A complementary diagnosis of naturally occurring tuberculosis in water buffaloes (Bubalus bubalis) in Rio de Janeiro using a MPB70-ELISA, Brazil

As tuberculosis is still a worldwide infection and buffalo breeding represents an important economic activity in various countries, the purpose of this study was to employ an enzyme-linked immunosorbent assay (ELISA) using MPB70 as a capture antigen for the diagnosis of naturally occurring tuberculosis in water buffaloes in Brazil. After the introduction of newly acquired cattle onto a tuberculosis (TB) free farm, an outbreak of TB was recorded in a mixed herd comprising water buffaloes (21) and cattle (46). The entire herd was tested by intradermal tuberculin injection (ITT) and positive animals were slaughtered and tested by culture, polymerase chain reaction (PCR), and ELISA. From the 21 buffaloes sampled, three were reactive by ITT. All the three had positive culture and ELISA, while PCR was positive in two of them. Besides that, one ITT-negative buffalo was slaughtered and presented positive results by both culture and ELISA, and was considered as anergic. Although there were only few animals, those findings demonstrate the diagnostic usefulness of an MPB70-ELISA to correctly detect Mycobacterium bovis tuberculosis in water buffaloes.     

Seroprevalence estimation and management factors associated with high herd seropositivity for <Emphasis Type="Italic">Babesia bovis</Emphasis> in commercial dairy farms of Puerto Rico

A cross-sectional study was conducted to determine individual cow seroprevalence of Babesia bovis in adult lactating dairy cattle of Puerto Rico (PR), to assess the associations of farm management factors on herd seroprevalence, and to document the species of ticks infesting cattle within these farms. Antibody activity against B. bovis was determined using an indirect fluorescent antibody test (IFAT). Serum samples were obtained from 2,414 adult lactating dairy cattle from 76 randomly selected commercial dairy farms. Herd seroprevalence ranged from 0 to 51% with an overall individual cow seroprevalence for B. bovis of 26%. Ticks were collected from animals on 7 (9%) of the 76 participating commercial dairy farms. All collected ticks (n = 87) were Rhipicephalus (Boophilus) microplus. Factors associated with high herd seropositivity were dairy farms with calf but not heifer raising facilities (OR = 16, 95% CI = 3.0-86), having more than 4 neighbors with cattle (OR = 17, 95% CI = 1.6-178), same producer owning more than one farm (OR = 7.2, 95% CI = 1.6-32), and use of government services to apply amitraz on cattle (OR = 5.5, 95% CI = 1.5-20).     

The use of MPB70 and MPB83 to distinguish between bovine tuberculosis and paratuberculosis

In order to demonstrate the potential to distinguish paratuberculosis (PTB) from bovine tuberculosis infection (TB), ELISAs with M. bovis-specific MPB70 or MPB83 as capture antigens were developed and tested on two groups of cattle: Group A comprised 23 animals positive for Mycobacterium avium paratuberculosis (Map) and TB free. Group B comprised 48 animals from a Map free herd during the previous 5 years, but confirmed as tuberculous by positive results on PPD testing and M. bovis culture. Results demonstrated a significant difference (p < 0.01) between reactivity of sera from these groups, encouraging the study of purified proteins to differentiate between both diseases.     

Estimates of within-herd incidence rates of Mycobacterium bovis in Canadian cattle and cervids between 1985 and 1994

We analysed the individual-animal data from six of the nine outbreaks of tuberculosis in Canadian cattle and cervids from 1985 to 1994. A “positive/reactor” animal was one which had either a positive culture or a positive or suspicious reaction on a mid-cervical, comparative cervical, or gross or histopathological test for tuberculosis. Individual-animal data were collected only for herds which had one or more positive/reactor animals. Data were collected from the outbreak records in the Regional or District offices of Agriculture and Agri-food Canada’s Animal and Plant Health Directorate. The within-herd spread of Mycobacterium bovis was studied by determining the most-likely date at which the herd was first exposed to M. bovis and the number of reactions which had developed by the time the herd was investigated. The animal-time units at risk in the herd were probably overestimated, resulting in conservative estimates of the within-herd incidence rates. Negative-binomial regression was used to investigate factors which might have influenced the within-herd spread of tuberculosis. Increasing age appeared to be a risk factor for being a positive/reactor animal. When compared to animals 0–12 months old, animals 13–24 months old had an incidence rate ratio (IRR) of 7.6, while animals >24 months old had an IRR of 10.4 (p=0.009). Actual and predicted incidence rates for tuberculosis in mature (>24 months old) animals were calculated. Actual and predicted incidence rates were similar for cervids, within an outbreak. There was more variability between actual and predicted rates in the dairy and beef animals. In the one outbreak (Ontario) where there were positive/reactor cervid, dairy and beef herds, the actual incidence rate for cervids (IR=9.3 cases per 100 animal-years) was almost twice that of dairy cattle (IR=5.0) and three times that of beef cattle (IR=3.1).     

Comparison of different testing schemes to increase the detection <Emphasis Type="Italic">Mycobacterium bovis</Emphasis> infection in Ethiopian cattle

Host immune responses to Mycobacterium bovis (M. bovis) infection are variable at the different severity stages of pathology of the disease. In countries like Ethiopia, where routine screening of bovine TB is not undertaken, the use of tests which measure cellular and antibody responses may help for the maximum detection of infection. In the present study, 701 cattle were tested for bovine tuberculosis (BTB) using comparative intradermal tuberculin (CIDT) test, interferon (IFN)-γ test, and lateral flow assay. The apparent prevalence was 32% when all the three tests were used, but varied from 23 to 25% when a pair of tests was used and from 9% to 15% when a single test was used. Agreement was observed between CIDT and IFN-γ tests both at a cut-off >2 mm (Kappa ± standard Error, k ± SE, 0.129 ± 0.045; 95%CI = 0.041,0.216) and a cut-off >4 mm (k ± SE, 0.094 ± 0.044, 95%CI = 0.008,0.179) while no agreement was observed either between CIDT test and lateral flow assay (k ± SE, −0.04 ± 0.033; 95%CI = −0.104,0.024) or between IFN-γ tests and lateral flow assay (k ± SE, −0.031 ± 0.032; 95% CI = −0.093,0.031). Thus, the use of more than one test leads to the detection of the maximum number of infected animals.     

Differential antigen recognition by serum antibodies from three bovid hosts of Mycobacterium bovis infection

Cattle, bison and buffaloes are susceptible to Mycobacterium bovis, the causative agent for bovine tuberculosis. Accurate and timely identification of infected animals is critical for improved management and control of disease in these species. Bovids develop humoral immune responses to M. bovis infection making serological tests attractive for tuberculosis screening. However, optimization and validation of antibody assays designed for various animal species require understanding of antigen recognition patterns in each target host. The objective of this study was to characterize serological reactivity profiles generated by cattle, American bison, and African buffaloes in tuberculosis. Serum samples from M. bovis-infected animals were tested for the presence of IgM and IgG antibodies to MPB70/MPB83 and CFP10/ESAT6 chimeric proteins using Dual-Path Platform technology. All three host species showed IgG responses of higher magnitude and frequency than IgM responses; further, IgM seroreactivity was limited to MPB70/MPB83, whereas IgG antibodies recognized both test antigens. In cattle, the IgM and IgG responses were elicited mainly by MPB70/MPB83, whereas bison and buffaloes showed similar IgG seroreactivity rates for MPB70/MPB83 and CFP10/ESAT6 antigens. The findings demonstrate distinct patterns of predominant antigen recognition by different bovid species in M. bovis infection.     

Mycobacterium kansashi infection in a bontebok (Damaliscus pygaragus dorcas) herd: diagnostic challenges in differentiating from the Mycobacterium tuberculosis complex

Two adult female bontebok (Damaliscus pygarus dorcas) were euthanized because of signs of pneumonia and weakness (case 1), and a nonresponsive lameness with draining fistula (case 2). Necropsy findings were similar in both cases and consisted of disseminated granulomatous lesions in the liver, kidneys, spleen, lungs, pleural surfaces, and multiple lymph nodes. Mycobacterium kansasii was isolated from both cases after multiple attempts on a variety of samples by two laboratories. The remaining four animals in the herd were tested for antibody responses using the Chembio ElephantTB STAT-PAK, DPP VetTB kits, and multi-antigen print immunoassay (MAPIA), for immune reaction using the intradermal tuberculin test, and by tracheal wash cultures, and thoracic radiographs. Banked serum samples collected in 2005 and obtained from the original institution, revealed 1/9 (11.11%) seropositive animals using the three immunoassays. Retesting the current herd in 2008 showed 2/6 (33.33%) seropositive animals by the three tests, with MAPIA demonstrating antibody reactivity to MPB83 and MPB70 proteins. Inconsistent intradermal tuberculin test results, cross-reactivity in serologic assays designed for tuberculosis detection, difficulty in obtaining definitive identification by culture, and inability to identify a source of infection created challenges in distinguishing the atypical mycobacteriosis due to M. kansasii from the initially suspected tuberculous infection in this herd. Owing to regulatory considerations, differences in host-to-host transmission, and source of infection between Mycobacterium tuberculosis complex and nontuberculous mycobacteria, correct diagnosis is crucial for management of these diseases in wildlife species.     

First-time detection of mycobacterium species from goats in Ethiopia

Tuberculosis (TB) is an important zoonosis affecting a wide range of hosts. An abattoir study was conducted on 1,536 randomly selected male goats slaughtered at Modjo Modern Export Abattoir to determine the prevalence of tuberculosis in slaughtered goats. Carcasses and organs of all the study animals were first examined by routine meat inspection followed by detailed meat inspection. Samples from tuberculous lesions were cultured for mycobacterial isolation and identification. Histopathology was done on 31 samples with tuberculous lesions. Detailed meat inspection detected 65 (4.2%; 95% confidence interval (CI) = 3.3–5.4%) tuberculous lesions. From these, 20 (30.8%) samples were confirmed mycobacterium positive on culture, out of which 18 were Mycobacterium bovis and two were Mycobacterium tuberculosis. Routine meat inspection failed to detect tuberculous lesions in 23% of carcasses with TB lesions detected by detailed examination. However, no statistically significant difference was observed between both methods in detecting tuberculous lesions (Kappa = 0.87). Origin and age of the goats did not statistically affect the disease prevalence (P > 0.05). Histopathologic lesions were observed in 21 samples (68%; 95% CI = 50.1–81.4%) out of the 31 carcasses with gross tuberculous lesions examined by histopathology. Eighteen (58%) tuberculous samples positive for histopathology were also culture positive. The sensitivity and specificity of histopathology were 90% (95% CI = 76.9–100%) and 72.7% (95% CI = 46.4–99%), respectively, using culture as a reference test. To the best of our knowledge, this is the first report of caprine tuberculosis from Ethiopia. Further studies are required at the farm level to determine the prevalence of tuberculosis in the general goat population.     

Tuberculosis in goats on a farm in Ireland: epidemiological investigation and control

This paper describes an outbreak of tuberculosis (TB) caused by Mycobacterium bovis in a dairy goat herd on a farm in Ireland, where 66.3 per cent of the herd tested positive to the single intradermal comparative tuberculin test (SICTT) at initial detection. An epidemiological investigation was conducted to determine the origin of the outbreak, considering issues such as animal movements and herd management practices. Infection was introduced with a consignment of goats, as determined by the variable number tandem repeat profile. Infection was eradicated using a test and cull programme involving the SICTT, the interferon-γ assay and a multiplex immunoassay (Enferplex TB).     

Prevalence and mortality rate of peste des petitis ruminant (ppr): possible association with abortion in goat

Present study was designed to investigate the prevalence and mortality (%) caused by Peste des Petitis Ruminant (PPR) and its possible association with abortion in goat flocks at different areas of Pakistan. A total of 140 animals were samples in the population of 650 which was having 185 deaths (Mortality rate = 28 %) from three different regions of the country. There were 58 abortions in the 140 pregnant goats of above said population One hundred & ten (110) serum samples from diseased, recovered and apparently healthy animals were tested for the presence of PPR antibodies by competitive ELISA (c ELISA). Eighty-four (84) animals were positive for PPR antibodies whereas in apparently healthy adult goats in the same flock, no PPR antibodies were detected. Twenty-four (24) tissue samples collected from the dead animals and six samples from aborted fetus were tested for the presence of PPR antigen by Immuno-capture ELISA (Ic ELISA). Nineteen (19) out of thirty (30) organ samples mainly from lung, spleen, lymph node were found positive for PPR antigen but negative from lungs of aborted fetus. There was a high rate of abortions (28–45 %) in each of the outbreak and it was highest in the outbreak of Golra Sharif, Islamabad (No. = 21 in total population of 100). As the serum samples from the aborted dams were found positive for PPR antibodies so the study provides the possible association of mortality and prevalence of PPR disease with high rate of abortions in goat.     

A cross-sectional study on bovine tuberculosis in Hawassa town and its surroundings,Southern Ethiopia

A cross-sectional study was conducted in Hawassa town and its surroundings from October 2007 to May 2008 to estimate the prevalence of bovine tuberculosis (BTB) based on comparative interadermal tuberculin test (CIDT) and abattoir survey. Accordingly, 39 herds comprising 413 cattle were subjected to CIDT, and the herd and individual animal prevalence were 48.7% (19/39) and 11.6% (48/413), respectively. One of the 16 milk samples collected from tuberculin-positive cows was culture positive. The prevalence significantly differed among the age group (P = 0.001) and management system (P = 0.001). Thus, age group over four (OR = 7.9) and animal with poor management system (OR = 4.1) had a higher odds for tuberculin reactivity compared to those with age group under four and cattle with good management system, respectively. Of the total 1,023 cattle subjected to postmortem examination, 11 (1.1%) were found to be positive for gross tuberculous lesions. Larger proportion (50%) of TB lesion was recorded in the respiratory pathway followed by digestive system (28.6%) and prescapular lymph nodes (21.4%). Of 14 tissue specimens collected from the gross lesions, four (28.6%) were positive for histopathological TB lesions. In conclusion, this study revealed the importance of BTB in the study area in particular and the region in general.     

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.     

Sero-epidemiological investigation of bovine toxoplasmosis in traditional and smallholder cattle production systems of Tanga Region,Tanzania

In view of the worldwide importance of Toxoplasma gondii and the fragmented information on the seroprevalence of the disease in animals in Tanzania, a study, using the modified Eiken latex agglutination test (LAT), was conducted from May 2003 to January 2004 to determine the prevalence of antibody to T. gondii in 130 randomly selected farms comprising 655 cattle. The overall seroprevalence of T.gondii antibodies in cattle and farms were 3.6% and 13%, respectively. Risk factors for animal and herd-level toxoplasmosis seropositivity were tested using multivariable logistic regression to control for confounding factors. Cattle managed under traditional husbandry practises were more likely to be seropositive than those managed under smallholder practises (48% versus 4.7%; p < 0.01). Herd size of ≥ 9 cattle were at greater risk of acquiring infection than herds holding fewer animals [≤ 9 cattle], (odd ratio [OR] = 3.99; 95% confidence interval [CI], 0.97–16.48; P = 0.001). We concluded that seroprevalence at herd level was high and relatively low at animal level, possibly due to the reduced susceptibility of cattle to T.gondii infection as compared to goats and sheep. The high seroprevalence in animals managed by traditional husbandry practise suggests that the parasite is widely distributed in the environment and could pose a public health threat to the people living in those areas.     

Seroprevalence estimation and management factors associated with high herd seropositivity for <Emphasis Type="Italic">Anaplasma marginale</Emphasis> in commercial dairy farms of Puerto Rico

A cross-sectional study was conducted to determine individual cow seroprevalence of Anaplasma marginale in adult lactating dairy cattle of Puerto Rico (PR) and to assess the associations of farm management factors on herd seroprevalence. Antibody activity against A. marginale was determined using the MSP-5 competitive enzyme-linked immunosorbent assay. Serum samples were obtained from 2,414 adult lactating dairy cattle from 76 randomly selected commercial dairy farms. Herd seroprevalence ranged from 3 to 100% with an overall individual cow seroprevalence for A. marginale of 27.4%. Factors associated with high herd seropositivity were pasture grazing as the main feed source (OR = 6.5, 95% CI = 1.2–34), observed monkeys on the premises (OR = 13, 95% CI = 1.2–138), use of 11% permethrin (OR = 17, 95% CI = 2.2–129), farmers who attended an acaricide certification program (OR = 0.18, 95% CI = 0.04–0.74), and lack of a fly control program (OR = 5.6, 95% CI = 1.3–24).     

Tuberculosis outbreak in a dromedary racing herd and rapid serological detection of infected camels

A recent outbreak of tuberculosis (TB) in a dromedary racing herd of 58 animals involved 3 infected animals. Disease was confirmed at necropsy by finding gross lesions from which Mycobacterium bovis (antelope type) was isolated. Sera collected from the camels in this herd were used to evaluate two new serological methods, Multiantigen Print Immunoassay (MAPIA) and rapid test (RT) developed using the lateral-flow technology, in comparison with the intradermal tuberculin tests. Antibodies were found in all three infected dromedaries by both RT and MAPIA, but not in the remaining 55 animals in the herd. With the limited number of animals tested in this study, the serological assays showed the potential for convenient, rapid, and accurate diagnosis of TB in live camels.     

Comparing recombinant MPB70/SahH and native 20-kDa protein for detecting bovine tuberculosis using ELISA

Bovine tuberculosis (bTB) is a zoonosis caused by Mycobacterium bovis. Test-and-cull protocols and gross pathological examinations of abattoir animals as well as milk pasteurization have been implemented to prevent the spread of tuberculosis from animals to humans worldwide. Despite the importance of precise and rapid diagnostic tests, conventional methods including intradermal skin tests and γ-interferon assays are limited by the high rate of false-negative results for cattle in the late infectious stage and due to laborious and time-consuming procedures. Therefore, antibody detection methods such as enzyme-linked immunosorbent assay (ELISA) are urgently needed to supplement the established approaches and expand the diagnostic window. This study was conducted to develop a bTB ELISA by evaluating recombinant and native proteins and various assay parameters. We produced recombinant MPB70 and SahH (M70S) and a native 20-kDa protein (20K) and optimized the ELISA protocol. The 20K ELISA showed 94.4% sensitivity and 98.2% specificity with an optimal sample-to-positive ratio cut-off of 0.531. The sensitivity and specificity of M70S ELISA were 94.4% and 97.3%, respectively, with an optimal sample-to-negative ratio cut-off of 1.696. Both assays showed acceptable diagnostic efficiency and could be used for bTB diagnosis in combination with established methods for herd screening and to expand the diagnostic window.     

Sensitive diagnosis of bovine tuberculosis in a farmed cervid herd with use of an MPB70 protein fluorescence polarization assay

After histopathological examination of a lesion found in a herd member returned a diagnosis of mycobacteriosis, a farmed herd (n = 47) of elk (Cervus elaphus nelsoni) and red deer (C. elaphus elaphus) was investigated for bovine tuberculosis with a battery of antemortem and postmortem diagnostic tests. Every animal was tested with the mid-cervical tuberculin skin test; all 47 had negative results. All of the 16 adult animals and 15 of the 31 calves (approximately 2-years-old) were blood-tested with a lymphocyte stimulation test (LST) and a fluorescence polarization assay (FPA), which detects antibody to the MPB70 protein antigen. At necropsy of the 31 blood-tested animals, tissues were harvested for histopathological examination and culture of mycobacteria. Mycobacterium bovis was isolated from 16 of the 31 animals, and a scotochromogen was also isolated from 1 of the 16 whose tissues yielded M. bovis. Each of these 16 animals, 15 of which were calves, also received a histopathological diagnosis of mycobacteriosis. Other species of mycobacteria, including those belonging to the M. avium and M. terrae complexes, were isolated from an additional 7 animals. The FPA was scored “positive” or “suspect” for 16 animals, 13 (81%) of which were culture-positive for M. bovis. The other 3 animals that were culture-positive for M. bovis had negative FPA results. Of the 3 FPA-positive or FPA-suspect animals that were culture-negative, 2 were suspected to have mycobacteriosis on the basis of the histopathological examination. The 7 animals from which Mycobacterium species other than M. bovis were cultured were all FPA-negative. The only animal with positive LST results was also FPA-positive and culture-positive for M. bovis. The M. bovis isolates had an identical spoligotype pattern, with an octal code of 664073777777600. This is the first report of the isolation and identification of this strain type in Canada.     

Tuberculosis Infection in Animal and Human Populations in Three Districts of Western Gojam,Ethiopia

Tuberculosis concurrent infection in cattle and their respective owners in North‐western Ethiopia had been investigated. Two hundred and ten cattle owners and 1220 heads of their cattle were included in the study to determine degree of tuberculosis infection in cattle owned by tuberculosis patients and tuberculosis patients. Comparative intradermal tuberculin test, bacteria culturing, acid fast staining and biochemical tests were used to conduct the study. The prevalence of tuberculosis was significantly (P < 0.001) higher in cattle owned by tuberculosis patients than in cattle owned by non‐tuberculosis owners, and infection with tuberculosis was threefold greater in cattle owned by tuberculosis‐positive owners. Further more, cattle owners who consumed raw milk were at higher risk (P < 0.001, OR = 3.23) for tuberculosis infection than those who consumed boiled milk. Mycobacterium tuberculosis (15.4%), Mycobacterium bovis (44.1%) and atypical mycobacteria (38.5%) were identified from milk collected from tuberculin‐positive cows using biochemical tests. Similarly M. tuberculosis (74.5%), M. bovis (14.9%) and atypical mycobacteria (8.5%) were identified from sputum and fine needle aspiration specimens of tuberculosis patient cattle owners. Mutual transmission of mycobacterium from animals to humans and vice versa has been signified.     

Evaluation of the gamma-interferon assay for eradication of tuberculosis in a goat herd

Objective To evaluate the usefulness of the gamma-interferon assay in the diagnosis of caprine tuberculosis in comparison with a single intradermal tuberculin test, and to obtain a group of animals free from this infection in a herd with a high prevalence.
Design An immunological study involving four serial comparative gamma-interferon and single intradermal tuberculin tests.
Animals A herd of 87 goats of Guadarrama breed.
Procedure Serial testing and segregation of animals.
Results We found that the number of infections detected by the gamma-interferon test was considerably greater than the number detected by the single intradermal tuberculin test. A group of 10 animals was negative to both tests in two consecutive rounds and three kids were negative in the last round of testing.
Conclusions Gamma-interferon assay is appropriate for diagnosis and eradication of tuberculosis in goats. This test is able to detect early Mycobacterium bovis infection. Avian reactors with simultaneous increased reaction to bovine PPD in the gamma-interferon assay (designated as avian^B reactors) should be considered test positive for M bovis . By serial testing with the gamma-interferon and the single intradermal tuberculin tests, and a policy of segregation of kids at birth, it is possible to achieve a group of animals test negative for tuberculosis from a herd of goats with high immunoreactivity to this infection.