Use of touch-down polymerase chain reaction to enhance the sensitivity of Mycobacterium bovis detection.

The confirmatory diagnosis of Mycobacterium bovis (M. bovis) in animal samples is carried out by culture in Stonebrink media. However, culture is very slow because of the extremely long duplication time of the bacillus and difficult because of the scarcity of bacilli in diagnostic samples. This study describes the development of a single-tube touch-down polymerase chain reaction (PCR) protocol for the detection of M. bovis using primers that target the IS6110 element. Spiked water and milk as well as routine diagnostic samples (milk and nasal swabs) from M. bovis-positive cattle were tested. This protocol allows the rapid and sensitive detection of M. bovis in bovine samples by enhancing the sensitivity of standard PCR amplification.     

Pneumatosis cystoides intestinalis in a rabbit doe (Oryctolagus cuniculus).

Pneumatosis cystoides intestinalis (PCI) is an infrequent condition of animals characterized by the existence of numerous thin-walled, gas-filled cystic structures within the intestinal wall and adjacent lymph nodes. Microscopically, the cystic structures appear to be dilated lymphatics located in the lamina propria, submucosa, muscularis, subserosa, mesentery, and mesenteric lymph nodes. This report describes a case of pneumatosis cystoides intestinalis in a rabbit doe from an organic farm where 20 rabbit does were fed ad libitum with a natural diet consisting of whole barley, pea beans, alfalfa hay, and a pelleted vitamin-mineral blend. A combination of nutritional, bacterial, and other factors are hypothesized as possible predisposing factors in the development of PCI.     

Use of avidity enzyme-linked immunosorbent assay and avidity Western blot to discriminate between acute and chronic Neospora caninum infection in cattle.

Avidity serological tests (avidity enzyme-linked immunosorbent assay [ELISA] and avidity Western blot) were developed and used to differentiate between acute (primary infection, reinfection, and recrudescence) and chronic Neospora caninum infection in cattle. In addition, the pattern of immunoglobulin G (IgG) avidity maturation against different specific antigens of N. caninum tachyzoites was studied. Sequential serum samples were collected from cattle naturally and experimentally infected with N. caninum. Four groups of experimentally infected cattle were included in the study and were representative of primary infection, reinfection, chronic infection, and noninfection. Serum samples were also collected from naturally infected cattle classified into nonaborting and aborting cows on the basis of clinical findings and serological profiles, and a third group composed of seronegative cows that seroconverted during the course of the experiment. All samples were tested by avidity ELISA and avidity Western blot. The IgG avidity ELISA allowed the discrimination between primary and chronic infection because all experimentally primary-infection cows showed low avidity indexes at week 4 postinfection (p.i.) compared with the high avidity values found at week 20 postinfection. However, this test did not allow the discrimination of reinfection or recrudescence from chronic infection. Regarding IgG avidity Western blot results, no antigenic markers correlating with acute (primary infection, recrudescence, and reinfection) or chronic infection were recognized. However, the 17-kD immunodominant antigen was mostly responsible for high avidity values obtained by avidity ELISA because it was intensively recognized by high-avidity antibodies in all chronically infected animals after urea treatment.     

Isolation of equine herpesvirus-2 from the lung of an aborted fetus.

This study describes the isolation of equine herpesvirus-2 (EHV-2) from the lung of an aborted equine fetus in Argentina. The isolated virus was confirmed as EHV-2 by indirect immunofluorescence using a rabbit anti-EHV-2 polyclonal antiserum and by virus-neutralization test using an equine polyclonal antibody against EHV-2. Restriction endonuclease DNA fingerprinting with BamHI also confirmed the identity of the virus as EHV-2. Furthermore, viral nucleic acid was detected by polymerase chain reaction from the original lung sample and from the DNA obtained from cells infected with the virus isolate. This work constitutes the first reported isolation of EHV-2 from an aborted equine fetus. The presence of EHV-2 in the lung of the aborted fetus would indicate that this virus is capable of crossing the placental barrier. However, no cause-effect relationship was established between the EHV-2 isolate and the abortion.     

An in vitro study of hematopoietic progenitor cells from peripheral blood of rabbits

The purpose of this study was to isolate and cultivate a subpopulation of pluripotent stem cells (PSCs) from the peripheral blood of rabbits, which are frequently used in veterinary research as an animal model. Pluripotent stem cells, as described in human beings, are fibroblast-like cells that exhibit a CD34 marker, specific from other hematopoietic stem cells. Commonly used human commercial media has been researched for culturing rabbit PSCs. These findings allow us to contemplate the direct application of this simple and standardized methodology in several areas of study, such as of the pharmacological effect of many drugs on hematopoietic cells, veterinary practice, and even the study of new strategies in cellular therapy for some human diseases.     

Using chlorophyll fluorescence induction for a quantitative detoxification assay with metribuzin and chlorotoluron in excised wheat (Triticum aestivum and Triticum durum) leaves

Chlorophyll fluorescence induction was used as a probe to detect herbicide detoxification in tolerant or susceptible wheat cultivars. Experimental conditions have been carefully examined for establishing detoxification kinetics of chlorotoluron and metribuzin, two photosystem-II-inhibiting herbicides. After a root treatment, leaves were cut, placed in glass tubes and maintained in the dark. The fluorescence induction rise was examined repeatedly and detoxification kinetics were established from these data for the same position on the individual leaves. The herbicide-dependent fluorescence rise decreased within hours in chlorotoluron-tolerant but not in susceptible Triticum aestivum cultivars. In contrast, no significant reversion could be detected after metribuzin application in both tolerant and susceptible cultivars of Triticum durum. Near the fluorescence-determined half-inhibition of photosystem II, linear detoxification kinetics were obtained in individual leaves, thus providing an accurate measurement of relative detoxification rates.     

Lactation curves in captive Iberian red deer (Cervus elaphus hispanicus)

This study examines milk production and the effect of milk production and sex of calf on body weights and gains of red deer calves and hinds of the Iberian subspecies (Cervus elaphus hispanicus). Milk production was assessed in 14 hinds by weighing calves before and after suckling and by adjusting these values to the Gamma function. Gamma estimates of total milk production up to d 105 were similar to the amounts computed directly from double weighing. Hinds showed two types of lactation curve: 1) the standard mammal lactation curve, with an asymmetrical peak at wk 2 to 4 (Type I) and 2) decreasing curves with no peak (Type II). Although there was great interindividual variability, hinds with Type I curves showed a trend to produce more milk than those with Type II. The type of curve did not seem to affect weight variables of the calf or those of the dam. Calves that gained more weight consumed greater amounts of milk (P<.05). Males were heavier than females at birth (P<.05), but males did not differ from females in their weight at 105 d, milk consumption, and gain. Gender did not affect hind weight, but dams of male calves showed a trend to be lighter (P = .063) at d 105 than dams of female calves. Our results suggest that suckling differences found in other studies between male and female calves may not involve differences in milk production, although other rearing costs seem to affect hind weight losses. They also suggest that the curve type may not depend only, as reported, on the nutrition plane.     

Risk factors associated with the seroprevalence of leptospirosis among students at the veterinary school of Zaragoza University

The prevalence and risk factors associated with leptospirosis were studied in veterinary students in Zaragoza. Sera were collected at the beginning and end of the academic year 1994 to 1995 and were tested by ELISA against a pool of Leptospira interrogans serovars bratislava, canicola, grippotyphosa, hardjo, icterohaemorrhagiae and pomona antigens. At the beginning of the study the prevalence was 8.14 per cent and at the end it was 11.4 per cent. The incidence of the disease during the study was 0.0394. Risk factors associated with leptospirosis included: taking the course specialising in food inspection and technology, on-farm work, contact with pets in general, and particularly carnivores, and contact with animal traders. The symptoms associated with the disease were myalgia and fever, and the treatment of the symptoms provided some protection.     

Capillary Supply During Development of Individual Regenerating Muscle Fibers

This study describes the capillary supply of individual regenerating muscle fiber during three stages of its development. Regeneration was induced by the injection of mepivacaine in the anterior tibial muscle of rats. Muscle fiber capillarization during regeneration was analysed in semithin sections, employing the following parameters: the number of capillaries around a fiber and the number of capillaries per fiber cross-sectional area. The results revealed that during development of regenerating fibers, the fiber cross-sectional area increased, the number of capillaries around each fiber increased and the number of capillaries per fiber cross-sectional area decreased. These data indicated a greater capillary supply of the individual regenerating muscle fiber during its early stage of maturation.     

Influence of age and purpose for testing on the cut-off selection of serological methods in bovine neosporosis

The aim of this study was to investigate the need for different cut-off points, according to animal age and the purpose of testing, for two of the most widely used serological techniques in bovine neosporosis, IFAT and a crude antigen ELISA (Civtest, HIPRA). Therefore, the population reference sera used were defined using a combination of multiple criteria such as epidemiological/clinical and histopathological parameters and an immunoblot test. Firstly, foetuses and breeding cattle (heifers and cows) were considered as separate subpopulations for serological evaluation. Secondly, cut-off points for each serological technique (IFAT and ELISA) according to age group (foetuses and breeding cattle) and the different practical applications (detection of infection and abortion) were calculated following the receiver operating characteristics (ROC) analysis. Cut-off points were defined, for IFAT and ELISA for aborted breeding cattle and for IFAT alone in the case of the foetuses, assuming an equivalent cost of false positive and negative results. In infected breeding cattle, for IFAT and ELISA and in foetuses for ELISA, two possible cut-off values were obtained, one for a maximum sensitivity and one for a maximum specificity and the intervals of unclear results were defined. In this case, a cut-off value for equal sensitivity and specificity was also estimated. When cut-off points for infected breeding cattle, 1:100-1:250 for IFAT and 0.306-0.451 for ELISA were applied to a target population, optimal and similar negative and positive predictive values together with similar apparent and true prevalence results were observed suggesting the possibility of using both tests interchangeably.