Evaluation of conventional and radiometric fecal culture and a commercial DNA probe for diagnosis of Mycobacterium paratuberculosis infections in cattle.

Radiometric (RCM) and conventional fecal culture (HEY) and a commercial polymerase chain reaction/DNA probe were evaluated as diagnostic tests for subclinical paratuberculosis in dairy cattle using fecal specimens from a repository of paratuberculosis specimens. The case definition of subclinical bovine paratuberculosis was isolation of Mycobacterium paratuberculosis, by conventional or radiometric culture, from fecal samples or internal organs of dairy cattle without diarrhea or chronic weight loss. Animals designated as free of the disease originated exclusively from certified paratuberculosis-free herds in Wisconsin. Among 182 infected cattle, RCM and HEY fecal culture and the DNA probe had test sensitivities of 54.4%, 45.1% and 33.5%, respectively. Fecal samples from only 111 of the M. paratuberculosis-infected cows tested positive by at least one of the three tests and these cows were designated as fecal shedders; the remaining 71 were considered to have prepatent infections. Among the 111 M. paratuberculosis fecal shedders, RCM, HEY and the probe detected the organism in 89.2%, 73.8% and 55.0% of the fecal specimens, respectively. Herd prevalence significantly affected the sensitivity of all three diagnostic tests (p less than 0.05) but only affected the fecal shedder detection efficiency of the DNA probe (p less than 0.01). No positive DNA probe results were found on 100 randomly selected fecal samples from cows in four certified paratuberculosis-free herds, thus the DNA probe was 100% specific. Probe analyses could be performed in 24 h or less. Time to complete the culture-based tests was 12 wk for HEY and 7 wk for RCM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparisons of the complement-fixation and indirect fluorescent antibody reactions in the detection of bovine babesiasis.

Complement-fixation (CF) and indirect fluorescent antibody (IFA) antigens were prepared from Babesia bigemina isolates obtained in Texas. These serologic procedures were evaluated on 130 serum samples sequentially collected from 5 B bigemina-infected mature cattle, beginning on the day of exposure and continuing for 175 day thereafter. Both tests were effective in detecting specific antibodies for the first 84 days of infection, with 57 of 60 (95%) serums tested being positive on the CF test and 57 of 57 (100%) tests being positive to the IFA test. During the interval from 98 to 175 days, 24 of 60 (40%) of the serums tested were positive with the CF test, and 53 of 56 (95%) were positive with the IFA test. During the first 84 days, a similar linear regression occurred in both CF and IFA serum titers, but after 98 days the IFA regression flattened out, whereas the CF titers decreased below the sensitivity threshold in 60% of the serums tested.     

A COMPARISON OF 4 SEROLOGICAL TESTS IN THE DETECTION OF HUMORAL ANTIBODIES TO ANAPLASMOSIS IN CATTLE

A capillary agglutination (CA), a complement fixation (CF), a plate agglutination (PT) and an indirect fluorescent antibody (IFA) test to detect humoral antibodies to Anaplasma marginale are described. Serums from 3, 4 or 5 groups of cattle were used to examine the efficiency of the tests. Agreement between all 4 tests was 86.6%. Agreement between pairs of tests was greater. The CF test was the most sensitive while the PT test was the least sensitive. However the PT could be carried out very rapidly and was suggested as the best screening test, providing improved antigen preparation techniques could increase sensitivity. The CA, CF and IFA tests all showed a stronger homologous antibody reaction when A. marginale antigen was tested against serums obtained from cattle infected with either A. marginale or A. centrale. Antibodies in the A. marginale serums were first detected by day 7 post-inoculation, rose to peak around day 29 and were still present on day 200. Antibodies in the A. centrale serums were first detected by day 29 rose to a peak around day 50 and had disappeared by day 150.     

Sensitivity and specificity of the complement fixation test for detection of cattle persistently infected with Anaplasma marginale.

The complement fixation (CF) test commonly is used to identify cattle infected with Anaplasma marginale prior to interstate or international movement. Estimates of the accuracy of the CF test in detecting animals persistently infected with A. marginale vary widely. In this study, the sensitivity and specificity of the CF test for detection of carrier animals was determined using serum from 232 cattle previously defined as A. marginale positive or negative by nested polymerase chain reaction methods and hybridization. Considering results from 2 independent laboratories and interpreting a 1:5 suspect reaction as positive, the best estimate of CF test sensitivity was 20%, with a specificity of 98%. Using a 1:10 cutoff, sensitivity decreased to 14% and specificity increased to 99%. Results of this study indicate that the CF test is ineffective for identifying cattle persistently infected with A. marginale and thus is inadequate for anaplasmosis regulatory and surveillance programs.     

Evaluation of a TaqMan real-time PCR for the detection of Theileria parva in buffalo and cattle

A real-time PCR assay based on TaqMan probe chemistry was developed for the detection of Theileria parva DNA in blood samples. It uses a Theileria genus-specific PCR primer set and a T. parva-specific probe to amplify and hybridize with a species-specific part of the 18S rRNA gene of the parasite. The test was evaluated using positive and negative reference blood samples and shown to be specific for T. parva. Analytical sensitivity was determined by testing a dilution series of T. parva positive blood. It was shown to be able to detect parasitaemia as low as 2 × 10(-6)%. The Taqman assay results were also compared with that obtained with the real-time hybridization probe PCR assay, which is currently employed as the official test for the diagnosis of T. parva infections in buffalo and cattle and was shown to be equally sensitive. A panel of 1164 field samples was screened using both assays and 164 samples tested positive in both tests, indicating a good correlation.     

The relationship between the isolation of Brucella abortus and serological status of infected, non-vaccinated cattle

Seventy two non-vaccinated cattle with various complement fixation (CF), rose bengal (RB) and enzyme-linked immunosorbent assay (ELISA) results at slaughter were examined bacteriologically and serologically. Brucella abortus was recovered from 49 (68.1%) of the cattle and the use of a biphasic culture medium was entirely responsible for the detection of 6 (12.2%) of the culture positive cattle. The supramammary and retropharyngeal lymph nodes were the most rewarding tissues to culture. A comparison of culture results and serological status demonstrated that B. abortus could be isolated from cattle with negative RB and CF tests and that the ELISA was useful in detecting these cattle and infected cattle with low CT titres. The RB test was also useful as it detected all but 4 of the cattle found to be infected.     

Cell-mediated immune responses in cattle adult-vaccinated with Brucella abortus strain 19 and in cattle infected with Brucella abortus field strain.

Cell-mediated immune responses in cattle adult-vaccinated with Brucella abortus strain 19, cattle infected with B abortus field strain, and nonexposed cattle were studied by an in vitro lumphocyte-stimulation test (LST). Lymphocytes were prepared from peripheral bovine blood by the Ficoll-diatrizoate technique, and results were assayed for [3H]thymidine incorporation into DNA by liquid scintillation spectrometry. Serotests and bacteriologic isolation attempts were conducted simultaneously with LST. Lymphocytes from cattle infected with field strains had significantly (P = 0.01) higher specific lymphocyte-stimulation inexposed controls. The LST, the serum standard-tube agglutination test (STT), the Rivanol (RIV) test, and the complement-fixation (CF) test correctly classified cattle from which field strains and strain 19 of B abortus were isolated. The LST was negative in cattle vaccinated with B abortus strain 19 (nonshedding), but the three serotests had many false-positive reactions. The CF test had the least false-positive reaction, followed by the RIV test, and the STT was the least specific. Well before the three serotests became positive, the LST was positive in samples from some cattle during the incubation period of the infection. There was little or no correlation between cell-mediated immune responses (as measured by LST) and serum antibody responses (as measured by STT, RIV test, and CF test) in vaccinated but culture-negative cattle and in some nonvaccinated cattle during the incubation period.     

Evaluation of the reverse transcription-polymerase chain reaction/probe test of serum samples and immunohistochemistry of skin sections for detection of acute bovine viral diarrhea infections.

Bovine viral diarrhea viruses (BVDV) cause both acute and persistent infections. While diagnostic tests have been designed to detect animals persistently infected (PI) with BVDV, the reliability of these tests in detecting acute BVDV infections is not known. It is also possible that acute BVDV infections may be confused with persistent infections in surveys for PI animals. In this study, 2 tests presently in use in diagnostic laboratories to test for PI animals, polymerase chain reaction amplification followed by probe hybridization (RT-PCR/probe) of serum samples and immunohistochemical detection of viral antigen in skin biopsies (IHC), were evaluated for their ability to detect acute BVDV infections. Sixteen colostrum-deprived, BVDV-free, and BVDV-antibody-free calves were infected with 6 different BVDV strains. Clinical signs, seroconversion, and virus isolation indicated that inoculated animals did replicate virus. Virus could be detected in 19% (3/16) of acutely infected animals by the RT-PCR/probe technique. No acutely infected animals were positive by IHC.     

Prevalence and molecular detection of Anaplasma marginale, Babesia bovis and Babesia bigemina in cattle from Puntarenas Province, Costa Rica

A study was conducted in 2008 to determine the prevalence of Anaplasma and Babesia infections in cattle in the Puntarenas Province of Costa Rica. Blood samples were taken from a total of 449 cattle during the month of March at 30 farms in the region of Espiritu Santu, Costa Rica. Commercially available enzyme-linked immunosorbent assays (ELISA) were used to determine presence of antibodies to Babesia bigemina and Anaplasma marginale, and real-time PCR was used to determine the presence of DNA from the disease-causing organisms. The ELISA results indicated that 87.5% of the cattle sampled were positive for antibodies to A. marginale, while 59.1% were positive for antibodies to B. bigemina. The real-time PCR results showed that 235 cattle were carrying A. marginale DNA (56.9%), 6 with B. bigemina DNA (1.34%), and 2 with B. bovis DNA (0.45%).     

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.     

Mycobacterium nonchromogenicum in nasal mucus from cattle in a herd infected with bovine tuberculosis

Skin test negative cattle from a herd containing an unusually high proportion (194/382) of tuberculin skin test positive cattle were investigated for remaining Mycobacterium bovis infected animals. Blood samples from the skin test negative cattle, analysed by an antibody ELISA and an interferon-gamma assay, were mostly test negative for M. bovis. Radiometric culture of nasal mucus samples from 48 of the cattle yielded 22 culture positives with acid-fast bacilli and cording in 6 of these. Subculture on solid media was successful for 7, including 2 with cording of the 22 radiometric culture positives. Mycobacterium tuberculosis complex DNA probe testing using the Accuprobe (Gen-Probe, Inc.) and M. tuberculosis complex-specific PCR amplification, performed on the solid media subcultures, were negative. 16S rRNA PCR and sequence analysis were successful for 6 of the 7 solid media subcultures obtained and revealed the presence of Mycobacterium nonchromogenicum in all 6 subcultures. This is the first report of M. nonchromogenicum in nasal mucus of cattle. The observation highlights the importance of integrating definitive tests such as the PCR for diagnosis of bovine tuberculosis and indicates a possible zoonotic risk.     

DNA探针、粪检菌和ELISA三种方法在诊断副结核病中的比较研究

本研究在已构建的副结核分枝杆菌Ｃ２株ＤＮＡ基因文库的基础上，应用正、反向杂交试验从基因文库中筛选出特异性片段ＰＴＰ３１，以光敏生物素标记制成ＤＮＡ探针。用此ＤＮＡ探针以及粪检菌和ＥＬＩＳＡ三种方法分别对３２份副结核菌素（ＰＰＤ）变态反应阳性牛粪便及相应血清样品进行检测，其检出率分别为４７％（１５／３２）、５６％（１８／３２）和３４％（１１／３２）。对随机采集的２７６份牛粪便及血清样品，３种方法的检出率分别为１０％（２７／２７６）、１３％（３６／２７６）和７％（７９／２７６）。本研究结果表明，ＤＮＡ探针与粪检菌呈现正相关性，ＤＮＡ探针比ＥＬＩＳＡ方法能够检出更多的阳性数。     

Indirect fluorescent antibody and complement fixation tests in the diagnosis of bovine theileriosis and babesiosis in Japan

Antibodies against Theileria sergenti and Babesia ovata were detected by indirect fluorescent antibody (IFA) and complement fixation (CF) methods. Both of the antibodies against T. sergenti and B. ovata could be detected with a single IFA testing procedure. Complement fixing antibody tests for T. sergenti in experimentally infected cattle were positive for about 70 days after the parasites could no longer be detected in the blood smear, and were negative thereafter. B. ovata CF antibody titres were negative on the 270th day post-infection, while the IFA tests against both parasites were positive for longer periods. B. ovata IFA-test titres remained relatively high until the 420th day after inoculation. In a survey of sera from naturally infected animals, the rate of detecting antibodies against both parasites was higher using the IFA test than using the CF test. The IFA method was more effective for diagnosis of Babesia infection than the CF technique.     

The bovine immune response to Brucella abortus IV. Studies with a double immunodiffusion test for antibody against A2.

A double immunodiffusion test for precipitins against Brucella antigen A2 was developed and applied to a variety of samples. The A2 precipitins were produced by a heifer infected with B. abortus strain 2308, cattle vaccinated with killed B. melitensis strain H38 or live B. abortus strain 19 and by a dog infected with B. canis. Precipitins were also detected in the second International Standard for anti-Brucella abortus serum, in several anti-B. canis sera and at low levels in one anti-B. ovis serum tested. Antisera produced in calves against Yersinia enterocolitica serotype 0:9 had no anti-A2 activity despite titers greater than or equal to 1/1024 and greater than or equal to 1/80 in standard Brucella agglutination and CF tests, respectively. The test for A2 precipitins lacked specificity as weak reactions were obtained with five of 295 sera from brucellosis-free herds. This test was relatively insensitive, detecting precipitins in only 16 of 24 sera from infected cattle and 27 of 54 sera positive by complement fixation and enzyme labelled antiglobulin tests performed with whole cell and smooth lipopolysaccharide antigens, respectively. The A2 precipitins were detected in nine sera from five cattle, in two infected herds, which were negative by agglutination and complement fixation tests.     

Evaluation of the indirect hemagglutination assay as a practical serodiagnostic test for mycoplasmal pneumonia of swine

Sera from swine experimentally or naturally infected with Mycoplasma hyopneumoniae (the etiological agent of mycoplasmal pneumonia of swine, MPS) were tested by the indirect hemagglutination assay (IHA), the enzyme-linked immunosorbent assay (ELISA) and the complement fixation (CF) test. The IHA detected antibody at comparable times and levels to the other 2 serological tests following experimentally-induced infection. In the late antibody response (greater than or equal to 86 days post-infection), the ELISA titres were higher than either the IHA or the CF test. The IHA appeared least satisfactory when it was used to test sera from commercial swine herds. When 1000 sera were tested, the IHA was positive for only 30 (22%) of 135 sera which were positive by the ELISA and the CF test. The IHA titres were low; 20 of the 30 sera had a titre of only 10. The end-points for the IHA were difficult to read for sera of this low titre. The relationship between positive IHA results for the herd sera obtained at necropsy, and the occurrence of gross or microscopic lesions typical of MPS was poor (41 and 50% agreement, respectively). An agreement of 39% was noted between positive IHA results and the localization of mycoplasmal antigens by an indirect immunofluorescence (IIF) test. However, IHA results correlated significantly (P less than 0.05) with gross and microscopic lesions, but not with the IIF test. No significant correlation was noted between the IHA (or the other 2 serologic tests) and the cultural isolation of M. hyopneumoniae or M. flocculare. On the basis of these results, the IHA appears to have limited promise as a practical test for the diagnosis of MPS in commercial swine herds because of the low titres observed, poor correlation of the IHA and other indicators of MPS, the necessarily subjective determination of end-points, and other inherent technical limitations of the test.     

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.     

An evaluation of the anamnestic test for brucellosis in cattle of the northern pastoral area

SUMMARY An investigation of the anamnestic test for brucellosis using Brucella abortus 45/20 vaccine was carried out in 3 groups of weaner cattle on 2 farms in western Queensland. Each group originally consisted of about 500 cattle. They were bled before and at 6 or 10 weeks after vaccination and again in the following year. The serums were tested by the complement fixation (CFT), Rose Bengal (RBT) and indirect haemolysis tests (IHLT). Most of the cattle reacting to one or more of the tests were killed and selected tissues were subjected to bacteriological examination for B. abortus. B. abortus was isolated from 19 of 30 (63%) pre-vaccinal reactors, 23 (24%) of 96 cattle reacting at 6 or 10 weeks after vaccination (the anamnestic test) and 1 (2%) of 50 cattle reacting one year after vaccination. The reactor found to be infected the year after vaccination had high serological titres in each of the 3 serological tests: RBT of 3, CFT of 128 and IHLT of 256. A subsequent test showed the group to be brucellosis-free. The CFT was the most efficient test. In the pre-vaccination tests 17 of 19 infected animals were positive in the CFT compared with 11 positive in the IHLT and 17 in the RBT. In post vaccination tests 22 of 23 infected animals were positive in the CFT compared with 18 in the IHLT and 19 in the RBT. At the pre-vaccinal and anamnestic tests (6 or 10 weeks after vaccination) 19 of 57 (33%) cattle with CF titre of 4 or 8 yielded B. abortus on culture compared with none of 26 cattle with similar titres in the year after vaccination. The interpretation of CF titres in cattle following 45/20 vaccination needs to be re-examined.     

Detection of antinuclear antibodies by the Inno‐Lia ANA update test in canine systemic rheumatic disease

Background: Certain systemic autoimmune diseases in dogs are characterized by high titers of circulating antinuclear antibodies (ANA), which can be demonstrated by indirect immunofluorescence (IIF). In an earlier study of IIF‐ANA–positive dogs, the Ouchterlony double immunodiffusion (DID) test was used to identify specific autoantibodies. The DID test has largely been replaced with line blot tests in human diagnostic settings. Objective: The objective of this study was to investigate whether the line blot assay Inno‐Lia ANA update test is a useful tool in demonstrating ANA specificities in canine patients with previously diagnosed IIF‐ANA–positive rheumatic disorders. Methods: Serum samples from 3 clinically healthy control dogs and 20 canine patients with clinical signs of systemic rheumatic disease and documented positive results for IIF‐ANA and DID tests were included in the study. The Inno‐Lia ANA update assay was performed with an anti‐canine detection antibody. Results: Six serum samples that had DID positivity with anti‐spliceosomal small nuclear ribonucleoproteins (snRNP) reactivity showed reactivity to multiple snRNP proteins in the Inno‐Lia test. Samples from 2 dogs that had other types of DID positivity also had clear SmB reactivity and 1 had weak reactivity to RNP‐70K. The other serum samples, including controls, were negative. Conclusions: Using the Inno‐Lia ANA update test, multiple snRNP specificities were demonstrated in some canine patients with autoimmune rheumatic disorders. Other canine autoantibodies may exist that are not detected by this test. Further studies are necessary to characterize the target antigen(s) of these remaining autoantibodies in canine sera.     

A Study of Complement-fixation and Serum Agglutination Tests on Vibrio fetus Infected and Immunized Cattle

Complement-fixation (CF) and tube agglutination (TA) tests for demonstration of Vibrio fetus antibodies were conducted on the sera of three groups of ten heifers. One group was vaccinated subcutaneously with a commercial V. fetus var venerealis bacterin and challenged intra-utero, at the external os cervicus one month later; the second was infected only and the third vaccinated only.

The vaccinated cattle developed high CF serum titers, but no such increase was observed in animals infected only. A moderate increase in serum antibody titers was demonstrated by the TA test following either infection or vaccination; although titers observed were not higher than those observed in the sera of some apparently normal uninfected animals. The group receiving both vaccine and challenge was the only one in which significant serum antibody titers were demonstrable by the TA test. The sera of these animals also had significant titers in the CF tests.

The CF and TA tests detected serum antibodies produced by the parenteral inoculation of V. fetus antigen. These two tests were of limited value in detecting serum antibodies from animals with genital V. fetus var venerealis infection, although the formation of local antibodies was demonstrable by the vaginal mucus agglutination test.

     

Evaluation of polymerase chain reaction and dot blot hybridisation tests in the diagnosis of pigeon circovirus infections

Polymerase chain reaction (PCR) and dot blot hybridisation (DBH) tests for detecting pigeon circovirus (PiCV) DNA were developed and evaluated using tissue samples obtained from diseased and clinically normal pigeons, which originated in Belgium and Northern Ireland. When PCR product was visually detected, the limit of detection of the PCR test was 31 fg, while that of the DBH was 1.6p g. For evaluation purposes, the results of the PCR and DBH tests, performed with DNAs extracted from samples of bursa of Fabricius (BF), were compared with those of in situ hybridisation (ISH) and histology. In 32 samples tested by all four tests, 27 (84%) were positive by PCR, 24 (75%) were positive by ISH, 20 (63%) were positive by DBH, and 13 (41%) were positive by histology. Additional PCR testing showed that in some disease-affected birds, PiCV DNA could be detected in a range of tissues including thymus, spleen, liver, kidney and brain. The PCR detection of PiCV DNA in BF samples from clinically normal birds indicated that PCR can detect infections in the absence of disease, a finding that mitigates against its use as a disease diagnostic. In addition, nucleotide sequence determinations indicated that PCR test performance was adversely affected by the sequence diversity exhibited by selected PiCVs. The application of the DBH test to dilutions of test samples indicated that the BF from some diseased pigeons contained very large amounts of virus DNA, as much as 10(13)genome copies/g tissue, and suggested that this test may be a convenient method of providing a semi-quantitative estimate of virus load.