Development of specific in vitro lymphocyte stimulation responses in horses infected with equine infectious anaemia virus

The response of lymphocytes, obtained from the peripheral blood of horses infected with equine infectious anaemia (EIA) virus, to the antigen of EIA virus was studied by measurement of uptake of tritiated thymidine in lymphocyte cultures. Lymphocyte response increased shortly after the primary infection. It decreased during the asymptomatic stage, but again increased to a high level after a recurrence of signs. Lymphocytes from horses with a long asymptomatic stage rarely showed a positive reaction. In chronic infections with EIA virus, however, horses occasionally showed a spontaneous temporary increase in lymphocyte response to EIA virus antigen without any other signs of EIA. Lymphocytes from horses infected with different strains of EIA virus were stimulated by both homologous and heterologous EIA virus antigen. The possible relationship between the stimulation of lymphocytes and cell-mediated immunity in EIA is discussed.     

Serological evidence of equine herpesvirus type 1 (EHV-1) activity in polo horses in Nigeria.

Serological evidence of Equine Herpes virus type 1 (EHV-1) activity in Polo horses in Nigeria is reported for the first time. Eighty-two percent of horses tested with known antigen had precipitating antibodies to EHV-1 while 43% of sera tested against antigen prepared from nasal discharges were positive suggesting that the virus was being excreted in the nasal discharges and probably acting as a source of infection for incontact animals as occurs in on-going acute infections. The result of this study indicates a high prevalence of EHV-1 activity among Polo horses in Nigeria and demonstrates the ubiquitous distribution of the virus in a country that has not been previously investigated.     

A SURVEY OF ANTIBODY TO AINO VIRUS IN CATTLE AND OTHER SPECIES IN AUSTRALIA

SUMMARY A serological survey of healthy cattle in Australia showed that antibodies to Aino virus were present in serums from cattle in northern Australia and down the east coast as far as central New South Wales in 1975, 1976 and 1977, but occurred with a lower frequency than antibodies to Akabane virus. in contrast to the findings with Akabane virus, no neutralising antibodies to Aino virus were detected in serums from camels, dogs or horses. Antibodies to both viruses were detected in buffaloes and sheep, but not in humans or any of the Australian indigenous species so far tested. All positive serums originated from within the known range of Culicoides brevitarsis.     

An enzyme-linked immunosorbent assay (ELISA) test for the diagnosis of equine infectious anemia in Brazil

An enzyme-linked immunosorbent assay (ELISA) test was developed for the detection of specific antibodies against the unique infectious anemia (EIA) virus in equine sera. The ELISA test was faster and more sensitive when compared with the classic test of agar gel immunodiffusion (AGID). A total of 200 sera were tested: 100 from negative horses and 100 from positive horses by AGID. The ELISA test showed 92 horse sera negative and 100 horse sera positive by AGID with values of optical density (OD) less than 0.139 and higher than 0.139, respectively. Eight horse sera were negative by AGID and higher than 0.139 by ELISA. Six of these became AGID positive also when re-tested 30 days later, and two were of the horses that showed clinical signs of EIA and died before re-testing.     

Equine antibody to bovine serum induced by several equine vaccines as a source of extraneous precipitin lines in the agar gel immunodiffusion test for equine infectious anemia.

Precipitin lines not associated with equine infectious anemia (EIA) were observed in routine agar gel immunodiffusion (AGID) testing for the infection. The serums which produced these lines were obtained from horses which had been given multiple vaccinations with commercially available cell culture-origin equine virus vaccines as part of a comprehensive herd health program. The lines formed against cell culture-derived, but not spleen-derived EIA viral antigens. Investigation revealed that bovine serum proteins in the vaccines induced precipitating antibodies which reacted with bovine serum proteins in cell culture-derived antigens. A vaccination trial, utilizing 4 commercially available vaccines in various combinations, indicated that as few as 2 vaccinations could induce AGID-detectable antibodies to bovine serum proteins in individual ponies. These antibodies were very transitory, usually lasting no longer than a week. Some horses, however, which had been given 4 vaccinations developed similar antibodies which persisted 3 months beyond the last vaccination. The extraneous precipitin lines produced by these antibodies in the AGID test for EIA were readily distinguished from true EIA-associated reactions and did not result in false-positive interpretations of the test. However, heavy percipitin lines due to strong antibovine serum activity did mask weakly positive EIA reactions.     

Use of enzyme immunoassay in a serological survey of leptospirosis in sheep

A total of 731 serums, all from Merino rams from 20 farms, were tested for antibodies against Leptospira interrogans serovars hardjo, pomona and tarassovi using the microscopic agglutination test (MAT). The enzyme immunoassay (EIA) technique was used to test all serums for IgM and IgG antibodies to serovar hardjo. In the MAT, reactions to serovar hardjo were most common with 236 rams (32.3%) reacting at 1/100 or greater. Only 1.9% of serums reacted against serovar tarassovi and 1.1% against serovar pomona. The percentage of sheep with positive MAT reactions to serovar hardjo ranged from 0 0 to 94.9 between farms. When using EIA, 46 (6.2%) of the serums were positive for IgM antibody and 246 (33.6%) were positive for IgG antibody. Correlation of the EIA for detection of IgG antibody with the MAT was good. The EIA detection of IgG antibody was considered to be a good alternative test to the MAT for epidemiological studies in sheep.     

A microplate enzyme immunoassay for detecting and measuring antibodies to Babesia bovis in cattle serum

A microplate enzyme immunoassay (EIA) is described for measuring IgG antibody to Babesia bovis in cattle serum. B. Bovis antibody status (whether positive or negative) and the amount of B. Bovis antibody (EIA score), were measured by comparison with reference serums. The EIA was shown to be specific for B. Bovis, and EIA score correlated well with EIA titre. Comparison of EIA with the Indirect Fluorescent Antibody Test (IFAT) showed more than 95% agreement between the methods and disagreement in only 1.6% of serum samples tested. The remaining 3.2% were positive by EIA and suspected positive by IFAT. The EIA was shown, by titrating positive serums, to be more sensitive than IFAT, which explained its tendency to detect more positive serums than IFAT. EIA detected B. bovis antibody in experimentally infected cattle by day 14 post infection (pi) and for at least 268 days pi. EIA score for B. bovis antibody in immune cattle increased significantly (p less than 0.05) following heterologous strain challenge.     

Detection of equine infectious anemia virus in horse leukocyte cultures derived from horses in various stages of equine infectious anemia viral infection

The enzyme-linked immunosorbent assay (ELISA) antigen-positive and agar-gel immunodiffusion test (AGID)-negative horses do not have infective equine infectious anemia (EIA) virus. The ELISA testing of horse leukocyte culture (HLC) supernatants did detect EIA virus in a HLC that was infected with the Wyoming strain of EIA virus and in HLC derived from horses in febrile, acute, or subacute stages of EIA infection. In supernatants of HLC derived from chronic and inapparent carrier horses, EIA virus was not detected with ELISA. Direct fluorescent antibody tests detected EIA virus in HLC infected with 10(6)TCID50 of the Wyoming strain of EIA virus and in 50% of the HLC from febrile acute or subacute horses. The direct fluorescent antibody testing of HLC derived from chronic and inapparent carrier horses did not detect cell-associated EIA virus. The pony inoculation test proved to be the most reliable and accurate method for detecting infective EIA virus in horses in various stages of EIA infection and accurately correlated with the AGID test.     

Evaluation of a serological test system for the diagnosis of natural Echinococcus granulosus infection in dogs using E. granulosus protoscolex and oncosphere antigens

Serum antibody responses in feral or domesticated dogs naturally infected with Echinococcus granulosus or/and other common helminths were examined in an enzyme-linked immunosorbent assay (ELISA) using antigens prepared from E. granulosus protoscoleces or oncospheres. The ELISA using the protoscolex antigen was optimised with serums from experimental dogs monospecifically infected with E. granulosus or other helminth parasites, and helminth-free dogs. Anti-protoscolex antibody was detected in 16 of 22 (72.7%) serums from feral dogs with E. granulosus burdens ranging from 300 to 302,600 worms per dog. Seven serums from feral dogs which did not harbour E. granulosus at autopsy but which originated from an endemic hydatid region were tested using protoscolex antigen, and 1 serum gave a positive reaction. One hundred and two serums from dogs known never to have been infected with E. granulosus all gave negative reactions to protoscolex antigen. The sensitivity of the ELISA test proved to be superior to that which has been achieved by arecoline purging as a method of diagnosis for E. granulosus infection in dogs. For use of the assay in hydatid control or eradication campaigns, its sensitivity can be increased by choosing a lower absorbance discrimination value above which serums are regarded as having positive reactions. However, this does introduce positive reactions of some serums from dogs infected with helminths other than E. granulosus. In further development of the assay, use of defined recombinant antigens may improve both sensitivity and specificity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunodiffusion test for ovine progressive pneumonia.

An agar gel immunodiffusion test was developed to detect precipitating antibody against ovine progressive pneumonia (OPP) virus. The test was conducted in plastic petri dishes containing 6 ml of 1% purified agar in tris buffer and 8% sodium chloride. Wells for serum and antigen were 8 mm in diameter and were cut in a hexagonal pattern 3 cm from a central well. Tests were read at 24 and 48 hours. Soluble antigen for the test consisted of concentrated nutrient medium removed every 2 weeks from a cell culture persistently infected with isolate WLC 1 of OPP virus. Specificity of results was verified by testing serums from experimentally exposed sheep and appropriate controls. Two lines of precipitate formed with some serums from experimentally inoculated sheep. Serums taken soon after exposure of sheep to the virus and those taken 3 to 4 years after exposure frequently formed only 1 line of precipitate. Of 37 lambs inoculated with OPP virus, 25% of those tested were positive by postinoculation (PI) month 1, 79% of those tested were positive by PI month 3, and all of those tested were positive by PI month 6. The test appears adequate to detect exposure of sheep to OPP virus.     

Enzyme-linked immunosorbent assay for diagnosis of equine infectious anemia

An enzyme-linked immunosorbent assay (ELISA) was elaborated for the detection of specific antibody to equine infectious anemia (EIA) antigen. Sera from horses experimentally infected with EIA virus were assayed by ELISA, complement fixation (CF) and immunodiffusion (ID) tests for antibody to EIA antigen. The ELISA technique was found to be much more sensitive than CF and ID tests. In addition, EIA specific antibody could be detected by ELISA at an earlier stage of infection than by CF or ID techniques. The applicability of the technique to diagnosis of EIA is discussed.     

Detection of cold-adapted vaccine-strain influenza virus using two commercial assays

Because of the contagious nature of influenza virus it is necessary to identify infected individuals after the virus is introduced into a population. The aim of this study was to characterise influenza virus detection with commercially available assays after intranasal vaccinating horses with cold-adapted influenza virus. Seven horses were vaccinated and placed with 3 unvaccinated horses. Nasal secretion samples were evaluated using 2 antigen detection assays. All 10 horses were positive in the Flu OIA assay during the study period, but only one horse was positive on one sample using the Directigen Flu A assay. Horses were most likely to be positive during the first 3 days following vaccination, and several horses were intermittently positive for several days after this. Obtaining positive test results from nonvaccinated, incontact horses suggests they became infected with vaccine-strain virus that was shed by vaccinated horses. These results are important for the correct interpretation of influenza antigen detection tests in situations when this modified-live intranasal vaccine has been used.     

Virus Diseases of The Respiratory Tract of Cats

Feline rhinotracheitis (FR) virus antigenically similar to the only known antigenic type of FR virus was isolated from nasal and pharyngeal swabs obtained from four cats with respiratory disease. The incidence of FR virus neutralising antibody in 45 cat serums obtained in the environs of Melbourne was approximately 50%. Data is presented that shows that the incidence of neutralising antibody to 2 antigenically distinct feline picornaviruses was 96% and 87% respectively. Complement fixing antibody to the Chlamydia (Psittacosis) group antigen was not found in any of 35 serums tested; it is suggested that the significance of Chlamydia sp as a cause of feline pneumonitis may require reappraisal.     

A SEROLOGICAL SURVEY FOR BLUETONGUE VIRUS ANTIBODY IN WESTERN AUSTRALIA

A serological survey was carried out to detect specific (serotype 20) and a group bluetongue virus antibody in cattle and sheep serums collected in Western Australia during the period January 1 1978 to June 30 1979. Of 18,849 cattle serums examined by the gel diffusion precipitin test (GDPT), 9.7% were positive and 6.1% gave doubtful results. All 1949 sheep serums tested were negative. Precipitin antibody was demonstrated in 22.5% of serums from Kimberley cattle and 3.6% of cattle serums from the Northwest. Serums collected from cattle in the South were consistently negative in GDPT. When 915 serums that reacted in the GDPT were further tested by the complement fixation test (CFT), 164 were positive. The percentage of CFT positive serums increased as the GDPT reaction became stronger. 2467 serums collected from cattle in Kimberley and Northwest areas and tested by the CFT, 175 (7.1%) were positive. These 175 positive serums were also examined by GDPT and 164 doubtful or positive reactions were obtained. The virus neutralisation (VNT) using serotype 20 virus was carried out on 3804 serums, including all serums that reacted in the GDPT, and 57 were positive. When the VNT positive serums were examined in the other 2 tests, 47 serums were either positive or doubtful in the GDPT and 8 were positive in the CFT. The presence of bluetongue virus group antibody in cattle serums closely followed the suggested distribution pattern of Culicoides brevitarsis but specific serotype 20 neutralising antibody was limited to cattle serums from stations situated north of latitude 17 degrees S in an area of mean annual rainfall higher than 700 mm.     

Detection of bovine respiratory syncytial virus using a heterologous antigen-capture enzyme immunoassay

Based on the marked antigenic similarities that exist between antigens of the human and bovine strains of respiratory syncytial virus (RSV), an enzyme immunoassay (EIA) designed to detect human RSV was used to detect bovine RSV. The commercial test kit (RSV EIA) consists of a solid phase (beads) coated with a capture antiserum prepared against the Long strain of human RSV. The RSV EIA test was compared with the method of inoculation of cell cultures and fluorescent antibody (FA) staining of lung tissue for the detection of bovine RSV. Using a cell culture-propagated stock of strain 375 of bovine RSV, the threshold of sensitivity of the EIA test for the cattle strain of RSV was determined to be less than or equal to 10(2.3) CCID50/ml. In addition, RSV EIA detected the bovine RSV in nasal samples obtained from 3 experimentally inoculated cattle. The RSV EIA exhibited a sensitivity of greater than or equal to 80% during the period that shedding of infectious virus took place. All of the bovine RSV FA-positive lung samples (n = 37) were positive by the RSV EIA. Twenty-six of the remaining 214 bovine RSV FA-negative lung samples were positive by the RSV EIA. The RSV EIA was also used to test 137 nasal swabs obtained from cases of bovine respiratory disease. Of these, 38 tested positive by RSV EIA. All samples that tested positive by EIA were confirmed by blocking assays using hyperimmune serum anti-bovine RSV and a pool of monoclonal antibodies specific for that virus.     

Development and evaluation of a microimmunodiffusion test for detection of antibodies to pseudorabies virus in swine serum.

A microimmunodiffusion test (MIDT) was developed for the detection of pseudorabies virus (PRV) antibodies in swine serum. The optimal medium for the MIDT was determined to contain 0.69% agarose in 0.05 M tris buffer (pH 7.2) with 0.025% sodium azide and no NaCl. The PRV antigen prepared by (NH4)2SO4 precipitation of viral fluids (42.5 g/100 ml), dialyzed against distilled water, and concentrated to approximately 100-fold of the original volume with polyethylene glycol (mol wt 20,000) provided a good reproducible antigen. The sensitivity of the MIDT was compared with the microtitration procedure of the virus-neutralization (VN) test by assaying 2,203 swine serums for PRV antibodies. An equal percentage of serums was positive in both tests; 419 had VN titers of greater than or equal to 4, and 421 were MIDT positive. Serums (314) that had VN titers of greater than or equal to 16 were all positive by the MIDT. Of serum samples with a VN titer of 8 (53), 50 were MIDT positive, a 94% correlation, and of 52 serums that had VN titers of 4, 36 were MIDT positive, a 69% correlation. In addition, 8 serums that had titer of less than 4 by VN test were positive by MIDT. Seventy-one serum samples were too cytotoxic, markedly hemolyzed, or contaminated to evaluate properly in the VN test; of these serums, 13 were MIDT positive. The MIDT is an accurate, rapid, economical, and sensitive diagnostic test for the detection of PRV antibodies in swine serums.     

Equine Infectious Anemia: Active Surveillance in Central Italy 2007-2009

Equine infectious anemia (EIA) is an infectious and potentially fatal viral disease of equids. EIA virus is usually transmitted from horse to horse by large biting insects, such as horseflies. The aim of this study was to evaluate the results of a national surveillance plan from 2007 to 2009 and evaluate the potential risk factors of EIA in horse populations in central Italy. In 2007, 18 of 6,773; in 2008, 30 of 7,940; and in 2009, 21 of 11,666 equines tested were seropositive for EIA. No statistical association was found between location or sex and the diagnosis of EIA. The seroprevalence rate (2007-2008-2009) was higher among older equids (older than 6 years) than among young (3 months to 5 years old) (P < .05). Likewise, the seroprevalence rate (2007-2008-2009) was higher among mules than among other horses (P < .05). Until 2007, the national equine register did not exist in Italy; therefore, it was difficult to measure the percentage of untested horses that presented a real but unquantified risk for continued EIA virus transmission. By introducing new laws governing the control and conducting active surveillance for EIA, it has been possible, in Italy, to develop a firm foundation of knowledge concerning the persistence and transmission of EIA and the risk factors and to better control the spread of this infection in horses.     

A propagating epizootic of equine infectious anemia on a horse farm

An epizootic of equine infectious anemia (EIA) involved 35 horses on a farm in south Georgia. During a 126-day period, 21 of these horses became seropositive for EIA. After the initial diagnosis in July, the horses were tested every 7 to 10 days. At least one additional horse was found to be seropositive on each testing day. As soon as they were determined to be seropositive, the horses were removed from the herd and sent to slaughter. The removal of the seropositive horses, however, did not stop the epizootic. We believe the initial infection was from a 7-year-old stallion that recently had been purchased or from 1 of 2 mares that were seropositive for EIA on the first test. None of the horses had been tested for EIA at the time of purchase or within 60 days before the epizootic.     

THE USE OF A FORMOLISED ANTIGEN AS A SCREENING TEST FOR LEPTOSPIRAL ANTIBODIES IN HORSES

Six hundred and thirty-two equine serums were examined for the presence of leptospiral antibodies. A positive reaction to one or more antigenic pools of a formolised leptospiral antigen (used in the rapid macroscopic slide agglutination test) was recorded in 41% of cases.
One hundred samples were tested with 5 formolised antigen pools and 19 live antigens (by the microscopic agglutination test). Of 20 samples in which the live antigen test suggested leptospiral infection with serotypes known to occur in the region, 17 (85%) were confirmed with the formolised antigens.
When the results of both tests were compared, there was agreement in 42 samples (30 positive and 12 negative). Forty-one samples produced equivocal results and 17 gave doubtful reactions to the formolised antigens, 15 of which were negative to the live antigens.
Dilution of the serums 1:1 with normal saline or heat inactivation had no effect in increasing the specificity between the formolised and live antigens. Agreement between operators in the use of the formolised antigen was poor. It is concluded that the formolised antigen has too wide a divergence to be of use for screening horse serums.     

Antibodies to bluetongue viruses in animals imported into United States zoological gardens.

Three hundred forty-five serum samples from 30 zoological animal species which had been imported into the United States were examined retrospectively for the presence of antibody to bluetongue viruses. Ninety eight (28.4%) were positive for antibody to bluetongue group antigen by the bluetongue agar gel immunodiffusion test. Bluetongue antibodies, most of which were against serotypes exotic to the United States, were detected in 13 animal species from Africa not previously reported to be infected by bluetongue virus. The lack of virus neutralizing antibody to any of the 20 known bluetongue virus types in four of the 28 positive serums studied may indicate the existence of new bluetongue virus serotypes, cross reactions with other orbiviruses or a more rapid decline of neutralizing than precipitating antibody. The possibility of recrudescence of bluetongue virus infection from some inapparently infected zoological animals and existence of a known bluetongue vector (Culicoides variipennis) in the United States would suggest that further assessment of bluetongue in zoological animals be made.