Use of pooled serum or milk samples for the epidemiological surveillance of bovine hypodermosis

An enzyme-linked immunosorbent assay (ELISA) was used on pooled serum and milk samples to determine whether hypodermosis could be detected where a larger sero-epidemiological survey was required. This study was undertaken to assess the potential of this assay for testing sera on milk samples, pooled from 10 cows, and determining the period of the year when detection was optimal. The sensitivity of the assay was determined by increasingly diluting a positive serum with pooled negative sera, from 1:10 to 1:100. The diagnostic lower limit of the assay requires at least two serological reactors within a herd of 100. The kinetic development and depletion of anti-Hypoderma antibody of individual and pooled sera or milk from 30 cows was evaluated from November to July. Anti-Hypoderma antibody levels of two groups of 8 calves, one control and one teated with ivermectin (Ivomec), were tested from October to June. These preliminary results indicate that an ELISA assay on serum or milk samples pooled from 10 cows can be used between February and April to evaluate the prevalence of hypodermosis within cattle herds in France, demonstrating the feasibility of using pooled serum already collected for bovine leucosis testing.     

Detection of conglutinin in bovine serum by complement-dependent agglutination of Escherichia coli

Agglutination of Escherichia coli (ECA) by normal bovine serum was shown to be prevented by heating serum to 56 degrees C for 30 min, but restored by normal horse, swine, rabbit or guinea pig sera. Further investigation of the ECA reaction using techniques to distinguish between conglutination and immunoconglutination indicated ECA to be a conglutination reaction. Testing of 264 sera obtained from 22 normal cattle over a period of 5 months did not show individual or seasonal variation in ECA. Changes in ECA and conglutination were detected in sera of periparturient cows. The ECA reaction is a simple technique for detecting conglutinin in bovine serum.     

Neutralization kinetics of bovine viral diarrhea virus by hyperimmune serum: one or multi-hit mechanism

The kinetics of bovine viral diarrhea (BVD) virus neutralization (VN) by hyperimmune serum followed first order kinetics at low dilutions (1:8 and 1:16) of hyperimmune bovine serum. A lag phase in the VN curve occurred when the serum was further diluted. Addition of rabbit anti-bovine IgG, but not anti-IgM, significantly increased the degree of VN after BVD virus was reacted with further diluted (1:256 dilution) anti-BVD bovine hyperimmune serum. Incubation of virus-hyperimmune serum (1:64 dilution) at 4 degrees C for 0 to 60 min, followed by incubation at 37 degrees C indicated VN occurred as a two-stage process: a binding (temperature-independent) phase that was followed by a triggering (temperature-dependent) phase. The data favor the thesis that neutralization of BVD virus occurs by a multi-hit mechanism and requires combination of at least two molecules of antibody with each virus.     

Addition of rabbit serum to EMJH medium improves isolation of Leptospira interrogans serovar hardjo

The addition of 2% pooled rabbit serum to semi-solid commercial EMJH medium with EMJH enrichment and 0,5 mg of 5-fluorouracil per ml was found to enhance the growth rate and success of isolation of Leptospira interrogans serovar hardjo from bovine urine. Cultures made on media without serum had to be kept for more than 130 days before being discarded as negative.     

Development and initial evaluation of an indirect enzyme-linked immunosorbent assay for the detection of Leptospira interrogans serovar hardjo antibodies in bovine sera.

Outer sheath antigen from Leptospira interrogans serovar hardjo type hardjoprajitno and acetic acid extracted antigens from serovar hardjo types hardjoprajitno and hardjobovis were evaluated in an immunoassay for ability to detect hyperimmune rabbit serum to serovar hardjo. The degree of cross-reactivity with hyperimmune rabbit sera to L. interrogans serovars pomona, copenhageni, grippotyphosa, canicola and sejroe, and Leptospira biflexa serovar patoc was also measured for each antigen. All of the antigens reacted with the antiserum to L. interrogans serovar hardjo. The outer sheath antigen however, also showed wide cross-reactivity with the antisera to all of the serovars of L. interrogans tested and with the antiserum to L. biflexa serovar patoc. The acetic acid extracted antigen from either type hardjoprajitno, or type hardjobovis, showed a high degree of specificity for serovar hardjo antiserum. The hardjobovis acetic acid extracted antigen was characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting, and was incorporated into an indirect ELISA for detection of anti-serovar hardjo antibodies in bovine serum. This ELISA showed a relative specificity of 100% with 156 bovine sera which were negative at a dilution of 1:100 in the microscopic agglutination test (MAT) for L. interrogans serovars hardjo, pomona, sejroe, icterohaemorrhagiae, copenhageni, canicola, and grippotyphosa. The relative sensitivity of this assay with 192 bovine sera which had serovar hardjo MAT titres of > or = 100 was 95.3% (95% confidence limit = 2.99%). The degree of cross-reactivity with 289 bovine sera which had serovar pomona MAT titres of > or = 100 (with no detectable serovar hardjo MAT titres) was approximately 1.0%. This assay was: easily standardized, scored objectively, repeatable, semi-automated and used a non-hazardous antigen that can be routinely prepared in gram amounts.     

Isolation, characterization, and quantitative measurement of serum alpha 1-acid glycoprotein in cattle

Bovine alpha 1-acid glycoprotein (alpha 1AG) was purified from pooled normal bovine sera by successive ammonium sulfate precipitation, ion-exchange chromatographies and gel filtration. Bovine alpha 1AG had a molecular weight of 42,000 +/- 2,000 and a sedimentation coefficient of 3.4S. It contained 26.6% carbohydrate. Gel isoelectric focusing revealed a microheterogeneity with 7 to 8 bands in a pI range of 3.2 to 3.7. It migrated to the alpha 1-globulin region upon immunoelectrophoresis. Single radial immunodiffusion was developed for the quantitative measurement of bovine alpha 1AG in serum. The mean serum value of alpha 1AG in 152 healthy Holstein cattle (1-12 years old) was 283.2 +/- 82.3 micrograms/ml. Elevated values (cut-off value = 450 micrograms/ml) were observed in cattle with traumatic pericarditis (100%), arthritis (100%), mastitis (91%), pneumonia (70%), and mesenteric liponecrosis (43%).     

Evaluation of alternative methods for the detection of bovine leukaemia virus in cattle

AIM: To evaluate commercially available enzyme-linked immunosorbent assays (ELISAs) and the polymerase chain reaction (PCR) for their ability to detect antibodies against or nucleic acid of the bovine leukaemia virus (BLV), the causal agent of enzootic bovine leukosis (EBL), and to assess their usefulness in a national eradication programme. METHODS: Eighty-two well-defined sera (including 18 from an OIE reference laboratory) and 399 field sera from New Zealand cattle were tested in five ELISAs and the results compared with the agar gel immunodiffusion (AGID) test and electrophoretic immunoblotting (EIB) results. A polymerase chain reaction-based technique, which could detect BLV-RNA and proviral-DNA, was also evaluated on a subsample of the field cases. RESULTS: Two commercial ELISAs classified 99% of the defined sera correctly, with the other three ranging in their correct classification between 88% and 95%. The ELISAs agreed in their general classification on the majority of the 399 blood samples (91.7%), and with the AGID for more than 95 % of the sera. In a dilution series of the international reference serum E4, the highest dilution with a positive (or suspicious) result ranged from 1:80 to 1:5120. A dilution series of 202 field positive samples tested in the preferred ELISA detected 98% of positive sera at a 15 and 1: 10 dilution, reducing to 78% at a 1:80 dilution of the sera. Agreement between serological tests and PCR was poor, mainly due to failure of the PCR to detect a number of serologically positive animals. CONCLUSION: ELISA tests detected about 10% more reactors than the AGID and the EIB combined. Some ELISA-positive animals were not detected by PCR, raising doubts about the usefulness of PCR-based technology in EBL eradication programmes.     

Evaluation of the critical parameters of a sensitive ELISA test using purified infectious bovine rhinotracheitis virus antigens

An enzyme-linked immunosorbent assay (ELISA) for the survey or titration of bovine sera for the presence of IgG antibodies against infectious bovine rhinotracheitis (IBR) virus was developed. The optimal conditions of serum dilution, antigen concentration, conjugate dilution, substrate concentrations, and reaction time were established using the signal/ noise (S/N) ratio as the determining criterion. Equilibrium density gradient purified IBR virus was used as antigen at an optimal concentration of 0.60 μg/cuvette. The use of purified antigen allowed the testing of sera at a 1 : 10 dilution without nonspecific reaction.The conditions of conjugate dilution, substrate concentration and reaction time were shown to have significant effects on the ELISA test. Results from 35 sera showed this optimized ELISA procedure to be as much as 1000-fold more sensitive than the serum neutralization plaque reduction assay. Numerous sera showing no neutralizing titer to IBR virus were found to be positive when examined by this ELISA method.     

Demonstration of circulatinf antibodies to Eimeria tenella by the indirect immunofluorescent antibody test using sprozoites and second-stage schizonts as antigen

In the indirect immunofluorescent antibody (IFA) test using sporozoites as an antigen, sera from chickens immunized via the natural route were positive in dilutions as high as 1 : 1 048. Serum from a rabbit, immunized only to sporozoites by subcuntaneous injection, was positive in a dilution of 1 : 4 4 096. Non-immunized chicken andn rabbit sera were positive in dilutions varying from 1 : 20 to 1 : 64.Sporozoites within sporocytes were not stained. In frozen sections of infected chorioallantoic membranes and ceca, sporozoites were not traced with the IFA test with immunized chicken or rabbit sera. However, with the rabbit serum specific diffuse intraepithelial and subepithelial fluorescence was observed in the ceca from 4 to 11 h after infection.Fluorescence was never associated with the first-stage schzonts and gamates, but second-stage schizonts were positive with both chicken and rabbit serum. The titres obtained with this antigen were about the same as those obtained with sporozoite smears. The possible presence of common antigens in sporozoites and second stage schizints is discussed.     

Anticytotoxin activity of bovine sera and body fluids against Pasteurella haemolytica A1 cytotoxin.

Toxin neutralizing activity of bovine sera and body fluids against Pasteurella haemolytica type A1 cytotoxin was evaluated by 51Cr release assay using bovine peripheral blood mononuclear leukocytes as the target cells. Sera collected from precolostral calves did not exert anticytotoxin activity at 10(-1) or higher dilutions, whereas randomly selected complement fixing antibody-negative sera neutralized on average over 90% of cytotoxin activity at the 10(-1) dilution and less than 50% of the toxin activity at 10(-2) or higher serum dilutions. Nasal secretions and lung washings of some of the cattle tested also contained cytotoxin neutralizing activity. The antibody nature of the cytotoxin neutralizing activity was demonstrated by its neutralization with bovine immunoglobulin G2 purified from pooled seropositive sera. Sera from a group of cattle which were vaccinated with a potassium thiocyanate extract of P. haemolytica, but which subsequently developed fibrinous pneumonia after aerosol challenge with bovine herpesvirus 1 and P. haemolytica, had significantly lower anticytotoxin activity than sera from another group of cattle which did not develop the disease after similar vaccination and challenge. Cattle which survived a natural outbreak of shipping fever had higher anticytotoxin activity than those having fibrinous pneumonia in the aforementioned experimental group, although there was no statistical difference between them and a randomly selected CF seronegative group. It is probable that this cytotoxin neutralizing antibody exerts a beneficial effect in protection of cattle against pneumonic pasteurellosis.     

Comparison of the 2-mercaptoethanol and dithiothreitol tests for determining Brucella immunoglobulin G agglutinating antibody in bovine serum.

The dithiothreitol test was evaluated as a substitute for the 2-mercaptoethanol test for determining Brucella immunoglobulin G agglutinating antibody in bovine serum. The tests were compared on 207 card-positive sera that showed a standard tube-agglutination titer of incomplete 1:50 or higher. The tests agreed within one dilution with 182 of the 207 sera tested for an 87.9% rate of agreement. When titers were not the same, those obtained with the dithiothreitol test were more frequently lower than higher than those obtained with the 2-mercaptoethanol test. Sixteen sera that showed a titer with the 2-mercaptoethanol test were negative with the dithiothreitol test and two that showed a titer with the dithiothreitol test were negative with the 2-mercaptoethanol test. Results suggest that the dithiothreitol test is not a reliable substitute for the 2-mercaptoethanol test to detect immunoglobulin G agglutinating antibody in bovine serum.     

Variations in sheep serum conglutinating and haemolytic activity for sheep erythrocytes sensitized by rabbit antibody

Based on conglutinating and haemolytic reactions with sheep erythrocytes (E) sensitized by rabbit antibody (A), three types of sheep sera were encountered. Type 1 sera do not conglutinate or haemolyse sheep E-rabbit A. Type 2 sera failed to conglutinate, but are haemolytically active. Type 3 sera have both activities. Serum from one type 1 sheep still failed to conglutinate 5 days after venepuncture but was now haemolytically active (i.e., type 2). Some sheep that initially had type 2 sera had, five days after an intraperitoneal injection of yeast cells, sera with conglutinating activity (type 3 sera). Type 1, 2 and 3 sera all had haemolytic activity with human E-sheep A indicator cells.Pooled type 3 sera have the highest conglutinating titres with sheep E-rabbit A after 10 min incubation at 39 °C. At this stage, the haemolytic titres were very low. From 10 min, the conglutinating titres decreased whereas the haemolytic titres gradually increased until 80 min. Optimal conglutinating activity required less rabbit A to sensitize sheep E than did haemolytic activity.     

The use of frozen cells in the microtitre serum neutralization test for infectious bovine rhinotracheitis.

Some parameters of the microtitre serum neutralization test were examined when using bovine fetal kidney cells derived from stocks stored in liquid nitrogen. In replicate tests with one serum there was no significant difference (p greater than 0.05) between titres calculated on the basis of cytopathic effect when either two or four wells were used per serum dilution. Also, there was no significant difference between titres calculated on the basis of a cell staining method when either two or four wells were used per serum dilution. When titres obtained by the 2-well-cytopathic effect method were compared with those obtained by the 2-well-stain method differences were not significantly different and it was concluded that the latter method using frozen cells constituted a practical and reproducible test. No significant titre variation occurred in cells serially passaged after having been frozen, or in cells derived from five different fetuses. Titres of sera from 41 cattle conformed to a log normal distribution pattern.     

Evaluation of enzyme-linked immunosorbent assay in comparison with complement fixation test for the diagnosis of subclinical paratuberculosis in cattle.

An enzyme-linked immunosorbent assay (ELISA) was evaluated and compared in parallel with the standard complement fixation test (CFT) for the diagnosis of bovine subclinical paratuberculosis. Bovine sera preabsorbed with the mixture of Mycobacterium phlei and kaolin suspension were assayed for antibody activities to the crude protoplasmic antigen of Mycobacterium paratuberculosis in the ELISA. ELISA antibody titer was expressed as ELISA antibody index (EAI) value: EAI = (At-An)/(Ap-An), where At, Ap and An are the absorbance values of a 1:200 dilution of unknown test sera, a 1:400 dilution of positive control serum, and a 1:200 dilution of negative control serum. An EAI of 0.6 or greater was established as a reasonable cutoff point for a positive antibody titer by ELISA. Of the 156 sera from cattle with subclinical M. paratuberculosis-infection, 106 (67.9%) were positive by ELISA and 41 (26.3%) by CFT. Of the 3,880 sera from cattle in the herds which had no history or evidence of paratuberculosis, 3,875 (99.9%) were negative by ELISA, and 3,787 (97.6%) by CFT. Positive ELISA titers were detectable 1 to 5 months earlier than positive CFT titers in experimentally infected cattle, and 7 to 10 months earlier in naturally infected cattle. These results indicate that the ELISA should replace the CFT as the routine test of choice for the diagnosis of bovine paratuberculosis.     

Serological reactions of leptospirosis-positive (MAR and CFT) bovine sera in ELISA

The collection of test sera for measuring ELISA results was composed of bovine sera with MAT titres of greater than or equal to 1:200 in the leptospirosis MAT and of greater than or equal to 1:5 in the CFT together with sera from a serologically negative and clinically non-suspicious cattle herd. To establish cut-off ODs, the geometric mean net-extinction of the negative serum collection plus 1, 2, and 3 standard deviations were calculated. By comparison of 3 different conjugates from rabbits, it was demonstrated that results from anti-total bovine Ig were superior to anti-IgG and anti-IgM conjugates. Considerations regarding sensitivity and specificity led to the recommendation to use a test serum dilution of 1:160, to apply anti-total bovine Ig conjugates, and to establish the cut-off OD at the geometric mean net-extinction of negative sera plus 3 standard deviations. Under such conditions, agreement between leptospirosis MAT/CFT positivity on the one side and ELISA positivity on the other was reached in 74%. This recommendation is made for cross-sectional studies but not for examinations of clinically suspicious cattle herds.     

A role for IgM in the in vitro opsonisation of Staphylococcus aureus and Escherichia coli by bovine polymorphonuclear leucocytes

The phagocytosis and killing of Escherichia coli (strain P4) and Staphylococcus aureus (strain M60) by bovine polymorphonuclear lymphocytes (PMN) suspended in phosphate buffered saline, requires the presence of calcium ions and opsonins. The highest dilution of normal decomplemented adult sera in which the opsonins are still active is approximately 1/1000 for S aureus and 1/200 for E coli. In fetal and precolostral sera heat labile factors are also required for opsonising E coli but these are not required for S aureus. IgG1 and IgG2 from adult sera did not opsonise the bacteria even though receptors for IgG2 anti-erythrocyte antibodies have been reported on bovine and ovine PMN. A systematic separation of adult serum proteins was carried out by salt fractionation, anion exchange and gel filtration chromatography. The results suggest strongly that the opsonin in adult bovine sera is IgM.     

Serological assays to discriminate rabbit haemorrhagic disease virus from Australian non-pathogenic rabbit calicivirus

Serological cross reactivity between the virulent rabbit haemorrhagic disease virus (RHDV) and the closely related but non-pathogenic rabbit calicivirus (RCV) makes it difficult to study the epidemiology of each virus and the interaction between them when both viruses co-circulate in wild rabbit populations. ELISA methods for the diagnosis of RHDV infection are well characterized, but no specific serological tests for RCV have been developed. Following the characterization of Australian non-pathogenic RCV-A1 strains, we used virus-like-particles (VLPs) and anti-RCV-A1 specific antibodies to establish a set of isotype ELISAs for detection of IgG, IgA and IgM in rabbit sera and secretory mucosal IgA in rectal swabs, and two competition ELISAs. These assays were used to discriminate between anti-RCV-A1 and anti-RHDV antibodies in rabbits. The isotype ELISAs were highly sensitive for detection of anti-RCV-A1 antibodies, but varying levels of cross reactivity from anti-RHDV antibodies occurred in the isotype ELISAs and one competition ELISA. However, the second competition ELISA specifically detected antibodies to RCV-A1 and showed no cross reactivity to anti-RHDV sera. These ELISAs provide important tools to monitor RCV-A1 infection when it occurs alone, and to discriminate between RHDV and RCV-A1 infection when they occur in the same rabbit population. When used in parallel with RHDV serology, they could be used to monitor the dynamics of these two closely related but pathogenically distinct viruses in wild and domestic rabbit populations.     

Antibody responses to rabbit haemorrhagic disease virus in predators, scavengers, and hares in New Zealand during epidemics in sympatric rabbit populations

AIM: To test for antibodies to rabbit haemorrhagic disease (RHD) virus (RHDV) in sera from mammals and birds associated with rabbit populations infected with RHDV. METHODS: Sera from feral and domestic cats, feral ferrets, stoats, hedgehogs, hares, harrier hawks, and black-backed gulls were taken (apart from some of the hares) from areas in New Zealand where RHD was active among rabbit populations. The presence of antibodies to RHD was investigated using a competition enzyme-linked immunosorbent assay (ELISA). RESULTS: Some individual animals of all species were seropositive. Thirty eight of 71 feral cats, but only 1/80 domestic cats were seropositive at a 1:40 dilution. The latter had not been exposed to RHDV. Also reactive in the ELISA were 2/8 stoats; 11/115 ferrets, with significantly more females having antibodies than males; 4/73 hedgehogs; 2/18 hawks, and 1/30 gulls. Three of 66 hares, comprising 3/14 from one population, were seropositive. CONCLUSIONS: Apart from the hares, all these species are known to prey upon rabbits or scavenge their carcasses, a possible means of exposure to RHDV. The possibility that the positive test reactions were due to cross-reactions with other caliciviruses cannot be ruled out, especially for the hares. Nor could the study differentiate whether the positive results were due to an antigenic reaction to ingestion of RHDV, as suggested by overseas work, or to infection of new species by RHDV. These possibilities are being investigated further.     

Detection of antibody to turkey coronavirus by antibody-capture enzyme-linked immunosorbent assay utilizing infectious bronchitis virus antigen

An antibody-capture enzyme-linked immunosorbent assay (ELISA) for detection of antibody to turkey coronavirus (TCV) utilizing infectious bronchitis virus (IBV) antigen was developed. Anti-TCV hyperimmune turkey serum and normal turkey serum were used as positive or negative control serum for optimization of the ELISA system. Goat anti-turkey immunoglobulin G (light plus heavy chains) conjugated with horseradish peroxidase was used as detector antibody. The performance of the ELISA system was evaluated with 45 normal turkey sera and 325 turkey sera from the field and the cutoff point was determined. Serum samples of turkeys experimentally infected with TCV collected sequentially from 1 to 63 days postinfection were applied to the established antibody-capture ELISA using IBV antigens. The optimum conditions for differentiation between anti-TCV hyperimmune serum and normal turkey serum were serum dilution at 1:40 and conjugate dilution at 1:1600. Of the 325 sera from the field, 175 were positive for TCV by immunofluorescent antibody (IFA) assay. The sensitivity and specificity of the ELISA relative to IFA test were 93.1% and 96.7%, respectively, based on the results of serum samples from the field turkey flocks using the optimum cutoff point of 0.18 as determined by the logistic regression method. The ELISA values of all 45 normal turkey sera were completely separated from that of IFA-positive sera. The ELISA results of serum samples collected from turkeys experimentally infected with TCV were comparable to that of the IFA assay. Reactivity of anti-rotavirus, anti-reovirus, anti-adenovirus, or anti-enterovirus antibodies with the IBV antigens coated in the commercially available ELISA plates coated with IBV antigens could be utilized for detection of antibodies to TCV in antibody-capture ELISA.     

The influence of normal guinea-pig serum and tissue culture assay system on foot-and-mouth disease virus neutralisation

The inclusion of normal guinea-pig serum in neutralisation reactions involving foot-and-mouth disease virus (FMDV) increased the neutralisation titre and rate of neutralisation by guinea-pig antiserum derived from animals convalescent from FMDV. Such inclusion had little or no effect on neutralisation involving guinea-pig antiserum collected early in infection or early or convalescent bovine antisera. Higher neutralisation titres and more rapid neutralisation were found from assay in bovine thyroid cells than in IB-RS-2 cells. Not all the increase in neutralising activity was removed by heating at 56 degrees C, indicating that other factors in addition to complement were responsible. In some tests a virus fraction resisting neutralisation was found and, when rabbit anti-guinea-pig gamma-globulin was added, an increase in neutralisation resulted, suggesting that most of the persistent virus fraction was due to the presence of infective virus-antibody complexes.