Duration of colostral antibodies to bovine leukemia virus by two serologic tests

The duration of detectable colostral antibodies to the glycoprotein antigen of bovine leukemia virus was studied in calves which were born to bovine leukemia virus-infected cows, but showed no serologic evidence of prenatal infection. Colostral antibodies detectable by an agar-gel immunodiffusion test (AGIT) persisted for less than 1 month to 6 months (mean 2.9 months) in the 139 calves examined. Colostral antibodies were detectable 1 to 5 months longer by radioimmunoprecipitation assay than by the AGIT in 22 of the 24 calves studied comparatively. The mean duration of colostral antibodies in those 24 calves was 3.8 months (min-max, 2 to 6 months) for the AGIT and 6.0 months (min-max, 4 to 9 months) for the radioimmunoprecipitation assay.     

Radioimmunologic detection of antibodies to bovine leukemia virus

The radioimmunologic assay (RIA) was elaborated for a demonstration of serum antibodies to bovine leukosis virus. The procedure makes use of the viral antigen bond to the fixed phase of a polystyrene carrier. The method was compared with the ELISA method and pseudoneutralizing and immunodiffusion tests. High congruence of the results of the RIA and ELISA methods was achieved, making 95%. The RIA method is more sensitive than the immunodiffusion test.     

Comparison and evaluation of four serological tests for detection of antibodies to Aujeszky's disease virus

Four methods used for the detection of antibodies to Aujeszky's disease virus in porcine sera were compared. The serum neutralisation test, countercurrent immunoelectrophoresis test, micro-immunodiffusion test and the enzyme-linked immunosorbent assay (Elisa) were assessed with particular regard to their use in large scale screening of porcine serum samples. The Elisa test was found to be the most suitable on the grounds of sensitivity, speed and cost.     

Comparison of the commercial agar-gel immunodiffusion test and radioimmunoprecipitation assay for detection of antibodies to bovine leukemia virus

In a double-blind study, the commercial agar-gel immunodiffusion test (AGID) was compared with a radioimmunoprecipitation assay (RIA) performed with glycoprotein (gp) antigen for detection of antibodies to bovine leukemia virus. Of 240 sera tested, 115 were from adult cows and 125 were from precolostral calves. Most adult animals were tested within 1 week of parturition. Sera from 74 cattle were positive and sera from 166 cattle were negative by gp RIA. Sensitivity of the AGID, compared with the gp RIA, was 85.1% when the test was read at 48 hours and was 94.6% when read at 72 hours. Specificity increased from 92.2% at 48 hours to 96.4% at 72 hours. Reading the AGID again at 72 hours also clarified most reactions that were questionable at 48 hours due to a haze around the test serum well. Of 3 RIA-positive precolostral calf sera, 2 were AGID-negative and 1 had a questionable reaction by the AGID at 48 hours. Of 5 RIA-positive sera that were AGID-negative at 48 hours, 2 were precolostral calves and 3 were cows tested at parturition. Of 166 RIA-negative reactions, none was falsely positive by the AGID at 48 or at 72 hours.     

Comparison and evaluation of three serological tests for detection of antibodies to infectious bovine rhinotracheitis

Abstract

AIMS: To retrospectively describe clinical features of dogs that were presented to a small animal clinic between 2003–10 with macroscopic haematuria, and investigate whether signalment of the dog and severity and duration of the haematuria at admission were associated with specific aetiologies.

METHODS: Medical records were evaluated of 162 dogs with macroscopic haematuria admitted to a University-based small animal clinic in Thessaloniki, Greece, from January 2003 to December 2010. The inclusion criteria were discolouration of the urine sediment combined with abnormal numbers of erythrocytes, when examined microscopically. Data collected from the medical records included signalment, severity, frequency and duration of haematuria, and diagnosis.

RESULTS: Between January 2007 and December 2010, 8,893 dogs were admitted to the clinic; of these 99 (1.1%) were admitted with haematuria. Of the 162 dogs with records of haematuria, 80 (49.4%) were aged between 5.1–10 years, presented with acute (96/162; 59.3%), constant (99/162; 61.1%) and mild/moderate (150/162; 92.6%) haematuria. Of 147 dogs with a recorded diagnosis, the commonest diagnoses were urinary tract infection (UTI, 42/147; 28.6%), urolithiasis (38/147; 25.9%), prostatic disease (25/147; 17.0%) and urinary tumours (13/147; 8.8%). The prevalence of UTI was higher in female (22/56; 39%) than male (20/91; 22%) dogs, and in medium sized (22/52; 42%) than small (6/40; 15%) dogs. Urolithiasis was most prevalent in small (21/40; 52.5%) dogs, and all dogs with urolithiasis presented with mild/moderate haematuria. The prevalence of prostatic disease was highest in large (11/46; 24%) and giant (3/9; 33%) sized dogs and in dogs aged >10 years (8/30; 27%).

CONCLUSIONS AND CLINICAL RELEVANCE: In this retrospective study from one small animal clinic, UTI, urolithiasis, prostatic disease and urinary tumours predominated among the causes of canine haematuria. The consideration of sex, age, and size of the dog and characteristics of haematuria were found to be useful parameters when forming the list of differential diagnoses.     

Detection of bovine leukemia virus antibodies using the cytotoxicity test in comparison with other serologic methods

In ninety-five serum samples taken in a herd of five-year to seven-year cattle that was heavily infected by bovine leukosis virus, the four serological assays were used for demonstration of the antibodies to bovine leukosis virus; cytotoxic test, immunodiffusion test in agar-agar, immunoenzymatic test and serum neutralizing test. The serum neutralizing test was found to be the most sensitive: further seven positive reagents were diagnosed in comparison with immunoenzymatic test; cytotoxic and immunodiffusion tests in agar-agar have the lowest sensitivity and the results of these tests are almost identical. It was found out in forty titrated samples that serum neutralizing test was by as much as 20 times more sensitive than immunoenzymatic test, the latter being about 50 times more sensitive than cytotoxic and immunodiffusion tests.     

Comparison of four RT-PCR assays for detection of bovine respiratory syncytial virus

RT-PCR assays for detection of BRSV, based on four different sets of primers were optimized and evaluated for their sensitivity and specificity. Primers used in this study were specific for genes encoding three BRSV proteins, nucleoprotein N and glycoproteins F and G. Our results indicated that RT-PCR with primers B7:B8 for G protein was the most efficient in detecting BRSV. Starters B7:B8 reacted specifically only with BRSV strains, no cross-reaction with other closely related viruses to BRSV was observed. RT-PCR sensitivity was also high and amounted to 10(1.66) TCID50. Starters for F and N genes of BRSV were not sufficiently specific and cross-reacted with RNA of HRSV. RT-PCR with primers for the genes F and N of BRSV was characterized by a lower sensitivity than RT-PCR with primers B7:B8. In conclusion, RT-PCR specific to a sequence of glycoprotein G gene, seemed to be the most useful for BRSV detection.     

Decay of colostral antibodies to bovine leukemia virus with application to detection of calfhood infection

An estimated weighted-regression method was used to describe the decay of colostral bovine leukemia virus (BLV) antibodies in the calf, as measured by agar-gel immunodiffusion with glycoprotein antigen. The prediction equation, based on 473 observations from 130 animals, was log10 inverse titer = 1.29 -0.012 age (days). The half-life of BLV antibodies was estimated to be 25.8 days. Ages at which colostral antibodies were last detected were between 51 and 187 days. Normal limits of antibody decay were estimated and used to identify virus-induced active antibodies in calves during the colostral antibody period. Calves known to be infected were identified between 2 and 180 days of age, using 95% limits. Application of this procedure for the early serologic detection of BLV-infected calves in eradication or control programs is discussed.     

Comparison of culling rates among dairy cows grouped on the basis of serologic status for bovine leukemia virus

OBJECTIVE: To determine the association between serologic status for bovine leukemia virus (BLV) and culling rates by use of survival times in a commercial Holstein dairy herd. DESIGN: Longitudinal study. ANIMALS: 593 milking cows. PROCEDURE: Cattle were tested for antibodies against BLV by use of agar gel immunodiffusion or ELISA 4 times each year from 1989 to 1993 and then annually through 1999. Dates of birth, first calving, and culling or death were obtained from Dairy Herd Improvement Association records. Most cows were enrolled in the study on the date of first calving. Survival times were compared among seropositive, seronegative, and seroconverted cows with the Kaplan-Meier method and a Cox regression model stratified on the basis of year of birth. RESULTS: Complete records were available for 593 of 685 (87%) cattle in the dairy herd during the study period. Median survival time for all cows was 31.7 months. Survival times, which correspond to cull rates, did not differ significantly between seropositive and seronegative cattle, whereas cattle that seroconverted during the study had a significantly longer survival time. Year of birth was positively and significantly associated with survival time. CONCLUSIONS AND CLINICAL RELEVANCE: BLV serologic status was not associated with cull rate as measured by survival time in this dairy herd. This finding is in contrast to results of studies that used survival analysis techniques; our results may influence management decisions concerning BLV.     

Comparison of four tests to evaluate the reactivity of rabbit sera against envelope or Gag-related proteins of bovine leukemia virus (BLV)

Bovine leukemia virus (BLV) has a long latency period during which animals are inapparently infected, may spread the disease, and are only detected by serological techniques or by the most cumbersome molecular biology techniques. We have compared techniques for detecting either total antibodies (ELISA), anti-p24 and Gag-related proteins (Western blot), or anti-gp51 (agar gel immunodiffusion, AGID, and syncytia inhibition, SI) in rabbits inoculated experimentally with inocula of variable immunogenicity. The two tests to detect antibodies to gp51 correlated well in sera clearly positive or clearly negative by either one, but correlation was poor in the intermediate groups. All sera positive by AGID were also positive by ELISA, but results did not agree in sera negative by AGID, ELISA proving to be more sensitive. Western blot was a good technique for detecting antibodies against Gag-related proteins. However, no band was identified to clearly correspond to anti-Env-related proteins. As for other retroviruses, testing of animals for infection with BLV should include the detection of antibodies anti-Gag and anti-Env proteins.     

An immunoblotting procedure for detection of antibodies against bovine leukemia virus in cattle.

A sensitive protein immunoblotting (Western blot) procedure has been developed for detecting anti-BLV antibodies in cattle sera. The antibodies against most of the major viral proteins could be detected. This procedure does not give any non-specific background staining and there is absence of any erroneous results due to utilisation of purified viral preparations. The procedure has been applied for detection of antibodies to BLV in a set of 74 sera samples and it has been compared with other commonly used serological tests like ELISA and agar gel immunodiffusion test.     

An improved immunodiffusion test for antibodies to bovine leukemia virus antigens

A new immunodiffusion (ID) test for antibodies to bovine leukemia virus (BLV) antigens was established. This test, refered to as the tannic acid enhanced, indirect, double immunodiffusion (IID-T) test, includes the following steps: (a) double micro-immunodiffusion using diluted references reagents, (b) treatment of the gel plate with antiglobulin serum, (c) treatment of the gel plate with 1% tannic acid. The IID-T test has proved to be eight times more sensitive than the double immunodiffusion test (ID) commonly used for anti-BLV. Diagnostic efficiency at individual levels of the IID-T test for anti-gpBLV is higher than this parameter of the ID test for anti-gpBLV and radioimmunoassay (RIA) for anti-p24BLV. The IID-T test is simple, reproducible, and more economic than the ID test in the amount of the reference reagents required. The IID-T test is more reliable than the ID test especially when weak, low titer sera are tested. Thus, the IID-T test seems to be suitable for large scale serodiagnosis of BLV infection in cattle.     

Syncytium-induction inhibition test with complement for detection of antibodies against bovine leukemia virus.

A modified syncytium-induction inhibition test which is more sensitive than the immunodiffusion test, was developed using rabbit complement. In this test, fetal lamb kidney cells continuously infected with bovine leukemia virus were used as effector cells, and the CC81 cat cells transformed with murine sarcoma virus, were used as indicator cells. The syncytium-induction inhibition effect of anti-bovine leukemia virus serum was enhanced significantly by the addition of rabbit complement. The syncytium-induction inhibition titers had a statistically significant correlation with the immunodiffusion titers and were four to 64 times higher than immunodiffusion titers. In 12 experimentally infected cattle, the syncytium-induction inhibition test detected the antibodies earlier than the immunodiffusion test and continuously detected them when immunodiffusion antibody changed to negative. In the 81 sera from naturally infected herds, 35 (43.2%) were positive by the immunodiffusion test and 55 (67.9%) by the syncytium-induction inhibition test.     

Isotype-specific ELISAs for the detection of antibodies to bovine respiratory syncytial virus

Isotype-specific ELISAs for the detection of antibodies to bovine respiratory syncytial virus (BRSV) are described. BRSV-specific IgG1 and IgG2 were determined in indirect double antibody sandwich assays. For IgA and IgM antibody capture assays were used. The isotype specificity of the assays was confirmed by the observation that samples with a high titre of BRSV-specific antibodies of particular isotype were negative in the assays for the other isotypes and vice versa. Comparison of the results obtained in the ELISAs and in the virus neutralisation test showed that acute phase antibodies were more efficiently detected in the latter. It also showed that the presence of BRSV-specific IgA was not correlated with neutralising activity in vitro. The serum antibody response of BRSV-infected seronegative calves from the field consisted of a nearly simultaneous increase of IgM, IgA and IgG1-antibodies in the acute phase of the disease, while the IgG2-response followed at various intervals thereafter. In young animals with maternal antibodies a different pattern was found. There was no increase in IgG1 and IgG2, but six of eight animals showed a weak IgM response and two of these six calves also showed a weak and short lasting IgA response. Because maternal antibodies are insufficiently effective in protecting calves against BRSV, the presence of such antibodies at mucosal surfaces was investigated. Maternal immunity was found to be restricted to IgG1 antibodies in serum. This agrees with the failure of maternal antibodies to protect mucosal surfaces against BRSV infection.     

Comparison of serologic tests for detection of antigen in canine heartworm infections

In 30 random-source dogs, we determined sensitivity and specificity of 5 serologic tests for detection of canine heartworm antigens. Seventeen of the dogs were infected naturally with adult Dirofilaria immitis, and 4 of the infected dogs were amicrofilaremic. The ability of the serologic tests to predict whether a dog was infected or uninfected (overall test accuracy) ranged from 73 to 97%. Sensitivity was not affected by circulating D immitis microfilariae, but was markedly influenced by the number of adult D immitis present. False-positive reactions were rare and were not associated with intestinal parasites or Dipetalonema reconditum microfilariae. Modifications of some of the test procedures were necessary to maximize test accuracy and reproducibility. These modifications and other technical details might limit the usefulness of some of the tests in a veterinary practice.