Multiplex real-time PCR for the detection and differentiation of equid herpesvirus 1 (EHV-1) and equid herpesvirus 4 (EHV-4)

A multiplex real-time PCR was designed to detect and differentiate equid herpesvirus 1 (EHV-1) and equid herpesvirus 4 (EHV-4). The PCR targets the glycoprotein B gene of EHV-1 and EHV-4. Primers and probes were specific to each equine herpesvirus type and can be used in monoplex or multiplex PCRs, allowing the differentiation of these two closely related members of the Alphaherpesvirinae. The two probes were minor-groove binding probes (MGB) labelled with 6-carboxy-fluorescein (FAM) and VIC for detection of EHV-1 and EHV-4, respectively. Ten EHV-1 isolates, six EHV-1 positive clinical samples, one EHV-1 reference strain (EHV-1.438/77), three EHV-4 positive clinical samples, two EHV-4 isolates and one EHV-4 reference strain (EHV-4 405/76) were included in this study. EHV-1 isolates, clinical samples and the reference strain reacted in the EHV-1 real-time PCR but not in the EHV-4 real-time PCR and similarly EHV-4 clinical samples, isolates and the reference strain were positive in the EHV-4 real-time PCR but not in the EHV-1 real-time PCR. Other herpesviruses, such as EHV-2, EHV-3 and EHV-5 were all negative when tested using the multiplex real-time PCR. When bacterial pathogens and opportunistic pathogens were tested in the multiplex real-time PCR they did not react with either system. The multiplex PCR was shown to be sensitive and specific and is a useful tool for detection and differentiation of EHV-1 and EHV-4 in a single reaction. A comprehensive equine herpesvirus disease investigation procedure used in our laboratory is also outlined. This procedure describes the combination of alphaherpesvirus multiplex real-time PCR along with existing gel-based PCRs described by other authors.     

Risk factors for Thoroughbred racehorse fatality in jump starts in Victoria, Australia (1989-2004)

REASONS FOR PERFORMING STUDY: The risk of fatality is greater in jump than in flat racing in Victoria, Australia. This is the first study to identify risk factors specific to jump starts in Victoria. OBJECTIVE: To identify risk factors for fatality of Thoroughbred racehorses in jump starts on all racecourses in Victoria, Australia between 1989 and 2004. METHODS: Fatalities comprised all horses that died during or immediately after a jump (hurdle or steeplechase) race or official jump trial and all horses that were subjected to euthanasia within 24 h of an event in which an injury was sustained. The retrospective study involved 191 case starts and 2324 control starts. Univariable and multivariable backward stepwise logistic regression was used to identify risk factors for fatality at any one start. A multiple level model was used with racecourse included as a random effect. RESULTS: In the final multivariable model, the duration of the racing career of the horse, the number of flat, hurdle and steeple starts accumulated in the 60 days prior to the case or control start, the number of flat and jump starts accumulated over the racing career, if the horse had had a start between 1 and 14 days prior to the case or control start, the type of jump race (hurdle or steeple), the calendar year of the start and the location of the racecourse were associated with fatality. CONCLUSIONS: The findings highlight the need to investigate further the differences between hurdle and steeplechase events and the adverse effect of prolonged prior flat racing careers on the risk of fatality in jump starts. POTENTIAL RELEVANCE: This is the first study to examine risk factors for fatality in jump starts in Victoria. The results should shape the development of interventions to reduce the risk in jump starts in the future.     

An inactivated vaccine for the control of bluetongue virus serotype 16 infection in sheep in Italy

Because no suitable products are at the moment available to safely control the spread of BTV-16 in Europe, an inactivated vaccine was produced from the reference field isolate of bluetongue virus serotype 16. One group of six sheep was vaccinated subcutaneously with the inactivated vaccine twice, on days 0 and 28, whereas a second group of eight sheep was inoculated with saline solution and used as mock-vaccinated control animals. Seventy-eight days after the first vaccination, all sheep were inoculated subcutaneously with a suspension containing 10(6.3) TCID(50) of a virulent reference BTV-16 isolate. Apart from a transient inflammatory reaction at the injection site, no adverse effects were reported following vaccination. All vaccinated animals developed high titres (7.3-9.3log(2)(ED50%/50 microl)) of virus-specific neutralising antibodies and were resistant to challenge with BTV-16. Conversely, following challenge, control animals developed hyperthermia and long lasting high-titre viraemia.     

Endometrial nitric oxide production and nitric oxide synthases in the equine endometrium: Relationship with microvascular density during the estrous cycle

Nitric oxide (NO) plays an important role in angiogenesis and in the regulation of the blood flow. This study was carried out to investigate (i) the effects of endogenous estrogens and progestins and exogenous progesterone (P₄) (5 ng/ml or 1 μg/ml) or estradiol 17β (E₂β) (50 pg/ml or 1 μg/ml) on in vitro endometrial NO synthesis; (ii) the presence of different isoforms of NO synthase; (iii) and their relationship to microvascular density in the equine endometrium during the estrous cycle. NOS expression was also evaluated in the myometrium. Expression of endothelial and inducible forms of NOS in the uterus was assessed by Western blot and immunocytochemistry. Vascular density in endometrial tissue was determined on histologic sections. In the luteal phase, compared to the follicular phase, endometrial NO production increased without exogenous hormones and with exogenous E₂β (1 μg/ml). Although immunocytochemistry revealed iNOS and eNOS expression in the endometrium, no positive signal for iNOS was detected by Western blot. Endothelial NOS was observed in endometrial glands, endothelial cells, fibroblasts, blood and lymphatic vessels. Endometrial eNOS expression was the highest in the follicular and mid-luteal phases while it was found to be the lowest in the early luteal phase. In the follicular phase, hyperplasia of endometrial tissue with respect to myometrium was detected. No difference in vascular density was present between phases. All together, NO may play some roles in both proliferative and secretory phases of endometrial development in the mare.     

The relation between ultrasonographic observations in the oviduct and plasma progesterone, luteinizing hormone and estradiol during the egg laying cycle in ostriches

In this study we investigated the temporal relationship between ovulation, egg formation, oviposition and the changes in plasma concentrations of progesterone, luteinizing hormone and estradiol-17beta during the egg laying cycle in farmed ostriches. In 10 egg-producing birds, transcutaneous ultrasound scanning was performed at 3h intervals and blood sampling at hourly intervals during a period of at least 48h (one egg laying cycle). In hens (n=8) that ovulated during the observational period, the ovulated egg was first detected 2h after oviposition; thus, ovulation occurred shortly after oviposition in all birds. During the period between two consecutive ovipositions, the developing egg remained for 9h in the proximal part (infundibulum, magnum or isthmus) and for 39h in the distal part of the oviduct (uterus). In ovulating hens, plasma progesterone concentrations showed a characteristic and consistent profile: from basal levels of around 0.1ng/ml concentrations started to increase 12h before oviposition, reached an average maximum of 3.5ng/ml at 3h before oviposition and returned to basal levels 3h and 30min after oviposition. Changes in plasma luteinizing hormone and estradiol-17beta concentrations showed comparable patterns of elevation and decline relative to the timing of oviposition and ovulation. However, variation in their individual basal concentrations was generally larger and peak values were less conspicuous than those of progesterone. In non-ovulating hens (n=2) neither progesterone, nor luteinizing hormone nor estradiol-17beta showed elevations to peak concentrations before oviposition. These data demonstrate that during the egg laying cycle of ostriches, events such as ovulation, egg development and oviposition evolve according to a rather strict time schedule, and that progesterone, luteinizing hormone and estradiol-17beta reach peak concentrations shortly before ovulation. Additionally, our findings also show that on-farm ultrasound scanning is a useful technique to discriminate between ovulating and non-ovulating hens.     

Bacteraemia in neonatal foals: clinicopathological differences between Gram-positive and Gram-negative infections, and single organism and mixed infections

REASONS FOR PERFORMING STUDY: Treatment for bacteraemia in foals must be started before the identity of the causative organism is known. Information aiding selection of effective antimicrobials should improve outcome. OBJECTIVES: To describe differences in clinical and clinicopathological data and outcome in foals with bacteraemia due to different classes of bacteria. METHODS: Records of foals with a positive blood culture, age < 10 days and presenting to a university hospital 1995-2004, were reviewed. Bacterial culture results, subject details, historical information, physical examination findings at admission and clinicopathological data generated during the first 48 h of hospitalisation were analysed. Results from foals with Gram-positive or Gram-negative organisms, single or mixed organism bacteraemias, and with bacteraemia due to 3 commonly isolated organisms were compared. RESULTS: Eighty-five foals met the inclusion criteria. Gram-negative organisms (n = 59) Gram-positive organisms (n = 13) or multiple organisms (n = 19) were cultured from individual foals. Foals with Gram-negative bacteraemia had lower total white blood cell and lymphocyte counts at admission than did those from which only Gram-positive bacteria were cultured. Mixed organism bacteraemia was associated with tachycardia, increased serum concentrations of sodium, chloride and urea nitrogen, acidosis, respiratory distress, recumbency on admission and nonsurvival. Actinobacillus spp. infections were associated with leucopenia, neutropenia, lymphopenia and depression on hospital admission. CONCLUSIONS AND POTENTIAL RELEVANCE: Recognising particular patterns of clinical and clinicopathological findings associated with infection with specific groups of bacteria may, in the future, aid antimicrobial selection and influence prognosis in bacteraemic foals.     

Fibroleiomyoma in elephant uterus

The result of the histological diagnosis from postmortal investigation of the Indian elephant (Elephas maximus) uterus structure is presented in the current study. The rare anatomical finding showed altered nodular part from the uterus wall. Through conventional and histochemical staining neoplasmatic formations were determined and diagnosis Fibroleiomyoma was established, which should not be accepted as the prime cause for the animal's death.     

Epidemiology of porcine non-specific colitis on Scottish farms

The objective of this study was to investigate epidemiological risk factors for porcine non-specific colitis (NSC). Forty-seven Scottish pig farms, with and without a clinical history of diarrhoea during the growing period (15-40 kg), were selected. The study included farm visits, clinical inspection of pigs, completion of farm management questionnaires, pathological tests into the cause of the diarrhoea and analysis of the non-starch polysaccharide (NSP) content of feeds. The results from 17 farms designated as NSC and 10 control farms suggest dietary associations with NSC. Farms with NSC fed diets with significantly higher levels of NSPs, especially containing the sugars glucose, arabinose and xylose. Few management factors were identified, although the high prevalence of infectious colitis reduced the power of the study.