Application of a new diagnostic approach to a bovine genital campylobacteriosis outbreak in a Saskatchewan beef herd

Abstract

A new real-time quantitative polymerase chain reaction (qPCR) test was used to diagnose Campylobacter fetus subsp. venerealis infection associated with dramatic reproductive losses in a commercial cow-calf herd. The results were verified with repeated culture, phenotypic characterization of the organism and DNA sequencing. This case demonstrates the need for a practical field test for C. fetus subsp. venerealis and the importance of considering this organism as a potential cause of pregnancy failure in beef herds.

In the spring of 2011, a practitioner contacted the disease investigation unit at the Western College of Veterinary Medicine to discuss a beef herd with reproductive failure. The history is as follows. On June 25, 2010, 3 bulls were turned out with 102 cow-calf pairs. The 2 yearlings (Bulls A and B) and a 4-year-old bull (Bull C) had each been examined for breeding soundness at the local veterinary clinic. On July 2, 2010, Bull A was diagnosed with a penile hematoma and Bull C with severe hind end lameness rendering him incapable of mounting. The remaining yearling (Bull B) was treated for foot rot following the second week of the breeding season. On July 7th, a 2-year-old bull (Bull D) was purchased and placed with the cows to replace the 2 injured bulls. Bull D had been examined for breeding soundness and was reported to have successfully bred 19 of 20 heifers for the previous owner. Bulls B and D were pulled from the pasture on September 11, 2010.

The herd owner did not pregnancy check the herd in the fall of 2010, but the local veterinarian was called to the herd on May 25, 2011 because 49 cows had not yet calved. The local veterinarian pregnancy tested the cows and found only 4 of 49 were pregnant. An additional 6 cows had been sold during the winter because the herd owner had observed them showing signs of estrus. The result was a calving rate of 50% (51/102) for the cows exposed to Bulls A, B, C, and D in the summer of 2010. The herd owner had observed only 1 abortion in February, 2011. Thirty-two bred cows were purchased in December 2010 from 2 sources and mixed with the herd. All the new cows calved as expected.

Case description

Initial contact was made with the disease investigation unit in May 2011. The two bulls (Bulls B and D) used in the summer of 2010 were retested for breeding soundness in the spring of 2011 by another local veterinarian using the protocol outlined by the Western Canadian Association of Bovine Practitioners (1). Scrotal circumference was > 36 cm for both bulls and both had greater than 90% normal sperm cells. The 3-year-old bull (Bull D) purchased in July 2010 was initially the focus of the investigation by the local veterinary clinic as there was concern, because he was an aggressive breeder, that he had limited the breeding activity of the remaining yearling (Bull B). A semen slide from this bull was sent for review to a specialist certified by the American College of Theriogenologists. Of the 200 sperm counted using eosin nigrosin stain, 91% were normal and there were no DNA condensation abnormalities reported in the 100 sperm examined using Feulgen’s stain (2). The bulls were also tested by the local veterinary clinic in April and again in May 2011 for Tritrichomonas foetus by culture and microscopic examination. All tests were negative. The herd owner was advised that 3 tests were necessary to give a high degree of certainty that the bulls were free of infection.

Two additional bulls were examined for breeding soundness in April 2011. One was a 3-year-old (Bull E), which had been used successfully with a group of heifers in 2010, and the second was a mature bull (Bull F), which had been purchased from the neighbors to be used in the summer of 2011. Both of these bulls were also reported to be satisfactory potential breeders.

The local veterinarian initially collected blood from 4 cows to be tested for evidence of exposure to bovine viral diarrhea virus (BVDV) and infectious bovine rhinotracheitis (IBR) because there was no recent history of vaccination in the herd. The samples were examined by a commercial diagnostic laboratory (Prairie Diagnostic Services, Saskatoon) using a previously reported laboratory procedure (3). Two of the cows had very low antibody concentrations to both IBR (1 and 11) and BVDV (1:54 and 1:108); 2 had moderate antibody concentrations for both IBR (39 and 45) and BVDV (1:1458 and 1:2918). Given the extensive diagnostic workup on the bulls, and the absence of any recent vaccination in the herd, the local veterinarian was suspicious of BVDV. The decision was made to not pursue additional testing for trichomoniasis or Campylobacter fetus subsp. venerealis infection at that time.

The herd was divided into 5 groups for the 2011 breeding season. Group 1 consisted of Bull D placed with 27 of the cows that did not calve in the spring of 2011. The cows were vaccinated with Bovishield Gold FP5 (Pfizer Animal Health, Montreal, Quebec) (a modified live virus vaccine consisting of strains of IBR, BVD, parainfluenza virus 3, and bovine respiratory syncytial virus) before the breeding season and were managed as a segregated group. Bull D was placed with these cows on July 5 and was removed on September 15, 2011. When these cows were pregnancy tested November 4, 2011, 19 of 27 cows (70%) were pregnant.

Group #2 consisted of 62 cows that had calved in the spring of 2011. They were vaccinated with Bovishield Gold FP5 and bred as a separate group to Bull E and Bull F from July 5 to September 15, 2011. The cows were pregnancy tested by the local veterinary clinic on November 4, 2011, and 15 of 62 cows (24%) were pregnant.

Group #3 consisted of 7 cows that had calved late and were kept at home, separate from the others. This group of cows was vaccinated with Bovishield Gold FP5 (Pfizer Animal Health) and bred to Bull B. These cows were pregnancy tested on November 15, and 6 (86%) were pregnant.

Group #4 consisted of 15 heifers vaccinated with Bovishield Gold FP5, bred to a new yearling bull (Bull G), and kept separate. On November 15, 13 of 15 (87%) were pregnant.

Group # 5 consisted of 24 of the bred cows purchased in December, 2010. These cows were vaccinated with Bovishield Gold FP5 VL5 (Pfizer Animal Health) (Bovishield Gold FV5 plus Campylobacter fetus, Leptospira canicola, L. grippotyphosa, L. hardjo, L. icterohaemorrhagiae, and L. pomona) then sent to a local communal pasture with their calves. Twenty-one of 24 (88%) of these cows were pregnant when tested November 4, 2011.

Given the poor pregnancy rates in groups #1 and #2, further diagnostics were undertaken in November 2011 with support from the outbreak investigation unit at the WCVM. Blood samples were collected from 21 open cows on November 6. All samples were negative for antibodies to Neospora caninum and negative (< 1:100) for Leptospira autumnalis, L. bratislava, L. canicola, L. grippotoyphosa, L. hardjo, L. icterohaemorrhagiae, and L. pomona. Serology data for BVDV and IBR were more complicated to interpret given the history of vaccination in the spring of 2011. No cows had high antibody concentrations to either IBR or BVDV; 8 of 21 (38%) cows had moderate antibody concentrations to IBR (between 41 and 80), 3 had BVDV antibody concentrations of 1:1458, and 4 had 1:972. The testing methods used by the veterinary laboratories that analyzed the samples have been reported previously (3,4).

Bulls B, D, E, and F were tested for infection with C. fetus subsp. venerealis using a SYBR green quantitative PCR (qPCR) test that is currently being evaluated for clinical application (5). Bull G and the bulls used at the communal pasture (group #5) were not tested in the fall of 2011. However, the bulls used on the communal pasture were tested in May 2012. Bulls D, E, and F were positive on the first test. All 4 bulls were retested a week later and the same 3 bulls were positive on the second test.

The positive bulls were purchased from the herd owner and transported to the Western College of Veterinary Medicine. Campylobacter fetus subsp. venerealis was cultured from all 3 bulls using 5% blood agar with 0.65-μm filters (5) and Skirrow’s agar. Colonies suspected to be C. fetus subsp. venerealis were subjected to Gram’s stain and to conventional PCR (6), and verified by sequencing of the resulting PCR products (5). Subspecies determination was performed using the phenotypic identification scheme described by Schulze et al (7), and all 3 isolates were identified as C. fetus subsp. venerealis. In addition, preputial samples were examined by the direct fluorescent antibody test (DFAT) (8) using a fluorescein isothiocyanate conjugated antibody (Laboratorio Azul Diagnostico, Azul, Bs. As., Argentina), and all 3 bulls were positive. The bulls were also tested for T. foetus using real-time PCR (9), and all were negative.

There was a significant difference in the occurrence of non-pregnancy between the breeding groups where the bulls were positive for C. fetus subsp. venerealis and those where the bulls were negative or untested [odds ratio (OR): 10.8, 95% confidence interval (CI): 3.4 to 34, P < 0.001]. The association was examined using logistic regression accounting for clustering by breeding group using generalized estimating equations (10) (Proc Genmod, SAS for Windows version 9.2; SAS Institute, Cary, North Carolina, USA).

The herd owner was advised to retest his remaining bulls for C. fetus subsp. venerealis before the next breeding season and to vaccinate both cows and bulls according to label directions with a C. fetus subsp. venerealis bacterin. All remaining bulls were culled before additional testing could be completed.

Discussion

Infection with C. fetus subsp. venerealis has been associated with infertility and early fetal death, and sporadic abortions after 4 mo of gestation resulting in poor pregnancy rates and prolonged calving seasons in affected beef herds (11). Campylobacter fetus subsp. venerealis is transmitted by carrier bulls to females during natural service or through infected semen during artificial insemination (AI). Bulls do not exhibit any clinical signs or changes in semen quality, and may carry the infection for life. The bacteria are well adapted to surviving in the low oxygen environment of the epithelial crypts found in the prepuce of older bulls (11). Susceptible females bred by carrier bulls will conceive at expected rates; however, endometritis often results in early embryonic death at about 7 to 10 wk of development (11).

Within 3 to 5 mo of being infected, most cows will develop a mucosal immune response and clear C. fetus subsp. venerealis from the uterus and oviducts. Normal fertility is usually re-established and a temporary immunity develops, providing resistance to subsequent infection for up to 2 y (11). However, vaginal C. fetus subsp. venerealis infections can persist after clearance of the uterine infection and females can also re-acquire a vaginal infection during the convalescent phase (11). Both scenarios can result in a transient female carrier state and lead to the infection of susceptible bulls.

There was a strong association between the diagnosis of C. fetus subsp. venerealis infection and reproductive performance in this herd in the summer of 2011. The apparent difference in reproductive success between the 2 groups with C. fetus subsp. venerealis positive bulls could potentially be explained by partial immunity in animals in group #1 which had failed to calve the previous season, potentially due to C. fetus subsp. venerealis infection. Whether this problem was present in 2010 is unclear as the numerous interruptions and potentially limited bull-to-cow ratio could explain at least some of the reported poor reproductive performance in the preceding year. For example, the semen quality of the yearling bull with foot rot during the second week of the breeding season could have been compromised in later weeks of the summer of 2010. However, 1 of the 2 bulls associated with the poor pregnancy performance in 2010 did test positive for C. fetus subsp. venerealis in 2011. The investigation of the poor pregnancy rate following the 2010 breeding season was complicated by failure to pregnancy test the cows in the fall. Pregnancy test results from the fall allowed for timely diagnostics and identification of C. fetus subsp. venerealis.

This case report is one of the first well-documented examples of poor pregnancy rates associated with C. fetus subsp. venerealis infection in beef herds from western Canada. The diagnosis was facilitated through the application of a new qPCR adaptation of a well-established laboratory technique that has recently been optimized for use on preputial samples. This test is currently used in a research setting but is also being examined for routine clinical application. Campylobacter fetus subsp. venerealis is difficult to culture in field settings and more so under the field environmental conditions in western Canada during the spring and fall when most bulls are tested for reproductive soundness.

Bacterial culture is the gold standard test for C. fetus and preputial samples from bulls are the most common sample tested (8,12). Campylobacter fetus is a fastidious organism and the accuracy of culture techniques depends on inoculum size, the presence of competing microflora in the sample, the environmental conditions during collection and transport of the sample to the laboratory, and experienced laboratory technicians (13,14). Samples are commonly inoculated into a transport enrichment medium (TEM) to encourage multiplication of small numbers of C. fetus subsp. venerealis organisms in the original sample (15). Various selective culture media and TEM’s have been compared and recommended for C. fetus subsp. venerealis(8,14,16). Even with adequate TEM, transport time and temperatures are critical. Monke et al (16) extrapolated from differences in C. fetus subsp. venerealis culture yields between 4 and 24 h and concluded that recovery of C. fetus subsp. venerealis is unlikely when transit times are longer than 24 h. Excessively cool or hot temperatures will also make recovery of the organism unlikely. These limitations are not manageable for western Canadian veterinarians testing bulls outdoors in February through April for sales and prebreeding evaluations, or in October through December, when examining bulls associated with poor pregnancy rates in herds at considerable distances from the nearest diagnostic laboratory.

Polymerase chain reaction and qPCR tests for the detection of C. fetus subsp. venerealis directly from preputial samples have been reported for research purposes in recent years (5,17,18). While each proposed test has variations in sample preparation and assay conditions, the qPCR test applied in this outbreak used a preputial scraping technique similar to that routinely used for trichomoniasis testing (5). The recovered preputial material was flushed into phosphate-buffered saline (PBS) (5). Vials of PBS (20 mM phosphate, 150 mM NaCl) are inexpensive to prepare relative to the complex TEM suggested in most standard protocols. Phosphate-buffered saline solution also has the advantage of relative stability and a longer shelf life than most TEM if it is sterilized and refrigerated until it is used. The sample should be kept cool to protect the bacterial DNA and shipped to the laboratory as soon as possible to optimize test sensitivity. However, while culture requires the sample to remain within a limited temperature range and arrival at the laboratory within a few hours, the qPCR has been successful after overnight or next day shipping with an ice pack using commercial courier services (5). Specific guidelines for handling and sensitivity to unusual transport and storage times have yet to be investigated.

Verification of the test results through repeated successful culture, DFAT, and product sequencing from these bulls after arrival at the WCVM supported the field diagnosis with the new protocol. While recommendation of this direct qPCR test for routine clinical application is pending assessment of clinical sensitivity and specificity, this case reminds practitioners to consider C. fetus subsp. venerealis as a potential cause of poor pregnancy rates in beef herds, especially when the other common causes such as management, bull breeding soundness, and poor nutrition have been ruled out. CVJ