Specific antibody responses to Mycobacterium bovis in infected cattle analysed with six mycobacterial antigens in enzyme-linked immunosorbent assays

A total of 23 (15.3 per cent) of 150 cattle infected with Mycobacterium bovis and which had never been tuberculin tested showed specific antibody responses to M bovis. Their sera may be important keys to the identification of unique M bovis antigens for use in specific serodiagnostic tests. Assessment of specific and non-specific responses was done by screening sera in six indirect anti-IgG enzyme-linked immunosorbent assays using whole cell sonicates of M bovis and five members of the Mycobacterium avium-intracellulare-scrofulaceum complex as respective antigens. Sera from 16 infected cattle that had been tuberculin tested positive and nine uninfected cattle (never tuberculin tested) were also assayed for specific and non-specific responses. Three other findings emerged. First, 43 of the 150 infected animals (28.7 per cent) showed no antibody responses to any of the mycobacterial antigens used. Secondly, the cattle showing the highest antibody levels were associated with the greatest cross reactivity. Lastly, the results indicated that tuberculin injections may increase antibody responses to shared, rather than specific, M bovis antigens in infected cattle.     

Immune responses of infected and vaccinated Hereford cattle to antigens of the cattle tick, Boophilus microplus

Responses of infested and vaccinated Hereford cattle to Boophilus microplus antigens were measured by enzyme-linked immunosorbent assay (ELISA), lymphocyte blastogenesis assay (LBA) and intradermal skin tests. Responses against soluble salivary gland extracts (SGS), salivary gland membrane (SGM), soluble gut extracts (GS), gut membrane (GM), soluble larval extracts (LS) and larval membrane (LM) antigens were tested. In one experiment, cattle infested with up to 160,000 ticks had positive cellular responses to SGS and significant antibodies against LM, GM, SGM, and SGS. Cellular responses to Concanavalin A were not depressed following infestation. Cattle vaccinated with GM, using Quil A as adjuvant, had positive cellular responses to gut and salivary gland antigens and significant antibody responses to all antigens tested. The antibody levels of vaccinated cattle were significantly higher than the antibody levels of infested cattle (P less than 0.05). In a second experiment, immune responses of cattle infested with 40,000 ticks were studied during 38 days. Cellular responses in LBA to several tick antigens were transiently elevated and significant levels of antibody were measured against LM, GM, SGM and SGS, from day 25 (P less than 0.05). Infested cattle had positive skin reactions following intradermal injection of larval and adult tick antigens (P less than 0.05).     

Acquired immune response of cattle exposed to buffalo fly (Haematobia irritans exigua).

Naturally acquired immunity to buffalo fly (Haematobia irritans exigua) infestation was examined in cattle. Animals exposed to flies had serum antibodies to buffalo fly antigens at levels that correlated with the intensity of exposure. Two weeks of intense exposure to buffalo fly induced an increase in peripheral blood eosinophil numbers and a concomitant rise in serum antibody levels in exposed animals. Antigens specific for antibody induced by natural exposure were identified using antisera from exposed cattle to probe Western blots of whole fly homogenate separated using SDS-PAGE. Similar immunoreactive bands were found with buffalo fly saliva. Immunoreactive proteins were partially purified from whole fly homogenates by anion-exchange chromatography. Fractions eluted from columns were screened using Western blots probed with serum from exposed animals. Exposed animals showed immediate hypersensitivity to partially purified antigens and to buffalo fly saliva. Flies which fed on exposed animals with high serum levels of antibody to fly antigens did not show greater mortality than flies fed on unexposed animals.     

The antibody response to bovine lymphocyte alloantigens

Antibodies were raised against lymphocyte cell-surface antigens by multiple immunisations with purified lymphocytes or by the exchange of skin allografts. Eighteen of 21 cattle immunised with lymphocytes raised a detectable cytotoxic antibody response. The serum antibody from 10 responders recognized only common lymphocyte antigens, those antigens which are present on all peripheral blood lymphocytes. One animal responded only to B lymphocyte antigens while 7 others responded to both classes of antigens. The amount of antibody produced varied greatly between individuals; antibody titres ranged from 1 to 1028. Antibody raised early in the response was sensitive to treatment with 2-mercaptoethanol (2-ME) suggesting that IgM was the predominant class of immunoglobulin. Subsequently antibody became resistant to this treatment suggesting the appearance of IgG. The antibody responses following the exchange of skin grafts were very similar in all 12 cattle studied. High titred antibody to common lymphocyte antigens was detected in the serum 14 days after grafting. The early antibody activity was sensitive to 2-ME treatment but became totally resistant within 14 days. Total peak antibody titres ranged from 128-2048. Antibody to B lymphocyte antigens was identified in 8 of the 12 cattle. The responses to B lymphocyte antigens were similar to those against the more widely distributed common lymphocyte antigens with respect to time of antibody appearance, time of peak titre and sensitivity to 2-ME. Peak titres ranged from 2 to 32. The change in antibody specificity with time was also studied. Sera from 11 of the 18 cattle which had responded against lymphocytes showed an increase or broadening in reaction frequency as immunisations increased, suggesting the production of antibody to secondary specificities. In the cattle which had been skin grafted, the broadest reaction patterns were seen 14 to 21 days after grafting. The broadest reaction patterns were seen when the antibody responses were at their highest titre levels and narrowed as titres decreased.     

Serological evidence of an Australian bovine lentivirus.

Recombinant 26 kDa capsid (CA) proteins of bovine lentiviruses, bovine immunodeficiency virus (BIV) and Jembrana disease virus (JDV), were expressed in Escherichia coli and utilised as antigens for an enzyme-linked immunosorbent assay (ELISA) and a western immunoblot (WIB) procedure for the detection of antibody in dairy cattle in Western Australia. A total of 690 serum samples, 30 from each of 23 farms, were tested by ELISA with a JDV CA protein antigen, and antibody was detected in 3.8% (p<0.05) of the sera. Nine sera from each farm were also tested by WIB with JDV CA protein antigens and antibody was detected in 15.9% of these samples. All ELISA-positive results were also WIB-positive, and all sera antibody-positive by WIB with JDV CA protein antigens were also antibody-positive by the WIB using recombinant BIV CA antigens. This study showed that recombinant protein antigens can be used for serological tests to detect bovine lentivirus infection in Australia.     

Enzyme-linked immunosorbent assay for differentiation of the antibody response of cattle naturally infected with Brucella abortus or vaccinated with strain 19

Purified O chain of Brucella abortus was passively attached to polystyrene to differentiate antibody responses of cattle vaccinated with B abortus strain 19 from those of naturally infected cattle. In the indirect assay, using O polysaccharide as antigen, a single serum dilution was used and mouse monoclonal antibody to bovine L chain conjugated with horseradish peroxidase was the detection reagent. Measurable antibody was not found in sera of vaccinated cattle, except for 3 sera from cattle that were persistently infected with strain 19. Sera from 25 cattle infected with pathogenic strains contained antibody on the basis of results of indirect enzyme immunoassay, using smooth lipopolysaccharide or O chain as antigens, or results of competitive enzyme immunoassay, using the O-chain antigen. Results in sera from calves with experimentally induced Yersinia enterocolitica serotype 0:9 infection or inoculated with a low dose of B abortus strain 2308 were comparable with those in sera of cattle that were vaccinated with strain 19. The data correlated with those from competitive enzyme immunoassay, using one serum dilution and horseradish peroxidase-conjugated mouse monoclonal antibody to smooth lipopolysaccharide. On the basis of results of the indirect enzyme immunoassay, all sera (except those samples obtained before inoculation) contained antibody to smooth lipopolysaccharide.     

Evaluation of the indirect fluorescent antibody test for Babesia major and Theileria mutans in Britain.

Use of the indirect fluorescent antibody (IFA) tests is described to detect antibodies to Theileria mutans and Babesia major in the sera of infected cattle. When antisera against T mutans and B major were tested against homologous antigens high antibody titres were recorded: when they were tested against each other or against Babesia divergens antigen insignificant titres (1/40 or less) were recorded. Thus the test was found to be species specific. Animals recovered from T mutans and B major infections retained significant levels of IFA titres for 22 and 11 months respectively. It is suggested that the IFA test could be used for field survey of the piroplasms of cattle in Britain.     

Seroprevalence survey for Anaplasma marginale-infection of Austrian cattle.

A serologic survey study of 5,076 Austrian cattle farming herds was carried out in the period of December 1988 till March 1990. One animal was randomly selected from each herd and the antibody titer against Anaplasma marginale in blood serum samples was evaluated by means of the complement fixation test. The number of these tested blood samples was 3.6% of 140,081 cattle herd farms of Austria. 109 (2.1%) of the tested animals showed positive titers (1:10) against Anaplasma marginale, in relation to the 140,081 cattle herds 0.08%, 4,786 (94.3%) blood serum samples were sero-negative, 188 (3.8%) reacted anticomplementary. The highest number of antibody-positive animals of 8 tested Austrian districts could be found in Carinthia (46 = 5.7%). In Burgenland all tested sera turned out to be negative. Concerning the distribution of sero-positive animals in Austria it can be stated that a decrease of positive reactors from southern to northern region is evident. A connection between the occurrence of anaplasmosis in Italy, Yugoslavia, Switzerland and Hungary, is postulated as a result of the different systems of keeping cattle in the provinces and the regional increase of tick invasion. Possibly an intensive animal transportation is of importance due to the introduction of the disease mentioned before. The results obtained show that anaplasmosis does occur in different areas of Austria. For control of this disease in Austria it is proposed that all imported cattle should be tested serologically for antibodies against Anaplasma marginale. Other diseases in connection with anemia should be excluded by clinical, serological, blood-, as well as pathological examinations.     

Antibodies to malignant catarrhal fever virus antigens in the sera of normal and naturally infected cattle in Kenya

Six different serological tests were used to examine Kenyan cattle sera for antibodies to the herpesvirus of malignant catarrhal fever. Significantly higher levels of indirect immunofluorescent antibody to early and late virus antigens and of complement fixing antibody were found in the sera of 13 naturally infected cattle than in 482 sera collected from four different groups of normal cattle. Virus neutralising and immunoprecipitating antibodies were also found in some infected cattle sera but not in normal cattle sera. Many non-specific reactions occurred using counterimmunoelectrophoresis. These preliminary results indicate that the serological diagnosis of wildebeest-associated malignant catarrhal fever may be possible.     

Evaluation of an indirect fluorescent antibody test, enzyme-linked immunosorbent assay and quantification of immunoglobulins in the diagnosis of bovine trypanosomiasis.

An indirect fluorescent antibody test (IFAT), a microscale version of the enzyme-linked immunosorbent assay (microELISA) and determination of IgM levels in serum were assessed for their comparative diagnostic value in the detection of bovine trypanosomiasis. Serum samples from drug-treated N'dama cattle and untreated N'dama and Zebu cattle from Liberia were examined for the presence fo antibodies to trypanosomes. In the untreated Zebu cattle, infections with T. vivax predominated and the prevalence of infection was higher than that found in untreated N'damas in which infections with T. congolense predominated. The proportion of animals which showed serological evidence of trypanosomiasis in the untreated Zebus was slightly higher than that found in the untreated N'damas. The prevalence of infection was low in N'dama cattle which had been treated with diminazene aceturate and homidium chloride but 50% of the animals showed serological evidence of trypanosomiasis. More serologically positive animals were detected by microELISA than IFAT, but both tests were equally sensitive in detecting antibodies in cattle in which trypanosomes were demonstrated by examination of peripheral blood. With both IFAT and microELISA it was necessary to carry out tests using antigens prepared from T. brucei, T. vivax and T. congolense in order to detect all serologically positive animals. Increases in serum IgM occurred in both N'dama and Zebu cattle but the levels were raised in only approximately half of the known infected animals. Overall, more animals gave positive reactions with IFAT and microELISA than showed raised IgM levels.     

An immunofluorescence test to determine the occurrence of type II collagen in muscle biopsies from cattle with rectovaginal constriction

Type II collagen occurs in the muscles of rectovaginal constriction (RVC) affected and carrier cattle but not in normal cattle. Muscle biopsies from known RVC affected and carrier cattle and normal cattle were examined for the presence of Type II collagen using affinity purified goat anti-collagen II serum in a fluorescent antibody test. Type II collagen was consistently found in RVC affected animals (22 of 23 samples score positive). Rectovaginal constriction carrier animals had variable staining for the Type II collagen (25 of 47 samples scored positive). Some positive staining was also observed in the control animals (8 of 34 samples scored positive). Because of the variable occurrence of Type II collagen, the value of fluorescent antibody staining to identify RVC carrier animals is uncertain.     

Evaluation of an enzyme immunoassay for serodiagnosis of infections with Theileria parva and T annulata

An enzyme linked immunosorbent assay (ELISA) was used to determine antibody levels in cattle infected with Theileria parva and T annulata, using antigens prepared from the intra-erythrocytic piroplasm stage of the parasites. Antibody levels in calves infected with T parva increased from the 16th day after infection to reach peak values at days 28 to 35 and then declined rapidly, but in calves infected with T annulata antibody levels rose steadily up to day 40. Similar patterns of antibody production were shown by indirect fluorescent antibody tests. Sera from animals infected with T parva gave higher ELISA values with the antigen prepared from the homologous parasite species than with the antigen prepared from T annulata, but sera from cattle infected with T annulata gave similar high ELISA values with antigens prepared from both T parva and T annulata. Sera from animals infected with T mutans, T sergenti, T velifera, Babesia divergens, B major and B bovis gave only slight or no cross reactions with the piroplasm antigens, but serum from a calf infected with B bigemina cross reacted at a significant level with both piroplasm antigens.     

Serologic cross-reactivity of antibodies against Borrelia theileri, Borrelia burgdorferi, and Borrelia coriaceae in cattle.

OBJECTIVE: To evaluate the immune response induced by Borrelia theileri infection and to determine whether B theileri induces cross-reacting antibodies to other bovine borreliae. ANIMALS: Two 3-month-old calves, 1 of which was splenectomized. PROCEDURE: Calves were exposed to Boophilus microplus infected with B theileri. Rectal temperature, PCV, bacteremia, and clinical signs of infection were monitored. Serum was obtained weekly and used to evaluate the humoral response to homologous antigen and B burgdorferi and B coriaceae, using an indirect fluorescent antibody (IFA) test, and to B burgdorferi, using a commercially available ELISA. The identity of cross-reacting antigens was explored, using monoclonal antibodies to genus- and species-specific antigens in an IFA test. RESULTS: B theileri-infected calves produced antibodies that cross-reacted with B burgdorferi and B coriaceae whole-cell antigens. Borrelia theileri whole-cell antigen was recognized by genus-specific monoclonal antibody H9724 but not by species-specific antibody H5332. False-positive reactions were not observed when serum from B theileri-infected calves was tested by use of the ELISA for B burgdorferi. CONCLUSIONS: B theileri induces humoral responses in infected cattle that can be confused with those of other borrelial infections. Care must be taken to definitively distinguish between the various borreliae that may cause disease in cattle. CLINICAL RELEVANCE: Serologic cross-reactivity must be taken into account when making a serodiagnosis of Lyme borreliosis or epizootic abortion in epidemiologic studies involving cattle.     

IgE responses to bluetongue virus (BTV) serotype 11 after immunization with inactivated BTV and challenge infection

To test the hypothesis that development of a BTV-specific IgE response plays a role in clinical disease manifestation, the humoral immune response of cattle to inactivated and virulent BTV was studied. Three calves received three sensitizing immunizations of inactivated BTV, 3 weeks apart. The BTV-sensitized animals, two non-sensitized BTV-seropositive and 4 BTV-seronegative control cattle. were challenge-exposed with BTV-11, UC8 strain. All cattle inoculated with inactivated BTV developed group-specific non-neutralizing and serotype-specific neutralizing antibodies. The development of post-challenge-exposure neutralizing antibody titers was inversely correlated with protective immunity. None of the BTV-challenged animals showed clinical disease. The levels of IgE were greatest in the sensitized calves after virus challenge in comparison with control groups. The sequential development, specificity and intensity of virus protein-specific humoral responses were evaluated using immunostaining. After challenge exposure of BTV-sensitized and non-sensitized cattle, total and IgE antibodies reacted consistently within BTV structural proteins VP2, VP5 and VP7. Although no correlation was found between clinical disease and IgE, results add support to the hypothesis that IgE may be involved in the pathogenesis of clinical disease, since infection with BTV causes an increase in serum IgE levels. However, these results suggest that the levels of virus-specific reactivity may be an important factor in determining whether or not clinical disease manifestation occurs.     

An experimental inactivated virus vaccine against bovine ephemeral fever 2. Do neutralizing antibodies protect against infection?

In order to develop a safe vaccine against bovine ephemeral fever (BEF) which could be used in areas normally free of the disease, studies were carried out on inactivated virus vaccines. Initial experiments were carried out in cattle using virus vaccines that had been inactivated with β-propiolactone or formalin and then made-up in aluminium phosphate gel or Freund's incomplete adjuvant. A minimum inactivated virus dose of 10⁶ PFU was necessary to stimulate a serum neutralizing antibody response in cattle. β-propiolactone inactivated BEF virus vaccines in Freund's incomplete adjuvant gave the best serum neutralizing antibody responses, producing high levels of neutralizing antibody with both high and low passage level virus. However, the magnitude of the antibody response bore little relationship to resistance of vaccinated animals to challenge with virulent BEF virus. A number of animals with high neutralizing antibody titres to BEF virus did not resist challenge. Using 500-fold less live virus at equivalent passage level to the low passage inactivated vaccine, similar or slightly lower antibody levels were attained, but most of the animals resisted challenge. It is suggested that the nature of the immune response and resistance to BEF infection may be complex and that reliance on serum neutralizing antibody as an indicator of resistance may give misleading results.     

Serological studies of Australian and Papua New Guinean cattle and Australian sheep for the presence of antibodies against bluetongue group viruses

Following isolation of a virus (CSIRO19) from insects in Australia and its identification as bluetongue virus serotype 20 (BTV20), a nationwide survey of antibodies in cattle and sheep sera was undertaken. Initial studies using the serum neutralization (SN) test showed that the distribution of BTV20 antibodies in cattle was confined to the northern part of Australia. Group-reactive antibody tests (agar gel diffusion precipitin, AGDP, and complement-fixation, CF) showed group-reactive cattle sera south of the BTV20 zone (northern Australia), and southwards from Queensland to New South Wales. Very few group-reactive sheep sera (45 out of 16213) were found and these were of doubtful epidemiological significance. Some of these BTV group-reactive, BTV20-negative, sera were tested in SN tests against BTV1 to 17 and Ibaraki (IBA) virus. The results indicated that BTV1, or a closely related orbivirus, was active in cattle in Queensland, northern Western Australia, and New South Wales, and that antibody to BTV15 was present in some of the cattle sera in northern Western Australia and the Northern Territory. Antibody to IBA virus was present in some cattle sera in Queensland, northern Western Australia and New South Wales. SN antibody titres ?60 were also found to a number of other BTV serotypes in cattle sera in northern Western Australia and Queensland (principally, BTV2 and BTV7). Low level reactions were commonly observed against these and a number of other BTV serotypes, often in the same serum samples. Further, 22% of the group-reactive cattle sera did not react with any of the viruses in the SN tests. Such results were difficult to interpret in terms of known Australian BTV or BTV-related isolates.     

Kinetics of immune response to foot-and-mouth disease virus (type Asia 1) in experimental cattle

Humoral and mucosal (secretory antibody)immune response to FMDV type Asia 1 in cattle was analyzed after vaccination and infection using virus neutralizing test (VNT). Vaccination (1/16th the usual dose) failed to protect cattle from generalized clinical disease following experimental FMDV Asia 1 infection. Our results showed that infection induced higher and prolonged serum antibody titres indicating antigen mass is important for optimal immune response. Experimental FMDV infection induced significant secretory antibody (mucosal) response in cattle. Though, there was no difference in the serum antibody response between the cattle that developed generalized infection (unprotected) and those with only localized infection (protected), secretory antibody response differed, wherein the unprotected cattle had higher secretory response than protected cattle. Thus, FMDV Asia 1 infection stimulates a similar serum antibody response and a unique secretory antibody response among the infected cattle. An erratum to this article can be found at     

三种抗牛运输应激技术的效果比较

本研究比较了3种抗应激处理措施防控肉牛运输应激的效果。74头试验牛分4组,分别在运输前用牛支原体弱毒疫苗滴鼻、肌内注射泰乐菌素或饲料中添加泰妙菌素,并设不做任何抗应激处理的对照。分别对试验牛起运前及运达后体温、血细胞数目、血清中酶含量、牛支原体抗体水平、生长速度、发病率、死亡率及治疗成本进行比较。结果显示,起运前及运达后试验牛体温、血细胞数及牛支原体抗体水平未见明显差异;泰乐菌素肌注组试验牛发病率、死亡率显著低于对照组（P〈0．01）;对照组试验牛治疗成本为抗应激处理组治疗成本的15．9倍。结果表明,运输前进行抗应激处理有利于降低肉牛运输应激,起运前肌注泰乐菌素抗运输应激效果最好。     

A survey for haemagglutination-inhibiting antibody to West Nile virus in human and animal sera in Nigeria

A survey for West Nile Virus (WNV) haemagglutination-inhibition (HI) antibody was carried out in humans and domestic animals. Human sera were collected from Ibadan, while the animal sera were collected from both Ibadan and Maiduguri. Out of 304 human sera tested, 123 were positive (40%). There was a higher prevalence of HI antibody in adults than children. Sex distribution of positive sera showed that 37% of males and 43% of females had WNV HI antibody. There was no significant difference in the prevalence of HI antibody in both sexes. On the 123 WNV HI positive sera tested, 104 (85%) and 78 (75%) had yellow fever and Potiskum HI antibody respectively. Monotypic WNV virus reactions were frequently found in children while polytypic reactions were frequently found in adults. A total of 200 animal sera were examined, 50 camels, 50 goats, 49 cattle and 51 sheep. The highest prevalence of HI antibody was found in camels (26%), followed by sheep (20%). Percentage of positive sera in other species were: goat (18%) and cattle (6%). Of the 35 WNV HI positive animal sera, 26 and 20% reacted with Yellow fever and Potiskum virus antigens respectively.     

Characterisation, experimental infection and serological response to caprine retrovirus

A virus (151) isolated from synovial membrane explant cultures from a goat with arthritis-synovitis was characterised with respect to cytopathic effect in synovial membrane cell cultures, virus morphology, buoyant density and presence of RNA dependent DNA polymerase. Virus 151 was shown to be a retrovirus with similar properties to caprine arthritis-encephalitis virus in the United States of America. Inoculation of the virus into uninfected goats caused the development of arthritis-synovitis lesions and the virus was recovered from affected joints and lung 361 days post-inoculation. The development of antibody to virus 151 was detected using an enzyme linked immunosorbent assay (ELISA). Other goats with arthritis-synovitis, progressive pneumonia or viral leukoencephalomyelitis all had antibody that reacted in this ELISA. Viruses similar to virus 151 were recovered from a number of cases. Goats inoculated with one of the viruses produced serum antibody that cross-reacted in ELISA using maedi-visna virus and virus 151 as antigens.