Specificity of an immunochromatographic test for anthrax

OBJECTIVE: To evaluate the specificity of an immunochromatographic test (ICT) for anthrax in cattle. DESIGN: A comparison of an ICT with blood smear and culture in uninfected cattle. PROCEDURE: Two hundred and forty blood samples were collected from dead cattle at two knackeries within Victoria and tested on-site with an ICT for the detection of protective antigen (PA) of Bacillus anthracis. Blood smears were prepared on-site and blood samples transported to the laboratory for aerobic and anaerobic culture. The results of the ICT were compared with blood smear and culture. Animals were regarded as not infected with B anthracis if the organism was not detected in a stained blood smear or on culture. Ten healthy yearling cattle were vaccinated with live spore anthrax vaccine and blood samples collected on days 0 to 7 and day 15 were tested in the ICT for the presence of PA. RESULTS: All blood samples from the 240 knackery cattle were ICT, smear and culture negative. All blood samples from the 10 vaccinated cattle were ICT negative. CONCLUSION: The ICT is a test with high specificity in cattle (98.5 to 100%; 95% CI) and recent vaccination of cattle does not give rise to positive reactions.     

Use of polymerase chain reaction to detect Brucella abortus biovar 1 in infected goats

The polymerase chain reaction (PCR) was used to diagnose goat brucellosis and compare its sensitivity against some of the most commonly used serological and bacteriological techniques. Twenty two female and one male out of 300 clinically healthy, mixed-breed goats were randomly chosen from a ranch located at Marín, Nuevo León, Mexico. Milk and blood samples were taken from each animal and used to obtain both microbiological cultures and DNA of the pathogen, and sera was tested against Rose Bengal antigen (RBT). Results showed that 86% of the blood samples were positive on the PCR test, while 60% were positive on the serological test. The pathogen was isolated from only one blood culture. Sixty four percent of the milk samples were positive on PCR tests, but failed to yield bacteria in culture. Biochemical and PCR specific assay demonstrated that Brucella abortus biovar 1 was associated with the infection. This study demonstrates the higher sensitivity of PCR over RBT and blood culture and its potential towards a rapid identification of Brucella strains.     

Enhancement of reactive oxygen species production from canine blood leukocytes by human recombinant interleukin-12

A novel biological activity of human recombinant interleukin-12 (rhIL-12) on canine peripheral blood mononuclear cells (PBMC) was investigated in vitro. Canine PBMC were cultured in the presence or absence of rhIL-12 for 3 days. The reactive oxygen species (ROS) production induced by opsonized-zymosan (OZ) was then measured by a luminol-dependent chemiluminescense assay and demonstrated that the ROS production was enhanced after culture with rhIL-12. A nitro blue tetrazolium test and flowcytometry analysis revealed that canine lymphocytes, eosinophils, and monocytes were capable of ROS production, but that monocytes had the highest capacity. These results suggest that rhIL-12 enhances ROS production from canine monocytes.     

The use of MPB70-ELISA for the diagnosis of caprine tuberculosis in Brazil

After one clinical case that evidenced the outbreak, a complete screening by intradermal tuberculin test was performed in one goat herd in Brazil. The herd was composed by 500 animals and 83 of them (16.6%) showed to be reactive to the comparative double cervical intradermal test. Four months after the test, all the 83 reactive animals were slaughtered and blood samples were collected from 45 of them, for serological assays. From those 45, 32 were randomly chosen for necropsy and histopathological and bacteriological procedures were conducted. Histopathology evidenced at least one characteristic lesion of tuberculosis in each animal, with typical granulommas where acid-fast bacilli (AFB) could be observed. Bacteriology was positive for Mycobacterium bovis in 22 samples (68.7%), therefore confirming the etiology of the outbreak. Sera of 45 animals plus 20 other from a certified free tuberculosis farm were tested in an ELISA using the recombinant M.bovis protein MPB70 as capture antigens. From those, 43 were reactive to the test, with high ODs results, considering a cut-off point established by ROC curve analyzing results (cut-off = 0.8; mean = 0.55; range: 0.157–1.357). These results suggest that MPB70-ELISA can be considered as a reliable tool to diagnose tuberculosis in goat herds, since this assay was capable to correctly detect 95.6% of the animals here examined.     

Brucella melitensis infection in dog: a critical issue in the control of brucellosis in ruminant farms

Canine brucellosis is a contagious disease associated with health implications for humans as well as for a wide range of wild and domesticated animals. In this study, 173 dog blood specimens were sampled from herding dogs in three different provinces including Tehran (n = 127), Qom (n = 40) and Alborz (n = 6) provinces. The presence of Brucella antibodies was determined using Rose Bengal plate test (RBPT), slow agglutination test (SAT) and 2-mercaptoethanol (2-ME), respectively. The seropositive samples were further screened using blood culture and PCR tests to identify and differentiate the implicated Brucella species. According to our results, 24.3% (42/173), 13.8% (24/173) and 6.3% (11/173) of blood samples were tested positive using RBPT, SAT and 2-ME, respectively. However, among 42 seropositive samples, only 38.1% (16/42) and 14.2% (6/42) were positive by PCR and culture, respectively. Brucella melitensis biovar 1 and biovar 2 was isolated from the bacterial cultures of 6 blood samples and confirmed by biotyping, AMOS PCR and Bruce-ladder PCR assays. These findings highlight the potential risk of Brucella transmission from dog to humans along with other livestock and reflect the critical role of infected dogs in the persistence of Brucella infections among ruminant farms. This study stresses the need for further epidemiological investigations on canine brucellosis among herding dogs and suggests the systematic screening of the disease among companion animals such as dogs in order to improve brucellosis surveillance and control programs.     

Comparison of agar gel immunodiffusion test, rapid slide agglutination test, microbiological culture and PCR for the diagnosis of canine brucellosis

The performance of the rapid slide agglutination test, with and without 2-mercaptoethanol (RSAT and 2ME-RSAT) and agar gel immunodiffusion test (AGID) was evaluated for the diagnosis of brucellosis in naturally infected dogs. The microbiological culture, PCR and clinical parameters were used as reference. A total of 167 dogs were clinically examined and tested by blood culture, culture of semen/vaginal swab and PCR in blood and semen/vaginal swab. According to the results observed the 167 dogs were divided into three groups: Brucella canis infected dogs (Group 1), B. canis non-infected dogs (Group 2) and dogs with suspected brucellosis (Group 3). The dogs were then tested by RSAT, 2ME-RSAT and AGID. Groups 1 and 2 were used to calculate the diagnostic sensitivity and specificity of the serological tests and the results observed in Group 3 were also discussed. The diagnostic sensitivity of RSAT, 2ME-RSAT and AGID was respectively 70.58%, 31.76%, and 52.94%. The diagnostic specificity of RSAT, 2ME-RSAT and AGID was respectively 83.34%, 100%, and 100%. In dogs with suspected brucellosis 15% were RSAT positive, none was 2ME-RSAT positive and 5% were AGID positive. Although the serological tests are the most commonly used methods for brucellosis diagnosis, a significant proportion of false-negative results were observed highlighting the importance of the direct methods of diagnosis, like blood culture and PCR to improve the diagnosis of canine brucellosis.     

Prevalence of bovine viral diarrhea virus infection in bulls in artificial insemination centers in Poland

This study was directed at the evaluation of the prevalence of bovine viral diarrhea virus (BVDV) infection in bulls in artificial insemination centers. Both serological and virological examinations were performed. Blood samples were tested in micro-seroneutralization test for BVDV antibodies. Virus isolation was performed in cell culture and BVDV antigen was detected in an indirect immunofluorescence assay with monoclonal antibodies. One hundred and seventy-five serum samples and 219 whole blood samples for virus isolation were tested. Neutralizing antibodies were found in 86% of the bulls. Persistent infection (PI) was detected in 0.9% of the analyzed blood samples.     

Comparison of conventional and non-conventional techniques for the diagnosis of bovine brucellosis in Sudan

The objective of the present study was to detect brucellosis in suspected dairy cattle in Khartoum State, Sudan using the conventional serological tests and tests done on milk in comparison to a PCR-based technique. Milk and blood samples collected simultaneously from suspected brucellosis cows (n = 147) in 12 different dairy farms around Khartoum State were used in the study. Overall, 54 (36.7%) of the total milk samples were positive according to the milk ring test (MRT), while 29 (19.7%) of the serum samples were positive according to the Rose Bengal test (RBT); microscopy on modified Ziehl-Neelsen-stained slides detected 13.6% of the cases, and recovery of Brucella species on both Brucella medium and tryptic soya agar was 7.5%. Thirty-three (22.4%) samples were found positive on PCR-amplified IS711 which were then taken as positive brucellosis cases. The differences of RBT and PCR-IS711 from MRT were highly significant (P < 0.05). MRT detected more cases of bovine brucellosis compared to RBT, PCR, microscopy, and culture. MRT is recommended as a noninvasive test compared to RBT, and it is less expensive compared to PCR and culture.     

The Establishment and Application of Metabolic Model of Blood Cells in Chicken

The experiment was aimed to establish a drug metabolic model of the blood cells in chicken. The effects of three kinds of culture medium (L-15,M199 and RPMI1640) and different concentrations of fetal bovine serum (FBS) were compared to optimize the culture condition of blood cells,and the proper time of medication was determined after the cell vitality was measured by MTT assay. The content of ribavirin and its metabolites (TCONH₂ and RTCOOH) were detected after the ribavirin was dosed. The results showed that the cell viability was highest when the blood cells cultured in L-15 medium,the state of blood cells was better when 10% FBS was added to the medium. The best time for medication was when blood cells were cultured for 3 h. The content of ribavirin was decreased with the time of administration,the metabolites of ribavirin were increased quickly after half an hour, it changed slowly after 3 h. In conclusion,the metabolic model of blood cells in chicken was successfully established,and the blood cells cultured in vitro were better using L-15 medium supplemented with 10% FBS. The metabolic transformation function of blood cells in chicken was indicated by the medication test of ribavirin and it could be used to study the drug metabolism in vitro.     

鸡血细胞药物代谢模型的建立与应用

试验旨在建立鸡血细胞药物代谢模型,并用利巴韦林进行验证。本试验比较了3种不同培养基（L-15、M199和RPMI1640培养基）及添加不同浓度胎牛血清的细胞培养效果,采用MTT法测细胞活力,确定最佳给药时间,之后用利巴韦林进行给药,检测培养液中利巴韦林及其代谢物（TCONH₂和RTCOOH）含量。结果发现,3种培养基中用L-15培养基培养时细胞存活率最高,胎牛血清添加浓度为10%时血细胞状态较好;血细胞活力检测表明其最佳给药时间为培养3 h;利巴韦林给药后,其含量随着时间的延长而降低,TCONH₂和RTCOOH在给药0.5 h时迅速产生,给药3 h后其浓度变化趋于平缓。综上所述,本试验建立的鸡血细胞代谢模型操作简便,用添加10%胎牛血清的L-15培养基培养效果较好,利巴韦林给药试验表明,鸡血细胞存在一定的代谢转化功能,该鸡血细胞代谢模型可用于某些药物的体外代谢研究。     

Increased generation of superoxide in erythrocytes infected with Babesia gibsoni.

The present study was conducted to clarify the mechanism underlying the oxidative process in erythrocytes infected with Babesia gibsoni. The parasite B. gibsoni was cultured together with erythrocytes from normal dogs for 7 days. When parasitemia reached 12.0-13.4% at Day 7. the production of superoxide in erythrocytes was significantly higher in the parasitized culture than in the control culture (p<0.005). The concentration of thiobarbituric acid reactive substances (TBARS) in erythrocytes in parasitized culture was also significantly increased compared with the control culture (p<0.005), indicating that lipid peroxidation was greater in infected erythrocytes than in non-infected cells. In addition, the rates of superoxide generation in the blood of B. gibsoni-infected dogs were also significantly higher than in non-infected dogs (p<0.001). These results indicate that superoxide anions are increased in erythrocytes parasitized with B. gibsoni. and suggest that oxidative damage, due to lipid peroxidation, might be caused in host erythrocytes by the parasite.     

Optimization of an in vitro assay to detect Streptococcus equi subsp. equi

Streptococcus equi is the etiologic agent of a highly infectious upper respiratory disease of horses known as strangles. Bacterial culture methods and polymerase chain reaction (PCR) of nasopharyngeal washes and guttural pouch lavages are used routinely to test clinical and carrier animals for the presence of S. equi but no definitive or gold standard test method has been shown to be optimal. We hypothesized that (i) a flocked swab submerged in ten-fold serial dilution suspensions of S. equi prepared in 0.9% NaCl would detect more colony forming units (CFU) than a rayon swab when used to inoculate a blood agar plate, (ii) centrifugation of a 1ml aliquot of each suspension would improve the limit of detection (LOD) by bacterial culture and PCR compared to the culture or PCR of submerged swab samples, (iii) PCR of the centrifuged samples from each suspension would be more sensitive than aerobic culture alone, and (iv) PCR of a 1ml aliquot directly from a sample would be more sensitive than PCR of a sample following submersion of a flocked swab in 1ml saline. Using 7 ten-fold serial dilutions of S. equi in 0.9% NaCl, the LOD for 4 bacterial culture methods and 3 PCR methods were compared. The LOD of direct PCR and flocked swab culture was determined at 1cfu/ml. All PCR methods were equivalent to each other and were more sensitive than any of the culture methods at the lower dilutions. At higher cell densities (>100cfu/ml) flocked swab culture was not statistically better than rayon swab culture, but it was superior to all other methods tested.     

Effects of positive results for Mycobacterium avium subsp paratuberculosis as determined by microbial culture of feces or antibody ELISA on results of caudal fold tuberculin test and interferon-gamma assay for tuberculosis in cattle

OBJECTIVE: To determine whether cattle testing positive for Mycobacterium avium subsp paratuberculosis as determined by microbial culture of feces or antibody ELISA were more likely to have false-positive responses on the caudal fold tuberculin (CFT) test or interferon-gamma (IFN-gamma) assay for Mycobacterium bovis than cattle testing negative for M paratuberculosis. ANIMALS: 1043 cattle from 10 herds in Michigan. PROCEDURE: Feces and blood samples for plasma were collected from cattle > or =24 months old on the day the CFT test was read. Fecal samples were submitted for microbial culture for M paratuberculosis. Plasma samples were tested for antibody against M paratuberculosis, and IFN-gamma after stimulation with purified protein derivative tuberculin from M bovis or M avium. RESULTS: Of 1043 cattle, 180 (17.3%) had positive CFT test results (suspects) and 8 (0.8%) had positive IFN-gamma assay results after stimulation with purified protein derivative tuberculin from M bovis. Forty-five (4.3%) and 115 (11.0%) cattle tested positive for M paratuberculosis as determined by microbial culture of feces and antibody ELISA, respectively. Cattle with positive responses for M paratuberculosis appeared to have an increased likelihood of false-positive results on the CFT test, although this association was not significant. CONCLUSIONS AND CLINICAL RELEVANCE: No significant association was detected among cattle testing positive for M paratuberculosis as determined by microbial culture of feces and antibody ELISA and positive CFT test and IFN-gamma assay results for M bovis.     

Continuous in vitro culture of erythrocytic stages of Babesia gibsoni and virulence of the cultivated parasite

Babesia gibsoni infected erythrocytes were collected from the blood of an experimentally infected dog. The parasite isolated could be continuously cultivated in vitro, with an average parasitemia of 18.2 +/- 2.4% on day 3 of culture, in RPMI-1640 medium supplemented with 7.5% normal dog serum in a humidified atmosphere containing 5% CO(2) at 37 degrees C. The parasites in the original culture were morphologically similar to those found in the peripheral blood of dogs, however, on the 4th generation of subculture, the large oval parasites, erythrocytes including many parasites and extracellular parasites were frequently observed. The B. gibsoni isolate was injected to the dog to test its infectivity after maintained in vitro for 738 days at the 214th subculture. The cultivated parasite did not cause a severe clinical sign in the dog.     

Evaluation of an Immunochromatographic Test to the Diagnosis of Canine Brucellosis Caused by Brucella canis

This study evaluated the performance of an immunochromatographic test (ICT) for the diagnosis of canine brucellosis caused by Brucella canis, comparing its results with that of the rapid slide agglutination test with and without the use of 2‐mercaptoethanol and the agar gel immunodiffusion test (AGID). The microbiological culture, PCR and clinical examination were used as reference. According to the results obtained in clinical examination, blood culture, culture of semen and vaginal swab and PCR in blood, semen and vaginal swab, a total of 102 dogs were divided into three groups: B. canis‐infected dogs (Group 1), B. canis‐non‐infected dogs (Group 2) and dogs with suspected brucellosis (Group 3). The diagnostic sensitivity of RSAT, 2ME‐RSAT, AGID and ICT in Group 1 was, respectively, 75%, 37.5%, 27.8% and 89.58%. The diagnostic specificity of RSAT, 2ME‐RSAT, AGID and ICT in Group 2 was, respectively, 91%, 100%, 100%, and 100%. In dogs with suspected brucellosis, 9.67% were RSAT positive, none was positive by 2ME‐RSAT, 3.22% were AGID positive and 6.45% were ICT positive. The main drawback concerning canine brucellosis diagnosis is the lack of a highly sensitive serological assay to be used as a screening test to the rapid identification of infected animals. The ICT showed a high diagnostic specificity and a diagnostic sensitivity value greater than that observed in the RSAT, 2ME‐RSAT and AGID. However, 10.41% of infected dogs had negative results by ICT. These dogs were positive by microbiological culture and/or PCR, indicating active infection and consequently a higher potential of spreading Brucella. Although rapid and simple to perform, the ICT lacked sensitivity to be used as a screening test.     

A comparative assessment of culture and serology in the diagnosis of brucellosis in dairy cattle

To investigate the usefulness of culture for the confirmation of brucellosis in cattle, a comparison of culture and serology was undertaken on 248 animals in four dairy herds where the disease was active. Paired supramammary (SM), retropharyngeal (RP), and internal iliac (IL) lymph nodes were cultured, and five serological tests were deployed: the microserum agglutination test (MSAT), complement fixation test (CFT), the indirect (iELISA) and competitive ELISA, and the fluorescence polarisation assay (FPA). Brucella abortus was isolated from 86.8% of animals on combined culture of all three lymph nodes. Individually, the highest isolation rate was from the RP (90.5% of culture positives). Of culture positive animals, 13.7% and 6.2% were positive from the RP and SM alone, respectively.Approximately half of the positive cultures yielded <10 colonies/culture plate. Although 80.9% of animals were positive in at least one serological test, only 45.2% were positive in all five. For culture-positive animals, the MSAT was the most sensitive test (71.8%). Of the culture-negative animals 67.7% were positive in at least one test, while 12.9% were positive in all five. Titres were higher in animals culture-positive from the SM, and there was a direct correlation between higher titres and higher colony counts in SM cultures. Only 8.9% of animals were both culture-negative and seropositive (in at least one test), while 16.5% were culture-positive and seronegative in all five tests. The results highlight and validate the sensitivity of bacteriological culture in confirming a diagnosis of bovine brucellosis. While the MSAT and FPA were the most sensitive serological tests, a significant percentage of infected animals were undetectable using these standard serological assays.     

An evaluation of a modified interferon-gamma assay for the detection of paratuberculosis in dairy herds

Whole blood samples were obtained from multiple dairy herds in Pennsylvannia and in Wisconsin which were previously determined to be infected with Mycobacterium paratuberculosis (MpS) (Johne's disease) by fecal culture. Blood samples were shipped overnight to the National Animal Disease Center (NADC) in Ames, IA for processing and interferon-gamma (IFN-gamma) analysis. Blood samples were incubated alone (non-stimulated) or with concanavalin A (ConA), a T-cell mitogen used as a positive control in the assay, for 18h. In addition, samples were incubated with M. avium purified protein derivative (AvPPD), M. bovis purified protein derivative (BoPPD), or a whole cell sonicate of M. paratuberculosis for 18h to elicit antigen-specific IFN-gamma production. After incubation, plasma was harvested and analyzed for IFN-gamma by ELISA. Values for IFN-gamma for non-stimulated blood samples (background) were consistently low for animals in all herds evaluated. In contrast, ConA stimulation of blood samples evoked a significant secretion of IFN-gamma regardless of infection status or fecal culture results for individual cows, indicating that immune cells were still viable after overnight shipment and capable of responding to stimulation. Antigen-specific IFN-gamma results were positively correlated with infection status as determined by previous fecal shedding and/or current fecal shedding of M. paratuberculosis. Accuracy of the IFN-gamma assay for correctly predicting infection status of individual cows in the herds with low levels of infection ranged from 50 to 75% when used as a single test. Combined use of the IFN-gamma test and a commercial ELISA antibody test accurately predicted infection status of 73% of cows from a dairy herd with a high level of M. paratuberculosis infection and 90% from a well-characterized group of dairy cows at the NADC. These results indicate that the antigen-specific IFN-gamma assay is a very sensitive diagnostic tool for detection of subclinical paratuberculosis in cattle and may be useful on an individual animal basis to remove infected animals from the herd.     

Isolation of Babesia divergens from carrier cattle blood using in vitro culture

Babesia divergens, the main causative agent of bovine babesiosis in Western Europe, was isolated from naturally infected cattle. Ninety-six blood samples were examined by means of an in vitro culture technique in sheep erythrocytes: 19 of them were collected from animals in the acute phase of the disease with visible parasitemia on blood smears, while the 77 remaining animals showed no microscopically detectable parasites. B. divergens was cultured from the 19 first blood samples as well as from 31 samples collected from asymptomatic animals. The time period before parasites could be detected in the culture varied in the latter samples from 6 to 20 days. The effects of sampling condition (anticoagulant used) and storage length were tested. A good correlation was obtained between immunofluorescent antibody test and culture, with identical results (positive or negative) for 89.6% of the samples collected from asymptomatic animals. The sensitivity of the in vitro culture method was determined and was about 10 parasites/mL of whole blood from three independent experiments performed with three different isolates, confirming its suitability to detect and culture diverse B. divergens isolates from carrier cattle. The parasites could indeed be isolated 9 months after the acute babesiosis phase in the blood of naturally infected animals. The 50 isolates collected in this study were successfully subcultured, cryopreserved and resuscitated using the same culture medium. The in vitro isolation of B. divergens from asymptomatic carrier cattle was achieved and will allow the analysis of parasite diversity within cattle herds.     

小肽与酵母培养物对舍饲牦牛生产性能、养分表观消化率和血清生化指标的影响

为研究添加小肽与酵母培养物对舍饲育肥牦牛生产性能、养分表观消化率和血清生化指标的影响,试验选取36头年龄为4周岁左右、体重水平相当、健康状况良好的麦洼牦牛随机分为4个处理组,对照组(TMR全混合日粮)、试验Ⅰ组(TMR+1%酵母培养物)、试验Ⅱ组(TMR+0.75%小肽)和试验Ⅲ组(TMR+1%酵母培养物+0.75%小肽)。结果表明:(1)小肽与酵母培养物可以显著提高舍饲育肥牦牛日粮中营养物质的表观消化率,其中粗蛋白质(CP)和中性洗涤纤维(NDF)的表观消化率极显著高于对照组(P<0.01),试验Ⅲ组的OM、CP、NDF和酸性洗涤纤维(ADF)表观消化率较对照组分别提高了10.87%、13.79%、17.52%、11.19%。(2)与对照组相比,各试验组平均日采食量(DMI)、平均日增重(ADG)和F/G差异极显著,试验Ⅰ、Ⅱ、Ⅲ组DMI较对照组分别提高了12.20%、6.51%、12.20%;ADG分别提高25%、10.74%、28.09%;F/G则分别降低了11.74%、5.39%、13.26%(P<0.01)。(3)与对照组相比,试验组尿素氮(UN)、甘油三酯(TG)、葡萄糖(Glu)、球蛋白(GLB)差异不显著(P>0.05);总蛋白(TP)和白蛋白(ALB)差异显著(P<0.05),试验Ⅰ、Ⅱ、Ⅲ组TP较对照组分别显著提高了13.02%、7.55%、18.48%;ALB分别提高了12.04%、5.41%、12.23%(P<0.05)。综上所述,小肽与酵母培养物组合可提高舍饲牦牛的生产性能与养分表观消化率,并改善血清中生化指标的含量。     

In vitro lymphocyte transformation as a herd survey method for bovine paratuberculosis.

The lymphocyte-transformation (LT) test was evaluated for its potential application as a field test for bovine paratuberculosis. Using a whole blood technique, samples from 3 consecutive collection periods were subjected to 3 mycobacterial antigens and to phytohemagglutinin. The results obtained from LT were compared with conventional serologic and cultural methods. A positive LT response to johnin purified-protein derivative (PPD) or avian PPD (or both) was noted in 40% to 60% of the animals tested. The complement-fixation test yielded 4% to 6.7% positive results, the immunodiffusion test between 1.2% and 1.4%, and the direct fecal culture between 2.4% and 6%. The mean of the stimulation indices of all positively responding animals was highest with johnin PPD. Specific stimulation to mammalian PPD occurred between 2.4% and 6% of the animals. The efficacy of the LT test for determining the incidence of infection with Mycobacterium paratuberculosis is discussed.