Experimental production of bovine respiratory tract disease with bovine viral diarrhea virus

Five 6-month-old calves were inoculated with bovine viral diarrhea (BVD) virus (n = 3) or Pasteurella haemolytica (n = 2) endobronchially with a fiberoptic bronchoscope. Five additional calves were inoculated sequentially with BVD virus followed by P haemolytica at a 5-day interval. Blood samples were collected daily from the calves for bacterial isolation. Clinical signs of respiratory tract disease in calves were recorded daily. If the calves survived, they were killed for necropsy 3 or 4 days after inoculation with P haemolytica (or 8 days after inoculation with BVD virus). The extent and nature of pulmonary lesions in the calves were determined, and the lower portion of the respiratory tract (lungs and trachea) was examined for both these organisms. The 3 calves, inoculated with BVD virus only, developed mild clinical signs mainly manifested as fever, nasal discharge, and occasional cough. Approximately 2% to 7% of the total lung capacity of these calves was pneumonic. Mild clinical signs and localized lesions involving about 15% of the lung volume developed in the 2 calves exposed to P haemolytica only. However, severe fibrinopurulent bronchopneumonia and pleuritis involving 40% to 75% of lung volume developed in the 5 calves inoculated sequentially with BVD virus and P haemolytica. The possible role BVD virus may have in bovine respiratory tract disease is discussed.     

Tiger,Bengal and Domestic Cat Embryos Produced by Homospecific and Interspecific Zona‐Free Nuclear Transfer

The aim of this study was to evaluate three different cloning strategies in the domestic cat (Felis silvestris) and to use the most efficient to generate wild felid embryos by interspecific cloning (iSCNT) using Bengal (a hybrid formed by the cross of Felis silvestris and Prionailurus bengalensis) and tiger (Panthera tigris) donor cells. In experiment 1, zona‐free (ZP‐free) cloning resulted in higher fusion and expanded blastocyst rates with respect to zona included cloning techniques that involved fusion or injection of the donor cell. In experiment 2, ZP‐free iSCNT and embryo aggregation (2X) were assessed. Division velocity and blastocyst rates were increased by embryo aggregation in the three species. Despite fewer tiger embryos than Bengal and cat embryos reached the blastocyst stage, Tiger 2X group increased the percentage of blastocysts with respect to Tiger 1X group (3.2% vs 12.1%, respectively). Moreover, blastocyst cell number was almost duplicated in aggregated embryos with respect to non‐aggregated ones within Bengal and tiger groups (278.3 ± 61.9 vs 516.8 ± 103.6 for Bengal 1X and Bengal 2X groups, respectively; 41 vs 220 ± 60 for Tiger 1X and Tiger 2X groups, respectively). OCT4 analysis also revealed that tiger blastocysts had higher proportion of OCT4‐positive cells with respect to Bengal blastocysts and cat intracytoplasmic sperm injection blastocysts. In conclusion, ZP‐free cloning has improved the quality of cat embryos with respect to the other cloning techniques evaluated and was successfully applied in iSCNT complemented with embryo aggregation.     

Immunoblot analysis of cyanogen bromide-cleaved Moraxella bovis pilin reveals presence of shared antigenic determinants on pili from heterologous strains

Moraxella bovis pilus proteins, collected and purified from four strains of M. bovis, were cleaved with cyanogen bromide. Two major fragments were produced. Antisera were produced in rabbits to the pilin protein fragments and to whole uncleaved pili from these strains. Immunoblots of whole and cyanogen bromide-cleaved pilin were reacted with the homologous and heterologous antisera to whole pili and cleaved pilin. Antisera to whole pili reacted strongly with homologous pilin. Weaker and inconsistent reactions were detected with heterologous pilin. Antisera produced to cyanogen bromide-cleaved pilin proteins reacted strongly with homologous and heterologous pilin fragments and uncleaved pilin proteins. These findings demonstrate the presence of conserved antigenic determinants on pili from heterologous strains that are non-immunogenic in the intact pilus but are immunogenic after treatment with cyanogen bromide. Cyanogen bromide-treated pilus preparation might have potential as a vaccine because antibodies are induced against heterologous strains of M. bovis, whether these cross-reactive antibodies are protective remains to be determined.     

Sequence variation identified in the 18S rRNA gene of Theileria mutans and Theileria velifera from the African buffalo (Syncerus caffer)

The African buffalo (Syncerus caffer) is a natural reservoir host for both pathogenic and non-pathogenic Theileria species. These often occur naturally as mixed infections in buffalo. Although the benign and mildly pathogenic forms do not have any significant economic importance, their presence could complicate the interpretation of diagnostic test results aimed at the specific diagnosis of the pathogenic Theileria parva in cattle and buffalo in South Africa. The 18S rRNA gene has been used as the target in a quantitative real-time PCR (qPCR) assay for the detection of T. parva infections. However, the extent of sequence variation within this gene in the non-pathogenic Theileria spp. of the Africa buffalo is not well known. The aim of this study was, therefore, to characterise the full-length 18S rRNA genes of Theileria mutans, Theileria sp. (strain MSD) and T. velifera and to determine the possible influence of any sequence variation on the specific detection of T. parva using the 18S rRNA qPCR. The reverse line blot (RLB) hybridization assay was used to select samples which either tested positive for several different Theileria spp., or which hybridised only with the Babesia/Theileria genus-specific probe and not with any of the Babesia or Theileria species-specific probes. The full-length 18S rRNA genes from 14 samples, originating from 13 buffalo and one bovine from different localities in South Africa, were amplified, cloned and the resulting recombinants sequenced. Variations in the 18S rRNA gene sequences were identified in T. mutans, Theileria sp. (strain MSD) and T. velifera, with the greatest diversity observed amongst the T. mutans variants. This variation possibly explained why the RLB hybridization assay failed to detect T. mutans and T. velifera in some of the analysed samples.     

卤虫无节幼体和配合饵料饲喂诸氏鲻虾虎鱼效果比较

为比较卤虫无节幼体(Arternia nauplii)和配合饵料的饲喂效果,分别采用两种饵料饲养30日龄的诸氏鲻虾虎鱼(Mugilogobius chulae)75d,比较存活、生长和全鱼成分。结果表明,卤虫组的存活率较高(≥52%),显著高于配合饵料组(14%～21%);配合饲料组的相对增重率(GW)为2 916.8%～3 998.7%,特定生长率(SGR)为4.5～5.0,生长效果优于卤虫组(GW为1 826.5%～2 101.7%,SGR为3.9～4.1);全鱼成分测定,卤虫组的水分、粗蛋白、粗脂肪、灰分含量分别为72.7%、19.5%、4.7%、3.0%,配合饵料组的分别为70.5%、18.7%、5.8%、3.5%,两组没有显著差异。适当延长饲喂卤虫无节幼体的时间,然后采用配合饵料进行饲养,能提高存活率并获得较好的生长效果。     

Enrichment of a fraction toxic to guinea-pigs from Pachystigma pygmaeum (Schltr.) Robyns

Pachystigma pygmaeum is one of several species of rubiaceous plants which cause delayed heart failure among ruminants after their ingestion at relatively high doses. Using guinea-pigs for toxicity determinations, we were able to separate and enrich a toxic fraction from a fermentation extract of the plant material by countercurrent distribution. It contained virtually no potassium salts, passed through a 500 dalton selective membrane, exhibited lability under acid conditions and was toxic at 1 g/kg per os, with a delayed response of 3-4 days.     

Efficacy of a Feline Leukemia Virus Vaccine in a Natural Exposure Challenge

A commercial feline leukemia virus (FeLV) vaccine was evaluated in a natural exposure system. All kittens were negative for FeLV antigen on two enzyme-linked immunosorbent assay (ELISA) tests and one indirect immunofluorescence antibody (IFA) test before vaccination or exposure. Twenty-three kittens were vaccinated subcutaneously at nine and 12 weeks of age. The vaccinated kittens and 14 unvaccinated littermates were housed in an infected environment starting at 14 weeks. The kittens were exposed for 24 weeks by living in a large room with one feline leukemia virus-positive, asymptomatic adult cat for each five kittens. Sixty-four percent of the unvaccinated kittens and 70% of the vaccinated kittens became infected as determined by ELISA. Forty-three percent of unvaccinated kittens and 39% of vaccinated kittens died. There was no difference between the infection and mortality of vaccinated kittens that developed antibodies to anti-FeLV glycoprotein 70-envelope antigen and those that did not. Consideration should be given to evaluation of feline leukemia virus vaccines using "street" virus in a natural exposure system.     

An electron microscopic study of intra-erythrocytic stages of Babesia bovis in the brain capillaries of infected splenectomized calves.

Splenectomized vaccine donor calves undergoing primary reactions to Babesia bovis infections may develop cerebral babesiosis which leads to death if not treated in time. A brain biopsy was performed on an artificially-infected animal showing nervous symptoms and the tissue was immediately processed for electron microscopic examination. Virtually every erythrocyte in the brain capillaries sectioned was infected with B. bovis. Intra-erythrocytic merozoites, trophozoites and dividing trophozoites were indentified. Important features of the piriform merozoites included a reduced apical complex consisting of the anterior polar ring, microtubules, rhoptries and micronemes. Unidentified membrane-bound bodies, mostly spherical in shape, were observed anterior to the nucleus. The trophozoites showed very little structural differentiation and no food vacuoles or micropores could be detected. Each trophozoite produced 2 identical merozoites and the parent cell became totally incorporated in the daughter merozoites in the multiplication process. Projections were seen radiating from the surface of infected erythrocytes which appeared to adhere to other surfaces on contact. This probably resulted in the sludging of infected erythrocytes in the capillaries. The latter observations coincide with those described for Babesia argentina.     

VP2-segment sequence analysis of some isolates of bluetongue virus recovered in the Mediterranean basin during the 1998-2003 outbreak

The complete nucleotide sequences of the VP2 segments of bluetongue virus (BTV) isolates recovered from Italy, Greece and Israel, from 1998 to 2003, were determined. Phylogenetic analysis of these sequences, those from related viruses and the South African vaccine strains, were used to determine the probable geographic origin of BTV incursions into Italy. Results indicated that viruses from each of the four serotypes isolated in Italy (2, 4, 9 and 16) possibly had a different origin. Analysis of the bluetongue virus serotype 2 (BTV-2) isolates gave evidence that this serotype probably moved from Tunisia. BTV-4 results showed probable incursion from the southwest and not from Greece or Israel. BTV-9 isolates clearly have an eastern origin (most probably Greece), whereas BTV-16 isolates are indistinguishable from the BTV-16 live attenuated vaccine strain. The phylogenetic findings were supported by polyacrylamide gel electrophoresis (PAGE) analysis of the complete amplified genome of each isolate except for BTV-16 Italian field isolate, which showed a slightly different PAGE profile. A combination of the complete VP2 sequencing and PAGE analysis of complete genomes, allowed not only phylogenetic analysis, but also vaccine detection and assessment of reassortment events.