Prevalence of Campylobacter Species on Cattle Breeding Farms in Zimbabwe

Infertility remains a challenge in breeding herds in most developing countries. In the current study, 104 penile sheath washes were collected from bulls of different breeds and ages from different cattle breeding farms in Zimbabwe. The samples were submitted to the Central Veterinary Laboratory, Zimbabwe, for screening of Campylobacter species using the polymerase chain reaction (PCR). Based on the PCR results, the animal-level prevalence was 25.96% (range 0–73.98%) and all the positive samples came from four (57.14%) of the 7 herds tested. The current study shows that Campylobacter spp. could be a causative agent in infertility observed in a number of herds in Zimbabwe. Strategies for treatment and control of campylobacteriosis should be enhanced in the country. More research and surveillance are needed to determine the epidemiology of Campylobacter species in Zimbabwean cattle herds.


Introduction
Campylobacteriosis is an infection caused by the bacteria Campylobacter spp. and is associated with a number of disease conditions in cattle, primarily related to poor fertility in breeding cows [1]. Campylobacter fetus subspecies fetus and Campylobacter fetus subspecies venerealis are most commonly associated with early embryonic death and abortion [2], the latter of which causes bovine genital campylobacteriosis (BGC). Reproductive diseases of cattle, which result in infertility and abortion, cause signifcant economic losses [3]. Financial losses arise from extended calving intervals, increased age at frst calving, low pregnancy rates, and temporary infertility in bulls [4]. In countries that maintain reproductive management based on natural mating with bulls such as Zimbabwe, mainly in beef herds where natural breeding prevails, the disease may be prevalent. In a study by Swanepoel et al. [5], BGC emerged as one of the most common identifed causes of infertility and abortion in Rhodesia (now Zimbabwe). In 1988, a prevalence of 100% was recorded on a farm in Harare, Zimbabwe [6].
In this study, the prevalence of Campylobacter spp. in breeding bulls was investigated in samples submitted to the Central Veterinary Laboratory for diagnostic testing.

Study Animals and Diagnostic
Tests. Te Central Veterinary Laboratory (CVL), Zimbabwe, provided veterinary diagnostic testing services to farmers and public and private veterinary practitioners. Te diagnostic tests carried out include tests for bovine sexually transmitted infections (STIs) such as Campylobacter spp. One hundred and four bulls of diferent ages and breeds were sampled from extensive beef production farms between September 2021 and February 2022. Te bulls came from a total of seven farms. Te farms are located in diferent provinces of Zimbabwe, that is, Mashonaland East, Harare, and Matabeleland North. Veterinarians submitted samples for diferent reasons including prebreeding examinations, surveillance, low pregnancy rates in cows, and abortions.
Skilled veterinarians carried out preputial scraping of the bulls to obtain samples for diagnostic testing. Te collected washes were put in a 10 ml container with phosphatebufered saline (PBS-pH 7.0) and sent to the CVL in cooler boxes for analysis within 24 hours.

DNA Extraction, Polymerase Chain Reaction, Gel
Electrophoresis, and Visualisation. DNA was extracted from the samples using the Zymo Quick DNA Miniprep Plus kit (Zymo, U S A) according to the manufacturer's instructions. Te concentration and purity of extracted DNA were checked using a Nanodrop spectrophotometer (Serva electrophoresis, Denmark).
25 μl reaction containing 9.3 μl of RNA-free water, 8.7 μl of the master mix (One Taq 2X Master Mix with standard bufer, New England Biolabs), 1 μl each of Campylobacter spp. specifc primers (primer sequence F-5′ GGC TGA TCT ACG ATT ACT AGC GAT 3′ and R-3′GCG CGC ATT AGA TAC CCT AGT AGT CC 5′), and 5 μl of the DNA template was used in each PCR tube. Nuclease-free water was used as a negative control. Te presence of Campylobacter spp. DNA was determined using PCR reaction undertaken as previously described [7], with modifcations in the number of cycles, that is, addition of 4 minutes of denaturation at 94°C to cycles 1-5 and reduction of annealing temperature from 37°C to 30°C. Final elongation was carried out at 60°C for 7 minutes instead of 10 minutes.
Te PCR test was carried out using a thermocycler (Eppendorf Mastercycler, Nexus Gradient Termal Cycler).
Gel electrophoresis was carried out using 10 μl PCR products alongside a 100 kb ladder on 2% agarose gel containing a fuorescent dye in 1X TAE bufer at 90 volts for 1 hour. Visualisation of the gel was performed using a gel documentation system (Kodak Gel Logic System 100). Te expected band size for Campylobacter spp. was 600 base pairs (bp).

Data Analysis.
Data were entered into MS Excel (Microsoft, USA), and animal prevalence was determined by calculating the number of positive bulls divided by the total number of sampled bulls. Herd prevalence was calculated by the number of herds, which had at least one bull testing positive for Campylobacter spp., divided by the total number of herds tested.

Prevalence of Campylobacter spp.
Te animal-level prevalence of Campylobacter spp. was 25.96%, and all the positive samples came from four (57.14%) of the 7 herds.
Only three (42.9%) herds out of 7 herds had all the bulls testing negative for Campylobacter spp. (Table 1).

Discussion
Te cattle population in Zimbabwe is estimated to be 5.2 million (Director, Division of Veterinary Technical Services). In recent years, farmers have lost cattle due to drought and diseases. Te current thrust of the Zimbabwean government is to replenish and increase the national herd [8]. One of the ways the government has been advocating for this increase is through improvement of cattle breeding through natural breeding and artifcial insemination. However, these eforts are being hampered by reproductive diseases such as those caused by Campylobacter spp. Te disease is known to hamper economic growth where farmers incur huge losses [9]. Te literature on the prevalence of Campylobacter spp. in Southern African and other developing countries is scarce.
Te current prevalence and impact of campylobacteriosis in Zimbabwe are unknown. Te last known study on prevalence of Campylobacter fetus subspecies venerealis was performed in 1989 [6], where 100% of the bulls had BGC. In recent years, veterinarians and farmers have reported existence of infertility problems in cows, and this is suspected to be due to sexually transmitted infections such as BGC. Furthermore, in the past years, movement of cattle increased due to the land reform program, resettlements, and social and cultural reasons [10]. Premovement permits were focusing mainly on foot and mouth disease, while production and reproductive diseases were not being checked. Tis could spread diseases such as campylobacteriosis across the country.
In this study, only 3 herds out of 7 had all bulls testing negative for Campylobacter spp. Te high prevalence of Campylobacter spp. infection noted in this study warrants further research on campylobacteriosis in Zimbabwe. It is important to note that the positive Campylobacter spp. samples identifed in the CVL laboratory were the frst in over ten years (Personal Communication, Technologist-incharge, CVL, Zimbabwe). In the past decade, farmers and animal movement regulatory authorities did not focus much on bovine sexually transmitted infections, and this may have resulted in the introduction of the infection into herds unknowingly through cattle movement, loaning, and sharing of bulls [9]. Te diference in prevalence across the herds in the country could be due to management systems. Previous studies have shown that prevalence of Campylobacter spp. is afected by climate change, locations, animal breeds, and age.
As STIs decrease productivity of cattle by inducing reproductive losses, reduced conception rates, extended calving seasons, and increased costs, replacing bulls and preventing loss of genetic potential due to culling the disease are of great economic importance. A comprehensive study should be carried out in Zimbabwe to determine the epidemiology of Campylobacter spp. with the aim of developing intervention to reduce such losses.

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Veterinary Medicine International

Conclusion and Recommendation
Te study reports a high prevalence of Campylobacter spp. in bull herds in Zimbabwe. Tere is a need for further surveillance of Campylobacter spp. in the country using advanced molecular techniques, which will be able to elucidate the subspecies responsible. Veterinary authorities should enforce strategies which can minimize the spread of cattle STIs in the country.

Data Availability
Raw data supporting this article are available from the corresponding author upon request.

Disclosure
Te research was carried out as part of the work performed at the Central Veterinary Laboratory, Zimbabwe.

Conflicts of Interest
Te authors declare no conficts of interest.