Retrospective diagnosis of bluetongue virus in stored frozen and fixed tissue samples using PCR

Stored frozen (-70 degrees C) and formalin-fixed tissue samples constitute a valuable resource for retrospective studies of infectious diseases, or for diagnostic investigations. The polymerase chain reaction (PCR) affords an accurate and rapid method for detection of viral nucleic acids. It was applied to stored tissue samples collected from sheep inoculated with two Australian serotypes of bluetongue virus, BTV 1 and 23, and two North American serotypes, BTV 11 and 17. Specific nested PCR products were detected in both frozen and formalin-fixed samples from the Australian sheep after storage for 3.5 years. The tissues from sheep inoculated with the North American serotypes yielded specific nested PCR products after storage at -70 degrees C for 14 years. No specific primary PCR products were detected in any frozen or formalin-fixed samples. The PCR assay offers a potential benefit for epidemiological studies, and for screening of stored semen, embryos and tissue banks.     

The HLD (CalMod) index and the index question

Twenty free-ranging guanaco (Lama guanicoe) in Chubut Province, Argentina, were immobilized for health evaluations. All but two animals appeared to be in good condition. Hematology, serum chemistry, and vitamin and mineral levels were measured, and feces were evaluated for parasites. Serology tests included bluetongue, brucellosis, bovine respiratory syncitial virus, bovine viral diarrhea/mucosal disease, equine herpesvirus 1, infectious bovine rhinotracheitis, Johne's disease (Mycobacterium paratuberculosis), foot and mouth disease, leptospirosis (17 serovars), parainfluenza-3, and vesicular stomatitis. Blood samples from 20 domestic sheep (Ovis aries) maintained in the same reserve with the guanaco were also collected at the same time for serology tests. No guanaco had positive serologic tests. Sheep were found to have antibody titers to bovine respiratory syncytial virus, Johne's disease, leptospirosis, and parainfluenza-3. There was no apparent difference in external appearance or condition, or statistical difference in blood test values, between the animals that were positive or negative for parasite ova.     

The impact of live animal importation on the epizootiology of foot-and-mouth disease in Saudi Arabia

Saudi Arabia imports annually more than 6 millions live ruminants for slaughter. The majority of these animals are imported from countries where foot-and-mouth disease (FMD) is enzootic. Serotypes of FMD virus not incorporated in the vaccine currently used in Saudi Arabia (e.g. SAT1 and SAT2) are prevalent in some of these exporting countries, and in others, the prevalent serotypes of FMD virus are not routinely typed. The previous exposure of imported animals to field FMD virus in the countries of origin has been confirmed by the detection of precipitating antibodies against virus infection associated (VIA) antigen and neutralizing antibodies against serotypes O, A, C and/or Asia1 of FMD virus in the sera of some imported animals. However, no isolation of FMD carrier virus could be made from 209 proband samples collected from sheep and goats imported from countries where FMD is enzootic. The significance of the obtained results is discussed. Particular emphasis has been placed on the possibility of importing either carrier animals which might act as potential source of infection or subclinically infected animals which might actively excrete FMD virus. In addition recommendations are made to reduce the risks of introducing exotic FMD virus strains to the Kingdom through live animal importation.     

Serogrouping of Bacteroides nodosus isolates from 62 sources in the United States

Bacteroides nodosus isolates from 62 sources in the United States were obtained from sheep with infectious foot diseases. Serotypic analysis of these isolates revealed 21 serotypes (designated I-XXI). These serotypes were compared with British and Australian/New Zealand B nodosus strains by use of reciprocal tube agglutination tests. These tests, as well as the cross-matching tube agglutination tests of the US serotypes, resulted in arranging the US serotypes into 11 serogroups, and comparing these serogroups with their Australian/New Zealand serogroup and British serotype counterparts. Three US serogroups and 1 additional British serotype had little or no relationship to any of the Australian/New Zealand serogroups A-H (the vaccine strains). One or more of these unrelated serogroups were found in 29% of the sources studied. The most frequently found US serotype was serotype XV at 29%. The most frequently found US serogroups were the serogroups analogous to serogroup B (43.5%) and serogroup H (37%); the other serogroups were found in 22.6% or less of the sources studied. Evaluation of 3 sources revealed that multiple serotypes in a single flock are common, multiple serotypes from a single lesion are possible, B nodosus isolates obtained from goats (unlike those from cattle) appear identical to the isolates obtained from sheep, and disease can appear in vaccinated animals, even in a flock that appears to be harboring only a single serogroup-B serotype (the serogroup for which there are 3 strains in the current vaccine).     

Delirium and persistent dyskinesia induced by a lithium-neuroleptic interaction

Vaccination with live cytopathogenic (cp) bovine viral diarrhoea virus (BVDV) is often used for control of this disease. In animals which are persistently infected with noncytopathogenic (ncp) BVDV this can lead to the outbreak of mucosal disease (MD). To simulate vaccination of such animals and to monitor the clinical-virological course after superinfection, nine clinically healthy calves which were persistently viremic were superinfected with different cp BVDV strains. One animal succumbed to early onset MD within three weeks after superinfection. During the observation period of 18 months four animals developed severe clinical signs. While two animals developed late onset MD, the other two had to be euthanized due to clinical signs which could not be related to the superinfecting BVDV. These results indicated that after superinfection or vaccination of persistently infected calves with cp BVDV the probability of developing early and/or late onset MD is significantly increased. The risks arising from uncritical vaccination of herds with unknown virological status in relation with the control of BVDV conforming to the actual official guidelines are discussed.     

The mosquitoes of Yellowstone National Park (Diptera:Culicidae)

Although antibodies to viruses in both the epizootic hemorrhagic disease virus (EHDV) and bluetongue virus (BTV) sero-groups have been reported from white-tailed deer (Odocoileus virginianus) in Texas (USA), there are few reports of hemorrhagic disease (HD) in these populations. To understand the extent and diversity of exposure to the North American EHDV and BTV serotypes in these deer populations, we serologically tested 685 white-tailed deer collected from November 1991 through March 1992 throughout their range in Texas. Overall, 574 (84%) of deer had antibodies to EHDV or BTV. Prevalence estimates varied according to ecological region, from 57% in the Gulf Prairies to 100% in the northwest Edwards Plateau. Based on serum neutralization tests, the deer had evidence of previous exposures to multiple EHDV and BTV serotypes, with evidence of exposure to two to five serotypes detected in each ecological region. The apparent lack of HD in relation to this high antibody prevalence cannot be explained, but may be related to enzootic stability in which a near perfect host-virus relationship exists.     

Level of viral antigen in blood leucocytes from cattle acutely infected with bovine viral diarrhoea virus

Blood samples from 24 calves undergoing experimental acute infection with a non-cytopathogenic bovine viral diarrhoea virus (BVDV) were examined for viral antigen in peripheral leucocytes with an enzyme-linked immunosorbent assay (ELISA), and for presence of virus in blood plasma in a cell culture assay. With the antigen ELISA, low positive values were detected in leucocytes sampled on days 3-4 from two of eight animals inoculated intranasally, and on days 11-13 from three of 16 animals inoculated intramuscularly. From 22 of the animals, low titres of BVDV were detected in blood plasma obtained 2-9 days after inoculation. All other samples, drawn between 2 and 21 days after inoculation, were negative for viral antigen. All animals seroconverted 3-4 weeks after inoculation, some after having shown mild and transient signs of disease.     

Large-scale use of liquid-phase blocking sandwich ELISA for the evaluation of protective immunity against aphthovirus in cattle vaccinated with oil-adjuvanted vaccines in Argentina

Specific serum activity levels against four reference strains of foot-and-mouth disease virus (FMDV) were evaluated from 1634 animals vaccinated with commercial quadrivalent oil vaccines and from 746 unvaccinated, naive animals, using the liquid-phase blocking sandwich ELISA (lpELISA) test. Cows from the FMDV-free area of Argentina were tested for the absence of specific FMDV antibodies (sp FMDV Abs) and those showing lpELISA titres < 1.0 were grouped in lots of 16 animals. They were vaccinated and challenged at 90 days postvaccination (DPV) with one of four virus strains used for vaccine production and control (prototype strains). Serum samples from vaccinated and control cattle were collected 60 and 90 DPV and the level of sp FMDV Abs was determined by lpELISA. Animals were examined for clinical signs of disease. Results show that serum lpELISA titre levels directly correlate with the percentage of protected animals. It was seen that 100, 98, 93 and 87% of the vaccinated cattle with antibody titre levels > or = 2.1 were protected against challenge with serotypes C85, A87,01 Cas and A79, respectively. Evidence is also presented of seroconversion in a sample of 3-5-month-old calves vaccinated in the field, showing lpELISA titres compatible with protection against the four vaccine viruses as long as 150 DPV. Results reported in this paper strongly support the use of the lpELISA test for a rapid and reliable evaluation of the efficacy of FMDV commercial vaccines as well as for the assessment of the immunological status of cattle in FMDV-free and enzootic regions of South America.     

Prolonged recovery of desiccated adenoviral serotypes 5, 8, and 19 from plastic and metal surfaces in vitro

BACKGROUND: Prevention of the spread of epidemic keratoconjunctivitis (EKC) at eye care facilities (doctors' offices, clinics, hospitals) has been a major public health goal for ophthalmology for more than 50 years. The authors explored a potentially contributing attribute of the adenovirus serotypes that cause EKC. Specifically, they investigated the capacity of different clinical and laboratory ocular serotypes (AD8, 19, and 5) to survive for extended periods of time in a desiccated state. METHODS: Twenty microliters containing 2000 plaque-forming units of different ATCC laboratory adenoviral ocular serotypes (AD8, 19, and 5) and clinical isolates (AD8 Cray, AD19 Kowalski, and AD5 McEwen) were inoculated onto 7-mm plastic disks and 6-mm aluminum foil disks and were allowed to completely desiccate. At weekly intervals up to 7 weeks, eight desiccated virus-inoculated plastic or metal disks per serotype were added to tissue culture medium, and the amount of recoverable virus was determined by plaque assay on A549 cells. RESULTS: Ocular adenoviral serotypes AD8, 19, and 5 could be recovered up to 49 days from plastic, and 35 to 49 days from metal. Sufficient virus concentrations (> 100 plaque-forming units/disk) to be clinically infectious were recovered up to 28 days. Differences in recovery among serotypes (AD19 > AD5, AD8) were demonstrated, but laboratory and clinical isolates of the same serotype were usually comparable. CONCLUSIONS: Ocular isolates of adenovirus that cause EKC are much harder than previously suspected, and the capacity to survive in a desiccated state may possibly play some role in office-based mini-epidemics of EKC.     

Biochemical and hormonal changes associated with experimental infection of chicks with infectious bursal disease virus

The inoculation of chicks with the infectious bursal disease (IBD) virus manifested typical clinical signs indicative of IBD viral infection. The inoculated birds seroconverted and showed significantly decreased total protein, lipid and a decrease in the albumin to globulin ratio. A significant increase was seen in the concentration of corticosterone and thyroxine but not in the triiodothyronine level.     

A nested PCR for detection of North American isolates of bluetongue virus based on NS1 genome sequence analysis of BTV-17

A nested polymerase chain reaction (PCR)-based assay, for detection of bluetongue virus (BTV) ribonucleic acid in cell culture and tissue samples, was developed. Two pairs of oligonucleotide primers (BTV1 and BTV4 and BTV2 and BTV3), selected from non-structural protein 1 (NS1) gene of BTV-17, were used for the nested PCR in two amplification steps. First a 826-bp product was amplified using an outer primer pair BTV1 and BTV4. The second amplification, using nested or internal primer pair BTV2 and BTV3, produced a 517-bp PCR product. RNA from North American prototype serotypes 2, 10, 11, 13 and 17, propagated in cell cultures, were detected by this nested PCR-based assay. The nested primers BTV2 and BTV3 increased the sensitivity of the BTV PCR assay, and as little as 0.1 fg of BTV RNA (equivalent to 5 viral particles) could be detected. Amplification products were not detected when the PCR-based assay was applied to RNA from a closely related orbivirus, epizootic hemorrhagic disease virus (EHDV) prototype serotypes 1 and 2; total nucleic acid extracts from uninfected BHK-21 cells; or whole blood from calves and deer that were BTV-seronegative and virus isolation negative. Application of this nested BTV PCR-based assay to clinical samples resulted in detection of BTV RNA from a variety of tissues collected from calves and deer with natural and experimental BTV infections. The described BTV PCR-based assay provides a valuable tool to study the epidemiology of BTV infection in susceptible wild ruminants and domestic livestock.     

Australian-Indonesian collaboration in veterinary arbovirology--a review

Australian-Indonesian collaboration in veterinary development programs has led to significant advances in the study of arboviruses. This paper reviews the resulting knowledge of arboviral infections of livestock in Indonesia. The first recognized arboviral disease of animals in Indonesia was bovine ephemeral fever. Serology indicates that the virus is widespread, as are related rhabdoviruses. Local sheep appear resistant to bluetongue disease, but imported sheep have suffered mortalities. Bluetongue viral serotypes 1, 7, 9, 12, 21 and 23 have been isolated from sentinel cattle; 1, 21 and 23 at widely separate locations. Bluetongue serotype 21 has been isolated from Culicoides spp. Serological reactors to Akabane virus are widespread, as are reactors to the flavivirus group. Japanese encephalitis, isolated from sentinel pigs, is the flavivirus of most veterinary importance but the limit of its easterly distribution is unknown. Many of the arboviruses present in Indonesia are also present in Australia and elsewhere in Asia. Their patterns of mobility among countries in the region are largely undescribed, but there are opportunities for further regional collaboration.     

Antibody responses and protective immunity to recombinant vaccinia virus-expressed bluetongue virus antigens

The role of individual viral proteins in the immune response to bluetongue virus (BTV) is not clearly understood. To investigate the contributions of the outer capsid proteins, VP2 and VP5, and possible interactions between them, these proteins were expressed from recombinant vaccinia viruses either as individual proteins or together in double recombinants, or with the core protein VP7 in a triple recombinant. Comparison of the immunogenicity of the vaccinia expressed proteins with BTV expressed proteins was carried out by inoculation of rabbits and sheep. Each of the recombinants was capable of stimulating an anti-BTV antibody response, although there was a wide range in the level of response between animals and species. Vaccinia-expressed VP2 was poorly immunogenic, particularly in rabbits. VP5, on the whole, stimulated higher ELISA titers in rabbits and sheep and in some animals in both species was able to stimulate virus neutralizing antibodies. When the protective efficacy of VP2 and VP5 was tested in sheep, vaccinia-expressed VP2, VP5 and VP2 + VP5 were protective, with the most consistent protection being in groups immunized with both proteins.     

Seroprevalence of infectious bursal disease virus in free-living wild birds in Japan

Serum samples collected from 739 free-living wild birds of 44 species from Gifu, Mie and Hyogo Prefectures in Japan during the period 1989 to 1997 were tested for antibodies to infectious bursal disease virus (IBDV) serotypes 1 and 2 by a virus neutralization test. Serological evidence of infection with serotypes 1 and 2 was found in 15 (2%) of the sera of 6 species and 36 (4.9%) of the sera of 11 species, respectively. Antibodies to IBDV were detected from both sedentary and migratory species. These findings suggest that free-living wild birds have an important role in the natural history of IBDV. These findings raise the possibility that the IBDV prevalent in the breeding grounds of these birds in other countries could be imported by the migratory species. This is the first report of an extensive serological survey of IBDV in wild birds.     

Application of enzyme-linked immunosorbent assay for epidemiological studies of diseases of livestock in the tropics of Mexico

This study was conducted at the Centre for Research, Teaching and Extension in Tropical Livestock (Centro de Investigación, Ense?anza y Extensión en Ganadería Tropical) of the Faculty of Veterinary Medicine of the National Autonomous University of Mexico. During the latter part of 1986 and throughout 1988 and 1989, the herd of Holstein x zebu cattle at the University was tested for IgG antibodies to twenty-one viral, bacterial, rickettsial and parasitic agents. Antigens prepared from twenty infectious disease agents were used as the solid phase in an enzyme-linked immunosorbent assay, and the agar gel immunodiffusion procedure was used to test for antibodies against bovine leukaemia virus. The prevalence of IgG antibodies was high (> 50%) for bluetongue virus, Anaplasma marginale and Mycoplasma bovis. Antibodies to Brucella abortus were absent and antibodies against bovine virus diarrhoea virus and infectious bovine rhinotracheitis virus showed a very low prevalence (< 5%). Antibodies to fifteen other antigens showed intermediate prevalence (15-46%). Antibodies to Campylobacter fetus, A. marginale, bluetongue virus, bovine leukaemia virus and Haemophilus somnus displayed seasonal variations. Levels of antibody to bovine leukaemia virus, M. bovis and Listeria monocytogenes exhibited increasing secular trends while antibodies to bovine virus diarrhoea virus and C. fetus showed declining trends. Prevalence of antibodies increased with the age of animals tested. No consistent difference in antibody prevalence was found between three genotypic groups examined.     

Computer-assisted myocardial thickening analysis of gated MIBI SPECT images

A total number of 2125 serum samples collected from indigenous sheep and goats and locally-raised cattle from different localities in Saudi Arabia, were screened for Akabane virus-neutralizing antibodies. The overall prevalence of antibodies was 32% in the animals examined. However the prevalence in cattle was 49%, while in sheep and goats it was 17%. No clinical cases of Akabane have been confirmed so far in Saudi Arabia; but virus isolation attempts are ongoing.     

Abattoir survey of sheep and goats in The Gambia

An abattoir survey of sheep and goats was carried out in The Gambia for one year. A total of 1248 goats and 438 sheep, predominantly young females, were slaughtered and sampled. Sixty per cent of the females of both species were pregnant. There were no significant differences between the dressing percentages of different breeds and age groups. Sex and stage of pregnancy had a significant influence on carcase yields in both species. In goats the highest carcase yields were obtained during the early dry season. Most of the animals were clinically healthy and there were few pathological findings postmortem. In both species, there was a seasonal fluctuation of packed cell volume (PCV), with a minimum during the rains, and although the prevalence of trypanosomiasis was low it reduced the PCV. Faecal egg counts of Trichostrongylidae were highest during the rainy season and goats had higher faecal egg and coccidial oocyst counts than sheep. In sheep, a breed difference was observed for PCV and an age difference for egg excretion. The peak or higher rates of egg excretion occurred during the rains in both species. The immune status against peste des petits ruminants was significantly lower in goats (39 per cent) than in sheep (49.5 per cent). Antibodies against bluetongue virus were found in 62.6 per cent of goats and 55.8 per cent of sheep.     

Acute arthritis of cats associated with feline calicivirus infection

Twelve specific pathogen-free cats were infected either by intra-articular inoculation or by contact exposure to one of two strains of feline calicivirus (FCV), either F65, a field strain originating from an outbreak of lameness in a group of cats, or a vaccine strain. Following either route of exposure, both strains induced signs typical of FCV infection including oral and nasal ulceration, conjunctivitis and ocular discharge. These signs were of equal severity for both virus strains, but overall, following either route of infection, F65 induced more severe disease than the vaccine strain, with marked pyrexia, lethargy and lameness. Vaccine virus only induced a relatively mild lameness following intra-articular inoculation. Gross pathological and histopathological lesions were seen in some of the joints, but again changes were more severe in the F65-exposed cats. Virus was isolated from both normal and affected joints from both groups of F65-exposed cats, and from a joint from each cat inoculated intra-articularly with vaccine virus. Mild transient lameness was also seen in one of two control cats inoculated intra-articularly, but no pathological changes were seen or virus isolated from joints. A cDNA probe used in RNA dot blot hybridisation experiments was found to be specific and more sensitive than virus isolation in detecting FCV in selected tissues. This may be useful in future studies on the pathogenesis of FCV disease and in studies on viral persistence in FCV carriers.     

Evaluation of bluetongue virus diagnostic tests in free-ranging bighorn sheep

Five bluetongue virus (BTV) diagnostic tests were evaluated for use in free-ranging bighorn sheep. We sampled one bighorn sheep population four times between 1989 and 1995. The tests evaluated included virus isolation (VI), polymerase-chain reaction (PCR), serum neutralization (SN), agar-gel immunodiffusion (AGID), and competitive enzyme-linked immunosorbent assay (c-ELISA). The c-ELISA, AGID and SN tests had high levels of agreement in determining serogroup exposure in bighorn sheep. We used maximum-likelihood algorithms to estimate the parameters of each diagnostic test used. Although the c-ELISA and AGID had high sensitivity and specificity, the SN had perfect specificity but lower apparent sensitivity. Due to the potential of cross-reactions among multiple serotypes, results of the SN must be interpreted with caution when assessing serotype exposure in an area where multiple serotypes are endemic. The PCR assay delineated convalescent antibody titers from more-recent infections, and consequently, was pivotal in distinguishing a different exposure pattern between the bighorn sheep and cattle in an adjacent herd. Based on an increasing seroprevalence (50% to 100%), BTV circulated through this bighorn sheep population between 1989 and 1993. This increase in seroprevalence coincided with a bighorn die-off due to BTV infection in June, 1991. An adjacent cattle herd was sampled in 1995 for comparison. The bighorn sheep and adjacent cattle had different patterns of exposure to BTV between 1994 and 1995. There was no evidence that BTV circulated through the bighorn sheep population from 1994 to 1995. In 1995, seroprevalence to BTV decreased to 72%, none of yearling bighorn was seropositive, and all of the 39 bighorn sheep were PCR-negative. In contrast, all adult cattle were seropositive to BTV by c-ELISA and SN, and 4 of the calves were seropositive; 11 of the 24 cattle were PCR-positive, including all five calves. Overall, the pattern of temporal herd immunity in the bighorn sheep appeared to follow a classic epidemic curve, with the appearance and subsequent disappearance of herd immunity coinciding with the 1991 die-off in this population. As low levels of herd immunity and high proportions of susceptible animals are key factors in the development of epidemics, this population of bighorn sheep may be at increased risk for a BTV epidemic in the future.     

Encephalitis in ferrets caused by a nonproductive strain of measles virus (D.R.) isolated from patient with subacute sclerosing panencephalitis

A nonproductive, syncytiogenic strain (D.R.) of measles virus, isolated from a patient with subacute sclerosing panencephalitis (SSPE), was inoculated intracerebrally into ferrets in an attempt to induce subacute encephalitis. Inoculation of freeze-thawed syncytia before immunization was the least effective procedure, and inoculation of live syncytia after immunization with measles virus vaccine was the most effective procedure, for induction of subacute or persistent subclinical encephalitis in the animals. After the latter procedure three of five ferrets developed subacute or subclinical encephalitis, whereas ferrets inoculated with live syncytia without prior immunization consistently contracted acute fatal encephalitis in one to two weeks. The subacute encephalitis in ferrets was characterized by high titers of antibody to measles virus in serum. At the time of sacrifice 1.25, 4.5, or 8.0 months after inoculation, brains of the ferrets showed histologic lesions similar to those characteristic of SSPE, and nonproductive syncytiogenic measles virus was recovered from the brains of two of the animals. All three ferrets had greatly increased concentrations of gamma-globulin in their brains and high levels of neutralizing and hemagglutination-inhibiting antibodies to measles virus. Only one of these animals developed clinical signs 1.25 months after inoculation.