Chlamydiaceae in riverine buffalo (Bubalus bubalis) and cows (Bos taurus) in Egypt with and without signs of reproductive disease

Abstract

AIMS: To obtain information and compare the prevalence of Chlamydiaceae in riverine buffalo (Bubalus bubalis) and cows (Bos taurus) in Egypt with and without clinical signs of reproductive disease.

METHODS: Vaginal swabs and blood samples were collected from animals attending Governmental Veterinary Clinics without (buffalo n=39, cows n=20) and with (buffalo n=63, cows n=53) signs of reproductive disease. Serum samples were tested for antibodies to Chlamydiaceae using complement fixation testing (CFT). Vaginal swabs were tested for Chlamydiaceae following inoculation into Vero cells and 6-day-old embryonated chicken eggs, using modified Giménez and immunoperoxidase staining, PCR analyses targeting the omp2 gene, and Restriction Fragment Length Polymorphism PCR (RFLP-PCR) for species identification.

RESULTS: Antibodies to Chlamydiaceae were detected in 30/39 (77%) and 50/63 (79%) buffalo without and with signs of reproductive disease, respectively, and 10/20 (50%) and 39/53 (74%) of cows with and without signs of reproductive disease, respectively. Positive samples from PCR analysis were identified in 31/39 (79%) and 37/63 (59%) buffalo without and with signs of reproductive disease, respectively, and 12/20 (60%) and 46/53 (89%) of cows without and with signs of reproductive disease, respectively. Using RFLP-PCR, 57/68 (84%) of samples from buffalo, and 47/58 (81%) from cows, were identified as Chlamydophila psittaci and the reminder as Cp. abortus. From the CFT and PCR results there was no significant difference in the prevalence of positive samples between species, or between animals without or with signs of reproductive disease.

CONCLUSION: The presence of anti-Chlamydiaceae antibodies in 77% of the animals with signs of reproductive disease and the detection of Chlamydiaceae in 72% of vaginal swabs of the animals suggest a pathogenic role by Chlamydiaceae in riverine buffalo and cows. The main Chlamydiaceae found in the genital tract of cattle in Egypt were Cp. psittaci and Cp. abortus.

CLINICAL RELEVANCE: Chlamydophila spp. should be included in diagnostic algorithms for reproductive disorders, in order to assess the real burden of Chlamydophila associated disease in buffalo and cattle and to evaluate the potential value of vaccines.     

Active Immunization against Gonadotrophin-releasing Hormone in Chinese Male Pigs

We have investigated, under the normal conditions of local Chinese pig farming, castration of young male pigs by vaccination with a newly developed vaccine against gonadotrophin releasing hormone (GnRH). Because of the very early onset of puberty, long fattening period and relatively harsh circumstances in Chinese pig production, an investigation of the endocrine response of Chinese breeds to this type of vaccination was of particular interest. Fifteen crossbred boars (Yorkshire × Yanan) from three different litters were randomly assigned to three groups of five animals each. The first group was immunized at 13 weeks of age with a GnRH tandem dimer OVA‐conjugate in Specol and received a booster immunization 8 weeks later. The second group was injected with Specol alone and served as untreated controls. The remaining group was surgically castrated at the time of weaning (at 6 weeks of age). Pigs were fed ad libitum from weaning onwards. All animals were slaughtered at 31 weeks of age. Immunized boars had undetectable or low serum testosterone (0.09 ± 0.12 ng/ml), low fat androstenone (0.05 ± 0.01 μg/g) levels and very low testes weights (19.1 ± 4.3 g). Intact controls had much higher serum levels of testosterone (9.76 ± 4.81 ng/ml), fat androstenone levels (2.26 ± 0.87 μg/g) and testes weights (114.3 ± 29.41 g) at slaughter. Both the immunized and castrated group grew significantly faster than intact boars (p < 0.01). Average daily gains in immunized, castrated and intact animals were 0.69 ± 0.08, 0.63 ± 0.05 and 0.42 ± 0.07 kg (mean ± SD), respectively. The present data demonstrate for the first time that the newly developed anti‐GnRH vaccine works very well under practical Chinese pig farming conditions, and can be an attractive alternative to surgical castration.     

Mycobacterium ulcerans infections in two horses in south-eastern Australia

Two horses were diagnosed as having Mycobacterium ulcerans infections. The first was a 21-year-old Quarterhorse-cross mare living in Mallacoota (a coastal town near the border of New South Wales and Victoria, Australia) that presented with lichenification, hair-loss and oedema on a fetlock, which subsequently ulcerated, as well as a non-healing ulcer on the wither. The second horse was a 32 year-old Standardbred gelding from Nicholson, near Bairnsdale, Victoria, that had an ulcerated lesion on its caudal thigh. Histologically, there were characteristic changes seen with M. ulcerans infections in other species, including extensive necrosis without associated granulomatous inflammation. The organisms were seen in Ziehl-Neelsen-stained smears or sections of the lesions from both horses and were isolated in culture from the first horse. A definitive diagnosis was provided by real-time polymerase chain reaction targeting the M. ulcerans-specific insertion sequence, IS2404. Delayed identification of the infectious agent in the first case led to the use of suboptimal antimicrobial therapy, resulting in failure to control the infection and the horse was subsequently euthanased. The second horse was successfully treated following surgical debulking of the centre of the lesion and one session of aggressive cryosurgery. Mycobacterium ulcerans should be considered in the differential diagnosis of unexplained lichenification with oedematous and ulcerated skin lesions in horses living in regions where this organism is endemic.     

Neonatal Clinical Evaluation, Blood Gas and Radiographic Assessment After Normal Birth, Vaginal Dystocia or Caesarean Section in Dogs

This study aimed to standardize signs and diagnostic criteria of respiratory function in newborn puppies delivered normally or after dystocia and caesarean operation. A total of 48 neonates were allocated into groups: eutocia (n = 20), dystocia (n = 8), caesarean (c)-section (n = 20). Neonatal health was assessed using the Apgar score and body temperature was determined at 0, 5 and 60 min after delivery. Venous blood gases (pO₂ and SO₂) was measured immediately and 60 min after delivery, and a thoracic radiograph was made between 0 and 5 min of life. The c-section group had significantly lower Apgar scores at birth and 5 min. Hypothermia was present at 5 min in the eutocia and c-section groups, and at 60 min in all groups. The eutocia group had an irregular respiratory pattern in 78% of puppies at birth, 27.7% at 5 min and 21% at 60 min compared with 87.5%, 62.5% and 12.5% of the pups in the dystocia group where there was irregular respiratory rhythm, moderate to intense respiratory sounds with agonic episodes. The c-section group had respiratory alterations in 70%, 45% and 16% of puppies at 0, 5 and 60 min, respectively. Radiographic abnormalities were present in 17% of the pups in the eutocia group, 25% of the pups in the dystocia group and 30% of the pups in the c-section group, respectively. The c-section group had significantly lower SO₂ values at 60 min than at birth. All puppies had hypoxaemia, but a significant decrease was observed in the c-section group. Newborn puppies had tissue hypoxia and irregular respiratory pattern at birth. Caesarean-section puppies had lower vitality; however, all developed satisfactory Apgar scores at 5 min of life, regardless of the obstetric condition.     

Influence of Ascorbic Acid and Glutathione Antioxidants on Frozen-Thawed Canine Semen

Poor sperm viability post-thaw has resulted in constant research into methods of cryopreservation of canine semen. One factor that may be involved in poor viability is sperm oxidative stress caused by excessive formation of reactive oxygen species. The present study was performed in order to evaluate the effect of different concentrations of ascorbic acid (AA) and glutathione (Glu) added to an extender for the freeze-thawing of dog sperm. Semen from five mature dogs was collected and frozen in two studies. Prior to and after freezing, sperm motility, morphology and membrane status were examined. In addition, sperm motility was examined up to 120 min after thawing to evaluate thermo-resistance. In study I, semen was collected twice from each dog. On both occasions, semen was divided into three aliquots: control, Glu 1 m m and Glu 5 m m . In study II, semen was collected twice and divided into three aliquots; control, AA 50 μ m and AA 250 μ m . Initial sperm motility was significantly higher in sperm diluted with AA 50 μ m ; sperm longevity, however, measured by a thermal-resistance test (TRT), was higher for Glu treatments. Higher concentration of Glu produced significant improvement in TRT and membrane status, whereas higher concentration of AA had a negative impact in sperm longevity. Antioxidant supplementation to semen freezing extenders improved semen quality post-thaw. Moreover, Glu had the most beneficial effect when supplemented at 5 m m .     

Evaluation of Trypsin Treatment on the Inactivation of Bovine Herpesvirus Type 1 on In Vitro Produced Pre-implantation Embryos

The aim of this study was to evaluate the efficiency of trypsin treatment on the inactivation of bovine herpesvirus type 1 (BoHV-1) on in vitro produced by fertilization and artificially infected bovine embryos. Bovine embryos on day 7 were exposed with 10 μl of BoHV-1, Los Angeles strain 10^7.5 TCID. These embryos and control embryos were divided in two groups: submitted to the sequential washes or to the trypsin treatment according to the International Embryo Transfer Society (IETS) guidelines. The embryos and the last washing drop of each group were used as inoculum to infect Madin Darby bovine kidney (MDBK) cells and submitted to nested PCR reaction using the primer that encodes the gene conserved region of virus glycoprotein gB. The data have shown that the control embryos and their last washing drop were negative. The exposed embryos that were treated with trypsin have shown positive results on the n-PCR and MDBK culture, and their last washing drop were negative. Our data have demonstrated that the trypsin treatment was not able to eliminate the BHV-1 of the embryos, suggesting an interaction between virus and embryo.     

Dimethylformamide Improves the In vitro Characteristics of Thawed Stallion Spermatozoa Reducing Sublethal Damage

A total of 42 ejaculates were used in the experiment; six ejaculates per stallion, obtained from seven Pure Spanish stallions (PRE), were split and frozen in freezing media with different concentrations and combinations of cryoprotectant (CPA): (i) Cáceres (skim milk based extender) containing 2.5% glycerol (2.5GL), (ii) Cáceres containing 1.5% glycerol and 1.5% dimethylformamide (1.5%GL–1.5%DMFA), (iii) Cáceres extender supplemented with 1.5% glycerol and 2.5% dimethylformamide (1.5%GL–2.5%DMFA) and (iv) Cáceres extender supplemented with 4% dimethylformamide (4%DMFA). After at least 4 weeks of storage in liquid nitrogen (LN), straws were thawed and semen analysed by computer‐assisted sperm analysis and flow cytometry (membrane lipid architecture (Merocyanine 540), integrity and sublethal damage (YoPro‐1) and mitochondrial membrane potential (JC‐1)). After thawing, better results were observed in samples frozen in 4%DMFA or in combinations of 1.5%GL–2.5%DMFA, in fact total motility increased by 16% in the 4%DMFA group compared to 2.5%GL (P < 0.05). Also, there was an increment in the percentage of progressive motile sperm in the 1.5%GL–2.5%DMFA group (9.8% 2.5GL vs 19% in the 1.5%GL–2.5%DMFA group p < 0.05); also, samples frozen in the 4%DMFA group had more intact (YoPro‐1 negative) sperm post‐thawing, 29.3% in 2.5%GL vs 36.7% in 4%DMFA group (p < 0.05). Membrane lipid architecture was not affected by any of the cryoprotectants tested, while samples frozen in 4%DFMA had a lower percentage of mitochondria with lower membrane potential. It is concluded that DMFA improves the outcome of cryopreservation of stallion spermatozoa mainly reducing sublethal cryodamage.     

Genotyping Shorthorn cattle for generalised glycogenosis

OBJECTIVE: To develop a procedure for routine genotyping of Shorthorn cattle for the generalised glycogenosis allele in exon 18 of the acidic alpha-glucosidase gene. PROCEDURE: Allele-specific amplification and double mismatch amplification procedures for the discrimination of the exon 18 alleles were evaluated using leucocytes and hair roots as sources of target DNA. RESULTS: Allele-specific amplification was effective for genotyping Shorthorn cattle at the 2454 site when purified DNA was used as target for the polymerase chain reaction. However, when the target DNA was derived from hair roots, differences in the relative yield of wild-type and mutant amplicons were observed. The double mismatch amplification procedure was effective in genotyping all subjects, independent of the source of DNA. The unique cleavage sites for Drd I and PshA I within exon 18 are present and absent respectively in the wildtype amplicon, and are lost and acquired, respectively, in the mutant amplicon. In addition, the Drd I and PshA I mismatching cleavage sites incorporated into the primers serve as internal controls for Drd I and PshA I cleavage. CONCLUSION: The double Drd I/PshA I mismatch amplification procedure using hair root samples as the source of DNA is a robust method for genotyping Shorthorns for generalised glycogenosis.