A Preliminary Comparative Study of a Dipstick Colloidal Dye Immunoassay and Two Antigen-detection ELISAs for Diagnosis of Trypanosoma evansi Infection in Cattle

A dipstick colloidal dye immunoassay (DIA) was developed for the field diagnosis of Trypanosoma evansi infection using affinity-purified polyclonal antibodies (PcAbs) and the monoclonal antibody (McAb) 8B9. PcAbs were adsorbed onto Palanil Red dye particles and used as dye reagents. Dipsticks were dotted with four different antibodies; normal rabbit and mouse IgGs as negative controls, and anti-T. evansi PcAb and McAb 8B9, which capture trypanosome antigens in the tested samples. Since the dye reagent bound to the captured antigens, the presence of coloured dots on the dipstick identified trypanosome infections. The sensitivity of the DIA was compared with two antigen detection ELISAs (Ag-ELISA); one was PcAb-based and the other was based on a combination of the same Mc- and PcAbs as were employed for the DIA. With a positive serum, the DIA detected trypanosomal antigen up to a dilution of 1:500 for both the PcAb and McAb dots, at which dilution the PcAb- and combination-based Ag-ELISA gave positive OD readings of 0.13 and 0.36, respectively. When 124 field sera were tested, circulating antigens were detected in 51 (41%) samples by the DIA, and 76 (61%) and 49 (40%) samples by the PcAb- and combination-based Ag-ELISAs respectively, of which 48 (63%) and 34 (69%) were also positive by the DIA.     

Antibody-ELISA for Trypanosoma evansi: Application in a serological survey of dairy cattle, Thailand, and validation of a locally produced antigen

Trypanosoma evansi is generally considered a mild pathogen in bovines. However, in Asia, acute and chronic signs have been observed in cattle, with high levels of parasitaemia, abortion and death. Investigations in Asian cattle are needed to better understand this epidemiological situation. To generate comparable data at a regional level, development and standardization of an antibody-enzyme linked immunosorbent assay for T. evansi (ELISA/T. evansi) was initiated and applied in an epidemiological survey carried out in dairy cattle in Thailand. A batch of 1979 samples was collected from dairy farms located throughout the country's four regions. Soluble T. evansi antigens initially produced in France were also produced in Thailand for comparison and technology transfer. Screening of 500 samples allowed us to identify reference samples and to determine the cut-off value of the ELISA. Seropositive animals – some of them confirmed by PCR – were found in the four regions, in 12 out of 13 provinces, in 22 out of 31 districts, in 56 farms out of 222 (25%, 95%CI ± 6%) and in 163 animals out of 1979 (8.2, 95%CI ± 1.2%). Estimated seroprevalence in 35 farms ranged between 1% and 30%, and in 21 farms it was >30%. Approximately 25% of survey cattle were exposed to the infection, in various situations. A sub-sample of 160 sera was tested on both antigens. Wilcoxon's (Z = 1.24; p = 0.22) and McNemars's tests (CHI2 = 3.55; p = 0.09) did not show any significant differences, showing that the locally produced antigen is suitable for further evaluation in the surrounding countries. Use of this standardized serological method will broaden knowledge of the prevalence and impact of the disease at the regional level in South-East Asia. Further validation of this ELISA will be necessary in other host species such as buffalo, horse and pig.     

Detection of foot-and-mouth disease virus infection in vaccinated cattle

The aim of this study was to evaluate the value of commercially available kits for the detection of foot-and-mouth disease (FMD) virus infection in vaccinated cattle. The cattle were vaccinated with a commercial aqueous FMD vaccine type A24 and subsequently challenged 28 days post vaccination with homologous FMD virus. Seven of eight animals were protected from clinical disease and all became carriers. They were bled sequentially for up to 130 days post infection and samples of sera were tested with three ELISA kits: CHEKIT FMD-3ABC, Ceditest FMDV-NS and SVANOIR FMDV 3ABC-Ab ELISA. The Ceditest kit appears to be relatively higher sensitive than the others. When examined with this ELISA, all cattle developed of FMDV nonstructural proteins (NSPs) antibodies and remained positive throughout the period of the experiment. The response of antibodies against 3ABC antigen delayed in two cattle challenged with FMDV A24 virus. One of the cattle reacted negatively in Svanoir ELISA kit and sera from two animals were found negative in CHEKIT ELISA. It can be concluded that all tested kits can be a promising tool for FMD control and eradication campaigns in situation where emergency vaccination was applied.     

Evaluation of an ELISA for the diagnosis of experimentally induced and naturally occurring Leptospira hardjo infections in cattle

An enzyme-linked immunosorbent assay (ELISA) for the diagnosis of Leptospira interrogans serovar hardjo (hardjo) infection in cattle was compared with the microscopic agglutination test (MAT). Glutardialdehyde was used in the ELISA to couple sonicated hardjo antigen to the microtiter plate. Mouse monoclonal anti-bovine IgG1 coupled to peroxidase was used as conjugate. Sera from calves experimentally inoculated with hardjo reacted positively in the MAT as early as 10 days after inoculation; these sera did not react positively in the ELISA until 25 days after the first inoculation. Positive and negative field sera from 704 adult cattle on 90 farms were examined by the MAT and the ELISA; a 90% correlation between the two tests was demonstrated. Eighty-six sera from calves inoculated with four Leptospira serogroups other than hardjo and 227 field sera from adult cattle with naturally occurring leptospirosis other than hardjo were examined by the ELISA. Fewer than 1% of these heterologous sera reacted with hardjo antigen in the ELISA. We concluded that the ELISA described in this report is an advantageous alternative to the MAT for diagnosing leptospirosis.     

Urogenital infection and seminal excretion after inoculation of bulls and rams with chlamydiae.

Five mature rams and 4 bulls were inoculated parenterally with bovine or ovine chlamydial strains of type 1 and 2. One to 3 days later, all animals developed a chlamydemia lasting 4 to 8 days. Chlamydial agents were isolated from the semen near the end of the chlamydemic phase. All rams and 3 of 4 inoculated bulls excreted chlamydiae in the semen for 22 to 29 days. From 8 to 39 days after inoculation, selected rams or bulls were killed to test for chlamydial infection in the urogenital tract and other organs. Chlamydiae were isolated in developing chicken embryos from testis, epididymis, and accessory sex glands. Bulls examined 29 and 39 days after inoculation did not harbor chlamydiae. Chlamydiae were also not isolated from 3 control bulls which were from the same herd as the principal bulls. All inoculated bulls and rams had a group-specific chlamydial antibody response within 7 days. The titers reached maximal levels of 128 to 512 at 14 days after inoculation. Subsequently, the antibody titers decreased gradually. Seminal plasma collected at different times after animals were inoculated did not fix complement in the presence of chlamydial group antigen. The number of polymorphonuclear leukocytes in the semen increased during the experiment. The semen was grossly purulent in 2 rams inoculated with the type 2 chlamydial strain of polyarthritis.     

Preliminary evaluation of diagnostic tests using horses experimentally infected with trypanosoma evansi

Seven surra negative horses were intravenously inoculated with 3 x 10(6)Trypanosoma evansi parasites derived from a camel. One horse was maintained as an uninfected negative control. Three antigen and three antibody detection tests were evaluated for diagnosis of infection in horses. The microhaematocrit centrifugation test (MHCT) was the most sensitive, first detecting parasites between one and three days (x 2.4) post infection (p.i.). The antigen (ag)-ELISA detected antigen between three and ten days (x 6.6) p.i. The latex agglutination test (LAT) first gave positive results on day 3 (x 3.0) p.i. Following the treatment of horses with trypanocidal drugs, the MCHT and the mouse inoculation test (MIT) became negative. Antigen levels using LAT declined and reached pre-infection levels in five out of six horses during the period of observation (92-279 days). Antigen levels using the ag-ELISA declined as well but did not reach pre-infection levels in any of the six horses.Three antibody detection techniques, ab-ELISA, card agglutination test (CATT), and immunofluorescent antibody test (IFAT) detected antibodies in the blood of all seven infected horses but not in the uninfected control. However, the ab-ELISA did not discriminate clearly between sera from infected and uninfected horses because unacceptably high ELISA background readings were detected in 15% of the surra negative horses shipped to the UAE from the UK. The ELISA antibody increased above pre-infection levels in the six horses experimentally infected, but not in one horse. In this horse the ELISA antibody level exceeded the cut-off level only after the reoccurrence of the T. evansi infection. The IFAT detected antibodies 15.7 days p.i. in all infected horses.     

Evaluation of Diagnostic Tests for <Emphasis Type="Italic">Trypanosoma evansi</Emphasis> in Experimentally Infected Pigs and Subsequent Use in Field Surveys in North Vietnam and Thailand

This study is concerned with the evaluation of established diagnostic tests for diagnosis of Trypanosoma evansi in pigs. The immune trypanolysis test (TL), card agglutination test (CATT), latex agglutination test (LATEX), enzyme-linked immunosorbent assay (ELISA), microhaematocrit centrifugation technique (MHCT) and mouse inoculation (MI) tests were initially evaluated in experimentally infected fattening pigs. All infected pigs were confirmed parasitologically positive with both MHCT and MI. Results of the serological assays indicated that the TL could be a reference test for the presence of RoTat 1.2 antibodies in pigs. The results of the CATT and LATEX were inconsistent with the TL while the ELISA results correlated with the TL results. The four serological assays were subsequently used in two field surveys in Vietnam and Thailand. Results of the two agglutination assays (CATT and LATEX) were not consistent and did not correlate with TL results. The ELISA at percentage positivity of 22 appeared to have good ability to discriminate between seropositive and seronegative animals. Of the 437 samples collected at smallholder pig premises in northern Vietnam, no positive pigs were detected with the TL test. In Thailand, 77 samples were collected from five farrowing farms with a history of surra. Two parasitologically positive sows were found and on each farm seropositive sows were detected.     

Molecular fingerprinting confirms extensive cow-to-cow intra-herd transmission of a single Mycobacterium bovis strain

In this study we have characterized M. bovis isolates from a herd of cattle in Uvalde, Texas in which 52 of the 193 animals selected at random in 1994 from a herd of 331 were caudal fold skin-test positive. Thirty-two of 52 skin-test positive cattle had gross lesions at slaughter, and isolations of M. bovis were made from 29 animals. The herd was comprised of Red Devon cattle purchased between 1978 and 1980 (n = 26) and breeding bulls (n = 3) introduced at later times, and all were tuberculosis test negative at the time of purchase. Other animals were natural additions (offspring) of these cattle. One additional animal, a Holstein present on the ranch at the time of purchase in 1976, was retained to nurse orphaned and weak calves. Using several molecular fingerprinting techniques we have verified a clonal relationship among the M. bovis isolates consistent with infection originating with a single strain. The molecular fingerprint patterns demonstrate the stability of the profiles despite persistence and spread of the organism within the herd for two decades and confirms their use in epidemiological tracing.     

Detection of carriers of foot-and-mouth disease virus among vaccinated cattle

To investigate and optimise detection of carriers, we vaccinated 15 calves with an inactivated vaccine based on foot-and-mouth disease virus (FMDV) A Turkey strain and challenged them and two further non-vaccinated calves with the homologous virus four weeks later. To determine transmission to a sensitive animal, we put a sentinel calf among the infected cattle from 60 days post-infection until the end of the experiment at 609 days post-infection. Samples were tested for the presence of FMDV, viral genome, specific IgA antibodies, antibodies against FMDV non-structural (NS) proteins or neutralising antibodies. Virus and viral genome was intermittently isolated from probang samples and the number of isolations decreased over time. During the first 100 days significantly more samples were positive by RT-PCR than by virus isolation (VI), whereas, late after infection more samples were positive by virus isolation. All the inoculated cattle developed high titres of neutralising antibodies that remained high during the entire experiment. An IgA antibody response was intermittently detected in the oropharyngeal fluid of 14 of the 17 calves, while all of them developed detectable levels of antibodies to NS proteins of FMDV in serum, which declined slowly beyond 34 days post-infection. Nevertheless, at 609 days after inoculation, 10 cattle (60%) were still positive by NS ELISA. Of the 17 cattle in our experiment, 16 became carriers. Despite frequent reallocation between a different pair of infected cattle no transmission to the sentinel calf occurred. It remained negative in all assays during the entire experiment. The results of this experiment show that the NS ELISA is currently the most sensitive method to detect carriers in a vaccinated cattle population.     

Measurement of antibody in serum and genital fluids of bulls by ELISA after vaccination and challenge with Tritrichomonas foetus

An enzyme-linked immunosorbent assay (ELISA) was developed for detecting antibody to Tritrichomonas foetus using both whole cell antigen (WCA) and membrane protein antigen (MPA). The test was used to detect specific antibody in serum, preputial washings and seminal plasma samples from 7 adult bulls which were vaccinated subcutaneously on 3 occasions with a membrane protein vaccine against T. foetus var brisbane in an oil adjuvant, and from 4 unvaccinated control animals. One month after administration of the third dose of vaccine, vaccinated and control bulls were repeatedly challenged with the live vaccine strain of the T. foetus. A steady increase in serum antibody titre was detected after each inoculation of vaccine when both antigens were used in the ELISA. However, MPA was more sensitive. After challenge, vaccinated bulls developed an increased titre. No specific antibody was detected in control bulls, except in one bull after challenge in which seroconversion was detected. The serum antibody titres of both groups of animals were also measured with the microagglutination test which proved less sensitive than the ELISA. Antibody titres to both antigens, although lower than in serum, were detected in the seminal plasma of vaccinated animals. The control bulls remained non-responsive. No antibody was detected by ELISA in preputial washings from either control or vaccinated bulls prior to challenge. Post-challenge, some of the vaccinated bulls were responsive with both antigens whereas the control bulls remained negative.     

Detection of serum antibodies in Eimeria tenella-infected chickens by enzyme linked immunosorbent assay (ELISA) with merozoite and oocyst antigens.

Soluble antigens prepared from sporulated oocytes and second generation merozoites of E. tenella were used for enzyme linked immunosorbent assay (ELISA) to investigate antibody in sera of two breeds of chickens, i.e. commercial broilers and SPF single comb white leghorn layers, which were experimentally infected with E. tenella. In broilers inoculated with oocysts at 15 days of age, ELISA values increased rapidly after day 19 post inoculation (PI) and reached the maximum lebel on days 29 and 32 PI against both merozoite and oocyst antigen. The values against merozoite antigen were significantly higher than those against oocyst antigen. In SPF layers infected at 15 days of age, the values increased gradually after 7 days PI. There were no significant differences between values against two antigens. Generally, the values in broilers tended to be higher than those in SPF layers, especially against merozoite antigen. In broilers inoculated with oocysts at 1 and 15 days of age, ELISA values increased rapidly and reached the maximum level on days 11 and 20 post second inoculation (PSI) against merozoite and oocyst antigens respectively and then the values against merozoite antigen decreased. The values against merozoite antigen were markedly higher than those against oocyst antigen. In SPF layers inoculated twice, the values reached the highest on day 11 PSI as in the case of broiler; however, after that day, the values against both antigens decreased. The sera reacted similarly against both antigens. The values against merozoite antigen were significantly higher in broilers than in SPF layers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of enzyme-linked immunosorbent assays for the detection of Fusobacterium necrophorum antibody in animal sera

Enzyme-linked immunosorbent assays (ELISA) for the detection of Fusobacterium necrophorum antibody in the sera of rabbits, cattle, and sheep were developed, using a ribosome-rich extract (RRE) from F necrophorum as the antigen. Test character, including optimal antigen dilution and substrate incubation periods, was established, using rabbit, bovine, and ovine antisera produced against RRE from isolates of F necrophorum. Rabbit antisera produced against 7 other species of bacteria were used to test the specificity of the F necrophorum RRE antigen. Cross-reactivity was not detected. Sera from 50 feedlot cattle were examined with the bovine ELISA. Of the 50 samples, 43 (88%) were positive for F necrophorum antibody. The ELISA developed in this study were sensitive and specific and appear to be readily adaptable to serologic investigations of F necrophorum.     

Optimization of in-house ELISA based on recombinant major sperm protein (rMSP) of Dictyocaulus viviparus for the detection of lungworm infection in cattle

The aim of this study was to optimize an in-house ELISA based on a recombinant version of the major sperm protein (MSP) of Dictyocaulus viviparus for routine diagnosis of lungworm infection in cattle. A recombinant MSP (rMSP) was cloned into pGEX-6P-1 vector and expressed as a glutathione-S-transferase (GST) fusion protein in Escherichia coli BL21 (DE3) chemically competent cells. The product was then employed as capture antigen in an ELISA, and validated against 304 samples of known status (216 negative and 88 positive) in which the antibody levels in sera had also been measured earlier with a commercial ELISA kit (Ceditest® lungworm ELISA). The receiver operating characteristic (ROC) curve analysis of the ELISA results estimated the optimized diagnostic sensitivity and specificity as 97.7% (95% confidence interval [CI]: 91.9–99.7%) and 98.1% (CI: 95.3–99.5%), respectively. The results from the in-house rMSP-based ELISA were compared with results obtained on both fecal examination and the Ceditest® lungworm ELISA. Rising antibody levels in sera of experimentally infected calves were observed between 21 and 28 days post infection, when patency was also confirmed by the presence of larvae in feces. Notably, using the in-house rMSP-based ELISA infection was confirmed in calves shedding larvae approximately 3–4 weeks post inoculation, while using the Ceditest® lungworm ELISA those animals remained negative. Additionally, 251 sera samples from calves naturally exposed to the parasites on pasture were used to evaluate the test. In in-house rMSP-based ELISA no cross-reactions were observed with sera from calves infected with the gastrointestinal nematodes (Ostertagia ostertagi and Cooperia oncophora), even though the presence of eggs in the feces was confirmed. Overall, the in-house rMSP-based ELISA optimized in this study showed excellent diagnostic performance for detection of lungworm infection in cattle.     

Detection of trichomonad species in the reproductive tracts of breeding and virgin bulls

Trichomoniasis is a sexually transmitted disease of cattle and a large bowel diarrheal disease of cats caused by Tritrichomonas foetus. Recently, other species of trichomonads have been identified from the prepuce of virgin bulls. It is not clear whether these non-T. foetus isolates are common (nor) or is it clear whether they are also present on the prepuce of breeding bulls. To answer these questions, we first developed an immunofluorescent assay (IFA) with T. foetus-specific monoclonal antibodies for comparison with a T. foetus-specific PCR assay. Results showed that all PCR positive isolates were also IFA positive, whether the isolates were from cats or cattle and PCR negative isolates were IFA negative. Bovine non-T. foetus (non-Tf) trichomonad isolates were detected by both assays in 14 virgin bulls, 10 breeding bulls, 21 bulls of undetermined breeding status (presumably breeding bulls) and 2 cows. These isolates from virgin bulls were mostly Tetratrichomonas spp. whereas the non-Tf isolates from most breeding bulls and the two cows were Pentatrichomonas hominis. All T. foetus isolates were from breeding bulls or bulls of undetermined breeding status. This IFA test which discriminates between T. foetus and non-Tf may be useful as a diagnostic assay, since no effective legal treatment is available, bulls positive for T. foetus are culled. With increasing reports of T. foetus large bowel infection in cats, these monoclonal antibodies may also be useful for diagnosis of feline infection. Since two isolates of non-Tf trichomonads were obtained vaginas of breeding cows, it may be that these parasites are sexually transmitted like pathogenic T. foetus.     

Serological studies on bovine besnoitiosis in Israel

Sera from 1,700 beef and dairy cattle in Israel were tested for besnoitiosis by indirect immunofluorescence. Over 90% of dairy animals were negative whereas about 50% of beef cattle were positive. Among beef bulls the percentage of positive reactors increased with age but in beef cows the pattern was less clear; 36% of young imported bulls negative upon arrival developed antibody titres after one season on the range. Cross-tests with Toxoplasma gondii antiserum and antigen indicated that the results of the survey for besnoitiosis were unlikely to have been influenced by Toxoplasma antibodies.     

Serum C-reactive protein and immune responses in dogs inoculated withBordetella bronchiseptica (phase I cells)

Eight Beagle dogs were inoculated intrabronchially with 5×10⁹ live, avirulent cells ofBordetella bronchiseptica L-414 strain (phase I cells) (B. bronchiseptica) to investigate the serum levels of their C-reactive protein, the white blood cell counts, the antibody responses toB. bronchiseptica in the sera and tracheal secretions, and the effects of prednisolone given to four of the dogs on C-reactive protein (CRP), white blood cells (WBC) and immune responses. In two Beagle dogs inoculated intrabronchially with sterile physiological saline, the concentrations of CRP and the WBC counts did not increase. CRP was markedly increased one day after inoculation in the dogs inoculated withB. bronchiseptica to 385.0–720.0 µg/ml (mean 498±132 µg/ml) in the group given theB. bronchiseptica inoculation only, and to 372.0–649.0 µg/ml (mean 551±106 µg/ml) in the group treated with prednisolone following inoculation ofB. bronchiseptica, as determined by an enzyme-linked immunosorbent assay (ELISA). The CRP levels were 23–95 times the pre-inoculation values, which indicated that prednisolone had no effect on the production of CRP. In the prednisolone-treated group, the WBC count increased and stayed at an increased level for approximately 12 days. An indirect fluorescent antibody test led to the detection of anti-B. bronchiseptica IgM and IgG antibodies in the sera from 5 days afterB. bronchiseptica inoculation and S-IgA and IgG anti-B. bronchiseptica antibodies in the tracheal secretions on the day after the challenge exposure toB. bronchiseptica. The increase in CRP after challenge exposure toB. bronchiseptica was significantly (p<0.05) smaller than that found after the first inoculation ofB. bronchiseptica.Abbreviations CRP C-reactive protein - ELISA enzyme-linked immunosorbent assay - FHA filamentous haemagglutinin - IFA indirect fluorescent antibody - WBC white blood cell(s)     

Increased risk of BVDV infection of calves from pregnant dams on communal Alpine pastures in Switzerland

Skin biopsies and blood samples from 117 calves, the offspring of dams that had been pastured on communal Alpine pastures while pregnant, were examined for bovine viral diarrhoea virus (BVDV) antigen. Immunohistochemical evaluation of skin biopsy samples revealed BVDV antigen in nine (7.7%) calves, and ELISA testing of serum samples was positive for BVDV antigen in six (5.1%). Three calves with positive skin biopsy samples and negative serum results were < 11 days old; it was assumed that maternal antibody interfered with the ELISA testing. Serum samples that were collected at a later date from two of the three calves were positive for BVDV antigen. These results were significantly different from those of a previous study in which the prevalence of persistently infected calves in an average Swiss cattle population was 0.64%. It was concluded that the risk of infection with BVDV is high in cattle sharing a communal Alpine pasture.     

Comparison of bovine cysticercosis prevalence detected by antigen ELISA and visual inspection in the North East of Spain

A cross-sectional serological study on cattle less than 2 years old, using an antigen ELISA for the detection of bovine cysticercosis was carried out between November 2009 and February 2010 in 10 slaughterhouses from the Catalonia region (North-Eastern Spain). Circulating antigen was detected in 23 of 2073 animals, i.e. a sero-prevalence of 1.11% (CI95%: 0.76-1.75%). The determined sero-prevalence was about 50 times higher than the prevalence obtained by visual inspection within the same period: 19 positive animals of 90,891 slaughtered animals (0.02%) in the same slaughterhouses. None of the animals with positive result in the Ag-ELISA was detected by meat inspection.     

Molecular characterization of Mycobacterium tuberculosis complex (MTBC) isolated from cattle in northeast and northwest China

We studied throat swabs and corresponding serum samples collected from 1067 protein purified derivative (PPD)-tuberculin skin test (TST) positive cattle from different regions of China. The 1067 throat swabs were inoculated onto modified Löwenstein–Jensen medium for the isolation and culture of Mycobacteria. Acid-fast bacilli were identified using traditional biochemical methods, polymerase chain reaction (PCR) amplification and multiplex PCR. They were distinguished as Mycobacterium tuberculosis complex (MTBC) and non-tuberculous mycobacteria (NTM) strains. An indirect Enzyme-Linked Immunosorbent Assay (ELISA) was applied to detect specific antibodies against bovine TB (bTB). Correlations among the ELISA, bacteriology and TST were analyzed and compared. Spoligotyping and variable number tandem repeats–mycobacterial interspersed repetitive unit (VNTR–MIRU) analysis were used to genotype the MTBC. In total, 111 strains of Mycobacteria were cultured from the 1067 throat swab samples, including 43 stains of MTBC (14 strains of Mycobacterium bovis and 29 of Mycobacterium tuberculosis) and 68 strains of NTM. Thirty-eight MTBC strains and four NTM strains were isolated from 72 throat swab samples that the ELISA determined were antibody positive; five MTBC strains and 64 NTM strains were isolated from 995 throat swab samples that were antibody negative on the ELISA. The positive isolation rates of MTBC and NTM were 38.7% (43/111) and 61.3% (68/111), respectively. The concordance rate of cultured MTBC with a positive result on the indirect ELISA for antibody was 52.8% (38/72), which was much higher than the positive rate for TST (4.0%; 43/1067). Genotyping of the 43 strains of MTBC isolated, using spoligotyping and VNTR–MIRU, showed that the 43 isolates had 26 genotypes; 16 strains had a unique genotype. Two groups of six strains and two strains, respectively, showed the same spoligotyping pattern, and belonged to the Beijing family and Beijing-like family, respectively. Combined application of spoligotyping and VNTR–MIRU typing would improve the molecular epidemiological investigation and monitoring of the etiology of bTB in China.     

Bovine viral diarrhoea (BVD) infections--control and eradication programme in breeding herds in Slovenia

A Slovenian BVD control and eradication programme was initiated in 1994, and the results from testing of bovine herds for antigen and antibodies in 1996 are presented. Samples originating from breeding herds, breeding herds for young bulls, and insemination stations were tested by antigen or antibody ELISA, or by PCR. Out of 7968 samples from 354 herds we found 18% of the animals antibody-positive. In one region situated in the north-east of Slovenia we found the herds to be almost nearly free of BVDV infections (5% prevalence). No positive antigen ELISA findings were done in 374 blood samples from recruitment herds for young bulls, whereas two out of 206 sera were investigated by PCR-reacted positive. The differences in seroprevalence found between regions is thought to be caused by differences in summer pasturing and husbandry practices.