Control of Campylobacter fetus in artificially contaminated bovine semen by incubation with antibiotics before freezing

Fresh, diluted semen containing 1.55 X 10(6) cfu/ml of Campylobacter fetus subsp. venerealis was incubated with 500 iu of penicillin, 500 micrograms of streptomycin, 160 micrograms of lincomycin and 300 micrograms of spectinomycin per ml at 35 degrees C for 0, 1, 2, 5, 10, 20 or 40 minutes. The semen was cooled to 5 degrees C, packaged in 0.25 ml French straws and then frozen in liquid nitrogen for 2 weeks. Immediately after thawing and removal of the antibiotics by centrifugation semen samples from each of the seven treatment groups were cultured as for C. fetus. Semen samples were also examined by in-vitro tests for sperm motility prior to and post-freezing. Incubation with the antibiotics for 5, 10, 20 or 40 min prior to freezing reduced the numbers of C. fetus in the semen to non-detectable levels in 38%, 69%, 88% and 100% of samples respectively. The incubated semen showed no significant reduction of sperm motility although fertility trials have not been done.     

Antimicrobial drug susceptibility of Staphylococcus aureus isolated from bovine mastitis

Isolates of Staphylococcus aureus (106) from bovine mastitis were tested for susceptibility to antimicrobial agents. beta-lactamase was produced by 69.8 per cent of isolates and 7.5 per cent were resistant to streptomycin (minimum inhibitory concentration more than 32 micrograms/ml). Resistance to other agents was rare. Intrinsic resistance or tolerance to beta-lactam antibiotics was not found.     

Effect of Antibiotics in Extender on Bacterial and Spermatozoal Quality of Cooled Buffalo (Bubalus bubalis) Bull Semen

The present study was designed to study the effect of traditional antibiotic combination (streptomycin and penicillin; SP) and relatively modern combination of antibiotics (gentamycin, tylosin, lincomycin and spectinomycin; GTLS) in extender on bacterial control and spermatozoal quality of liquid buffalo bull semen stored at 5°C. Semen collected from Nili‐Ravi buffalo bulls (n = 10) was diluted with skim milk extender containing either SP (streptomycin 1000 μg/ml and penicillin 1000 IU/ml), GTLS (gentamycin 500 μg/ml, tylosin 100 μg/ml, lincomycin 300 μg/ml and spectinomycin 600 μg/ml) or negative control with no antibiotics (NA). Liquid semen was stored at 5°C for 5 days. Aerobic bacteria isolated from buffalo semen were Pseudomonas aeruginosa and Staphylococcus aureus. The only facultative anaerobic bacterium isolated was Klebsiella pneumoniae. In vitro antibiotic sensitivity test revealed that Ps. aeruginosa and Staph. aureus were susceptible to gentamycin. Staphylococcus aureus and K. pneumoniae were susceptible to tylosin and linco‐spectinomycin. Total aerobic bacterial count was significantly lower in semen samples treated with GTLS than those of SP on third and fifth day of storage at 5°C. There was no difference (p > 0.05) in sperm motility, longevity and plasma membrane integrity (PMI) in extender containing SP or GTLS combination until the third day of storage at 5°C. On fifth day of storage sperm motility, longevity and PMI was significantly better in extender containing SP compared with GTLS and NA. Intact acrosomes, and sperm head, mid piece and tail abnormalities remained similar (p > 0.05) because of antibiotics up to 5 days of storage. In conclusion, GTLS is more capable than SP for bacterial control of buffalo bull semen. Moreover, GTLS and SP are equally efficient in preserving spermatozoal quality of extended buffalo bull semen for 3 days at 5°C.     

Isolation of mycoplasmas from bovine semen in Northern Ireland

In a survey of 332 fresh and 137 processed bovine semen samples and 25 preputial washes, mycoplasmas and, or, ureaplasmas were isolated from 46 per cent, 31 per cent and 80 per cent, respectively. Intermittent isolation from different semen collections from the same bull indicated that at least three collections per bull were necessary to determine whether infection was present. When stored processed samples were examined Mycoplasma canadense and M bovigenitalium were isolated from straws taken as long ago as 1975. Addition of lincomycin and spectinomycin to the semen extender eliminated the isolation of mycoplasmas and reduced the rate of isolation of ureaplasmas.     

Kill kinetics of the cephalosporin antibiotics cephalexin and cefuroxime against bacteria of veterinary importance

Kill kinetic studies for two cephalosporin antibiotics, cephalexin and cefuroxime were carried out against veterinary strains of Escherichia coli, Pasteurella multocida, Streptococcus suis, Erysipelothrix rhusiopathiae and a laboratory culture of Staphylococcus aureus. In more than 90 per cent of cases a kill of more than 99 per cent was achieved within four hours of antibiotic treatment at concentrations of 2 or 4 micrograms/ml. Although cefuroxime was effective at lower concentrations than cephalexin the rates of kill of the two antibiotics were comparable. The results are discussed in relation to in vivo dosage regimens.     

Selective medium for isolation of Haemophilus somnus from cattle and sheep

Incorporation of vancomycin (5 micrograms/ml), neomycin (5 micrograms/ml), sodium azide (50 micrograms/ml), nystatin (100 iu/ml) and cyclohexamide (100 micrograms/ml) into 5 per cent horse blood agar results in a selective medium for the primary isolation of Haemophilus somnus from cattle and sheep. Addition of thiamine monophosphate (1 microgram/ml) to the medium enhanced growth of this bacterium. Gram-positive bacteria did not grow on the medium and colonies of many Gram-negative bacteria were eliminated or reduced in numbers and size. Colonies of H somnus were larger on the selective medium than on sheep blood agar but retained typical morphology. Recovery of 18 laboratory strains was 73 to 166 per cent (mean 112) on selective medium compared to sheep blood agar. H somnus was isolated from the vagina of a total of 136 (28.6 per cent) of 476 cows surveyed, 79 (16.6 per cent) on sheep blood agar and 129 (27.1 per cent) on selective medium. The selective agents and thiamine were stable indefinitely as a freeze dried mixture while prepared plates were stable for two weeks.     

The effectiveness of gentamicin or polymyxin B for the control of bacterial growth in equine semen stored at 20 degrees C or 5 degrees C for up to forty-eight hours.

Semen from three stallions was used to evaluate the effectiveness of two antibiotics added to semen extender for samples stored at 20 degrees C or 5 degrees C for up to 48 hours. Each ejaculate was divided into six different treatments: semen+extender (SE); SE+gentamicin (100 micrograms/mL); SE+polymyxin B (1000 units/mL); and each of the above treatments inoculated with Pseudomonas aeruginosa ATCC 27853. Sampling of diluted semen for bacteriological analysis was performed after 2, 8, 24 and 48 hours of preservation at either temperatures. The presence of nonspecific bacteria was noted after two hours in all SE aliquots. The number of bacteria did not change in samples stored at 5 degrees C, while in samples preserved at 20 degrees C, it increased by three to four times after 48 hours. In semen aliquots treated with either of the antibiotics, the number of nonspecific bacteria was very low after two and eight hours at both temperatures. This number remained stable up to 48 hours at 5 degrees C, while an increase was noted at 24 and 48 hours at 20 degrees C. At 5 degrees C, the number of P. aeruginosa cells tended to decrease between 24 and 48 hours in SE aliquots. The presence of gentamicin or polymyxin B appeared to rapidly inhibit growth of P. aeruginosa. At 20 degrees C, growth of P. aeruginosa increased between 8 and 24 hours in SE, while the presence of antibiotics almost completely inhibited the growth of the bacterium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Equine mastitis--a review of 28 cases

Mastitis was diagnosed in 28 mares. Cytological evaluation of milk samples showed large numbers of neutrophils in 72 per cent and bacteria in 33 per cent. Aerobic bacteria were cultured from 71 per cent of samples. Streptococcus zooepidemicus was the most common isolate (37 per cent). Gram-negative species accounted for 42 per cent. Determination of the probability for antimicrobial susceptibility of bacterial isolates from mares with mastitis indicated that a trimethoprim-sulphonamide combination would be active against more than 75 per cent of isolates while penicillin would be active against less than 60 per cent. Systemic antibiotics, frequent milking, hotpacks and/or hydrotherapy and, if possible, infusion of an intramammary antibiotic preparation is recommended for treatment of equine mastitis.     

Effect of selenium and vitamin E addition to the extender on liquid stored capercaillie (Tetrao urogallus) semen quality

Captive breeding has become an important tool in species conservations programmes, maintaining genetic diversity and restoring wild, endangered populations. In order to improve the reproductive efficiency of captive kept capercaillie, the purpose of the study was to determine the effect of selenium and vitamin E addition to semen extender on sperm characteristic during short‐term storage. Ejaculates collected individually from four capercaillie were divided into two parts, diluted threefold with basic EK extender and EK enriched with 1 mg/ml of organic selenium and 8 mg/ml of vitamin E (EK+Se+E) and stored 24 hr at temp. +4°C. Spermatozoa morphology, motility and motility parameter were evaluated in net, diluted and stored semen samples. Significant (p < .05) differences between individual males were stated in relation to the majority of traits evaluated in the freshly collected semen. Comparing to the fresh semen, a significant (p < .05) decrease in percentage of live sperm in total (by 3.8% points on average) has been observed in samples diluted by EK extender, while in semen diluted with EK+Se+E extender this decrease was lower (1.5%pts on average) and not significant. Also per cent of motile sperm in EK+Se+E extender was higher (p < .05) then in EK (71.6% vs. 58.9%), but taking into account the values of individual males, both extender and male effect on liquid semen storage become apparent. Obtained data allow concluding that selenium and vitamin E addition to EK extender had positive effect on morphology and motility of capercaillie semen stored 24 hr at 4°C and can be recommended for similar studies carried out on other Galliformes species.     

Effect of vaginal douching and different semen extenders on bacterial load and fertility in turkeys

1. A study on artificial insemination of Beltsville Small White turkeys investigated the effect on bacterial load and fertility of vaginal douching with diluents containing Gentamicin 400 microg/ml and different semen extenders. 2. Irrespective of the extenders used, vaginal douching with Gentamicin reduced the microbial load of the vagina with resultant improvement in fertility and hatchability and corresponding reduction in embryonic mortality. 3. Eggs from hens inseminated with semen extended with Beltsville Poultry Semen Extender (BPSE) diluent along with vaginal douching showed a trend towards higher per cent fertility and per cent hatchability of total and fertile eggs set compared to other extenders, though this was non-significant.     

Simple transport medium for the isolation of Chlamydia psittaci from clinical material

Infectious elementary bodies of Chlamydia psittaci in tissue samples from field cases of enzootic abortion were placed in five different transport media (A to E). In one medium, in the absence of refrigerative storage, the organism remained viable for 30 days and at 4 degrees C for 34 days. This was medium D; it consisted of sucrose (74.6 g/litre), K2HPO4 (1.237 g/litre), L-glutamic acid (0.721 g/litre), fetal calf serum (10 per cent v/v), vancomycin and streptomycin (100 micrograms/ml) and nystatin and gentamicin (50 micrograms/ml). Samples of this transport medium were supplied to veterinary investigation centres throughout the UK. Of 1862 samples submitted for diagnosis of enzootic abortion only 1.55 per cent were so contaminated that chlamydiae could not be detected. This transport medium permits the isolation of C psittaci from clinical material for up to about one month, even in the absence of conventional storage facilities.     

Susceptibility of obligate anaerobes to trimethoprim-sulfamethoxazole

Susceptibilities of trimethoprim and sulfamethoxazole were tested alone and in combination (1:20) against 94 isolants of obligate anaerobes from clinical specimens. Ninety percent of the isolants were inhibited by less than 0.25 microgram - less than 4.75 micrograms trimethoprim-sulfamethoxazole/ml; 5.4 micrograms trimethoprim/ml and; 900 micrograms sulfamethoxazole/ml. We concluded that trimethoprim-sulfamethoxazole would be effective in the treatment of infectious processes containing species of obligate anaerobes, including those resistant to the penicillin and cephalothin groups of antibiotics.     

Frozen Turkey Semen

Semen samples from Bronze turkeys, extended with Brown's buffer and antibiotics, and protected with combinations of ethylene glycol-glycerol and N,N-dimethylacetamide-glycerol were frozen at the rate of 8°C per minute down to -196°C. Similar treatments were used as controls. Five White virgin hens were inseminated with semen from each group before and after dialysis. The per cent fertility of the frozen semen samples after dialysis was lower than the corresponding control groups.     

Serological surveillance of equine viral arteritis in the United Kingdom since the outbreak in 1993

Serological analysis of blood samples submitted to the Animal Health Trust showed that during 1995, 185 of 9203 unvaccinated horses (2.0 per cent) tested positive for antibodies to equine arteritis virus (EAV), and that during 1996, 46 of 8851 unvaccinated horses (0.52 per cent) tested positive. During both years thoroughbreds were the predominant breed tested and only a small proportion of these (<0.3 per cent), consisting predominantly of imported mares, were seropositive. In contrast, among standardbred horses, from which samples were actively solicited in 1995, 84 of 454 (18.5 per cent) were seropositive. Among standardbreds there was a difference in prevalence between types of horses, with 3.7 per cent of racing horses, 25 per cent of non-racing horses and 41 per cent of stallions testing seropositive. Investigations of seropositive stallions identified during 1994 and 1995 demonstrated that clinically inapparent equine viral arteritis (EVA) had occurred previously in the UK. Of 50 seropositive stallions, nearly half were standardbreds and nearly all had been imported from either North America or the European Union. Whether 34 seropositive stallions were shedding virus in their semen was established either by test mating, by the serology of the covered mares, or by investigation by MAFF following the introduction of the Equine Viral Arteritis Order 1995. Nine of the stallions (26.5 per cent) were identified as presumptive shedders of EAV in semen and among specific breeds, viral shedding was identified in six of 15 (40 per cent) standardbreds and three of nine (33 per cent) warmbloods. In contrast with the outbreak of EVA in the UK in 1993, no signs of disease typical of EAV infection were reported during these investigations, even in mares test mated to stallions shedding the virus.     

Effect of dilution temperature on boar semen quality

As boar semen is very sensitive to cold shock and changes in temperature during semen processing can have a profound impact on semen quality, the effect of the extender temperature at the time of dilution was investigated in a two-step dilution protocol for boar semen being processed for liquid storage. Fifteen boars of different breeds and ages from a commercial artificial insemination centre were included. One ejaculate per boar was collected and processed with Beltsville Thawing Solution semen extender. Each ejaculate was diluted (1 : 1) at 30 °C, and subsequently, the samples were diluted (30 × 10(6) sperm/ml) with either preheated extender [29.3 °C ± 0.2 °C, group A (GA)] or extender at room temperature [22.7 °C ± 0.6 °C, group B (GB)]. Samples were transported to the Faculty of Veterinary Medicine (University of Ghent, Belgium) in two isotherm boxes (one per group), stored at 17 °C and investigated for three consecutive days (D0 to D2). At D0, D1 and D2, motility parameters [computer-assisted semen analysis (CASA)] and the per cent of sperm with intact membrane (% IM) by eosin nigrosin staining were evaluated. At D0 and D2, the % of sperm with intact acrosome (% IA) was studied by Pisum sativum agglutinin staining. The average temperature of the 1 : 1 dilution was 29.4 °C ± 1.1 °C immediately after extender addition. No significant differences were found between groups for per cent motility [79.3 ± 9.0 for GA and 81.1 ± 9.2 for GB (p = 0.372)], % progressive motility [56.5 ± 13.3 for GA and 58.4 ± 13.8 for GB (p = 0.737)] or any CASA parameter. No differences were found for % IM [85.1 ± 10.7 and 84.5 ± 3.8 for GA and GB, respectively (p = 0.761)] and % IA [72.2 ± 9.4 for GA and 68.3 ± 16.6 for GB (p = 0.792)]. In conclusion, when a two-step dilution is performed, preheating the extender for the second dilution to match the semen temperature did not result in better semen quality compared to a dilution at a moderate room temperature.     

Antimicrobial Resistance in Some Gram‐Negative Bacteria Isolated from the Bovine Ejaculate

The bacterial load and degree of antibiotic resistance present in untreated and antibiotic‐treated semen samples were investigated in five bulls standing at a cattle‐breeding centre. Bacterial load was determined by colony counts from semen samples cultured on brain heart infusion and nutrient agar plates. Antibiotic resistance in these bacteria was assessed by measuring the diameter of bacterial growth inhibition zones around discs containing different concentrations of antibiotics. Representative antibiotic‐resistant bacterial isolates were selected for identification. Untreated semen contained few culturable bacteria, and all were completely sensitive to gentamycin, spectinomycin and lincomycin: six of the isolates showed some resistance to tylosin. In semen to which antibiotics had been added as part of the routine production process, two isolates were sensitive to all of the antibiotics tested, and the remainder were resistant to all. Resistant Gram‐negative isolates that were identified included Pseudomonas and Stenotrophomonas spp. both in the class Gammaproteobacteria and a Sphingomonas sp. which is in the class Alphaproteobacteria.     

Testing for antibiotic residues in milk

Milk from dairy farms in England and Wales has been tested regularly for antibiotic residues since 1965. The sensitivity of the test organism was 0.02 iu/ml penicillin or equivalent until the change to 0.01 iu/ml in January 1986. In 1984-85, 99.6 per cent of the 2,000,000 milk samples tested passed the test and there was an average of 695 failures per month. From 7500 on-farm investigations over the two years 1983-85 the most frequent reasons suggested by farmers for their test failures were not withholding milk for the full withdrawal period (19.3 to 16.5 per cent) and accidental transfer of milk (16.3 to 16.7 per cent). Lactating and dry cow intramammary antibiotic preparations were held responsible for rather over 50 per cent and 25 per cent respectively of the failures in both years.     

Applicability of a New Cell Culture Device for Cooled‐Storage of Stallion Semen

A new device for storage and shipping of cell cultures – the Petaka G3 cell management device – was tested for its applicability for cooled‐storage of equine semen. Semen from three stallions was processed with EquiPro extender either without antibiotics (three ejaculates per stallion) or with gentamicin (250 mg / l; three ejaculates per stallion). Semen was either stored at five (anaerobic conditions) or 15°C (aerobic conditions) in syringes or cell culture devices. Total and progressive motility, as well as membrane integrity of spermatozoa, were evaluated from days 1 to 7 after collection with computer‐assisted semen analysis. In experiment 1 (extender without antibiotics), total motility, progressive motility and viability of spermatozoa significantly decreased over time (p < 0.05). The decrease was significantly faster at 15°C than at 5°C (p < 0.05). In the presence of gentamicin (experiment 2), this difference was no longer present. It can be concluded that cooled‐storage of equine semen in sophisticated devices for cell culture is not advantageous to syringes for successful maintenance of semen longevity.     

Antimicrobial susceptibility of Pseudomonas aeruginosa isolates from dogs with otitis externa

Pseudomonas aeruginosa is a common cause of otitis externa in dogs, and treatment of these infections is becoming problematic because of the increasing number of multiresistant strains. The aim of the present study was to compare the in vitro activities of cefepime, ceftazidime, enrofloxacin, ciprofloxacin, gentamicin and ticarcillin/clavulanic acid against 104 strains of P aeruginosa isolated from dogs with otitis externa. Antimicrobial susceptibility and minimum inhibitory concentrations, in μg/ml, were evaluated by the E test (bioMérieux). The most active compound was ceftazidime, with 100 per cent efficiency. The majority of tested strains were susceptible to ticarcillin/clavulanic acid (89.4 per cent), followed by ciprofloxacin (88.5 per cent) and cefepime (60.6 per cent). The highest resistance was observed to enrofloxacin (51.9 per cent) and gentamicin (43.3 per cent). Large numbers of strains were intermediately susceptible to antibiotics registered for use in veterinary medicine in Croatia--enrofloxacin (47.1 per cent) and gentamicin (41.3 per cent).     

Antibiotics for the preservation of koala (Phascolarctos cinereus) semen

Aim To determine the normal microbial flora of the koala ejaculate and prepuce in order to select appropriate antibiotics for addition into diluents designed for the preservation of semen.
Procedure Bacteriological samples of the koala prepuce (n = 12) and ejaculate (n = 20) were submitted for microbial culture and sensitivity testing. Microbial flora of ejaculates collected by electroejaculation and artificial vagina were compared. The effects of varying concentrations of penicillin G and gentamicin on sperm motility and on the growth of bacteria in diluted semen stored at room temperature and 16°C over a 24 h period were investigated.
Results A range of bacteria was isolated from the koala prepuce and ejaculate. The predominant organisms in semen collected by electroejaculation and artificial vagina were Corynebacterium spp, none of which could be assigned to any recognised species. The addition of penicillin G and gentamicin to a PBS-based diluent at dose rates of 1000 to 2000 IU/mL and 100 to 200 m g/mL respectively, resulted in no adverse effect on sperm motility over a 24 h incubation period. Penicillin G (1000 IU/mL) and gentamicin (100 m g/mL) prevented growth of bacterial contaminants in diluted koala semen.
Conclusion By controlling the growth of bacteria in extended koala semen, penicillin G and gentamicin are likely to lengthen the period by which spermatozoa can be stored at 16°C and reduce the possibility of disease transmission during artificial insemination procedures.