Prevalence of bovine campylobacteriosis in indigenous cattle of three states in Nigeria

Summary A survey of bovine campylobacteriosis in breeding bulls and cows was carried out in the states of Kaduna, Kano and Borno. Six hundred and eighty nine cattle composed of 585 and 104 breeding bulls and cows respectively were sampled.Campylobacter fetus subsp.venerealis was isolated from 12 bulls whileCampylobacter fetus subsp.fetus was isolated from three of them. Campylobacter fetus subsp.fetus was isolated fromfour cows while Campylobacter fetus subsp.venerealis was isolated from one cow. The overall prevalence of campylobacteriosis in the three states was 2.9% (20/689). The result of the study identifiesCampylobacter fetus subsp.venerealis as the agent of enzootic infertility in Nigeria and suggests that it may be a significant problem.

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Resumen Un estudio sobre la prevalencia de campilobacteriosis en reproductores machos y hembras bovinos se llevó a cabo en los estados de Kaduna, Kano y Borno. Seiscientos ochenta y nueve animales, compuestos de 585 y 104 toros reproductores y vacas respectivamente, fueron examinados.Campylobacter fetus subsp.venerealis fue aislado de 12 toros, mientras queC. fetus subsp.fetus, se aisló de tres de ellos.C. fetus subsp.fetus se aisló de cuatro vacas, mientras queC. fetus subsp.venerealis, se aisló de una vaca. La prevalencia general de campilobacteriosis en los tres estados fue de 2.9% (20/689). El resultado de este estudio identifica alC. fetus subsp.venerealis, como el agente de infertilidad enzoótica en Nigeria, y sugiere que este puede ser un problema significativo.

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Résumé Une enquête sur la campylobactériose bovine a été menée sur du bétail reproducteur dans les états de Kaduna, Kano et Borno au Nigeria. Elle a porté sur 689 bovins dont 585 taureaux et 104 femelles.C. foetus subsp.veneralis a été isolé sur 12 taureaux etC foetus subsp.foetus sur 3 d'entre eux.C. foetus subsp.foetus a été décelé sur 4 vaches tandis queC. foetus subsp.foetus ne 1'a été que sur une seule. L'a prévalence globale de la maladie dans les 3 états représente 2,9 p. 100, soit cas sur 689 examinés. Cette enquête a permis d'identifierC. foetus subsp.veneralis comme étant responsable d'une infertilité enzootique au Nigeria. Elle indique que cette affection peut constituer un important problème.

     

Application of a multiplex PCR assay for Campylobacter fetus detection and subspecies differentiation in uncultured samples of aborted bovine fetuses

Campylobacter (C.) fetus (epsilonproteobacteria) is an important veterinary pathogen. This species is currently divided into C. fetus subspecies (subsp.) fetus (Cff) and C. fetus subsp. venerealis (Cfv). Cfv is the causative agent of bovine genital Campylobacteriosis, an infectious disease that leads to severe reproductive problems in cattle worldwide. Cff is a more general pathogen that causes reproductive problems mainly in sheep although cattle can also be affected. Here we describe a multiplex PCR method to detect C. fetus and differentiate between subspecies in a single step. The assay was standardized using cultured strains and successfully used to analyze the abomasal liquid of aborted bovine fetuses without any pre-enrichment step. Results of our assay were completely consistent with those of traditional bacteriological diagnostic methods. Furthermore, the multiplex PCR technique we developed may be easily adopted by any molecular diagnostic laboratory as a complementary tool for detecting C. fetus subspecies and obtaining epidemiological information about abortion events in cattle.     

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

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.     

Evaluation of a Campylobacter fetus subspecies venerealis real-time quantitative polymerase chain reaction for direct analysis of bovine preputial samples

The detection and subspeciation of Campylobacter fetus subsp. venerealis (CFV) from veterinary samples is important for both clinical and economic reasons. Campylobacter fetus subsp. venerealis is the causative agent of bovine genital campylobacteriosis, a venereal disease that can lead to serious reproductive problems in cattle, and strict international regulations require animals and animal products to be CFV-free for trade. This study evaluated methods reported in the literature for CFV detection and reports the translation of an extensively tested CFV-specific polymerase chain reaction (PCR) primer set; including the VenSF/VenSR primers and a real-time, quantitative PCR (qPCR) platform using SYBR Green chemistry. Three methods of preputial sample preparation for direct qPCR were evaluated and a heat lysis DNA extraction method was shown to allow for CFV detection at the level of approximately one cell equivalent per reaction (or 1.0 × 10³ CFU/mL) from prepuce. The optimized sample preparation and qPCR protocols were then used to evaluate 3 western Canadian bull cohorts, which included 377 bulls, for CFV. The qPCR assay detected 11 positive bulls for the CFV-specific parA gene target. DNA sequence data confirmed the identity of the amplified product and revealed that positive samples were comprised of 2 sequence types; one identical to previously reported CFV parA gene sequences and one with a 9% sequence divergence. These results add valuable information towards our understanding of an important CFV subspeciation target and offer a significantly improved format for an internationally recognized PCR test.     

Pulsed-field gel electrophoresis typing of Campylobacter fetus subsp. fetus isolated from sheep abortions in New Zealand

AIMS: To genotype Campylobacter fetus subsp. fetus isolates cultured from sheep abortions submitted to diagnostic laboratories in New Zealand during the year 2000 breeding season. To compare the types found nationally with those found in the Hawke' Bay region in 1999, and strains held in the New Zealand Reference Culture Collection, Medical Section (NZRM) from a study published in 1987.

METHODS: Campylobacter fetus subsp. fetus isolates cultured by veterinary diagnostic laboratories in the year 2000 breeding season, from sheep abortions from throughout New Zealand, were typed using pulsed-field gel electrophoresis (PFGE). In addition, seven freeze-dried C. fetus subsp. fetus isolates (strain numbers 2939–2945) from the NZRM, representing restriction types a–g found amongst sheep abortion isolates in a study published in 1987, were typed using PFGE.

RESULTS: In total, 293 C. fetus subsp. fetus isolates from 200 farms were obtained from veterinary diagnostic laboratories. Twenty-two distinct PFGE profiles were identified amongst the isolates. PFGE type B1 was predominant in each region of New Zealand and was identified from 66% of farms overall. Of the C. fetus subsp. fetus restriction types a–g lodged with the NZRM, 3/7 had PFGE profiles indistinguishable from profiles found in the current study. The other four restriction types had PFGE profiles that were unique but similar to those found in the current study.

CONCLUSIONS: PFGE type B1 was predominant amongst the C. fetus subsp. fetus isolates cultured from sheep abortions in each region of New Zealand in the year 2000, as was found in Hawke' Bay in 1999. The similarity between PFGE profiles of C. fetus subsp. fetus sheep abortion isolates from 1987 and 2000, and the relative prevalence of the PFGE groups, suggests that there has been no major genotypic shift in the population of C. fetus subsp. fetus implicated in sheep abortion in New Zealand during this time.     

The association between antibody titres againstCampylobacter fetus and reproductive efficiency in dairy cattle

The relationship between the antibody titres againstCampylobacter fetus and various indices of reproductive efficiency was studied in a cross-sectional study of 178 dairy cows from three California Dairy Herd Improvement Association herds. Blood samples were collected from the lactating cows during December 1986. Enzyme-linked immunosorbent assays were used to determine the antibody titres of the cow againstCampylobacter fetus, Haemophilus somnus andLeptospira hardjo and were classified as either negative or positive. The status of a cow as either negative or positive againstCampylobacter fetus andHaemophilus somnus represents serological evidence of natural exposure to the corresponding bacterial agents. However, the status againstLeptospira hardjo was assumed to reflect a vaccinal titre since all the cows studied had been routinely vaccinated against this organism in September 1986. The data on demographic and reproductive parameters pertained only to the current lactation of the cows and were obtained from Dairy Herd Improvement Association individual cow records of December 1986. Five indices of reproductive efficiency were used, namely the recent calving interval, the calving-to-conception interval, the calving-to-last-service interval, the number of services per conception, and the number of services since last calving. The serological status againstHaemophilus somnus, Leptospira hardjo and other covariates suggested by the results of previous studies were included in modelling the relationships of interest. Stepwise multiple linear regression analyses were carried out to study the adjusted relationship ofCampylobacter fetus with each measure of reproductive efficiency.Multivariate analyses revealed that the adjusted relationship forCampylobacter fetus with all five measures of reproductive efficiency was non-significant (p > 0.05). Among the covariates,Leptospira hardjo had a strong and independent relationship with recent calving interval, the unstandardized partial regression coefficient being -0.77. The possible biological mechanisms of these associations are discussed.Abbreviations BMDP biomedical computer programs - DHIA dairy herd improvement association - ELISA enzyme-linked immunosorbent assay - ICR individual cow record - PSS physiological saline solution - SD standard deviation; Other abbreviations are shown in Table I     

The association between antibody titres againstCampylobacter fetus and milk production efficiency in dairy cattle

The association between serological evidence of exposure toCampylobacter fectus and milk production performance was studied in 178 lactating cows from three California Dairy Herd Improvement Association herds using a cross-sectional study design in December 1986. ELISAs were used to determine the antibody titres againstCampylobacter fetus, Haemophilus somnus andLeptospira hardjo, which were classified as either negative or positive. The status of a cow as negative or positive againstC. fetus andH. somnus represents the serological evidence of natural exposure to the corresponding bacteria. However, the status againstL. hardjo was assumed to be the level of vaccinal titre against this organism since all the cows studied had been vaccinated against this agent. The data on demographic and productivity variables relating to the current lactation of the cows were obtained from Dairy Herd Improvement Association individual cow records for December 1986. Four measures of milk production efficiency for the current lactation were used. The status againstL. hardjo and other covariates suggested by previous studies were included in modelling the relationships of interest. Stepwise multiple linear regression was used to study the adjusted relationship ofC. fetus with each measure of milk production efficiency.Multivariate analyses revealed that the adjusted relationships ofC. fetus with the test-day's milk production, the extended 305-day milk production and the relative value of milk production were not significant (p>0.1). However, after adjusting for possible covariates,C. fetus-positive cows had an average of 7.43% lower mature equivalent milk production thanC. fetus-negative cows (p=0.02). Among the covariates, the serological status againstL. hardjo had a strong and independent relationship with the percentage mature equivalent milk production, the unstandardized partial regression coefficient being 4.53.We conclude from this cross-sectional study that the association ofC. fetus seropositivity with one index of milk production is the first indication that latentC. fetus infection may be associated with subclinical mastitis, perhaps through a hypersensitivity reaction. However, this needs further investigation.Abbreviations BMDP Biomedical Computer Programs - DHIA California Dairy Herd Improvement Association - ELISA enzyme-linked immunosorbent assay; other abbreviations are shown in Table I     

‘Soya Milk Tris‐based Phytoextender Reduces Apoptosis in Cryopreserved Buffalo (Bubalus bubalis) Spermatozoa’

The objective of this study was to investigate the effect of newly developed soya milk Tris (SMT)‐based phytoextender as an alternative to egg yolk Tris (EYT) extender used for cryopreservation of buffalo (Bubalus bubalis) spermatozoa on apoptosis. Fresh buffalo semen (control without dilution) was cryopreserved in conventional EYT (20% egg yolk v/v in Tris) and SMT (25% soya milk v/v in Tris) extender and used for the assessment of expression of apoptotic proteins. Proteins extracted from a total number of nine ejaculates from three individual buffalo bulls chosen at random were separated using SDS–PAGE followed by immunoblotting against caspase‐8, caspase‐9, caspase‐3, poly(ADP‐ribose)polymerase (PARP), cytochrome c and apoptosis inducing factor (AIF). In addition, fluorescence microscopy was used for the detection of mitochondrial membrane potential (JC‐1 assay) and apoptotic cells (annexin V‐FITC/PI assay). The results obtained clearly indicate the significant (p < 0.05) reduction in the expression of caspase‐3 (27 kDa), caspase‐8 (53 kDa), caspase‐9 (50 kDa) precursor and cytochrome c (17 kDa) in semen cryopreserved in SMT extender in comparison with EYT extender. A non‐significant (p > 0.05) reduction in expression of PARP‐DNA‐binding subunit (24 kDa) was observed in SMT extender. No expression of AIF was found in cryopreserved semen samples. A significant (p < 0.05) increase in the mean percentage of cells having high mitochondrial membrane potential and a non‐significant (p > 0.05) decrease in late apoptotic cells (AN+/PI+) was observed in SMT extender when compared to EYT extender. The results demonstrated that cryopreservation of buffalo semen in SMT‐based phytoextender can replace the traditional egg yolk extenders as it reduces the expression of apoptotic proteins maintaining high mitochondrial membrane potential and gives better protection to sperms in terms of its non‐animal origin.     

Development and evaluation of an indirect enzyme-linked immunosorbent assay for the detection of antibodies against Campylobacter fetus in cattle

Campylobacteriosis is a zoonosis that occurs worldwide. Infection with Campylobacter fetus (C. fetus) causes infertility and abortion in sheep and cattle. The current study focuses on the SapA gene of C. fetus that encodes surface array proteins and plays an important role in the virulence of C. fetus. The SapA-N (1398 bp) and SapA-C (1422 bp) fragments were amplified from the C. fetusSapA gene using polymerase chain reaction (PCR), and the corresponding recombinant proteins rSapA-N and rSapA-C were expressed in Escherichia. coli BL21 cells. Results of Western blotting and enzyme-linked immunosorbent assay (ELISA) showed that the immunological activity of rSapA-N was higher than that of rSapA-C (P < 0.05). Therefore, rSapA-N was selected to establish an indirect ELISA for detecting antibodies against C. fetus. The diagnostic criteria were as follows: S/P ? 0.45: positive; S/P < 0.4: negative; 0.45 > S/P ? 0.4: suspected. The specificity and sensitivity of our method were 94.3% and 88.6%, respectively. Moreover, no cross-reactions were observed between rSapA-N and serum samples that were positive for other bovine bacterial pathogens diseases such as Mycobacterium avium subspecies paratuberculosis. One hundred and two serum samples from cows that had experienced abortion were tested. Four and 2 C. fetus-positive serum samples were found among the 70 bovine brucellosis-positive samples and the 32 infectious bovine rhinotracheitis (IBR)-positive samples, respectively. The findings suggest that the rSapA-N-based ELISA method has immense potential in future applications.     

Epidemiology of Bovine Venereal Campylobacteriosis: Geographic Distribution and Recent Advances in Molecular Diagnostic Techniques

Bovine venereal campylobacteriosis (BVC) is a major cause of economic loss to the cattle industries in different parts of the world. Camplylobacter fetus subsp. venerealis (Cfv), the main causative agent of BVC, is highly adapted to the genital tract of cattle and is transmitted by carrier bulls. However, infertility and abortions can also be caused by the intestinal pathogens C. fetus subsp. fetus (Cff), and C. jenuni, which are not venereally transmitted. Bovine venereal campylobacteriosis, caused by Cfv associated with lowered fertility, embryo mortality and abortion, repeated returns to service, reduced pregnancy rates and extended calving intervals, has the highest prevalence in developing countries where natural breeding in cattle is widely practised. The epidemiology, pathogenesis and diagnosis of the disease have been the subject of previous reviews. The main focus of this review is to highlight the epidemiology of this disease with particular reference to geographical distribution and recent advances in molecular diagnostic techniques. It is hoped that further research interest of scientists will be stimulated with a view to finding lasting solutions to the reproductive problems associated with the disease for better livestock productivity, particularly in developing endemic countries.     

Bovine Campylobacteriosis: A Review

Campylobacteriosis (vibriosis) is a venereal disease of cattle caused by the organism Campylobacter fetus subspecies fetus previously known as Vibrio fetus subspecies venerealis. Characteristically the disease causes infertility in the female with an increased number of services necessary for conception. Abortions late in gestation are also occasionally seen. Most cases or outbreaks occur after the recent introduction of an infected bull or cow into a susceptible breeding herd. Often the disease remains undetected until late fall when the livestock owner recognizes that he has a number of females exhibiting estrus. A tentative diagnosis can be made by a study of the herd history and can often be confirmed by laboratory means.

In recent years many advances have been made towards establishing an understanding of the immune response that occurs with infection and systemic immunization. In this review, recommendations are made regarding the appropriate time to immunize the breeding herd against campylobacteriosis.

     

Experimental infections with Campylobacter fetus in bulls of different ages

Factors affecting the establishment of a carrier state of Campylobactor fetus subsp. fetus (venerealis) in preputial cavities of bulls were investigated by following infection in bulls of two different age groups until slaughter, 9–18 weeks post infection. Each of the four older bulls (66–74 months at infection) and three of the four younger bulls (41–49 months at infection) were culturally positive until slaughter, indicating no increased susceptibility with age. Relative proportions of immunoglobulin classes and albumin in preputial fluids were generally similar to those determined prior to infection, although protein concentrations decreased and ratios of IgG/IgA and IgG₁/IgG₂ increased in most of the bulls. An unexplained diminution of sample volumes occurred with progressive sampling. Changes in concentrations of proteins and in sample volumes bore no apparent relationship to infection of bulls with C. fetus. Low levels of C. fetus agglutinins were detected in sample obtained both before and after infection, but no appreciable rise in antibody titers occurred following infection. Alterations in superficial antigens of C. fetus isolates obtained during the course of infection were demonstrated in the majority of animals. The capacity of the organism to undergo antigenic variation and to provoke a minimal immune response may contribute to its prolonged survival in the preputial cavity.     

Unique genotypes of Mycobacterium avium subsp. paratuberculosis strains of Type III

Mycobacterium avium subsp. paratuberculosis (MAP) strains with two new IS900 restriction fragment length polymorphism (RFLP) BstEII types intermediate suspected to belong to the MAP Type III group were isolated from migrating sheep in Germany. Such strains have only been sporadically identified in a few studies. For a better understanding of the genomic diversity of MAP with regard to specific host associations, geographic origin, and the discussed classification into Type I, Type II and Type III, these isolates were further characterized.Using IS900-RFLP, the isolates showed unique fingerprint patterns after BstEII-, PstI-, PvuII- and BamHI-digestion which had not been published before. Additionally, using gyrB-PCR-restriction endonuclease analysis (PCR/REA) and mycobacterial interspersed repetitive unit (MIRU)-PCR, the two strains showed differences to known patterns of the Type I as well as the Type II group. Unique genotypes were also obtained with multilocus short sequence repeat (MLSSR) sequencing and MIRU-variable-number tandem-repeat (VNTR) typing.As expected, genomic profiles identical to the Type I and different from the Type II group were detected by IS1311-PCR/REA, IS1311 sequencing as well as by Large Sequence Polymorphism analysis (LSP^A 8, 17, 20, 4-II, and 18).In addition to distinct growth characteristics, the unique genotypes of the studied sheep strains support their affiliation to the assumed third group within the MAP subspecies and suggest the existence of different genotypes within this Type III group. The results could serve as further evidence that Type I and Type III groups are more closely related to each other than to the bovine Type II group.     

Detection of flagellar antigen ofCampylobacter jejuni andCampylobacter coli in canine faeces with an enzyme-linked immunosorbent assay (ELISA) — New prospects for diagnosis

A new diagnostic procedure was developed to detect the flagellar antigen ofCampylobacter jejuni andCampylobacter coli in canine faecal specimens and was tested on faecal samples from random-source dogs obtained from the local dog pound. Extraction of acid-soluble proteins was performed on faecal specimens and the extracted material was evaluated using species-specific monoclonal antibodies in an enzyme-linked immunosorbent assay. The assay detected allC. jejuni orC. coli infected specimens compared with direct selective faecal culture. One of 18 faecal specimens culture-negative forC. jejuni was identified as positive by the assay, i.e. a false positive rate of 1 of 18 (5.6%) and a corresponding specificity of 94.4%. These results suggest that the screening procedure developed to detect flagellar antigens ofC. jejuni andC. coli in canine faecal samples should be further investigated as a diagnostic alternative to culture.Abbreviations ELISA enzyme-linked immunosorbent assay - PBS phosphate-buffered saline - OD optical density     

Prevalence of bovine genital campylobacteriosis and trichomonosis of bulls in northern Nigeria

Background

A survey was conducted to determine the prevalence of campylobacteriosis and trichomonosis, and their concurrence with brucellosis, in cattle in three states of northern Nigeria.

Methods

A total of 602 preputial samples was collected from bulls in 250 herds and tested using culture and identification. Various indigenous and exotic breeds were studied and four major management systems were encountered. Age of the cattle was estimated using dentition, farm records or cornual rings.

Results

The estimated true animal-level prevalence of Campylobacter fetus infection was 16.4% (95% CI: 13.0-20.7), of which 18.5% was C. f. fetus and 81.5% was C. f. venerealis. Of the latter, 92% were C. f. venerealis biovar intermedius strains. Animal-level prevalences in Adamawa, Kano and Kaduna states were 31.8%, 11.6% and 8.3% respectively, and were highest in bulls >7 years old (33.4%) and in the Gudali breed (28.8%). Of the 250 herds, 78 (25.5%, 95% CI: 19.4-32.7) had at least one infected bull, and herd prevalence was highest in the pastoral management system (43.5%). After adjustment for confounding using multivariable analysis, the odds of C. fetus infection were highest in Adamawa state (P < 0.01), in the pastoral management system (P < 0.01), and in bulls >7 years old (P = 0.01), and tended to be higher in Bos taurus breeds (P = 0.06). There was a strong positive association between the presence of campylobacteriosis and brucellosis (P < 0.01), both within bulls (OR = 8.3) and within herds (OR = 16.0). Trichomonosis was not detected in any herds.

Conclusion

Bovine genital campylobacteriosis is prevalent particularly in the pastoral management system in northern Nigeria, with C. f. venerealis biovar intermedius as the major aetiology. There was a strong positive correlation between the occurrence of campylobacteriosis and brucellosis. No evidence of trichomonosis was found in herds in this study.     

Optimization of Ram Semen Cryopreservation Using a Chemically Defined Soybean Lecithin‐Based Extender

The purpose of the present study was to investigate the effects of a chemically defined soybean lecithin‐based semen extender as a substitute for egg yolk‐based extenders in ram semen cryopreservation. In this study, 28 ejaculates were collected from four Zandi rams in the breeding season and then pooled together. The pooled semen was divided into six equal aliquots and diluted with six different extenders: (i) Tris‐based extender (TE) containing 0.5% (w/v) soybean lecithin (SL0.5), (ii) TE containing 1% (w/v) soybean lecithin (SL1), (iii) TE containing 1.5% (w/v) soybean lecithin (SL1.5), (iv) TE containing 2% (w/v) soybean lecithin (SL2), (v) TE containing 2.5% (w/v) soybean lecithin (SL2.5) and (vi) TE containing 20% (v/v) egg yolk (EYT). After thawing, sperm motility and motion parameters, plasma membrane and acrosome integrity, apoptosis status and mitochondrial activity were evaluated. The results shown that total and progressive motility (54.43 ± 1.33% and 25.43 ± 0.96%, respectively) were significantly higher in SL1.5 when compared to other semen extenders. Sperm motion parameters (VAP, VSL, VCL, ALH and STR) were significantly higher in SL1.5 compared to other extender, with the exception of SL1 extender. Plasma membrane integrity (48.86 ± 1.38%) was significantly higher in SL1.5 when compared to other semen extenders. Also, percentage of spermatozoa with intact acrosome in SL1.5 (85.35 ± 2.19%) extender was significantly higher than that in SL0.5, SL2.5 and EYT extenders. The results showed that the proportion of live post‐thawed sperm was significantly increased in SL1.5 extender compared to SL0.5, SL2 and EYT extenders. In addition, SL1, SL1.5 and SL2.5 extenders resulted in significantly lower percentage of early‐apoptotic sperm than that in EYT extender. There were no significant differences in different semen extenders for percentage of post‐thawed necrotic and late‐apoptotic spermatozoa. Also, the results indicated that there are slight differences for percentage of live spermatozoa with active mitochondria between extenders. In conclusion, SL1.5 extender was better than other extenders in most in vitro evaluated sperm parameters.     

Detection of Campylobacter Antibodies in Sheep Sera by a Dot‐ELISA Using Acid Extracts from C. fetus ssp. fetus and C. jejuni Strains and Comparison with a Complement Fixation Test

In this study, a dot‐enzyme‐linked immunosorbent assay (Dot‐ELISA) was evaluated in comparison with a complement fixation test (CFT) for the detection of Campylobacter antibodies in sheep sera. Acid glycine extracts (AGE) of both Campylobacter fetus ssp. fetus and Campylobacter jejuni strains that had been isolated from the gall‐bladder of slaughtered sheep was used as antigen in both tests. A total of 153 sheep sera from aborted (74) and slaughtered (79) sheep were examined by both Dot‐ELISA and CFT. Twenty‐two sera showed anti‐complementary activity were not suitable for CFT. Of the 22 sera showing anti‐complementary activity, two sera were found to be positive in Dot‐ELISA. Eighty‐eight (67.2%) of the remaining 131 sera were negative by both Dot‐ELISA and CFT using AGE of both Campylobacter strains whereas 43 sera (32.8%) gave different reaction patterns in Dot‐ELISA and CFT with the extracts of both Campylobacter strains. Twelve sera were positive by both tests using AGE of C. fetus ssp. fetus but CFT failed to detect antibodies in nine of these sera when AGE of C. jejuni was used. Twelve sera were positive by both tests only when AGE of C. fetus ssp. fetus was used. Eleven sera were positive only by CFT. Seven of these reacted only with the AGE of C. fetus ssp. fetus and four sera were positive by using AGE of both Campylobacter strains. The remaining eight sera were found to be positive only by dot‐immunobinding assay either with the AGE of both Campylobacter strains or with the AGE of one of the Campylobacter strains. It is concluded that Dot‐ELISA using AGE from C. fetus ssp. fetus could be employed for the detection of Campylobacter antibodies in sheep sera and the additional use of AGE from C. jejuni as antigen appeared not to be profitable for this purpose.     

Investigation of in vitro measurable sperm attributes and their influence on electroejaculated bull semen with a fixed‐time artificial insemination protocol in Australian Bos indicus cattle

Increasing use of fixed‐time artificial insemination (FTAI) in beef cattle production has presented an opportunity for the use of fresh or chilled semen as an alternative to standard cryopreserved semen. The objective of this study was to examine in vitro sperm function and pregnancy rate of electroejaculated semen, chilled and stored for 48 hr, compared to conventionally cryopreserved semen with an optimized FTAI protocol in Brahman cattle. Semen from three Brahman bulls was collected, and aliquots were extended in either chilled (at 5°C) or frozen (LN₂) in a Tris‐egg yolk extender base with 2.4% or 7.0% glycerol, respectively. Semen samples were assessed 48 hr after collection or post‐thaw and warming, for sperm motility, in vitro sperm function and fertilizing ability, and used in a FTAI programme. The overall pregnancy rates was significantly different (p < .01) after FTAI with frozen (n = 173; 53.2%) and chilled semen (n = 174; 31.6%). In contrast, the in vitro sperm assessment showed that the chilled semen had significantly faster motility (p < .05), a higher proportion of progressively motile spermatozoa (p < .05), with significantly higher proportions of acrosome intact, viable spermatozoa (p < .01). This study showed that reasonable pregnancy rates in Brahman cattle can be achieved using FTAI with chilled semen collected using electroejaculation and stored for up to 48 hr. However, improvements in semen extenders are required in consideration of semen collection method to improve the longevity of sperm fertilizing ability to significantly increase FTAI output using chilled storage of bull semen.     

Comparison of Different Glycerol and Egg Yolk Concentrations Added to Tris‐based Extender for the Collared Peccaries (Tayassu tajacu) Semen Freezing

This study aimed to evaluate various concentrations of egg yolk (5, 10, or 20%) in combination with different concentrations of glycerol (3% or 6%) added to a Tris‐based extender on the post‐thaw characteristics of sperm obtained from Tayassu tajacu. For this purpose, semen from 10 sexually male mature collared peccaries was collected by electroejaculation and evaluated for sperm motility, vigour, viability, morphology and functional membrane integrity. The ejaculates were initially extended in Tris‐fructose plus egg yolk (5%, 10% or 20%). After cooling, the semen was added to Tris‐egg yolk plus glycerol (6% or 12%), resulting in a final concentration of 3% or 6% glycerol of the extender. Straws were frozen using liquid nitrogen and thawed in a water bath at 37°C for 30 s. The frozen–thawed semen was evaluated as reported for fresh semen. After thawing, a significant decrease was verified for sperm motility and vigour, for all the samples in comparison with fresh semen. However, no differences were evidenced among treatments for any sperm characteristics evaluated (p > 0.05), except for the combination between 10% egg yolk and 6% glycerol, which provided the worst preservation of functional membrane integrity (p < 0.05). The interactions between higher concentrations of egg yolk (20%) and glycerol (6%) and also between lower concentrations of the same substances (5% egg yolk and 3% glycerol) added to the Tris‐based extender negatively affected the preservation of the normal sperm morphology after thawing (p < 0.05). In conclusion, the use of Tris‐based extender added to 10% or 20% egg yolk plus 3% glycerol is recommended for effective sperm cryopreservation in collared peccaries.