Preputial Washings Research Articles

Identification and Antimicrobial Susceptibility Profile of Preputial Bacterial Flora from apparently Healthy Bulls, Rams, and Bucks in Maiduguri, Northeastern Nigeria

The aim of this study was to identify the preputial aerobic bacterial flora from bulls, rams and bucks and to determine their antimicrobial sensitivities. Preputial swabs were collected randomly from twenty each of clinically healthy post pubertal bulls, rams and bucks. Using standard bacteriological techniques, the following bacteria were identified; Escherichia coli (bulls 26.5%, rams 19.2% and buck 22.7%), Staphylococcus aureus (bulls 25.0 %, rams 26.0 % and bucks 24.2 %), Streptococcus (bulls 20.6%, rams 26.0 % and bucks 25.8%), Salmonella (bulls 14.7%), Shigella (rams 15.1% and bucks 15.2%) and Klebsiella (bulls 13.2%, rams 13.7 % and bucks 12.1%) species. There was no statistically significant difference (p>0.05) between the proportions of bacterial species and different animal species. In bulls, all bacterial isolates were susceptible to Ciprofloxacin (100%) and Levofloxacin (100%) while 60% of the bacterial isolates showed resistance to amoxicillin, ampiclox and streptomycin. In rams, all the bacterial isolates were susceptible to ciprofloxacin (100%) and gentamycin (100%) while 40% of bacterial isolates were resistant to chloramphenicol. In bucks, all bacterial isolates were susceptible to amoxicillin (100%), ampiclox (100%) and gentamycin (100%) while 20% bacterial isolates were resistant to streptomycin. This study shows that a few bacteria colonize the prepuce of ruminants in Maiduguri and there is need for preputial washing prior to breeding to reduce contamination of semen during natural mating or collection. Amoxiciillin, ampiclox, ciprofloxacin and gentamycin could be used as therapeutic agents to diseases that could be caused by the bacteria in prepuce of these animal species.

Preputial bacterial flora and antibacterial susceptibility in Camels in Maiduguri, North-eastern, Nigeria

This study aimed to investigate preputial aerobic bacterial flora from Camels in the arid zone of Northern Nigeria. Preputial swabs were randomly collected from twenty (20) clinically healthy adult Camels (Camelus dromedarius). Using standard bacteriological techniques, the following bacteria were identified; Escherichia coli (22.9%), Streptococcus species (22.9%), Staphylococcus aureus (21.4%), Klebsiella Species (17.1%) and Shigella (15.7%). These isolated bacteria were found to be susceptible to Amoxicillin, Ampiclox, Ciprofloxacin, Gentamycin and Levofloxacin. However, Klebsiella and Shigella species showed intermediate susceptibility to streptomycin and Shigella further showed resistance to chloramphenicol. The results of this study showed that some bacteria colonized the prepuce of camel and there is a need for preputial washing before breeding to reduce the transmission of venereal disease. Amoxicillin, Ampiclox, Ciprofloxacin, Gentamycin and Levofloxacin could be used as therapeutic agents for venereal diseases that could be caused by the aforementioned bacteria in Camelids.

Detection of Tritrichomonas foetus by RT-rtPCR in pooled bovine preputial washings.

Trichomonosis is a venereal disease of cattle caused by the protozoan Tritrichomonas foetus. T. foetus infection in cattle herds can be economically costly for cattle producers; therefore, testing is important for detection of the agent. Given that bulls are considered to be subclinical carriers of T. foetus, it is important to detect T. foetus infection prior to movement and/or breeding season. We have described previously the development of an updated set of PCR primers and probes that offer increased sensitivity of T. foetus detection in preputial washings collected in PBS by utilizing reverse-transcription real-time PCR (RT-rtPCR) that targets the 5.8S ribosomal RNA of the T. foetus organism. Here, we report improvements in the updated RT-rtPCR reagents as well as the evaluation of testing of pooled preputial washings. We found that up to 5 preputial washings can be pooled, similar to routine testing practices (InPouch culture), without reducing the sensitivity of detection of T. foetus.

Molecular diagnosis of bovine genital campylobacteriosis using high-resolution melting analysis.

Bovine Genital Campylobacteriosis (BGC) is a worldwide spread venereal disease of cattle caused by Campylobacter fetus subsp. venerealis (Cfv). Although several real-time PCR assays were developed for Cfv identification, most target mobile genetic elements, which may lead to false-positive diagnosis. In this study, a real-time PCR assay coupled with High-Resolution Melting analysis (HRM) was developed for the identification of Campylobacter fetus subspecies and application in BGC diagnosis. Two HRM assays targeting different single nucleotide polymorphisms were validated using 51 C. fetus strains, including 36 Cfv and 15 C. fetus subsp. fetus (Cff). The specificity was assessed in 50 preputial samples previously tested as negative for C. fetus and in 24 strains from other Campylobacter species. The analytical sensitivity was determined with ten-fold dilutions of Cfv genome copies and in preputial samples spiked with Cfv cells. Both HRM assays accurately identified the 51 C. fetus strains, showing 100% concordance with the previous identification. C. fetus subspecies identification by HRM showed concordant results with the glycine test in 98.0% of the isolates. No amplification was obtained in C. fetus negative preputial samples as well as in strains from other Campylobacter species. The assays were able to detect 102 genome copies of Cfv, while for preputial washing samples the limit of detection was 103 CFU/mL. These novel HRM assays represent a highly specific and sensitive tool for the identification of C. fetus subspecies and show potential for direct use in bull preputial samples for BGC diagnosis.

Detection of Tritrichomonas foetus in cattle at the Kumasi abattoir

The study was conducted to determine the prevalence of Tritrichomonas foetus in slaughtered cattle at the Kumasi abattoir. A total of one hundred (100) slaughtered cattle were sampled randomly (60-bulls and 40 cows) with preputial wash and vaginal lavage collected after slaughter for laboratory analysis using Wet-mount technique. Data obtained were analyzed by descriptive statistics using MS Excel and the results expressed in percentages and graphs. The prevalence of bovine Tritrichomoniasis was 34% with the cows recording 20% and the bulls 14% of the sampled population. Out of the cows sampled, twenty (20) were positive representing 50% and out of the bulls sampled, fourteen (14) were positive representing 23.3%. This clearly shows that cows had the highest prevalence compared to the bulls, therefore sex has a significant effect on the infection, since p˂0.05. The WASH and the Zebu cattle had relatively higher % positivity of 41.7% and 40.7%. N’dama cattle recorded 28.6% positivity which was same as the 28.6% positivity in the White Fulani, while the Sanga breed recorded the least positivity (21.4%) of the infection. Breed of cattle however had no significant effect on infection (p>0.05). The highest prevalence of Tritrichomonas infection was 47.2% in adult cattle of 4 years old whiles adult cattle of 3 years old had a prevalence rate of 28.6% whereas cattle of 2 years of age recorded the lowest prevalence rate of 22.7%. However, age had no significant effect (p>0.05) on the infection. Further studies should be conducted to ascertain the rate of infection in the country by using a larger sample size. In order to reduce the level of infection it is advisable to screen all breeding bulls and cull the affected ones.

Evaluation of PCR assays for Campylobacter fetus detection and discrimination between C. fetus subspecies in bovine preputial wash samples

Campylobacter fetus is a zoonotic pathogen found in cattle, in which it is one of the main causes of infectious infertility. Most diagnostic laboratories use PCR as quick easy tool for C. fetus identification. However, there is no standardized PCR assay for C. fetus detection and subspecies differentiation, hindering the comparison of results. In this study, we evaluated selected PCR assays targeting the 16S rRNA, gyrB, cpn60, cstA, cdtB and nahE genes for C. fetus identification and ISCfe1, sapB2, parA and virB11 for subspecies differentiation. Analytical sensitivity and specificity were assessed for each PCR assay, and the assays were then tested on 289 bull preputial samples that had also been analysed by 16S rRNA barcode metagenomics. In total, 41 C. fetus-positive samples were included. The P12 PCR assay targeting the gyrB gene performed best, detecting the pathogen in 95.1% of positive samples. For the discrimination of C. fetus subspecies, we were able to identify a proportion (85.4%) of the C. fetus-positive samples correctly as C. fetus venerealis with at least one subspecies-specific PCR, but C. fetus fetus was not detected in any of the samples tested. Remarkably, C. fetus subspecies amplification was observed following PCR on some samples (33.1%) considered C. fetus-negative, highlighting the need for rigorous criteria for discriminating between C. fetus subspecies, to improve understanding of the role of the two C. fetus subspecies in the epidemiology and pathogenesis of bovine infectious infertility.

Whole-Genome Sequencing of Multidrug-Resistant Campylobacter fetus subsp. fetus NWU_ED24, Isolated from a Bovine Sheath Wash Sample.

Campylobacter fetus subsp. fetus is an opportunistic human pathogen that is frequently identified as a cause of intestinal infections as well as bloodstream infections. This bacterium is well known to cause spontaneous abortions in sheep and cows. The strain reported in this study was isolated from a preputial wash sample from a bull in South Africa.

Genotyping and antibiotic resistance patterns of Campylobacter fetus subsp.venerealis from cattle farms in India.

Bovine genital campylobacteriosis caused by Campylobacter fetus subsp. venerealis (Cfv) is of considerable economic importance to the cattle industry worldwide. Cfv causes syndrome of temporary infertility in female cattle, early embryonic mortality, aberrant oestrus cycles, delayed conception, abortions and poor calving rates. In the present study, a total of 200 samples obtained from vaginal swabs, cervicovaginal mucous (CVM), preputial washes and semen straws were investigated that were obtained from organized cattle farm of MLRI, Manasbal and unorganized sectors. Out of a total of 200 samples, 49 (47·57%) vaginal swabs, 1 (3·33%) preputial wash and 8 (25%) carried out CVM samples were positive for Cfv, whereas none of the semen straws were positive for Cfv. A total of eleven isolates of Cfv were recovered. PFGE (Pulse field gel electrophoresis) analysis revealed four different pulsotypes (I-IV) circulating in the screened farms. A common pulsotype circulating among farms could not be established. Insertion element (ISCfe1), a 233bp amplicon of Cfv, was sequenced and the sequence was deposited in GenBank (accession no: MK475662).

Trichomonosis in dromedary bulls

During a routine health check of 12 breeding dromedary bulls using microscopy two of them were found to harbour trichomonad flagellates in their preputial washings. A PCR conducted on one of the samples did confirm trichomonads but not Tritrichomonas foetus. DNA sequencing identified the organism as Tetratrichomonas species. After repeated careful washings of the prepuce with oxidised water resampling revealed negative results.

Estimation of Bacterial Load from Preputial Washing of Jaffrabadi Bulls

Estimation of Bacterial Load from Preputial Washing of Jaffrabadi Bulls

Application of Multiplex PCR Assay for Campylobacter fetus Sub sp. Detection and Differentiation in Bovine Preputial Wash

The aim of this research was to evaluate the detection and differentiation of C. fetus subspecies through multiplex PCR (mPCR) method from the bovine preputial wash. The evaluation was done by comparing 2 extraction methods, which was by commercial kit extraction and boiling method, and also by measuring the effect of Transport Enrichment Media (TEM) addition as bacterial enrichment. Spiked bovine preputial washes with 2 extraction methods (commercial kit and boiling), with and without TEM addition were used as a sample. The results showed that the highest sensitivity/specificity was by boiling extraction without TEM addition, which reached 83.3%/100% for all or grouped sample. The results also supported by Poisson regression analysis which showed the comparison between sample with and without TEM addition which had 1.3 Incidence Rate Ratio (IRR). The obtained IRR score indicated that sample without TEM addition had 30% better result compared to the sample with TEM addition. Furthermore, a comparison between commercial kit and boiling extraction showed no significant difference (1.029 IRR). The research concluded that boiling extraction without TEM addition is the most accurate, fast and economical extraction method regarding its high sensitivity and specificity value, with less than 24 hours testing time and without any commercial kit requirements. However, a validation and verification with field sample were still needed before applied in the laboratory.

Role of preputial washing in reducing microbial load and improving bovine semen quality

Quality semen production remains the main focus and objective of semen processing laboratories throughout the world. Bacterial and other microbial contaminants affect the semen quality and hence the fertility, and also lead to reproductive disorders as well as lower conception rates and increased embryonic mortality, abortion and other complications in females. Microbial contamination affects the semen adversely, by exerting direct spermicidal effect, formation of reactive oxygen species, toxin production, adherence with spermatozoa, deriving nutrients and oxygen from the medium and thus competing with spermatozoa for the factors of growth and normal functioning. Despite hygienic measures, several ubiquitous and opportunistic microbes find their ways into semen during collection, processing, and storage of semen, and survive even during freezing. Stringent sanitary precautions are therefore required at every step of collecting semen and its processing. Preputial cavity is considered as main source of semen contaminating microorganisms. Flushing the preputial cavity with normal saline or any suitable liquid combination with antimicrobial activity, prior to semen collection reduces the microbial load and thereby improves the semen quality.

Effect of preputial washing on bacterial load and preservability of semen in Murrah buffalo bulls.

Aim:To study the effect of preputial washing on bacterial load, preservability and semen quality in Murrah buffalo bullsMaterials and Methods:A total of 36 collections of three Murrah buffalo bulls maintained at Artificial Breeding Research Centre, ICAR-National Dairy Research Institute, Karnal, were collected at weekly intervals from each bull without preputial washing and latter ejaculates from same bull with preputial washing by infusing normal saline (0.85%), KMnO4 (0.02%) and savlon (2.0%) to first, second and third bull, respectively. The microbial load and semen quality were evaluated during different hours of storage at refrigerated temperature (0, 24 and 48 h) and after thrawing of cryopreserved (at −196°C) semen.Results:The results of preservation of semen at refrigerated temperature showed that bacterial load was markedly lower in ejaculates of bulls subjected to preputial washing. Semen preserved at refrigerator temperature and cryopreserved, the effect of washing solution was significant for individual motility (IM), non-eosiniphilic count, hypo-osmotic swelling reactivity (HOST), total plate count (TPC) and acrosome integrity. KMnO4 was found to be the best in lowering bacterial load, sperm abnormalities and in improving semen quality such as motility, non-eosinophilic count, HOST and acrosome integrity even up to 48 h of preservation and cryopreserved semen. Effect of duration of preservation and stage of cryopreservation was also significant for IM, non-eosiniphilic count, HOST, sperm abnormalities and acrosome integrity.Conclusion:Overall the results suggested that preputial washing with KMnO4 solution improved the semen quality and reduced microbial load of Murrah buffalo bull’s semen preserved at refrigerated temperature and cryopreservation.

Evaluation of the microbial quality of fresh ejaculates of camel (Camelus dromedarius) semen

A series of five factorial arranged experiments were conducted to investigate the effect of different management during semen collection on the microbial quality (bacterial load, type of microbes and frequency of isolation) of dromedaries’ semen. Microbial analysis of seventy-nine fresh ejaculates from twenty-two camels showed the presence of nine variant colonies. The most prevalent organisms in the dromedary semen were species of Staphylococcus, Bacillus and Streptococcus. One yeast species was represented among the isolates. The middle aged camels (9–13 years) had significantly (P>0.05) higher mean bacterial loads than young (4–8 years) and old aged (14–18 years) animals. The mean bacterial populations of ejaculates collected by an electro-ejaculator were significantly (P>0.05) higher than those collected by an artificial vagina. Candida spp. was identified in 53.8% of the samples collected by an electro-ejaculator and was not detected in ejaculates collected by an artificial vagina. The mean semen bacterial load detected during the breeding season was significantly (P<0.01) higher than that collected during the non-breeding season. No fungi were isolated from semen samples collected in the non-breeding season. The difference between the mean semen bacterial loads in the first and the second ejaculate was highly significant (P<0.01). The preputial wash significantly (P<0.01) reduced the bacterial load. This study revealed that the microbial contamination of dromedaries’ semen is found in different intensities during different management procedures of semen collection.

DETECTION OF BOVINE TRICHOMONIASIS OF BULLS IN BASRAH SLAUGHTERHOUSE

Bovine trichomoniasis is a venereal disease of cattle caused by the protozoan Trichomonas fetus . The study based on collection and diagnosis 200 samples of preputial wash from bulls that send to the Al-Tuaisa slaughter house in Basrah private by made a slide smear stained with Gemza stain . The study starts at 2011 and extend to 2012 . The result shows that the percentage of infection in bulls that slaughtered in a slaughter house of Basrah private was 2% .

The prevalence of bovine venereal campylobacteriosis in cattle herds in the Lake Chad basin of Nigeria

The prevalence of bovine venereal campylobacteriosis (BVC) was investigated in the Lake Chad basin of Nigeria. Preputial washings and cervico-vaginal mucus samples were obtained from 270 cattle presenting a history of abortion and lowered fertility, kept in traditional and institutional farms. All the samples investigated were cultured using standard bacteriological technique. Campylobacter fetus was isolated from six bulls and four cows. In all cattle sampled, the isolation rates were 2.2% for C. fetus subsp. venerealis and 1.5% for C. fetus subsp. fetus; the herd and within-herd prevalence rates for C. fetus were 22.2% and 3.4%, respectively, while the overall active infectivity rate was 3.7%. BVC probably contributes to lowered fertility and abortions found in cattle in the Lake Chad basin of Nigeria, associated more with C. fetus subsp. venerealis than C. fetus subsp. fetus.

Prevalence of Arcobacter spp. in Humans, Animals and Foods of Animal Origin Including Sea Food from India

The present study reports the prevalence of Arcobacter, an emerging pathogen in human, animals and foods of animal origin in India. A total of 600 samples from various sources, viz. diarrhoeal stools of humans and dogs, faecal swabs of animals (pig, poultry), preputial washings of breeding bulls and food samples (chicken, pork, fish) were examined for presence of Arcobacter spp. Using cultural methods, a total of 63 Arcobacter spp. were isolated of 600 (10.50%) samples with highest isolation rate were from pig faeces (21.33%) followed by sea foods (17.33%), poultry faeces (14.67%), pork (16.00%), chicken meat (12.00%) and human stools (2.67%). The isolates were confirmed as arcobacters by genus-based PCR. PCR screening of all the enriched samples revealed the overall prevalence of Arcobacter spp. to be 12.00% with highest in pig (25.33%), followed by sea food (21.33%), poultry (17.33%), pork (16%), chicken meat (12%) and human stools (4.00%). No Arcobacter spp. was isolated or detected from diarrhoeal faecal samples of dogs and preputial washings. With multiplex PCR, three different species were detected (A.butzleri, A.cryaerophilus and A.skirrowii) with most of the samples showing mixed infections. There are only two recent reports from India; one with cultural isolation and another with PCR detection of Arcobacter spp. in stool samples of humans with clinical diarrhoea. In this context, our present report is the first report of isolation and detection of Arcobacter spp. from various sources of animals and foods including diarrhoeic human stool samples, utilizing both cultural and molecular tools identifying arcobacters at genus and species level. These results support the importance of arcobacters as an emerging food-borne pathogen, possessing zoonotic potential.

Development and evaluation of a species-specific PCR assay for the detection of Brucella ovis infection in rams

Brucella ovis infection is a major cause of epididymitis and infertility in rams, resulting in reproductive failure and significant economic losses worldwide. The goal of this study was to develop a PCR test targeting specific B. ovis genomic sequences. Specific primer pairs were designed targeting 12 of those ORFs. Samples of blood, serum, semen, urine, and preputial wash were collected from experimentally infected rams ( n = 9) every other week up to 180 days post infection (dpi), when tissue samples were obtained. Blood, serum, semen, urine, and preputial wash samples were obtained, in weekly intervals for 1 month, from eight rams belonging to a B. ovis-free flock. Semen samples were also obtained from rams belonging to naturally infected flocks ( n = 40). The limit of detection of this PCR protocol was 100, 10, and 1 CFU/mL for semen, urine and prepucial wash samples, respectively. Sensitivity and specificity values obtained with this PCR method were similar to that of bacteriology when evaluating biological samples. Agreement between PCR and bacteriology results was greater than 90%. These results clearly indicate that this species-specific PCR method is highly efficient for the diagnosis of B. ovis infection in semen, urine, preputial wash and tissue samples from infected rams.

Detection and identification of Tetratrichomonas in a preputial wash from a bull by PCR and SSCP

Motile Tritrichomonas foetus-like trichomonads were found during microscopic examination of a wet mount sample of a preputial wash collected from a bull. Staining of the organisms with a modified Wright-Giemsa stain revealed that several had four anterior flagella of unequal length instead of the three anterior flagella of equal length characteristic of T. foetus. Limited propagation of these organisms was achieved in InPouch ® medium but no growth occurred in modified Diamond's medium. The 5.8S rRNA gene and the flanking internal transcribed spacers were amplified from the trichomonad gDNA of two preputial wash samples and a fecal sample taken from the affected bull. Amplicons were subjected to single-strand conformation polymorphism (SSCP) analyses. The SSCP banding patterns of the amplicons from gDNA of the preputial wash samples were different from those of a T. foetus control sample. These unknown trichomonads were not detected in the fecal sample. The gDNA extracted from preputial washes was also subjected to PCR using primers developed to amplify the 16S rDNA of the non- T. foetus trichomonads, Tetratrichomonas and Pentatrichomonas spp. Amplicons were produced from the gDNA of the two preputial washes but not from the T. foetus gDNA control sample. The 16S rDNA sequences obtained from the trichomonads in the two preputial washes samples were 100% similar to that of a Tetratrichomonas species previously isolated from an Angus bull from the United States.

Morphological and Molecular Identification of Non‐Tritrichomonas foetusTrichomonad Protozoa from the Bovine Preputial Cavity

Tritrichomonas foetus is the causative agent of bovine trichomonosis. This protozoan is found in the preputial cavity of bulls and is transmitted to cows during coitus. Currently, the diagnosis of this parasite is based on microscopic examination of preputial washings or scrapings, but it was recently recognized that other trichomonads similar in size, shape, and motility to T. foetus can be present in preputial samples. Despite the serious consequences of an incorrect diagnosis for bovine trichomonosis, the precise speciation of these other trichomonads has remained uncertain. Here, a total of 12 non-T. foetus isolates were microscopically examined. On the basis of morphological criteria, seven of these isolates were identified as Tetratrichomonas sp., whereas four other isolates coincided with the description of Pentatrichomonas hominis. In the last isolate, a third non-T. foetus species was identified as belonging to the genera Pseudotrichomonas or Monocercomonas: the first time that species of either of these genera have been reported in preputial samples. To confirm these data, small subunit rRNA gene sequences were obtained by PCR from the 12 trichomonad isolates. These new sequences were analysed in a broad phylogeny including 72 other parabasalid sequences. From our phylogenetic trees, we confirmed the taxonomic status of non-T. foetus organisms isolated from preputial samples (Tetratrichomonas, Pentatrichomonas, and Pseudotrichomonas) and suggested the existence of two Tetratrichomonas species, despite their morphological similarity. The route of transmission of the non-T. foetus organisms identified in the bovine preputial cavity is discussed and we confirm that the PCR assay using the previously described T. foetus-specific primers TFR3 and TFR4 could be a useful alternative method for the diagnosis of bovine trichomonosis.