Infectious disease continues to cause significant problems on reproductive efficiency in the cattle industry. The purpose of this project is to evaluate new testing strategies for Tritrichomonas foetus and Campylobacter fetus subsp. venerealis. This thesis describes the result of three studies that evaluated the use of real-time PCR for the identification of Tritrichomonas foetus and Campylobacter fetus subsp. venerealis in carrier bulls. The first study evaluated the specificity of a real-time PCR test for T. foetus in individual culture enriched samples, and the sensitivity of the assay for use in pooled samples of up to 25 bulls. Specificity estimates were 98.8% (95% CI 97-99.4) and 100% (95% CI 98.9-100) for culture and real-time PCR, respectively. The sensitivity of the real-time PCR assay for pooled preputial samples was: 96.8% (83.8-99.4) for pool ratios 1/3 and 1/5; 93.5% (79.3-98.2) for pool ratios 1/2, 1/15, 1/20 and 1/25; and 90.3% (75.1-96.6), and were not significantly different. However, 13 of the 217 pools tested were negative and 9 of these negative testing pools contained the same positive sample. The media in this positive sample showed evidence of contamination and could potentially explain the failure to detect T. foetus. The second study evaluated the sensitivity of a real-time PCR for the detection of T. foetus in individual and pooled direct preputial samples. Sensitivity of individual samples tested by culture, real-time PCR in direct and culture enriched samples were determined from 121 samples obtained from 9 infected bulls. Sensitivity estimates were: 95.0% (95% CI: 89.6% to 97.7%) for culture, 95.9% (95% CI: 90.7 to 98.2) for real-time PCR in cultured enriched samples, and 90.1% (95% CI: 83.5 to 94.2) for direct preputial samples and did not differ (P=0.12). Sensitivity estimates for direct pooled samples in groups of 5 or 10 were: 83.6% (95% CI: 75.6 to 89.4) and 77.3% (95% CI: 68.6-84.1), respectively and were not significantly different (P=0.08). The use of repeat sampling tested in pools by real-time PCR increased the sensitivity to 100% and 96% for 3 consecutive samples (pools of 5 or 10, respectively). The use of pooled direct preputial samples although sensitive, still requires the use of repeated sampling. The third study determined the sensitivity and specificity of a recently developed real-time PCR (qPCR) tests for Cfv. A total of 300 virgin bulls were tested by both culture and qPCR. Specificity estimates were 85% (95% CI: 80.5 to 88.6) for qPCR and 100% (95% CI: 98.7 to 100) for culture, and were significantly different (P<0.01). A total of 4 naturally infected bulls and 9 artificially infected bulls were sampled serially to obtain positive samples for a sensitivity analysis. Sensitivity estimates and 95% confidence intervals are as follows: qPCR (85.4%, 95% CI: 80.6-89.2); direct culture on blood agar (82.3%, 95% CI: 77.2-86.5), DFAT (72.1%, 95% CI: 66.2-77.4), direct culture on Skirrow agar (32.7%, 95% CI: 27.2-38.7), TEM and blood agar (30%, 95% CI: 23.4-37.5), and TEM and Skirrow agar (38.1%, 95% CI: 31-45.9). The sensitivity of the different tests evaluated varied significantly with different ambient temperatures (P<0.01). The sensitivity of the qPCR was significantly higher than any other test when temperatures exceeded 5°C. The use of repeated sampling at weekly intervals significantly improved the sensitivity of the qPCR. The real-time PCR assay for the detection of T. foetus in both individual and pooled samples appears to be highly sensitive and specific. Moreover, the possibility of using direct preputial samples provides a cost-effective diagnostic strategy. Real-time PCR in direct preputial samples for BGC diagnosis in bulls has good sensitivity and specificity. However, the use of repeated sampling maybe needed in order to maximize the ability to detect carrier bulls.

The pathogen Campylobacter fetus (CF) is classified into two subspecies, Campylobacter fetus subspecies fetus (CFF) and Campylobacter fetus subspecies venerealis (CFV). Even though CFF and CFV are genetically closely related, they exhibit differences in their host adaptation; CFF inhabits the gastrointestinal tract of both humans and several animal species, while classical CFV is specific to the bovine genital tract and is of particular concern with respect to international bovine trade regulation. Traditionally, differentiation between the two subspecies has been achieved using a limited number of biochemical tests but more rapid and definitive genetic methods of discrimination are desired. A recent study suggested that the presence of a genomic island only in CFV could discriminate between the two sub- species but this hypothesis could not be confirmed on a collection of isolates originating in Canada. To identify alternative gene targets that would support accurate subspecies discrimination, this study has applied several approaches including suppression subtractive hybridization and whole genome sequencing supplemented with optical mapping. A subtractive hybridization screen, using a well-characterized CFV isolate recovered during routine screening of bulls in an Artificial Insemination center in western Canada and that lacked much of the genomic island and a typical Canadian CFF isolate, yielded 50 clones; characterization of these clones by hybridization screening against selected CF isolates and by nucleotide sequence BLAST analysis identified three potentially CFV-specific clones that contained inserts originating from a second genomic island. Further screening using a larger CF sample set found that only Clone #35 was truly CFV-specific. Optical maps (NcoI digest) of the Canadian CFF and CFV isolates used for the subtractive hybridization showed that certain regions of these genomes were quite distinct from those of two reference strains. Whole genome sequencing of these two isolates identified two target genes (PICFV5_ORF548 and CFF_Feature #3) that appear to be selectively retained in the two subspecies. Screening of a collection of CF isolates by PCRs targeting these three loci (SSH_Clone #35, PICFV5_ORF548 and CFF_Feature #3) supported their use for subspecies discrimination. This work demonstrates the complex genomic diversity associated with these CF subtypes and the challenge posed by their discrimination using limited genetic loci.

The pathogen Campylobacter fetus (CF) is classified into two subspecies, Campylobacter fetus subspecies fetus (CFF) and Campylobacter fetus subspecies venerealis (CFV). Even though CFF and CFV are genetically closely related, they exhibit differences in their host adaptation; CFF inhabits the gastrointestinal tract of both humans and several animal species, while classical CFV is specific to the bovine genital tract and is of particular concern with respect to international bovine trade regulation. Traditionally, differentiation between the two subspecies has been achieved using a limited number of biochemical tests but more rapid and definitive genetic methods of discrimination are desired. A recent study suggested that the presence of a genomic island only in CFV could discriminate between the two sub- species but this hypothesis could not be confirmed on a collection of isolates originating in Canada. To identify alternative gene targets that would support accurate subspecies discrimination, this study has applied several approaches including suppression subtractive hybridization and whole genome sequencing supplemented with optical mapping. A subtractive hybridization screen, using a well-characterized CFV isolate recovered during routine screening of bulls in an Artificial Insemination center in western Canada and that lacked much of the genomic island and a typical Canadian CFF isolate, yielded 50 clones; characterization of these clones by hybridization screening against selected CF isolates and by nucleotide sequence BLAST analysis identified three potentially CFV-specific clones that contained inserts originating from a second genomic island. Further screening using a larger CF sample set found that only Clone #35 was truly CFV-specific. Optical maps (NcoI digest) of the Canadian CFF and CFV isolates used for the subtractive hybridization showed that certain regions of these genomes were quite distinct from those of two reference strains. Whole genome sequencing of these two isolates identified two target genes (PICFV5_ORF548 and CFF_Feature #3) that appear to be selectively retained in the two subspecies. Screening of a collection of CF isolates by PCRs targeting these three loci (SSH_Clone #35, PICFV5_ORF548 and CFF_Feature #3) supported their use for subspecies discrimination. This work demonstrates the complex genomic diversity associated with these CF subtypes and the challenge posed by their discrimination using limited genetic loci.