Cloning and sequencing of the ovine gamma-interferon gene

Cytokines are major modulators of the immune system of all animals. The cloning and expression of recombinant cytokine genes have permitted the analysis of their immune function and role in the control of the immune response to disease and vaccination. While human, murine, and bovine genes have been cloned and sequenced, the cloning of ovine cytokine genes has not yet been reported. As sheep are of dominant economic importance to the Australian farming industry, it is of significance to clone and express these genes to facilitate the development of new and better vaccines and pharmaceuticals. We have initially selected ovine gamma-interferon (gamma-IFN) as a target cytokine gene. By the use of the polymerase chain reaction (PCR), using primers based on the bovine gamma-interferon sequence, we have amplified the ovine gamma-interferon gene from crude messenger RNA extracted from lymphocytes. After cloning and DNA sequencing the gene, we found that ovine gamma-IFN is 93% identical to bovine gamma-IFN in amino acid sequence. This result indicates that the PCR method will be a rapid and efficient means for cloning other ovine cytokine genes.     

In vitro Evaluation of in vivo Fertilizing Ability of Frozen Rabbit Semen

The present work studied different spermatozoa parameters and the ability of frozen rabbit spermatozoa to fertilize, in vitro, in vivo‐matured oocytes, as a test to predict their in vivo fertility and prolificacy. Semen from rabbit bucks was frozen using two freezing protocols [in a freezer at ?30°C or in liquid nitrogen vapour (LNV)]. For the in vivo trial, females were inseminated with frozen‐thawed spermatozoa. Oocytes used for in vitro testing were recovered 14 h after ovulation induction from donors and co‐incubated with 2 × 10⁶ frozen‐thawed spermatozoa during 4 h at 37°C in Tyrode's medium under an atmosphere of 5% CO₂ in air with maximal humidity. After co‐incubation period, presumptive zygotes were cultured in TCM199 supplemented with 20% foetal bovine serum (FBS), under the same conditions described above. Although no statistical differences were observed between freezing protocols in seminal parameters [motility rate: 40 and 35%, VCL: 35 and 46 μm/s, amplitude of lateral head displacement (ALH): 1.7 and 2.4 μm, for semen frozen at ?30°C and in LNV, respectively], significant differences were noted in the fertilizing ability in vivo and in vitro. Semen frozen at ?30°C showed the highest fertilizing ability in vitro (26.7% vs 6.2 and 8.7% for semen frozen at ?30°C, in LNV and fresh semen, respectively) and the lowest fertility rate in vivo (21.7% vs 64.2% and 70.6% for semen frozen at ?30°C, in LNV and fresh semen, respectively). Sperm frozen at ?30°C seemed to be more capacitated.     

Development and trial of a bovine herpesvirus 1 - thymidine kinase deletion virus as a vaccine

SUMMARY An Australian bovine herpesvirus 1 (BHV1) isolate with a defined (427 base pair) deletion in the protein coding region of the thymidine kinase gene was obtained by standard marker rescue procedures. After selection in the presence of the nucleotide analogue 5iodo-deoxy-uridine the virus was analysed by hybridisation with three differential oligonucleotide probes, restriction endonuclease profile studies and DNA sequence analysis. The virus elicited an immune response in recipient animals after either intramuscular or intravenous administration and produced no significant deleterious side-effects when administered at a dose sufficient to stimulate the host immune response. The safety and immunogenicity of the recombinant BHV1 virus 39B1 were similar to those reported for other registered BHV1 vaccines and the virus would appear to be suitable for the production of a vaccine seed lot and more exhaustive field trials as a prelude to commercial vaccine production and registration.     

Molecular epidemiology of clinical isolates of Pseudomonas aeruginosa isolated from horses in Ireland

Clinical isolates (n = 63) of Pseudomonas aeruginosa obtained from various sites in 63 horses were compared using ERIC2 RAPD PCR to determine their genetic relatedness. Resulting banding patterns (n = 24 genotypes) showed a high degree of genetic heterogeneity amongst all isolates examined, indicating a relative non-clonal relationship between isolates from these patients, employing this genotyping technique. This study characterised 63 clinical isolates into 24 distinct genotypes, with the largest cluster (genotype E) accounting for 10/63 (15.9%) of the isolates. ERIC2 RAPD PCR proved to be a highly discriminatory molecular typing tool of P. aeruginosa in isolates recovered from horses. With the adoption of several controls to aid reproducibility, this technique may be useful as an alternative to PFGE, particularly in epidemiological investigations of outbreaks where speed may be a significant parameter. This is the first report of clonal heterogeneity amongst P. aeruginosa from horses and demonstrated that ERIC RAPD PCR is a rapid method for the examination of this species in horses, which may be useful in outbreak analysis.     

Frequency of pancreatic amyloid deposition in cats from south-eastern Queensland

Summary Stereological procedures were used to estimate the amount of amyloid deposition in the pancreatic islets of 83 cats from random sources in south-eastern Queensland. Most had only minor deposits of less than 20% of islet volume (median 9%), but deposits equal to more than 50% of the islet volume were found in 10% of the cats. Amyloid deposition in pancreatic islets was correlated with the age of the cat. Although similar observations have been made previously in cats from the USA, the frequency of amyloid deposition was higher in this population of cats from south-eastern Queensland.     

Pregnancy Rates in Angus Cross Beef Cows Bred at Observed Oestrus With or Without Second GnRH Administration in Fixed‐Time Progesterone‐Supplemented Ovsynch and CO‐Synch Protocols

Crossbred cows (n = 1073) from five locations had oestrous cycles synchronized with 100 μg of GnRH IM and insertion of controlled internal drug release device (CIDR) on Day 0 followed by 25 mg of PGF_2α IM and CIDR removal on Day 7. Kamar^® patches were placed on all cows at CIDR removal. Cows were observed three times daily for oestrus after PGF_2α administration. In the Ovsynch‐CIDR group, cows detected in oestrus (n = 193) within 48 h after PGF_2α were inseminated using the AM–PM rule. Among these cows, 80 received and 113 did not receive a second GnRH at 48 h after PGF_2α. Cows (n = 345) not detected in oestrus received a second GnRH at 48 h after PGF_2α on Day 9, and fixed‐time AI 16 h after the GnRH on Day 10. In the CO‐Synch‐CIDR group, cows detected in oestrus (n = 224) within 48 h after PGF_2α were inseminated using the AM–PM rule. Among these cows, 79 received and 145 did not receive a second GnRH at 64 h after PGF_2α. Cows (n = 311) not detected in oestrus received a second GnRH on Day 10 at the time of AI, 64 h after PGF_2α. The AI pregnancy rates were not different between the Ovsynch‐CIDR and CO‐Synch‐CIDR groups (p = 0.48). There were no differences in the AI pregnancy rates for cows inseminated at a fixed time (p = 0.26) or at detected oestrus (p = 0.79) between the treatment groups. Among cows inseminated in oestrus, there were no differences in the AI pregnancy rates between cows that received or did not receive the second GnRH (p = 0.47). In conclusion, acceptable AI pregnancy rates can be achieved with or without inclusion of oestrus detection in the Ovsynch‐CIDR and CO‐Synch‐CIDR protocols. Among cows detected in oestrus, cows that received a second GnRH yielded similar pregnancy rates when compared with cows that did not receive the second GnRH.     

Multiple viral plaques with sebaceous differentiation associated with an unclassified papillomavirus type in a cat

Abstract

CASE HISTORY AND CLINICAL FINDINGS

A 15-year-old neutered male domestic short-haired cat was presented due to multiple 0.5–2?cm-diameter crusting plaques in the left preauricular region, over the bridge of nose, and in the right periocular region. The plaques did not appear to cause discomfort.     

Distribution of prion protein genotypes in breeds of sheep in New Zealand

AIM: To use an established high through-put genotyping procedure to gain an estimate of the frequency of alleles of the prion protein (PrP) gene in some common sheep breeds in New Zealand.

METHODS: Using a genotyping procedure based on matrix-assisted laser desorption ionisation-time of flight (MALDI-TOF), DNA samples from 3,024 sheep from New Zealand, including breeds such as Romney, Texel, Coopworth, Merino and mixedbreed, were isolated, genotyped and the results analysed.

RESULTS: The 15 scrapie genotypes commonly reported, and derived from the five commonly reported allelic variants (ARR, ARQ, AHQ, ARH and VRQ), were all observed in the samples analysed. The estimates were indicative of the frequencies in the population of alleles present in breeds of sheep in New Zealand. There was a significant difference between the frequencies of alleles between breeds, but the ARQ, followed by the ARR allele, were, except in Carwell sheep, the most common alleles present.

CONCLUSION: This study gave an indication of the percentages of PrP gene alleles in sheep in New Zealand, including data previously unreported from breeds in this country. It is of interest because of the relatively large size of the sheep population in New Zealand compared with many countries, and it provides some useful information on the genetic susceptibility or resistance of the sheep population in New Zealand to scrapie. The frequencies of the alleles can be different for an individual breed compared between countries.