Assessment of bluetongue viraemia in sheep by real-time PCR and correlation with viral infectivity.

Inoculation of embryonated chicken eggs is the standard method for the titration of infectious Bluetongue virus (BTV). Here, six RNA extraction methods coupled with optimised dsRNA denaturation and real-time RT-PCR were evaluated for the quantitation of BTV in blood samples from experimentally infected sheep and results were correlated to infectious virus titres. An exogenous dsRNA internal control (IC) from the closely related Epizootic hemorrhagic disease virus (EHDV) was used to assess the efficiency of BTV genome extraction, dsRNA denaturation, RT, and PCR amplification. Recovery rates of IC and BTV dsRNA copies from extracted blood samples were highly correlated. Adjustment of BTV concentrations according to the IC recovery reduced variation in sample analyses among the different extraction methods and improved the accuracy of BTV quantitation. The EID(50)/ml titre, determined in blood samples from sheep infected experimentally with BTV-1 or BTV-9, correlated highly with the assessed concentration of BTV dsRNA copies. However, this correlation was consistent only during the first 28 days post-infection. The optimised extraction methods and quantitative RT-PCR could be useful for experimental studies of BTV transmission, pathogenesis and vaccine efficacy, or adapted further for the detection and quantitation of EHDV, African horse sickness virus and other dsRNA viruses.

A pathogenic myxoma virus in vaccinated and non-vaccinated commercial rabbits.

A case of a myxoma virus strain in vaccinated and non-vaccinated rabbits is described, and genetic identification of that strain was performed in this study. In two commercial farms being 150km apart, myxomatosis has been occurred after the import of animals from a common supplier. The disease was manifested firstly in the existing non-immune population of does and fatteners, and later in all vaccinated animals, being 2-3 months immune at the time of first symptoms. Morbidity was almost 100% with nasal discharge, listlessness, fever, eyelid swelling, eye and nasal purulent discharge, papules in the ears, facial oedema, and swelling of the anagenital region, with result always the death of the animals. Examination by PCR had shown the presence of a 492-bp specific product in all the symptomatic animals tested from both farms, having 100% nucleotide sequence identity with the homologous region of the myxoma virus Lausanne strain. The simultaneous occurrence of myxomatosis in the vaccinated and non-vaccinated rabbits of both farms, suggests that the supplier was possibly the source of a viral isolate with increased virulence.

Assessment of serological response of young and adult sheep to conjunctival vaccination with Rev-1 vaccine by fluorescence polarization assay (FPA) and other serological tests for B. melitensis.

The serological response of young and adult sheep vaccinated conjunctivally with Rev-1 vaccine was assessed by fluorescence polarization assay (FPA), Rose Bengal test (RBT), complement fixation test (CFT), modified Rose Bengal test (m-RBT), indirect ELISA (i-ELISA) and competitive ELISA (c-ELISA), at different post vaccination intervals. One hundred and thirty six adult sheep and 64 lambs were used in the study. The vaccinated animals were bled prior to vaccination (0 day) and thereafter at 21st, 42nd, 35th, 63rd, 91st, 125th, 159th, and 223rd and 330th day post vaccination. The majority of animals (young and adult) showed positive reaction by FPA, RBT, CFT, m-RBT and c-ELISA 21 days post vaccination, whereas by i-ELISA at 42 days. All tests perform equal when animals vaccinated as young are tested 125 days (4 months) post vaccination. In case of animals vaccinated at adulthood, FPA, RBT, CFT and c-ELISA perform equal if the animals are tested 223 days (approximately 8 months) post vaccination. I-ELISA and m-RBT show low specificity if ewes vaccinated at adulthood are tested 330 days (11 months) post vaccination. If control of brucellosis in sheep is based on conjunctivally vaccination of lambs with Rev-1, the vaccinated animals can be tested by any test used for diagnosis of B.melitensis infection accurately at least 4 months post vaccination. If brucellosis control is based on mass vaccination the use of m-RBT and i-ELISA is not recommended for testing adult animals at least for 330 days (11 months) post vaccination due to tests low specificity. Further research is needed so the appropriate cut-offs to be established for FPA, c-ELISA or i-ELISA to become valuable tools for the eradication of Brucella spp. infection in small ruminants in areas where vaccination is practiced.

Long-term administration of a commercial porcine reproductive and respiratory syndrome virus (PRRSV)-inactivated vaccine in PRRSV-endemically infected sows.

The purpose of this study was to investigate the safety and efficacy of a commercial European porcine reproductive and respiratory syndrome virus (PRRSV)-inactivated vaccine after 18-month use in gilts/sows at a farm with high seroprevalence. In a farrow-to-finish farm with 1100 sows, all sows and gilts were systematically vaccinated with the PRRS-inactivated PROGRESSIS vaccine for a period of 18 months. Farm's reproductive and litter characteristics were longitudinally recorded for this period and historically compared with those of the year prior to vaccination. Serology, employing immunoperoxidase monolayer assay, had confirmed a high prevalence of PRRS-specific antibodies in most age groups within the farm prior to vaccination. Seroprevalence during the experiment ranged between 0% and 100% in weaners and growers, but remained at stable high levels (> 93%) in finishing pigs and gilts throughout all 2-year period of serology measurements. No local or systemic vaccine side effects were noted throughout the trial period. Vaccinations had resulted over time in a significant improvement of sow reproductive performance (e.g. reduction of premature farrowings, abortions and increase of farrowing rate) and litter characteristics (e.g. increase of the number of live born and weaned pigs and decrease of stillborn, mummified, weak and splay-legged piglets). It has also been observed that the higher the degree of immunization of a sow, the better the improvement of her reproductive parameters. Sows after vaccination have shown improved characteristics compared to homoparous sows prior to the application of vaccinations in the farm.

Development of a semi-nested PCR using degenerate primers for the generic detection of small ruminant lentivirus proviral DNA.

A PCR assay was developed for the reliable detection of small ruminant lentivirus (SRLV) proviral DNA. The method involved the use of degenerate deoxyinosine-substituted primers and a second semi-nested PCR step that increased the polyvalency and sensitivity of the detection, respectively. Primers were designed from the pol gene conserved motifs of 85 SRLV isolates and were evaluated using different SRLV isolates together with Maedi-Visna virus (MVV) and caprine arthritis-encephalitis virus (CAEV) reference strains. The method successfully detected SRLV proviral DNA in total DNA extracts originating from whole blood samples, separated peripheral blood mononuclear cells (PBMCs) and tissue cultures. The semi-nested PCR was compared with the agar gel immunodiffusion test and proved to be highly sensitive, specific and capable of detecting many SRLV variants in infected or suspect animals. Therefore, it would be useful in the diagnosis of natural SRLV infections, in eradication programs and epidemiological studies. Whole blood samples can be used directly, thus alleviating the need for PBMC separation, and thereby enables a simple, fast and cost-effective analysis of a large number of samples.

First partial characterisation of small ruminant lentiviruses from Greece.

Small ruminant lentivirus (SRLV) infections are widespread in Greece, but SRLVs have never been isolated and characterized. In this study, we present the sequence of a 574-nucleotide (191-amino acid) region of the gag gene of SRLV strains from four sheep and one goat from a single geographic area of Greece. All five sequences appeared to be closely related at both nucleotide (2.1-14.2% variation) and deduced amino acid (1.6-4.2% variation) level. Greek SRLV strains were closer to ovine prototypic strains (average divergence 16.8%) than to the caprine strain CAEV-Co (21% divergence). By amino acid composition, the Greek SRLVs were on the average more than twice as distant from CAEV-Co as from other ovine strains. Phylogenetic analysis suggested that Greek strains segregate into a unique group, separate from, but related to, other ovine prototype sequences.

Equine infectious anemia in mules: virus isolation and pathogenicity studies.

There appears to be a lack of information concerning responses of mules to natural infection or experimental inoculation with equine infectious anemia virus (EIAV). In the present study EIAV was isolated from mules, for the first time, and its pathogenicity in naturally infected and experimentally inoculated animals was investigated. Two naturally infected (A and B) and three EIAV free mules (C, D and E) were used for this purpose. Mule A developed clinical signs, whereas mule B remained asymptomatic until the end of the study. Mules C and D were each inoculated with 10ml of blood from mule A and developed signs of the disease; they were euthanatized or died at day 22 and 25 post-inoculation, respectively. Mule E served as a negative control. The virus was isolated from the plasma samples of mules with clinical signs of the disease (A, C and D), but not from the asymptomatic mule B. Both proviral DNA and viral RNA were amplified from blood and tissues of the infected animals by nested polymerase chain reaction (nPCR). Antibodies were not detected in the two experimentally infected mules until their natural death or euthanasia. Clinicopathological and laboratory findings showed that, in mules, EIAV produced clinical signs similar to those observed in horses and ponies. Nested PCR proved to be a rapid, sensitive and specific diagnostic method for the detection of EIAV, regardless of the disease stage.

Sheep poxvirus identification from clinical specimens by PCR, cell culture, immunofluorescence and agar gel immunoprecipitation assay.

Some 40 clinical specimens of skin lesions from sheep pox suspected cases were investigated by four different diagnostic assays: PCR, virus isolation in lamb testis cell cultures, direct immunofluorescent assay (DIFA) and antigen detecting agar gel immune precipitation test (AGIPT). All the specimens were positive by PCR and virus isolation, 29 were positive by DIFA and 16 by AGIPT. Using virus isolation on cell cultures as the gold standard, the PCR sensitivity was 100%, while that of DIFA and AGIPT was 73% and 40%, respectively. Skin samples with orf lesions or normal skin biopsies were PCR-negative. Cross-reactions with orf virus were observed in three samples only in the AGIPT assay. The PCR described combines high specificity and sensitivity with speed. PCR was therefore shown to be the method of choice for sheep poxvirus diagnosis directly from clinical specimens.

Utilisation of bacteriophage display libraries to identify peptide sequences recognised by equine herpesvirus type 1 specific equine sera.

Three filamentous phage random peptide display libraries were used in biopanning experiments with purified IgG from the serum of a gnotobiotic foal infected with equine herpesvirus-1 (EHV-1) to enrich for epitopes binding to anti-EHV-1 antibodies. The sequences of the amino acids displayed were aligned with protein sequences of EHV-1, thereby identifying a number of potential antibody binding regions. Presumptive epitopes were identified within the proteins encoded by genes 7 (DNA helicase/primase complex protein), 11 (tegument protein), 16 (glycoprotein C), 41 (integral membrane protein), 70 (glycoprotein G), 71 (envelope glycoprotein gp300), and 74 (glycoprotein E). Two groups of sequences, which aligned with either glycoprotein C (gC) or glycoprotein E (gE), identified type-specific epitopes which could be used to distinguish between sera from horses infected with either EHV-1 or EHV-4 in an ELISA using either the phage displaying the peptide or synthetic peptides as antigen. The gC epitope had been previously identified as an immunogenic region by conventional monoclonal antibody screening whereas the gE antibody binding region had not been previously identified. This demonstrates that screening of phage display peptide libraries with post-infection polyclonal sera is a suitable method for identifying diagnostic antigens for viral infections such as EHV-1.

Selenium and vitamin E effect on antibody production of sheep vaccinated against enzootic abortion (Chlamydia psittaci).

The effect of selenium (Se) and vitamin E (vit E) on antibody production of sheep vaccinated against Chlamydia psittaci (ovis) was investigated. Thirty-two sheep, one year old, seronegative to Chlamydia infection, vaccinated against enterotoxemia and dewormed were used. Injectable sodium selenite (0.1 mg/kg b.w.) was given twice to animals of the first group (gSe), with a three week interval. The sheep of the second group (gE) received 1 g vit E each orally, six times at weekly intervals. The animals of the third group (gSeE) were given Se and vit E in doses and routes of administration as in gSe and gE. The animals of the fourth group served as controls (gC) and injected normal saline. The first vaccination was made at the time that the second Se injection was given. Revaccination was made two weeks later. The experiment lasted 29 weeks. The results indicated that Se alone led to a significant increase of Chlamydia antibody response (P < 0.05), but not when it was given in combination with vit E. Animals that received vit E (gE) had much lower titres, just above of those of the controls.

Detection of sheep poxvirus in skin biopsy samples by a multiplex polymerase chain reaction.

The development of a multiplex polymerase chain reaction (PCR) method with amplification of capripoxvirus in a single-step procedure from skin biopsies using three primer pairs, two specific for capripoxvirus and one specific for alpha-tubulin is described. A sensitive multiplex PCR was achieved by optimization of parameters such as the primer concentrations, magnesium and dNTPs concentrations. False negative results that sometimes arise due to inhibitors of DNA amplification may be avoided by the inclusion in the assay of alpha-tubulin primers. The results reported on 42 skin biopsies from sheep suspected to have poxvirus infection, indicated that the assay could monitor simultaneously DNA extraction from skin biopsy samples and allow improved detection of capripoxvirus within 24 h of specimen receipt in the laboratory.

Effects of alpha-tocopherol and selenium on pregnant sows and their piglets' immunity and performance.

Four groups of 12 pregnant sows of the same genetic background, with similar litter size, living under the same housing conditions and having the same hygienic and nutritional standards, were used. In control animals (gC), a basic feed was provided, in which the dietary level of alpha-tocopherol was 20 mg/kg of feed and that of selenium (Se) was 0.45 mg/kg of feed (standard ration). Sows in the second group (gE) received basic feed supplemented with 30 mg alpha-tocopherol per kg (50 mg/kg of feed, in total). To those in the third group (gSe) basic feed was provided but additionally they were injected with 30 mg Se (sodium selenite) on days 30, 60 and 90 of pregnancy. For the sows in the fourth group (gESe), basic feed was supplemented with 30 mg alpha-tocopherol per kg. In addition they received 30 mg injectable Se (sodium selenite) on days 30, 60 and 90 of pregnancy. The experiment started on day 30 of pregnancy and lasted until weaning day (28 days post-farrowing). It was found that alpha-tocopherol and selenium act synergistically. Piglets born from sows in gESe had the highest immunoglobulin concentration level up to weaning day. All productive and clinical parameters (number of piglets born/litter, number of weaned piglets/litter, and piglets' average body weight at birth and on weaning day) were greater in these piglets in comparison with the animals of the other groups. Diarrhoea problems were minimal in the piglets in gESe.

A Salmonella abortusovis inactivated vaccine protects mice from abortion after challenge.

Two types of Salmonella abortusovis vaccines were prepared, one with aluminium hydroxide (vaccine A) and the other with water in oil (vaccine B) adjuvants. They were compared in a pregnant mouse model, aiming at protecting them from abortions after challenge with a virulent strain of S. abortusovis. The protection for vaccine A was from 74% to 77.6% and that for vaccine B from 71% to 79.6%. Abortions occurred 5-10 days post challenge and S. abortusovis was isolated from all aborted fetuses and from the liver and the spleen of their mothers at the end of the experiment (18 days post challenge). The presence of salmonella in the liver and the spleen of vaccinated non-pregnant but challenged mice was studied in a separate experiment. The bacterium was isolated from one out of 12 vaccinated mice 6 days post challenge as well as from the six controls.

Preparation of a high titre Salmonella abortusovis H-antigen and antiserum and improvement of the microagglutination test.

Serological diagnosis of small ruminant abortions due to Salmonella abortusovis (S.a.o) was improved by using modified procedures to produce high titre H-antigens and anti-H serum. While the titres of the two standard antigens were 1:8 and 1:20, both modified antigens had a titre of 1:50. Similarly, the use of adjuvanted and non adjuvanted antigens for the production of anti-H hyperimmune serum resulted in a titre of 1:160,000 as compared to 1:20,000 when standard procedures were used. In addition, the slow micro-agglutination test has been modified and results can now be read within the same day.

Sheep poxvirus identification by PCR in cell cultures.

A simple, rapid and specific diagnostic polymerase chain reaction (PCR) method was developed for sheep poxvirus identification. The primers used were from the sequenced genomes of the capripox viruses KS-1 and InS-1. Six different sheep pox isolates were tested against two orf (parapox) and three animal herpesviruses as controls. Material from uninfected cell cultures was also used as control. The sensitivity of the PCR was approximately equivalent with each of the two primers and for the six sheep pox isolates. All the negative control virus DNAs were negative and differed clearly from those of the sheep pox strains.

Immunosuppression in goats by dexamethasone and cyclophosphamide.

The influence of dexamethasone and cyclophosphamide on the goat immune system was investigated. Seven goats, with a previous contact with caprine herpesvirus type 1 (CHV-1), were used. All had been vaccinated with live Mycobacterium paratuberculosis vaccine. Six goats were injected intravenously (i.v.) with dexamethasone daily for 5 days (2.5-4 mg/kg BW per day). Three also received 25 mg/kg BW of cyclophosphamide on day 0. The seventh goat was not treated. Dexamethasone alone caused depression, slight lymphopenia and fall in tuberculin reaction. Dexamethasone plus cyclophosphamide caused a severe clinical reaction, marked leukopenia (lymphopenia and polymorphopenia), fall in tuberculin reaction and significant increase in CHV-1 neutralizing antibody titres. M. paratuberculosis antibody reaction was variable and thus difficult to be assessed. CHV-1 was not isolated.

Ovulation and lambing rates in Karaguniki ewes after treatment with follicular fluid.

The aim of the present study was to examine the effect of steroid-free bovine follicular fluid (bFF) on both ovulation and lambing rates. For this purpose, 30 adult ewes of the Karaguniki breed were randomly allocated to three treatment groups (A,B and C; n=10 ewes each) during the breeding season of 1988. The ewes in Group A received bFF (6 ml iv) twice daily during their luteal phase, starting on Day 5 and lasting until Day 9. The ewes in Group B received a mixture of bFF/arachid oil (3 ml sc, 2:1) on Days 3, 4, 5, 10 and 12 of the estrous cycle. The ewes in Group C (Controls) were treated subcutanecusly with a mixture of steroid-free bovine plasma and arachid oil (2:1) on the same days as the ewes in Group B. Plasma concentrations of progesterone showed that the luteal function during the treatment cycle was normal in all treated and control ewes. The ovulation and lambing rates, however, were greater in Group A (2.5 +/- 0.2 and 1.9 +/- 0.3, respectively) and in Group B (2.1 +/- 0.2 and 1.6 +/- 0.1, respectively) than in Group C (1.5 +/- 0.2 and 1.2 +/- 0.3, respectively). Precipitating antibodies were detected in the plasma of Group B ewes only.

An experimental study on the pathogenicity of the caprine herpesvirus type 1 (CHV-1).

The pathogenicity of caprine herpesvirus type-1 (CHV-1) in goat kids and lambs was studied. Two experiments were carried out. In the first, two Saanen goat kids and four lambs of a local breed were infected intravenously with the Swiss strain E/CH of the virus. Clinical reaction was severe in the kids, but it was very mild in the lambs. Virus was excreted from the kids in higher titres and longer than in lambs. Virus was also isolated from tissue specimens but only from a kid that died on post inoculation day 4 (PID 4). The gross- and histopathological lesions were more severe in kids. In the second experiment only lambs were used. They were dexamethasone treated and then virus inoculated. A very mild infection developed. The lambs did not shed the virus, neither the virus was isolated from their tissues collected at necropsy. Nevertheless histopathological lesions were seen. In both experiments the animals seroconverted on PID 10.