Evaluation of the sensitivity and specificity of bovine tuberculosis diagnostic tests in naturally infected cattle herds using a Bayesian approach

Test-and-slaughter strategies have been the basis of bovine tuberculosis (BT) eradication programs worldwide; however, eradication efforts have not succeeded in certain regions, and imperfect sensitivity and specificity of applied diagnostic techniques have been deemed as one of the possible causes for such failure. Evaluation of tuberculosis diagnostic tools has been impaired by the lack of an adequate gold standard to define positive and negative individuals. Here, a Bayesian approach was formulated to estimate for the first time sensitivity (Se) and specificity (Sp) of the tests [single intradermal tuberculin (SIT) test, and interferon-gamma (IFN-γ) assay] currently used in Spain. Field data from the first implementation of IFN-γ assay (used in parallel with SIT test 2-6months after a first disclosure SIT test) in infected beef, dairy and bullfighting cattle herds from the region of Castilla and Leon were used for the analysis. Model results suggested that in the described situation: (i) Se of SIT test was highly variable (40.1-92.2% for severe interpretation, median=66-69%), and its Sp was high (>99%) regardless interpretation criteria; (ii) IFN-γ assay showed a high Se (median=89-90% and 83.5% for 0.05 and 0.1 cut-off points respectively) and an acceptable Sp (85.7% and 90.3% for 0.05 and 0.1 thresholds) and (iii) parallel application of both tests maximized the combined Se (95.6% using severe SIT and 0.05 cut-off point in the IFN-γ assay). These results support the potential use of the IFN-γ assay as an ancillary technique for routine BT diagnosis.     

Eradication of bovine leukemia virus infection in commercial dairy herds using the agar gel immunodiffusion test.

Demands for bovine leukemia virus test negative breeding cattle and for semen from bovine leukemia virus test negative bulls by several countries have encouraged the eradication of bovine leukemia virus infection from selected herds in Canada. This project was undertaken to evaluate the suitability of the agar gel immunodiffusion test, standardized to detect anti-bovine leukemia virus glycoprotein antibodies, for eradication of bovine leukemia virus from commercial dairy herds. Of nine participating herds, the prevalence rate of bovine leukemia virus infection was low (less than 10%) in three, medium (11-30%) in four and high (greater than 30%) in two. The herds were tested by the agar gel immunodiffusion test, reactors were removed and the herds were then retested at regular intervals. The results indicate that it is possible to eliminate bovine leukemia virus infection from the herds after two to three cycles of agar gel immunodiffusion tests and prompt removal of the reactors.     

The gamma-interferon assay for diagnosis of bovine tuberculosis in cattle: conditions affecting the production of gamma-interferon in whole blood culture

The recently developed gamma-interferon (IFN-gamma) assay system for the diagnosis of bovine tuberculosis in cattle has been accredited by the Standing Committee on Agriculture for use in Australia. In this test system, whole blood is incubated with tuberculin purified protein derivative (PPD) antigens for 16 to 24 h. The plasma is then collected and assayed for IFN-gamma production using an enzyme immunoassay (EIA). The assay system has proven to be a rapid, sensitive and inexpensive method for measuring antigen specific cell-mediated reactivity when compared with the more traditional lymphocyte proliferation assay. The IFN-gamma assay is the first in-vitro cellular assay to be used as a routine diagnostic test in veterinary medicine. While the IFN-gamma EIA has been optimised, several conditions affecting the production of IFN-gamma in whole blood culture needed investigation. We determined that optimal IFN-gamma production required the use of heparinised blood, cultured with 20 micrograms/ml of PPD within 8 h of collection. The use of blood collected post mortem resulted in reduced sensitivity for the assay. The kinetics of IFN-gamma release were established as were the effects of intradermal tuberculin testing on the IFN-gamma assay.     

The management of bovine reproduction in elite herds

The management of bovine reproduction is the cornerstone of health provision in elite herds. Aims and objectives for reproductive performance should be herd specific and data to monitor progress should not only be frequently collected, but also analysed and reported. Strategic monitoring of animals should include a vaginal examination for evidence of uterine disease, as well as transrectal ultrasonography of the genital tract. There has been considerable advancement in our ability to intervene in the reproduction of cattle during the last 50 years. However, it is salutary to note that during this time fertility has consistently declined, despite increasing veterinary intervention. Most elite herds use artificial insemination and success depends on accurate detection of oestrus expression, but this appears to be less overt than 25 years ago. In addition, half the cattle have abnormal oestrous cycles after parturition and conception rates are decreasing by 1% per year. Risk factors for abnormal oestrous cycles include puerperal problems, negative energy balance, which can be evaluated by body condition scoring, and uterine disease. Bacterial contamination of the uterus is ubiquitous after parturition in cattle and disease disrupts ovarian follicle growth and function. Reproduction is also disrupted by stress associated with clinical disease, pain or a sub-optimal environment. The challenge for veterinarians providing reproduction control programmes to elite herds is to transfer our knowledge of the problems underlying subfertility to the farm, in order to provide effective solutions.     

Detection of swine torque teno virus in Italian pig herds

Anellovirus is a recently created, floating genus of viruses. Torque teno virus (TTV), the type species in the genus, was first discovered in a human patient with a post-transfusion hepatitis of unknown aetiology. Recently, TTV genetically related to but distinct from those discovered in humans have also been found in animals, including pigs. The aims of this study were to estimate the prevalence of swine TTV in Italian pig herds and some risk factors possibly associated with this infection. Serum samples from 179 healthy pigs from 10 farms located in north-central Italy were tested by polymerase chain reaction for the presence of swine TTV DNA. Viral DNA was found in the sera of 43 pigs (24.0%), coming from eight of the 10 farms examined. Prevalence was significantly higher in finishing herds (40.1%) than in farrow-to-finish herds (11.0%) and did not depend on the size of the herd. Within the finishing herds the prevalence was significantly higher in weaners (57.4%) than in fatteners (22.9%), but this difference was not observed in farrow-to-finish herds. No relationship was observed between the prevalence of swine TTV and the implementation of some general hygiene practices and biosecurity procedures within the herds.     

Longitudinal study of Salmonella infection in Italian farrow-to-finish swine herds

A longitudinal study of Salmonella enterica infection was carried out in five Italian farrow-to-finish swine herds previously known to be infected by Salmonella. Five litters were randomly selected from each herd and in each litter six piglets were randomly selected and individually identified. Thus, the study included 30 pigs from each farm. At weaning, individual blood samples were collected for serological examination from all selected piglets and on the same day from all sows in the farrowing unit. Piglets were bled again at approximately 60, 90, 150, 210 and 270 days of life whereas the last blood sample was collected at slaughtering. In one of the herds, in which the duration of productive cycle was about 12 months, the last blood samples were collected at 350 days of life. With the same time scheduling, five pen pooled faecal samples were collected from each herd for bacteriological examination. At slaughtering, mesenteric lymph nodes were collected from each ear-tagged pig. Sero-prevalence (cut off S/P ratio 0.25) in sows varied from 93.8% to 100%. In four herds, sero-prevalence in piglets showed a similar profile with complete decline of maternal antibodies at day 60 and clear sero-conversion between day 90 and day 150. In one herd, sero-conversion was observed earlier and 56% of piglets were positive at day 90. The peak of sero-prevalence was observed between day 210 and day 270. Sero-prevalence at slaughtering varied from 66% to 100%. Salmonella was isolated from faecal samples in four of five herds. No Salmonella was isolated from mesenteric lymph nodes at slaughter in two of the herds. Culture prevalence from mesenteric lymph nodes in the other three herds ranged from 3.3% to 30%. This longitudinal study provides original information about epidemiological dynamics of Salmonella enterica infection in Italian swine herds in consideration of the unique extended fattening period typical of the Italian production.     

Study on prevalence of bovine viral diarrhoea virus (BVDV) antibodies in 29 Italian dairy herds with reproductive problems

An epidemiological survey on prevalence distribution of antibodies to BVDV was carried out in dairy cattle herds during 1995-1996 in northern Italy. A total of 704 serum samples from 29 non-vaccinated herds reported to have reproductive problems were tested for serum neutralising antibodies. In each herd, sampling was based on the stratification by age into five classes (< 6 months old calves, 6-12 months old calves, pregnant heifers, uniparous, pluriparous). Overall, 53.3% of samples were serologically positive, with the lowest ratio in 6-12 months old calves (37.9%) and the highest in pluriparous cows (71.2%).     

Improving the specificity and yield of the contagious bovine pleuropneumonia complement fixation test antigen.

Several methods for increasing yield and specificity of the contagious bovine pleuropneumonia complement fixation test antigen which is derived from Mycoplasma mycoides subsp mycoides, strain V5, were examined. Changes in culture conditions that increased the cell mass per unit volume of culture did not result in comparable increase in antigen yield.Sixteen to 60-day-old cultures yielded more boiled cell antigen than younger cultures. Some antigen in young cultures appeared to be masked, probably by galactan. The yield of antigen extracted from boiled cells with ethonol was as much for two to eight-day-old cultures as for older cultures. The ethanol extract antigen was less reactive with false positive bovine sera than standard boiled antigen while reactivity with anti Mycoplasma mycoides sera was similar to that of standard antigen. Adsorbed gamma globulin was not detected in either boiled or ethanol extract antigen. The data suggest that several complement fixing antigens were present in antigens derived from older cultures.     

Evaluation of four commercial enzyme-linked immunosorbent assays for the diagnosis of bovine paratuberculosis in Chilean dairy herds.

The accuracy of 4 commercial enzyme-linked immunosorbent assays (ELISAs) for diagnosis of bovine paratuberculosis was compared using sera from 53 Mycobacterium avium subsp. paratuberculosis (MAP) fecal culture-positive dairy cows (cases) and sera from 345 dairy cattle resident in 11 fecal culture-negative herds on 2 consecutive occasions 1 year apart (controls). The specificity of all 4 ELISA kits was >99%, and their diagnostic sensitivity ranged from 30.2% to 41.5%. Pairwise comparison of ELISAs found no significant differences (McNemar's chi-square test > 0.05), and assay agreement for categorical assay interpretation (positive or negative) was high (>98%) with kappa values ranging from 0.84 to 0.95. Receiver operating characteristic (ROC) curve analysis and the corresponding area under the ROC curves indicate that kit B had the highest overall accuracy. Thus, all 4 ELISA kits for bovine paratuberculosis had comparable accuracy when tested on Chilean dairy cattle, with kit B having a slight statistical advantage based on ROC area under the curve analysis. This suggests that any of the 4 kits could be appropriate for herd certification and for paratuberculosis control programs on Chilean dairy cattle.     

Multiple apparent Sarcocystis abortion in four bovine herds

Foetuses recovered from multiple abortions in four dairy herds had multifocal nonsuppurative encephalitis, myocarditis and hepatitis. Focal placentitis was usually present. Sarcocystis-like protozoa were found in the brains of foetuses from two of the outbreaks. Apart from excess salivation in a few cows in one herd, farmers reported no clinical abnormalities prior to the abortions, and all cows remained normal after the abortions. Dogs and cats fed an affected foetus and neonatal calves from the affected herds failed to excrete protozoa in their faeces. The identity of the protozoa in the foetal brain was not confirmed.     

Production and characterization of monoclonal antibodies specific for bovine gamma-interferon

Nine stable hybridoma cell lines were established which secreted specific monoclonal antibodies (MAbs) to bovine gamma-interferon (BoIFN-gamma). Specific binding of each of the MAbs to recombinant BoIFN-gamma (rBoIFN-gamma) was demonstrated in an indirect ELISA, whilst none of the MAbs bound to rBoIFN-alpha or rBoIFN-beta. In a Western blot the MAbs reacted with the 16 kDa and 32 kDa polypeptides present in rBoIFN-gamma preparations. Competitive ELISA's showed that four MAbs bound to one epitope on rBoIFN-gamma, and the other five MAbs bound to a separate epitope. Two MAbs, each recognising different epitopes, were shown to neutralise the anti-viral activity of natural BoIFN-gamma.     

The gamma-interferon test: its usefulness in a bovine tuberculosis survey in African buffaloes (Syncerus caffer) in the Kruger National Park

A survey to determine the bovine tuberculosis status of buffalo herds north of the Olifants River in the Kruger National Park was conducted, using a new diagnostic approach. Diagnosis of Mycobacterium bovis infection was accomplished using the gamma-interferon assay technique in 608 adult buffaloes out of a total of 29 discreet herds. The animals were immobilized in groups of 10-15, bled, individually marked and then revived and released on site. As soon as test results were available (after 26-36 h), the same buffalo herd was relocated by tracking the frequency of a radio-collar previously fitted to one adult cow per group during the initial operation. Bovine reactors were identified, darted and euthanased from the helicopter. Necropsy and culture findings of all culled buffaloes showed excellent correlation with the results of the ante-mortem gamma-interferon test. The survey revealed that over and above the two positive herds that had been identified during a previous survey carried out in 1996, there were three additional, but previously unidentified, infected herds in the region north of the Olifants River.     

Duration of bovine intramammary infections in commercial dairy herds

Data on the infection status of cows on seven commercial dairy farms were collected over 492 full lactations. Foremilk samples were taken at an average interval of five weeks. A total of 249 streptococcal and 433 staphylococcal infections were diagnosed. Spontaneous elimination occurred in 49 per cent of all streptococcal infections and in 54 per cent of Staphylococcus aureus infections. The average duration of spontaneously eliminated infections was 10.8 weeks for Streptococcus agalactiae, 9.9 weeks for Strep dysgalactiae, 10.4 weeks for Strep uberis and 12.8 weeks for Staph aureus. The average duration of infections persisting until drying off was 19.3 weeks for Strep agalactiae, 18.7 weeks for Strep dysgalactiae, 18.5 weeks for Strep uberis and 25.2 weeks for Staph aureus. The method and rate of elimination of infection as found in this analysis are of value for estimating new infection rates and selecting quarters for dry cow therapy.     

Effect of a test and control program for bovine Johne's disease in Victorian dairy herds 1992 - 2002

OBJECTIVE: To report on progress in Johne's disease (JD) control in infected dairy herds participating in the Victorian Johne's disease Test and Control Program (TCP). PROCEDURE: Clinical histories and JD testing data recorded by the Department of Natural Resources and Environment (now called Department of Primary Industries) were analysed for 542 dairy herds participating in the TCP. The herds were required to conduct annual herd tests of cattle 2 years old and older with an enzyme linked immunosorbent assay (ELISA), cull the reactors and manage the younger cattle to minimise infection. RESULTS: Testing of over 680,000 animals identified over 10,000 reactors giving an average prevalence of reactors at the first whole-herd test (T1) of 1.78%. There was a relatively rapid increase in the incidence of clinical disease before the TCP started and then it markedly declined. There was a slow and interrupted decline in reactor prevalence, with a marked peak occurring at the fourth herd test (T4). The average age of reactors and clinical cases was 5.7 and 5.9 years, respectively. Of the reactors and clinical cases detected during the TCP, 87% and 95% respectively, were born before the TCP started. Thirty herds completed the program by achieving three successive negative whole herd tests and 91 herds dropped out because of inability to comply with the agreed requirements of the program. There were no home-bred reactors born after the start of the program in 253 (47%) herds and of the 522 herds that were tested more than once, there were 319 (61%) herds in which no home-bred reactors were detected after the first year of testing. The number of ELISA positive animals detected at T1 appeared to be only about 26% of the animals from that round that subsequently became positive or developed clinical disease at later test rounds. CONCLUSION: The TCP caused a marked decline in the number of clinical cases, probably because animals in which clinical disease was imminent were detected by testing and removed. A reduction in prevalence of reactors occurred only when most herd members were born after the TCP started. The sensitivity of the ELISA appears to be low based on the large number of reactors that were negative at T1 but were positive at later tests. Low sensitivity of diagnostic tests and the long incubation period of the disease limits meaningful analysis of the program until it has continued for some years. Measures adopted in the TCP have not broken the cycle of infection in many participating herds. It is unsure if this was because of poor compliance with control recommendations or a poor understanding of methods of transmission by scientists. Eradication is not feasible in the short-term.     

Control of bovine leukemia virus infection in dairy herds by agar gel immunodiffusion test and segregation of reactors.

Canadian cattle intended for export, in future, may have to originate from herds which are serologically negative for bovine leukemia virus, in addition to being negative individually by the agar gel immunodiffusion test as currently required. In this study, agar gel immunodiffusion testing of herds and segregation of reactors were examined. The results demonstrated that bovine leukemia virus infection could be controlled when three groups: 1) bovine leukemia virus-positive, 2) bovine leukemia virus-negative and 3) replacement cattle were maintained at separate locations.