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 150 km 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.     

Development of an ELISA for detection of myxoma virus-specific rabbit antibodies: test evaluation for diagnostic applications on vaccinated and wild rabbit sera.

An enzyme-linked immunosorbent assay (ELISA) was developed and compared with 2 reference diagnostic tests (indirect immunofluorescence [IF] and complement fixation) to detect myxoma virus-specific antibodies in sera from 50 rabbits experimentally vaccinated with an attenuated strain of myxoma virus or with a Shope fibroma virus. The ELISA was highly specific (100% specificity) and sensitive (100%, 21 days after homologous vaccination). In a comparison of the ELISA with the IF test in 128 wild rabbits from France, discrepant results were obtained in only 11 (8.6%) animals, which were positive with the ELISA and negative with the IF test. The higher sensitivity and the good specificity of the ELISA was confirmed in a serologic survey of 118 rabbits from 2 Kerguelen (Indian Ocean) islands, where the prevalence of myxomatosis varied considerably. The ELISA is an alternative serologic test for diagnosis, vaccine evaluation, and seroepidemiologic surveys of myxomatosis.     

Novel bivalent vectored vaccine for control of myxomatosis and rabbit haemorrhagic disease

A novel, recombinant myxoma virus-rabbit haemorrhagic disease virus (RHDV) vaccine has been developed for the prevention of myxomatosis and rabbit haemorrhagic disease (RHD). A number of laboratory studies are described illustrating the safety and efficacy of the vaccine following subcutaneous administration in laboratory rabbits from four weeks of age onwards. In these studies, both vaccinated and unvaccinated control rabbits were challenged using pathogenic strains of RHD and myxoma viruses, and 100 per cent of the vaccinated rabbits were protected against both myxomatosis and RHD.     

Program of vaccination and antibiotic treatment to control polyserositis caused by Haemophilus parasuis under field conditions

The present study investigated the effects of vaccinating sows and piglets or piglets alone against Haemophilus parasuis on the prevalence of H. parasuis in nasal swabs, on the humoral and cellular immune responses, and on the production parameters of piglets at 3 Korean farms with a clinical history of polyserositis caused by H. parasuis. Piglets born to vaccinated or non-vaccinated sows were subdivided into 3 groups: vaccinated sows and vaccinated pigs (VS-VP), non-vaccinated sows and vaccinated pigs (NVS-VP), and non-vaccinated sows and non-vaccinated pigs (NVS-NVP). The proportion of piglets with positive nasal swabs was significantly lower (P < 0.05) in the vaccinated animals (VS-VP and NVS-VP groups) than in the non-vaccinated animals (NVS-NVP group) at 35 and 60 d of age at the 3 farms. The overall growth performance (from 7 to 60 d of age) of the vaccinated piglets was significantly better (P < 0.05) than that of the non-vaccinated piglets at the 3 farms. Piglets in the VS-VP group had significantly higher levels (P < 0.05) of H. parasuis-specific IgG antibodies, lymphocyte proliferation, and interferon-γ-secreting cells than piglets in the NVS-VP and NVS-NVP groups on days 1, 7, 21, 35, and 60 after birth at the 3 farms.     

Prevention of bovine respiratory syncytial virus infection and clinical disease by vaccination

A double blind field trial was carried out with a live attenuated bovine respiratory syncytial virus vaccine. The trial involved 530 calves, two to 10 months old, on 27 dairy farms, where respiratory problems due to bovine respiratory syncytial virus infections had been observed during the preceding year. In 17 herds either all calves were vaccinated (nine groups) or all calves received a placebo (eight groups). In 10 herds half the number of calves were vaccinated and the other half kept as non-vaccinated controls. Calves were vaccinated intramuscularly twice with an interval of four to five weeks. These groups were under regular clinical observation and animals were tested periodically for antibodies to bovine respiratory syncytial virus and parainfluenza type 3 virus. Serological examination indicated that no bovine respiratory syncytial virus infection had occurred prior to the first vaccination in August. Vaccination did not cause adverse reactions. Low concentrations of neutralising and complement fixing antibodies were induced by vaccination and a sharp increase of antibody titres was observed after natural infection of vaccinated animals. Infections with bovine respiratory syncytial virus occurred in six out of eight non-vaccinated groups, in nine out of 10 partly vaccinated groups and in only two out of nine completely vaccinated groups. Virus infection in completely vaccinated groups was significantly reduced compared with partly vaccinated and non-vaccinated groups. The incidence of bovine respiratory syncytial virus lower respiratory disease was significantly reduced in completely vaccinated groups compared to non-vaccinated groups. Generally only mild signs of upper respiratory disease were present in completely vaccinated groups after bovine respiratory syncytial virus infection.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of a mutant bovine herpesvirus-1 (BHV-1) in post-arrival outbreaks of IBR in feedlot calves and protection with conventional vaccination.

Outbreaks of infectious bovine rhinotracheitis (IBR) have recently been observed in vaccinated feedlot calves in Alberta a few months post-arrival. To investigate the cause of these outbreaks, lung and tracheal tissues were collected from calves that died of IBR during a post-arrival outbreak of disease. Bovine herpesvirus-1 (BHV-1), the causative agent of IBR, was isolated from 6 out of 15 tissues. Of these 6 isolates, 5 failed to react with a monoclonal antibody specific for one of the epitopes on glycoprotein D, one of the most important antigens of BHV-1. The ability of one of these mutant BHV-1 isolates to cause disease in calves vaccinated with a modified-live IBR vaccine was assessed in an experimental challenge study. After one vaccination, the majority of the calves developed humoral and cellular immune responses. Secondary vaccination resulted in a substantially enhanced level of immunity in all animals. Three months after the second vaccination, calves were either challenged with one of the mutant isolates or with a conventional challenge strain of BHV-1. Regardless of the type of virus used for challenge, vaccinated calves experienced significantly (P < 0.05) less weight loss and temperature rises, had lower nasal scores, and shed less virus than non-vaccinated animals. The only statistically significant (P < 0.05) difference between the 2 challenge viruses was the amount of virus shed, which was higher in non-vaccinated calves challenged with the mutant virus than in those challenged with the conventional virus. These data show that calves vaccinated with a modified-live IBR vaccine are protected from challenge with either the mutant or the conventional virus.     

Occurrence of atypical myxomatosis in Central Europe: clinical and virological examinations

An outbreak of the atypical form of myxomatosis struck a rabbit farm in Hungary. The animals had previously been vaccinated with a vaccine containing Shope rabbit fibroma virus strain. The disease appeared in winter when the presence of mosquitoes and fleas is not common. The virus was isolated from an eyelid specimen of a naturally infected rabbit. The surviving animals were observed for four weeks, blood samples were collected and, after euthanasia, organ specimens were also examined by morphological methods including pathology and electron microscopy. Serum samples were examined by virus neutralisation for antibodies. Genetic analysis of the isolated virus was carried out by polymerase chain reaction (PCR) and direct sequencing. The primers were designed on the basis of the major envelope gene (Env) of the Lausanne reference strain in the GenBank. The viral proteins were examined by SDS-PAGE. The isolated virus (ref. no.: BP04/2001) was able to infect the susceptible animals directly, by contact. The disease was characterised by respiratory symptoms of the upper tracheal tract, conjunctivitis and high mortality by the 11th-14th day. Aerogenic infection with strain BP04/2001 resulted in 100% morbidity among the susceptible animals. Sequencing of the amplified 400-bp-long DNA revealed 97% homology with the Env gene of the Lausanne strain, which proves that strain BP04/2001 is a variant of the Lausanne strain having been enzootic throughout Europe. The live vaccine strain used in Hungary against myxomatosis, which is also a Lausanne-derived strain, protected the animals. According to the protein analysis a protein of 200 kDa in size is not expressed in strain BP04/2001. This is the first report on atypical myxomatosis in Central Europe. The virus spreads by airborne transmission and may cause severe losses in the rabbit population.     

Atypical myxomatosis--virus isolation, experimental infection of rabbits and restriction endonuclease analysis of the isolate

Atypical form of myxomatosis, which caused non-lethal and clinically mild disease in domestic rabbits 1 month after immunization with a commercially available vaccine MXT, is described. The isolated myxoma virus designated as Litovel 2 (Li-2) did not induce systemic disease following subcutaneous and intradermal applications in susceptible experimental rabbits but led to the immune response demonstrated by ELISA. No severe disease was induced in those Li-2 inoculated rabbits by challenge with the virulent strains Lausanne (Lu) or Sanar (SA), while the control animals showed nodular form of myxomatosis with lethal course of the illness. Restriction fragment length polymorphism (RFLP) of genomic DNA with KpnI and BamHI endonucleases was used for genetic characterization of the Li-2 isolate, the vaccine strain MXT and both virulent strains Lu and SA, respectively. In general, RFLP analysis has shown to be informative for inferring genetic relatedness between myxoma viruses. Based on restriction endonuclease DNA fragment size distribution, it was evident that the pathogenic strain SA is genetically related to the reference strain Lu and the isolate Li-2 is more related, but not identical, to the vaccination strain MXT.     

Possible interaction between myxomatosis and calicivirosis related to rabbit haemorrhagic disease affecting the European rabbit

Serological data on myxoma virus, rabbit haemorrhagic disease (RHD) virus and RHD-like viruses in juvenile rabbits (Oryctolagus cuniculus) trapped in 1995, 1996 and 1997 in two areas of France were analysed. For each disease, the effects of bodyweight, year, month and seropositivity for the other disease were modelled by using logistic regressions. In one area, a model including RHD seropositivity was selected to explain the myxoma virus seropositivity. Models including myxoma virus seropositivity were selected to explain the RHD seropositivity in both areas, and the odds of a rabbit being seropositive to both viruses were 5.1 and 8.4 times higher than the odds of a rabbit being seronegative to myxoma virus and seropositive to RHD. The year and bodyweight had significant effects for myxomatosis in one area and for RHD in both areas.     

Impact of field vaccination with a Theileria annulata schizont cell culture vaccine on the epidemiology of tropical theileriosis

Tropical theileriosis, caused by Theileria annulata, is an important tick-borne disease of cattle. A cell culture attenuated vaccine has been developed in our laboratory by long-term in vitro propagation of the schizont stage of the parasite. A longitudinal study was conducted at selected farms housing indigenous, cross-bred and exotic animals to investigate the effect of vaccination on the epidemiology of the disease. A total of 120 animals in 4 age groups were vaccinated with the vaccine before the onset of disease season. An equal number of age-matched animals were kept as controls at the same sites. Animals were monitored for 14 months at monthly intervals. The 97.5% vaccinated animals showed a rise in antibody titres 1 month post-vaccination, as determined by single dilution ELISA. The 78.3% of non-vaccinated animals became sero-positive over the period of observation. Mean antibody titres were significantly higher in vaccinated than non-vaccinated animals. Cross-bred animals showed higher antibody titres followed by exotic and indigenous animals in both the vaccinated and non-vaccinated groups. However, the antibody titres in animals of different ages were similar. The 36.7% vaccinated and 64.2% non-vaccinated animals became carriers (<0.5% piroplasms in erythrocytes) during the observation period. Clinical cases of theileriosis were recorded only in the non-vaccinated group suggesting that vaccinated animals were sufficiently immune to withstand field tick challenge for at least 14 months.     

Atypical Myxomatosis – Virus Isolation,Experimental Infection of Rabbits and Restriction Endonuclease Analysis of the Isolate

Atypical form of myxomatosis, which caused non‐lethal and clinically mild disease in domestic rabbits 1 month after immunization with a commercially available vaccine MXT, is described. The isolated myxoma virus designated as Litovel 2 (Li‐2) did not induce systemic disease following subcutaneous and intradermal applications in susceptible experimental rabbits but led to the immune response demonstrated by ELISA. No severe disease was induced in those Li‐2 inoculated rabbits by challenge with the virulent strains Lausanne (Lu) or Sanar (SA), while the control animals showed nodular form of myxomatosis with lethal course of the illness. Restriction fragment length polymorphism (RFLP) of genomic DNA with KpnI and BamHI endonucleases was used for genetic characterization of the Li‐2 isolate, the vaccine strain MXT and both virulent strains Lu and SA, respectively. In general, RFLP analysis has shown to be informative for inferring genetic relatedness between myxoma viruses. Based on restriction endonuclease DNA fragment size distribution, it was evident that the pathogenic strain SA is genetically related to the reference strain Lu and the isolate Li‐2 is more related, but not identical, to the vaccination strain MXT.     

Immunogenicity in Aujeszky's disease virus structural glycoprotein gVI (gp50) in swine.

Aujeszky's disease virus (ADV) envelope glycoprotein gVI (gp50) was purified from virus-infected Vero cells by ion-exchange and immunoaffinity chromatography and its usefulness as a subunit vaccine was evaluated in active and passive immunization studies. Four-week-old piglets were immunized intramuscularly (IM) with purified gVI twice two weeks apart and challenged intranasally (IN) 10 days after the second immunization with 30 LD50 (10(8)PFU) of a virulent strain of ADV. Pigs, vaccinated with 100 micrograms of purified gVI, produced virus neutralizing antibodies and did not develop clinical signs after challenge exposure. The challenge virus was not isolated from nasal swabs and tonsils of gVI-vaccinated pigs, whereas non-vaccinated control pigs developed illness after challenge exposure with the same virulent ADV strain which was later recovered from their nasal swabs and tonsils. Pregnant sows vaccinated twice with purified gVI (IM) at a three week interval produced virus neutralizing antibodies in colostrum. Four-day-old sucking piglets born of vaccinated sows were passively protected by colostral antibodies against intranasal challenge with a lethal dose of virulent ADV. Sera from gVI-vaccinated pigs were distinguished from experimentally infected swine sera by their differential reactivity in enzyme-linked immunosorbent assay (ELISA) using four major viral glycoproteins (excluding gVI) as antigen purified by the use of lentil-lectin.     

Influence of vaccination on Aujeszky's disease virus and disease transmission

Parenteral vaccination of fattening pigs with either modified live or inactivated Aujeszky's disease virus did not prevent infection with field strain virus or the development of clinical disease. The duration and severity of the clinical syndrome was, however, reduced and vaccinated pigs did not suffer the severe weight loss and high mortality experienced by non-vaccinated pigs in the acute phase of disease. The range of tissues in which challenge virus replication took place was more restricted in vaccinated animals and the concentration of virus in infected tissues was reduced. Vaccination shortened the duration of field virus excretion and carriage in the tonsil. Replication of modified live vaccine virus was restricted to the site of inoculation in the neck and associated lymph nodes for two days after vaccination and it was not excreted by vaccinated pigs. Attempts to infect pigs by feeding them tissues taken from non-vaccinated or vaccinated pigs soon after challenge infection were unsuccessful.     

ISCOM of BHV-1 envelope glycoproteins protected calves against both disease and infection.

A subunit vaccine in the form of immunostimulating complex (iscom) was prepared to contain the envelope glycoproteins of bovine herpesvirus type 1 (BHV-1). This iscom preparation was tested in a vaccination experiment on 4-month-old calves seronegative to BHV-1. In this experiment, four groups with three animals per group were used. Two groups were vaccinated with the iscom preparation twice, four weeks apart, one group with 50 micrograms and the other with 100 micrograms per calf. The third group received a commercial inactivated whole-virus vaccine applying the same vaccination program. The fourth group served as control. Two weeks after the second vaccination, all the animals were challenge-infected intranasally with a virulent BHV-1 strain and four days later with a virulent Pasteurella multocida--this in order to mimic hard field conditions. When exposed to challenge infection, all the animals vaccinated with the iscom were fully protected, i.e., no virus could be recovered from their nasal secretions and no clinical symptoms were recorded. In contrast, the animals vaccinated with the commercial vaccine, responded to challenge with moderate fever and loss of appetite, and virus was isolated from the nasal secretions. The animals in the control group developed severe clinical symptoms. In the sera of iscom-vaccinated animals, the virus neutralization titers reached levels of 1/3500 or higher.     

Foot and mouth disease virus transmission during the incubation period of the disease in piglets, lambs, calves, and dairy cows

Transmission of foot and mouth disease (FMD) virus by infected animals may already occur before clinical signs are evident. Quantitative data for FMD transmission rates during this so-called high-risk period are currently lacking and would provide useful information to develop surveillance systems in which the number of new outbreaks is an outcome variable. In order to address this, we used experimental data to quantify transmission in cattle, swine and sheep during the non-clinical phase of the disease. Groups consisted of vaccinated or non-vaccinated animals of one species; half of each group was inoculated with FMDV, the other half was contact-exposed. We estimated the reproduction ratio R(nonclin) using a mathematical SIR model. R(nonclin) was defined as the average number of secondary infections caused by one infectious individual in its non-clinical phase. Animals not showing clinical signs shed lower amounts of virus than clinically affected ones. Therefore, we estimated transmission proportionally to the virus excretion. Low estimates for R(nonclin) in groups with non-vaccinated and vaccinated calves; 0.30 [0.03; 3.43] and 1.03x10(-8) [0; infinity] respectively and 0.21 [0.02; 2.48] for the non-vaccinated and 0.16 [0.009; 2.96] for the vaccinated lambs, were observed. These results indicate that only few secondary infections are to be expected from infected calves and lambs when they are not clinically affected. In groups of non-vaccinated piglets estimates were R(nonclin)=13.20 [4.08; 42.68], and in vaccinated piglets R(nonclin)=1.26 [0.18; 8.96]. The estimate for R(nonclin) for non-vaccinated dairy cows was R(nonclin)=176.65 [80.38; 388.24], whereas R(nonclin) in the vaccinated groups could not be estimated. Our findings suggest that a large number of individuals might have been infected before clinical signs are noticed, especially in non-vaccinated swine and dairy herds. These findings suggest that after clinical recognition of FMD, priority should be given to trace back contacts with swine and dairy farms, as they may already have been infectious in the herd's incubation period.     

Construction and testing of a novel host-range defective myxoma virus vaccine with the M063 gene inactivated that is non-permissive for replication in rabbit cells

Deletion of the M063 gene from myxoma virus produces a virus that is unable to replicate in rabbit cells in vitro or in live rabbits but can be propagated in non-rabbit cell lines. A targeted M063 deletion mutant was constructed in the attenuated Uriarra strain of myxoma virus and the ability of this virus to act as a safe, non-transmissible vaccine against myxomatosis was tested in outbred laboratory rabbits. Immunization with the M063 deletion vaccine provided good short-term protection against lethal challenge with virulent myxoma virus. Long-term protection was similar to reported results with heterologous live virus, with some rabbits protected but others succumbing to challenge. Replication-deficient poxvirus vaccines, like the Modified Vaccinia Virus Ankara (MVA) in man and the myxoma virus vaccine described here in rabbits, are very attractive from a safety perspective. Seasonal boosting would be predicted to provide long-term protection. Targeted host-range gene deletions could have potential for rapid development of poxvirus vaccines in general.     

Efficacy of vaccination with porcine reproductive and respiratory syndrome virus following challenges with field isolates in Japan

The objective of this study was to evaluate the influences of genetic and antigenic variations in field isolates of porcine reproductive and respiratory syndrome virus (PRRSV) on vaccine efficacy. Four-week-old pigs were vaccinated with a commercial modified live virus vaccine. Four weeks after vaccination, pigs in both the vaccinated group and the non-vaccinated group were challenged intranasally with 10(7) TCID(50) of PRRSV wt-11 (Experiment 1) or PRRSV wt-7 (Experiment 2). Based on genome sequencing of ORF5 and cross neutralization test results, PRRSV wt-11 is similar to the vaccine strain, whereas wt-7 is distinct from the vaccine strain. In the vaccinated challenged groups, clinical signs were less severe, the mean rate of weight gain was greater, and gross lung lesions were less severe when compared with the non-vaccinated challenged groups in both experiments. In Experiment 1, the virus was isolated from serum at 3 days post-challenge, and the mean virus titers in broncho-alveolar lavage fluids (BALF) and tissues were lower in pigs in the vaccinated challenged groups compared with those in the non-vaccinated challenged group. In Experiment 2, virus isolation from serum, BALF and tissues showed no significant differences between the groups. These results suggest that commercial PRRSV vaccine could be effective in reducing clinical disease following a challenge with field isolates of PRRSV. However, with regards to virological protection, the efficacy of the vaccine may be affected by the nature of the PRRSV isolates.     

Influence of vaccination route on the efficacy of Aujeszky's disease deletion-mutant vaccine.

In order to compare the effect of the route of immunization on the efficacy of a modified live Aujeszky's disease (AD) vaccine, which had deletions in both thymidine kinase (TK-) and glycoprotein gIII genes (gpIII-), 20 six-week-old pigs were vaccinated by either the intramuscular (IM) (n = 10) or subcutaneous (SC) (n = 10) route. All the animals, including five non-vaccinated control animals, were challenged with virulent AD virus 22 days after vaccination. Four of five non-vaccinated animals died within 12 days after challenge. Although none of vaccinated animals died, three of animals in the SC group exhibited clinical signs, and average daily gains in the SC group were depressed. The animals in the IM group were not found to shed challenge virus, but those in the SC group shed the virus up to 9 days. Virus neutralizing antibody titers in the vaccinated animals were low or non-detectable by 21 days after vaccination. A glycoprotein gII (gpII) screening ELISA detected gpII antibody in all animals in the IM group. While, only 30% of animals in the SC group were positive by the same test. The results of this study indicate that TK-, gpIII modified live AD virus vaccine is effective against challenge with virulent AD virus; however, vaccination by the SC route reduced vaccine efficacy in comparison with IM route.     

Field evaluation of the protective efficacy of Mycobacterium bovis BCG vaccine against bovine tuberculosis

The protective efficacy of Mycobacterium bovis BCG (1 × 10⁶ single dose) was evaluated under field conditions. A total of 140 male Holstein Friesian calves, one to two week-old were selected. Two groups of 70 each were formed, one group was vaccinated and the other was injected with a placebo during their second week of age and followed until 12 months of age. The study considered a positive case of tuberculosis to be an animal that had a positive reaction to the three following tests in a row: tuberculin, IFNγ PPD-B and IFNγ ESAT6-CFP10 during the 12 months of exposure. The results showed a 59.4% efficacy (IC95%: 47.64-71.16). The non-vaccinated calves were 2.4 times more at risk of becoming infected (IC95%: 1.07-5.68) compared to vaccinated animals. As a complementary test a PCR test was performed using nasal exudates in some animals from both groups using a Mycobacterium complex detection kit. All the positive PCR reactions (5/44) were found in the non-vaccinated animals. These findings suggest that the use of the BCG vaccine, even though it is not capable of protecting 100%, does prevent TB vaccinated animals from excreting bacilli in their nasal secretions at their first year of age.     

THE EFFECT OF CALFHOOD VACCINATION WITH STRAIN 19 ON THE SEROLOGICAL DIAGNOSIS AND ERADICATION OF BOVINE BRUCELLOSIS

Data from 42,224 cattle from 694 herds collected during the brucellosis eradication campaign were used to examine the effects of calfhood strain 19 vaccination. The prevalence of infection in vaccinated herds was 1.8% compared with 9.1% in non-vaccinated herds (p< 0.005). The mean titre in the former group was lower (p< 0.001). Vaccinated herds required 3.3 herd tests to achieve a provisionally free status compared with 4.8 in non-vaccinated herds (0.001 < p < 0.005). Vaccination did not significantly reduce the number of herd tests in herds with less than 100 breeding females. In tests after the initial herd test only 0.5% reactors were found in vaccinated herds compared with 6.9% in non-vaccinated herds (p< 0.005). There were 0.9% false positive to the Rose Bengal plate test in non-vaccinated and 2.1% in vaccinated animals (p< 0.005) in non-infected herds. In infected herds this percentage was 3.0% and 4.2% respectively by (p< 0.05). In the non-infected herds there were 0.04% false positives to the complement fixation test out of 10,506 non-vaccinated cattle tested and 0.2% out of 24,734 vaccinated cattle.