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.     

Accidental Mycobacterium bovis infection in a veterinarian

CASE: A veterinarian developed tenosynovitis and secondary carpal tunnel syndrome following accidental inoculation of Mycobacterium bovis during the necropsy of a tuberculous possum from Westland, New Zealand.

CLINICAL RELEVANCE: M. bovis infection is a zoonotic disease, and occupational exposure to tuberculous animals places people at risk of contracting the disease.

CONCLUSIONS: Adhering to safe work practices reviewed in this article is important to minimise the risk of infection to people handling tuberculous animals.     

Oestrous sheep serum balances ROS levels to supply in vitro capacitation of ram spermatozoa

Reactive oxygen species (ROS) are fundamental for intracellular signalling. In spermatozoa, they are involved both to apoptosis and to capacitation, and changes in ROS levels can alter the balance between these two processes. Oestrous sheep serum (OSS) is considered an efficient agent for in vitro capacitation of ram spermatozoa. We have explored the effects of OSS on ram sperm physiology, especially on ROS production, during in vitro capacitation. Semen samples from 15 rams were cryopreserved. After thawing, samples were submitted to four treatments: control (CTL), 10% OSS supplementation for in vitro sperm capacitation, caspase inhibitor (INH, Z‐VAD‐FMK 100 μM) and OSS (10%) plus caspase inhibitor (I + E). Sperm samples were incubated for 30 min at 38.5°C and 5% CO₂ and evaluated motility and kinetic parameters by computer‐assisted semen analysis (CASA) and viability (propidium iodide), apoptotic‐like membrane changes (YO‐PRO‐1), acrosomal status (PNA‐FITC), intracellular calcium (FLUO‐3), membrane fluidity (M540) and ROS production (CM‐H₂DCFDA) by flow cytometry. OSS induced changes in kinetic parameters compatible with capacitation, with a decrease in the percentage of progressive motility and linearity, and an increase in the amplitude of the lateral displacement of the sperm head (p < .05). Moreover, OSS increased the proportion of M540+ viable spermatozoa, YO‐PRO‐1+ and acrosome‐reacted spermatozoa (p < .05). After incubation, OSS and I+E achieved lower ROS levels (p < .05). Ca²⁺ levels did not change with the incubation, but were slightly higher (p < .05) when both OSS and the inhibitor were present. We suggest that OSS may modulate ROS levels, allowing intracellular signalling for capacitation to occur while preventing higher levels that could trigger apoptosis.     

Efficacy of metoclopramide for treatment of ileus in horses following small intestinal surgery: 70 cases (1989–1992)

Summary: In this retrospective study, postoperative ileus was studied in horses having resection of the small intestine followed by a jejunojejunal (n=35) or a jejunocaecal (n=35) anastomosis. Twenty-six horses received no metoclopramide, 27 received metoclopramide as an intermittent intravenous infusion and 17 horses received metoclopramide as a continuous intravenous infusion (0.04 mg/kg/hour). Horses receiving a continuous infusion of metoclopramide had a reduced total volume (P< 0.001), shorter duration (P< 0.001), and a slower rate (P< 0.001) of postoperative gastric reflux, and a shorter postoperative hospital stay (P< 0.01) when compared to horses receiving no metoclopramide and horses receiving metoclopramide as an intermittent infusion. Horses having jejunocaecostomy performed had a larger volume (P< 0.05), longer duration (P< 0.05), and a greater rate (P< 0.05) of postoperative gastric reflux, and a longer postoperative hospital stay (P< 0.001) than horses undergoing jejunojejunostomy. Horses that had an abdominal drain placed during surgery had a longer length of intestine resected (P< 0.05) and a longer postoperative hospital stay (P< 0.05) than horses without an abdominal drain. Horses that died or were euthanased during the postoperative period had a greater total volume (P< 0.05), longer duration (P< 0.05), and greater rate of postoperative gastric reflux (P< 0.01), a longer length of small intestine resected (P< 0.01), and a shorter postoperative hospital stay (P< 0.05) than horses that survived until discharge from the hospital. In a multivariate regression analysis the continuous infusion of metoclopramide was the only variable that contributed significantly to predicting the total volume (P< 0.001; r²= 0.24), duration (P< 0.001; r²= 0.24) and rate (P< 0.001; r²= 0.25) of postoperative gastric reflux. We concluded that metoclopramide given as a continuous intravenous infusion decreased the incidence and severity of ileus following small intestinal resection and anastomosis in horses.     

The resistance to chemotherapeutic agents of Escherichia coli from domestic dogs and cats

The prevalence of antibiotic resistant Escherichia coli in the rectal flora of 168 healthy dogs and 93 cats in the Brisbane area was investigated. Rectal swabs were plated on MacConkey agar with and without antibiotics, and 690 isolates confirmed as faecal E. coli were tested for resistance to tetracycline, streptomycin, chloramphenicol, ampicillin, neomycin, furazolidone and sulphanilamide. Resistant isolates were obtained from 101 (60%) of the dogs and 24 (26%) of the cats sampled. A high percentage of the isolates was resistant to tetracycline, streptomycin, ampicillin and sulphanilamide. Multiple resistance to 3 or more of the drugs was exhibited by the majority of isolates and a total of 31 different multiple resistance patterns was demonstrated. Of the 50 strains tested for transfer of resistance, 30 (60%) transferred some or all of their resistance determinants to an E. coli K12F - recipient.     

A latex cryptococcal antigen agglutination test for diagnosis and monitoring of therapy for cryptococcosis

SUMMARY: A latex cryptococcal antigen agglutination test (LCAT) was performed on sera obtained during the first 14 days of treatment from 58 animals (46 cats, 9 dogs, 2 koalas and 1 long billed corella) with cryptococcosis. The same commercial kit was used for all samples, and most serum samples were treated with pronase before testing. Sera from all 58 cases tested positive with the qualitative LCAT protocol (using undiluted sera), while sera from all 26 cats without cryptococcosis tested negative. Titres determined using the quantitative protocol ranged from 1 to 131 072 (median titre between 2048 and 4096), with 57 of 58 cases (including all 8 animals that presented for neurological signs) having titres 2 and thus considered positive according to the manufacturer's recommendations. The LCAT titre was positively correlated with disease severity (r = 0.4169; P = 0.0011), and patients with disseminated skin and/or lymph node involvement had significantly higher titres than those that did not (P = 0.0157). The presence of neurological signs, the species of the patient, concurrent viral disease (in cats) and the biotype of the isolate had no significant association with the LCAT titre. Cats that died of active cryptococcosis despite treatment did not have significantly higher titres (P = 0.3010) than those that responded to treatment. Sequential LCAT determinations obtained in 37 patients during treatment provided a useful quantitative indication of clinical progress, although the decline in titre lagged somewhat behind clinical improvement. Generally, the antigen titre declined by 2 to 4 fold per month during successful therapy. Although there are insufficent data to make unequivocal recommendations, we suggest that either antifungal therapy be continued until the LCAT titre declines to less than 1, or therapy be discontinued after a 32 fold or greater reduction in titre, with periodic monitoring of the serum antigen titre. Treatment of serum samples with pronase substantially increased the sensitivity of the LCAT.     

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.     

Corpus Luteum–Endometrium–Embryo Interactions in the Dairy Cow: Underlying Mechanisms and Clinical Relevance

Conception rates of dairy cows are currently declining at an estimated 1% every year. Approximately, 35% of embryos fail to prevent luteolysis during the first three weeks of gestation. Interactions between the corpus luteum, endometrium and embryo are critical to the successful establishment of pregnancy and inadequacies will result in the mortality of the embryo. For example, as little as a one day delay in the post-ovulatory rise of progesterone has serious consequences for embryo development and survival. Recently, we found that LH support, degree of vascularization and luteal cell steroidogenic capacity were not the major factors responsible for this luteal inadequacy, but are nevertheless essential for luteal development and function. Progesterone acting on its receptor in the endometrium stimulates the production of endometrial secretions on which the free-living embryo is dependent. However, their exact composition and effects of inadequate progesterone remains to be determined. The embryo is recognized through its secretion of interferon tau (IFNT), which suppresses luteolytic pulses of prostaglandin F_2α. In the cow, it is most likely that IFNT inhibits oxytocin receptor up-regulation directly and does not require the prior inhibition of oestrogen receptor α (ESR1). Unravelling the precise luteal-endometrium and embryo interactions is essential for us to understand pregnancy establishment and development of strategies to reverse the declining fertility of dairy cows.