuberculosis test status in paratuberculosis-positive dairy herds monitored from May 1996 to November 1997 in Michigan, USA Case definition

Infection status

Number of cows

Quartiles

ELISA‡ and culture‡

‡ ÿ

11 41

60, 89, 149 48, 84, 142

ELISA‡ and cultureÿ

‡ ÿ

23 41

63, 94, 132 56, 74, 126

ELISAÿ and culture‡

‡ ÿ

27 41

58, 74, 122 48, 85.5, 133

ELISA‡ or culture‡

‡ ÿ

75 41

58, 77, 124 48, 84, 142

ELISA‡

‡ ÿ

34 68

63, 97, 132 48, 84, 142

Culture‡a

‡ ÿ

52 72

57, 73, 121 48, 84, 142

a

Significant at p0.25.

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Table 6 Model summaries of the relationship between M. paratuberculosis test status and days open (Multivariable linear regression models including days in milk and parity as covariates, with herd included as a fixed effect from a prospective cohort study of the effect of M. paratuberculosis test status in paratuberculosis-positive herds, May 1996 to November 1997 in Michigan, USA.) Case definition

Parameter estimatea

S.E.

Wald's p

Adjusted model, R2

Model, Fb

ELISA‡ and culture‡ ELISA‡ and cultureÿ ELISAÿ and culture‡ ELISA‡ or culture‡ ELISA‡c Culture‡

27.4 22.1 ÿ7.56 3.93 27.9 ÿ11.9

23.9 12.8 11.2 11.1 11.4 10.7

0.26 0.09 0.50 0.72 0.02 0.27

0.36 0.48 0.36 0.41 0.43 0.41

6.91 12.78 9.75 16.89 18.62 17.22

a

Parameter estimate is the adjusted mean difference in days open (positive vs. negative). Full model, F, all significant (p0.001); 118 degrees of freedom for each model. c Parameter estimate for M. paratuberculosis test status significant at p0.05 level. b

after parturition (28 days, pˆ0.02). Conversely, in the two models in which a case was defined as RFC positive (either culture positive and ELISA negative or culture positive with no regard to ELISA results), days open was actually negatively associated with M. paratuberculosis test-positive status although these differences were not statistically significant. Thus, when fecal culture alone, or ELISA and fecal culture in parallel were used to identify cases and controls, the effect of M. paratuberculosis test status on days open was not significant, and in some cases the direction of the association was reversed. Despite finding a statistically significant increase in days open for ELISA-positive animals, further studies are necessary to determine whether the observed association is really an indication of a cause-and-effect relationship or merely a spurious association. Is there a biologically plausible explanation for the observed differences in results when the method of case diagnosis is changed? In herds with high paratuberculosis prevalence, the risk of false-positive fecal results may be quite high. The false-positive risk with fecal culture is increased in herds with high prevalence because the environment is very contaminated and animals might be culture positive due to organisms simply transiting through the gut, while actual infection has not occurred. In this circumstance, the ELISA might be a better determinant of infection status because a positive result indicates that an immune response had occurred to the agent. This might be more clearly associated with actual infection rather than just passage of M. paratuberculosis through the intestinal tract. 5. Conclusion The increase in days open is an indication that perhaps reduced estrus expression or an increased post-partum anestrous period are occurring in the subclinically infected ELISA-positive animal. We speculate that this is due to a negative energy balance secondary to M. paratuberculosis infection.

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