A Universal `One-tube' RT-PCR Protocol for Amplifying Isolates of Bovine Viral Diarrhoea Virus

The increase in the knowledge of the genetic variability of BVDV and the identification of some of the genetic determinants of its pathogenicity require robust and practical tools for rapid molecular characterization of the various genotypes of this virus. This study was undertaken to develop a standard protocol for RT-PCR that allows the amplification of various parts of the genome of BVDV without the need for optimizing each individual reaction. The reaction set-up is very flexible because it consists of two pre-mixes. These are a master mix, with all the required reagents except the desired primers, which are the components of the second pre-mix and are therefore easily interchangeable between the different reactions. After adding any primer-containing pre-mix to the fixed master mix, a non-interrupted cycling protocol led to the generation of amplicons of up to 4 kbp in size in amounts sufficient for subsequent sequencing reactions. The method was applied to five different regions of the BVDV genome: (i) the well-known 5-UTR to differentiate genotypes I and II; (ii) the entire E2 gene, or an approximately 550 bp region within the E2 gene, in order to find the molecular equivalent of antigenic varieties; (iii) the entire structural protein coding region covering the Npro, capsid, E ^RNS, E1 and E2 genes; (iv) a 2.1 kbp region embracing the NS2/3 junction which is known to be cleaved in cytopathic biotypes of BVDV; and (v) the region covering the entire NS4B and NS5A/B genes. All six RT-PCRs were successfully applied using (i) primers with lengths of between 20 and 52 nucleotides, (ii) an aliquot of RNA extracted from either 10⁶ infected bovine embryonal lung cells or the same number of leukocytes from viraemic cattle, and (iii) all the genotype I and II strains of BVDV tested. The technique described was used to generate various Sindbis virus/BVDV recombinants. The correct processing of the amplicon-derived E2 glycoprotein of BVDV strain PT810 was demonstrated by its reaction with a monoclonal antibody in an immunofluorescence assay. Given the variety of RT-PCRs tested, we conclude that this universal protocol may be useful with other RNA viruses.     

Mortality in sahelian goats in Nigeria

The cause of mortality in sahelian goats was investigated in three local government areas of Borno State (Kukawa, Maiduguri and Mongonu) that are known for high goat production. A total of 150 selected flocks (50 flocks from each of the local government areas) were administered questionnaires through spot visits and interviews of the flock owners. On the whole, 644 (21.8%) goats died between May 1996 and April 1997 out of the 2956 goats in the 150 flocks. Mortality (41.4%) was higher in kids (<6-month old) than in adults (14.4%). Gastrointestinal disorders (dyspepsia), and respiratory diseases were the most-common causes of mortality. Cause-specific mortality risks did not differ between kids and adults.     

Persistent activity of doramectin and ivermectin in the prevention of cutaneous myiasis in cattle experimentally infested with Cochliomyia hominivorax

A study was conducted to evaluate the activity of a single administration of doramectin or ivermectin against severe, induced infestations of Cochliomyia hominivorax. Twenty-four Holstein bull calves were allocated to four groups of six animals each and treated either with saline, doramectin 1%, or either one of two formulations of ivermectin 1% at a dose rate of 200 microg/kg. On Day 12 after treatment, each calf was anesthetized and two wounds were created on the left side of the shoulder and rump of each calf and 2 h later, each wound was implanted with 100 newly hatched larvae of C. hominivorax. On Day 15 after treatment, the procedure was repeated on the right side of each calf. Wounds were examined daily for 5 days and evidence of live larvae was recorded. Doramectin provided reduction in myiasis of 90.9 and 83.3% at 12 and 15 days after treatment, respectively, compared to the saline control treatment (P < 0.0001). In contrast, there were no significant differences in the number of calves with myiasis between those treated with either of the ivermectin formulations and the saline control.     

Intake of heavy metals (lead, cadmium, zinc) originating from colored paints and their incorporation into organs and tissues of heifers

In a feeding trial the effect of heavy metals as lead, cadmium and zinc originating from colour paints was investigated. The study included 30 early pregnant heifers subdivided in five groups with six animals each with an average body weight of 419 kgs. During the testing period the trial was carried out in a three factorial fashion with two time sections: section one--mineral supplementation and lead preexposure; section two--Exposure to heavy metal containing colour paint pigments. The investigation led to the following results: A short time uptake of daily 10 g lead containing colour paint (i.e. 22-24 mg Pb/per animal and day) did not cause any change of general health and normal behavior of the respective animals. A storage of lead in the body tissue could be observed by way of a hint. An additional prestress with lead (145-158 mg Pb/animal/day over a period of 84 days did not influence the general health of the animals significantly. Both the fodder uptake and the body weight gain was inconspecious. A storage of lead could be traced in the skeleton, kidney and liver and--on a reduced level--also in the muscle. Only the content of lead in the kidney exceeded the maxmium tolerated value (> 1.0 mg/kg) of the German Meat Hygiene regulation. A mineral deficient feeding regimen (especially lack of calcium) enhance an increased storage of lead in the tissues (skeleton).     

Retrospective evaluation of occlusion of patent ductus arteriosus with hemoclips in 20 dogs

Twenty dogs with patent ductus arteriosus occluded with Hemoclips were evaluated with a mean follow-up time of 799.4 days (range, 83 to 3,580 days). Significant decreases were found between pre- and postsurgical means for vertebral heart size and for echocardiographic left atrial/aortic-root ratios and percent fractional shortening (%FS). Despite a good clinical outcome, six of 20 dogs had persistent cardiomyopathy of overload with diminished %FS (28% or less) at follow-up. One dog had residual ductal flow identified five days postoperatively. Subsequent evaluations in this case at 60, 144, 226, 344, and 560 days postoperatively demonstrated gradually diminishing ductal flow. The remaining 19 dogs did not recanalize.     

Lateral musculature in the whitemouth croaker (Micropogonias furnieri): its characterization with respect to different gonadal conditions

Histochemical and ultrastructural studies were performed on the lateral musculature from individual female whitemouth croaker, Micropogonias furnieri, at the anterior, medium and posterior regions. Based upon histochemical myosin-ATPase (m-ATPase) determination, diverse types of red, pink and white fibres were discerned. Red muscle had abundant mitochondria and stained intensely for aerobic enzymes, white muscle scarcely stained for the same enzymes and pink muscle responded in an intermediate manner. Both white and pink muscle had few mitochondria. The relative proportion of red muscle increased towards the caudal region; pink muscle diminished towards this region and white muscle modified its proportion only in the anterior region. m-ATPase activity showed differences in relation to the gonadal condition along the body, particularly in the white fibres at the anterior and medium regions.     

Comparison of bacterial cultivation, PCR, in situ hybridization and immunohistochemistry as tools for diagnosis of Haemophilus somnus pneumonia in cattle

The aim of the present study was to compare the potential of bacterial cultivation (BC), PCR, in situ hybridisation (ISH), and immunohistochemistry (IHC) in the diagnosis of Haemophilus somnus, when applied to pneumonic bovine tissue. Lungs from 65 field cases submitted for bacteriological examination were included in the study. The PCR-detection was performed on three different samples: plate-PCR (detection on plate washes after incubation of lung tissue on agar plates); swab-PCR (direct detection on a swab from the cut surface); and, whenever possible, a bronchus-PCR (direct detection on a swab from the main bronchus of the right cranial lung lobe). In order to examine the pathological significance of the findings, a histopathological examination of the cases was performed. H. somnus was detected by one or more techniques in 33 cases in total. By BC the bacterium was isolated from 10 cases, IHC and ISH were positive in 17 and 19 cases, and plate- and swab-PCR were positive in 21 and 29 cases, respectively. The bronchus-PCR was positive in 30 out of 61 cases examined. The PCR-technique was the most sensitive method, and as this technique is fast and relatively inexpensive, it should be considered as a supplementary tool in the diagnosis of H. somnus induced calf pneumonia.     

Induction of early immunopotentiation to Fimbriae of Salmonella Enteritidis (SE) by administering thymulin and zinc to SE-vaccinated chicken breeders: relationship to protection

The purpose of this study is to attempt the induction of early immunopotentiation of antibodies specific to fimbriae of Salmonella enterica serovar Enteritidis (SE), by administering thymulin and zinc to SE-vaccinated chicken breeders, and the improvement of protection against a controlled-live challenge by SE. The first two groups of breeders were administered subcutaneously at 15 and 19 weeks of age a killed SE vaccine. Breeders of the third and fourth groups were left unvaccinated. Breeders of the first group, immunopotentiated by thymulin and zinc, were able to induce the earliest antibodies in their pooled sera at 2 weeks post the first SE-vaccination, specific to fimbriae (approximately 21 KDa) of SE. However, the second group that was only vaccinated with the same SE-vaccine produced specific antibodies to fimbriae at 3 weeks following the second vaccination (22 weeks of age). Breeders of the third group, that were neither SE-vaccinated nor immunopotentiated by thymulin and zinc, but were challenged by live SE at 22 weeks of age, were able to show specific antibodies to fimbriae at 3 weeks post challenge (25 weeks of age). The fourth group that was deprived of SE-vaccination, immunopotentiators, and challenge didn't show any background antibodies specific to SE-fimbriae. The presence of the earliest antibody-immunopotentiation to fimbriae of SE in breeders of the first group, administered thymulin and zinc, was associated with the lowest frequency of SE-infected ceca (10%) among the challenged groups. In addition, breeders of the first group were the only challenged birds resulting in absence of SE infection in their cecal tonsils. The first group-vaccinated, immunopotentiated, and challenged, and the second group-vaccinated and challenged only resulted in breeders with absence of SE infection in their oviducts and spleens. In conclusion, immunopotentiation of chicken breeders by thymulin and zinc induces the earliest specific antibodies to fimbriae of SE associated with the lowest frequency of SE-infected ceca, and absence of SE infection from cecal tonsils, oviducts and spleens.     

Bush cover and range condition assessments in relation to landscape and grazing in southern Ethiopia

Progressive growth of bush cover in dry savannahs is responsible for declines in range conditions. In southern Ethiopia, the Booran pastoralists assisted our understanding of spatial patterns of bush cover and range conditions in 54 landscape patch types grouped into six landscape units within an area of 30000 km². The size of landscape patches sampled was 625 m². We assessed the relationships between bush cover, grass cover and bare soil and grazing pressure and soil erosion and changes in range condition. Externally, political conflicts and internally, break down of land use, and official bans on the use of fire promoted bush cover and the decline in range conditions. Bush cover was negatively correlated with grass cover, and positively correlated with bare soil. Grass cover was negatively correlated with bare soil and grazing pressure in most landscape patch types. Grazing pressure was not significantly correlated with bush cover or bare soil, while soil erosion was directly related to bare soil. Soil erosion was absent in 64% of the landscape patch types, and seemingly not a threat to the rangelands. The relationship between bush cover, grass cover, bare soil and soil erosion is complex and related to climate, landscape geology, and patterns of land use. Main threats to range conditions are bush climax, loss of grass cover and unpalatable forbs. Currently, >70% of the landscape patch types are in poor to fair range conditions. Decline in range conditions, unless reversed, will jeopardise the pastoral production system in southern Ethiopia.     

Reproductive performance of crossbred and purebred male rabbits

The effect of buck genetic type and crossbreeding parameters on fertility and prolificacy were estimated using two rabbit sire lines and their reciprocal crosses. The relationship between the reproductive performance of inseminated multiparous does and several semen quality traits was also investigated. The semen characteristics evaluated were: pH (pH), mass and individual motility (MM, IM), percentage of viable spermatozoa (Vi), spermatozoa with normal apical ridge (NAR), normal spermatozoa (NSP), spermatozoa with morphological abnormalities of head (HAP), neck-midpiece (NAP), and tail (TAP), spermatozoa with the presence of proximal (PD) and distal (DD) cytoplasmic droplets.

Fertility was analysed as a continuous trait (kindling rate) or as a binary trait (success or failure of kindling). In the first case, the analysis was performed using GLM procedures of SAS v.8 according to a model that included the fixed factors of buck genetic type, number of ejaculates per pool and week of insemination. In the second case, fertility was analysed using GENMOD procedures of SAS v.8 according to a mixed model including the same fixed factors as before plus the physiological status of the does and the permanent random effect of female. Number of kits born alive and number of stillborn were analysed with MIXED procedures of SAS v.8 with the same model used for the analysis of fertility as a binary trait. Estimates of the estimable functions of crossbreeding genetic parameters of the lines were obtained from the solutions of the corresponding models by generalized least squares using GLM, GENMOD and MIXED procedures. Crossbreeding parameters were estimated according to the model of Dickerson. A linear regression was used to determine the relationship between fertility and litter size and the semen characteristics evaluated.

Significant differences in fertility were observed among buck genetic types, which were favourable to type R. Differences between lines in maternal genetic effects were relevant and favourable to type R for fertility. Individual heterosis was important but unfavourable for fertility.

A slight correlation was obtained between all semen quality traits and fertility and prolificacy. Two multiple models were found for fertility, including NAP, IM, NSP, buck genetic type and Vi in one model or NAR in other model. Individual motility had an important positive effect, while NAP had a small negative effect. When MM, TAP and buck genetic type were included in a multiple model for the number of kits born alive, both MM and TAP had significant small effects. Individual motility and DD appeared to be related to number of kits stillborn, but only DD had a significant although negligible effect.