Mycobacterium Avium Subspecies Paratuberculosis Shedding Research Articles

Long-term outcomes of the Italian Mycobacterium avium subspecies paratuberculosis control programme for dairy cattle.

The considerable epidemiological and economic implications of paratuberculosis, caused by Mycobacterium avium subspecies paratuberculosis (MAP), have placed importance on control efforts aimed at preventing MAP transmission. In this context, Italy issued national guidelines for the control and status certification of MAP in dairy cattle in 2013. We assessed the long-term outcomes of the Italian MAP control programme for 14 dairy farms located in northern Italy by retrospectively reviewing the results of yearly serological tests, presence of clinical cases, MAP faecal shedding in serologically positive animals, farm management and health ranking as indicators of herd health between 2014 and 2021. A significantly higher number of serologically positive animals were observed between 2014 and 2016 than between 2017 and 2021, as well as an improving trend in the paratuberculosis health ranking for nine of the 14 farms. No clinical cases were reported. MAP shedding was detected in 9.4% of serologically positive animals. Discarding colostrum and prioritised culling of seropositive animals assisted by adoption of standardised serological testing were presumed to have a key role in MAP control, despite the reluctance of some farmers to address hygienic issues and improve the separation of calves from adult animals. The small number of farms included in this study and the fact that these were not randomly selected may limit the generalisability of the findings. The Italian paratuberculosis control plan has provided measures to limit the uncontrolled spread of MAP infection within and between herds by promoting animal trading between farms certified as negative or low risk.

Estimation of the specificity of an antibody ELISA for paratuberculosis generated from a sector of the UK cattle population using results from a paratuberculosis control programme

In the United Kingdom (UK) a voluntary programme to control paratuberculosis in cattle based on herd management and serological screening has been operating since 1998. The programme assigns a risk level to each participating herd according to the within herd seroprevalence and the confirmation of the presence of infection with Mycobacterium avium subspecies paratuberculosis (MAP) by faecal culture or polymerase chain reaction (PCR). From the outset a general concern over the specificity of the paratuberculosis antibody enzyme-linked immunosorbent assay (ELISA) resulted in the use of a faecal screen for the causal organism to negate or confirm infection in individual seropositive animals. Progress in improving the diagnostic tests has been gradual throughout the life of the programme and the under-pinning approach to using tests to determine the risk of paratuberculosis for a herd required to be re-examined. This study used a large data set of more than 143,000 test results over five years from the lowest paratuberculosis risk level category of herds to estimate the specificity of a commercially available paratuberculosis antibody ELISA for cattle. In each year of the study the estimated specificity reached or exceeded 0.998. We also examined the apparent impact that annual or more frequent application of the single intradermal comparative cervical tuberculin (SICCT) test for tuberculosis (TB), using purified protein derivatives of Mycobacterium bovis and Mycobacterium avium subspecies avium, had on specificity of the antibody ELISA for paratuberculosis. We found a statistically significant difference in three of the five years with herds that were officially tuberculosis free and not subject to frequent SICCT testing. This difference was small and considered to be of little practical importance for the paratuberculosis assurance programme. We concluded that, in the UK the mandatory TB surveillance programme of cattle herds is not a limiting factor in the use of serological testing to support herd-level assurance schemes for paratuberculosis. Furthermore, in paratuberculosis, where shedding of MAP is intermittent and the sensitivity of the commercially available PCR tests for detection MAP is highly variable, faecal screening of seropositive animals is an unreliable method for negating infection in seropositive cattle.

Analysis of Colostrum and Udder Skin Swabs from a Dairy Goat Herd in Germany regarding the Occurrence of Mycobacterium avium Subsp. paratuberculosis.

Simple SummaryThe analysis of transmission routes for paratuberculosis as well as their prevention are critical for effective disease control. The aim of the present study was to assess the importance of colostrum and the udder skin as routes for transmission of Mycobacterium avium subspecies paratuberculosis (MAP) within a dairy goat herd. Swabs of the udder skin and colostrum samples were collected from goats of a paratuberculosis-infected herd during lambing season and analyzed for MAP DNA. Additionally, cultivation of the swabs was performed. MAP could not be detected in the colostrum. A low number of udder skin swabs was positive for MAP DNA but no bacteria could be cultured. Because the detection methods are imperfect, the occurrence of MAP in colostrum or on the udder skin can never be completely excluded. Therefore, present recommendations concerning colostrum and youngstock management should still be re-evaluated, but applied in goat herds within a paratuberculosis control program until the role of colostrum and udder skin for within-herd transmission of MAP is further investigated.Oral intake of Mycobacterium avium subspecies paratuberculosis (MAP) in first days of life is considered to be the main route of infection for paratuberculosis. This can be related to a direct contact to contaminated feces or feeding of MAP containing colostrum. Colostrum is believed to become contaminated either by lactogenic shedding or introduction of MAP from environmental sources. In this pilot study, the presence of MAP in individual and bulk colostrum samples from a paratuberculosis-infected, vaccinated dairy goat herd in Germany and the effect of udder skin disinfection on the MAP load of colostrum were examined. In order to distinguish between lactogenic shedding and fecal contamination, 49 udder skin swabs were cultivated on solid medium whereas 29 swabs were additionally analyzed by qPCR. qPCR was applied on 110 individual colostrum samples collected from 55 goats, one before and one after disinfection with a mycobactericidal disinfectant, and 14 bulk colostrum samples. MAP DNA was detected in 10.3% (3/29) of the swab samples, but no viable MAP was cultivated from any sample. These results indicate a low-level MAP contamination of the udder skin and colostrum of milking goats suggesting a low risk of MAP transmission via these routes.

Is the transmission of Mycobacterium avium subspecies paratuberculosis (MAP) infection through milk intended to feed calves an overlooked item in paratuberculosis control programs?

As in many parts of the world, Chile shows a high Mycobacterium avium subsp. paratuberculosis (MAP) infection rate. Evidently, the control recommendations have been inefficient. In the author's opinion, the potential risk of MAP transmission to susceptible calves through milk consumption is largely overlooked. Two observational studies were performed, one to confirm MAP shedding in young stock in a high MAP-infected dairy herd and MAP load in milk intended to feed these calves was estimated. In parallel, in a second study, we estimated the relationship between MAP herd seroprevalence and the cow MAP shedding level as well as the presence of this pathogen in milk used to feed dairy calves. In the first study, 53.7% of cows and 22.5% of calves showed positive culture results. Among all cows tested, 9 (2.19%) animals had a positive serum ELISA, and MAP load in milk reached 106bacteria/mL. In the second study, three seroprevalence categories were established as follows: high (≥ 9%), medium (> 5% and ≤ 9%), and low (≤ 5%). Statistical significant differences among these categories were observed. Animals from the high seroprevalence category shed significantly more MAP than the others. However, in the low category, heavy shedder animals were also observed. Finally, in all study herds, MAP presence in milk intended to feed calves was reported, even from herds without ELISA-positive animals.

Defining resilience to mycobacterial disease: Characteristics of survivors of ovine paratuberculosis

Paratuberculosis is an insidious, chronic disease of ruminants that has significant animal welfare implications and reduces on-farm profitability globally. Not all animals exposed to the causative pathogen, Mycobacterium avium subspecies paratuberculosis (MAP), succumb to disease and this unique, long-term trial was designed to track animals that were resilient. The advantages of understanding immune protection include the management option to retain resilient individuals in a herd/flock and the potential for deliberate manipulation of the host immune response using novel vaccines. Twenty sheep experimentally exposed to MAP and 10 controls were monitored for 2.5 years during which the condition progressed, resembling natural disease development. Cellular and humoral immune parameters and faecal MAP shedding were examined regularly and disease outcomes were classified at necropsy, based on the presence of viable MAP and histopathological lesions in intestinal tissues, either at the termination of the trial or when animals were culled due to weight loss. There were distinct characteristics, such as an early strong IFNγ response, that differentiated resilient sheep from susceptible individuals prior to the onset of clinical disease. Faecal MAP shedding and serum antibody level, commonly used to diagnose disease, were more ambiguous. The former was transient in the majority of resilient animals and therefore should not be used for diagnosis of MAP infection in younger animals. Remarkably, the serum antibody level in some resilient animals was higher than the usual positive-negative cut-off for disease diagnosis at multiple samplings throughout the trial. Consequently the antibody response in resistance to paratuberculosis requires further investigation.

Prevalence of Mycobacterium avium subspecies paratuberculosis and hepatitis E in New World camelids in Austria

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of paratuberculosis in domestic ruminants and New World Camelids (NWC). Hepatitis E virus (HEV) is an important public health concern worldwide....

Variation in susceptibility of different breeds of sheep to Mycobacterium avium subspecies paratuberculosis following experimental inoculation

Exposure to Mycobacterium avium subspecies paratuberculosis (MAP) does not always lead to Johne’s disease. Understanding differences in disease susceptibility of individual animals is a key aspect to controlling mycobacterial diseases. This study was designed to examine the susceptibility or resistance of various breeds of sheep to MAP infection. Merino, Suffolk first cross Merino, Border Leicester, and Poll Dorset sheep were orally inoculated with MAP and monitored for 14 months. Clinical disease occurred more frequently in the Merino (42%) and Suffolk first cross Merino (36%) compared to the Border Leicester (12%) and Poll Dorset (11%) breeds. Infection risk, as determined by culture of gut and associated lymphoid tissues, ranged from 75% for the Suffolk first cross Merino to 47% for the Poll Dorset sheep. Significant differences were identified in the site in the intestines of the most severe histopathological lesions and the immune responses to infection between the breeds. However, there was no difference in faecal MAP shedding by clinical cases between breeds. All breeds tested were susceptible to MAP infection, as determined by infection and clinical disease development, although there were differences in the proportions of diseased animals between the breeds. Poll Dorset and Border Leicester sheep were more resilient to MAP infection but there was evidence that more animals could have developed disease if given more time. These findings provide evidence of potential differential disease susceptibility between breeds, further our understanding of disease pathogenesis and risks of disease spread, and may have an influence on control programs for paratuberculosis.

Faecal shedding of Mycobacterium avium subspecies paratuberculosis reduces before parturition in sheep?

Paratuberculosis is a disease affecting mainly ruminants, and it is caused by Mycobacterium avium subspecies paratuberculosis (MAP). Control of the disease relies primarily on test-and-removal that is not easy to apply effectively for reasons associated with its pathogenesis. Therefore the aim of this preliminary study was to investigate whether positivity of sheep to MAP-specific antibody and DNA, detected by ELISA and faecal real time polymerase chain reaction (PCR) respectively, varies between specific stages of breeding i.e. before and after parturition, and before the mating season.Rectal faeces (n=120) and whole blood (n=120) samples were collected from a flock of sheep with no record of clinical paratuberculosis. The sheep under study are maintained throughout the year, in isolated confinement with no access to pasture or other animals, and a continuously monitored diet.The proportion of positive animals to real time PCR before parturition (13.9%) was found to be significantly lower (χ2=10.67, P=0.0015) than the proportion of positive animals after parturition (59.5%), and before the mating season (47.6%). The proportion of positive animals after parturition and before the mating season did not differ significantly. PCR analysis of one sample collected from each animal after parturition, allowed detection of more than 71% of the reactors.In conclusion, faecal shedding of MAP detected by real time PCR seems to be decreased before parturition, which probably makes the specific period less suitable for detection of shedders in sheep with no clinical signs of paratuberculosis.

Can Immune Response Mechanisms Explain the Fecal Shedding Patterns of Cattle Infected with Mycobacterium avium Subspecies paratuberculosis?

Johne’s disease (JD) is a chronic disease in ruminants and is caused by infection with Mycobacterium avium subspecies paratuberculosis (MAP). At late stages of the disease, MAP bacilli are shed via feces excretion and in turn create the potential for oral-fecal transmission. The role of the host immune response in MAP bacteria shedding patterns at different stages of JD is still unclear. We employed mathematical modeling to predict if the variation in MAP shedding could be correlated to the immune response in infected animals. We used a novel inverse modeling approach that assumed biological interactions among the antigen-specific lymphocyte proliferation response (cell-mediated response), antibody/humoral immune responses, and MAP bacteria. The modeling framework was used to predict and test possible biological interactions between the measured variables and returns only the essential interactions that are relevant in explaining the observed cattle MAP experimental infection data. Through confronting the models with data, we predicted observed effects (enhancement or suppression) and extents of interactions among the three variables. This analysis enabled classification of the infected cattle into three different groups that correspond to the unique predicted immune responses that are essential to explain the data from cattle within these groups. Our analysis highlights the strong and weak points of the modeling approach, as well as the key immune mechanisms predicted to be expressed in all animals and those that were different between animals, hence giving insight into how animals exhibit different disease dynamics and bacteria shedding patterns.

Specific faecal antibody responses in sheep infected with Mycobacterium avium subspecies paratuberculosis

Many studies have examined the serum antibody response to Mycobacterium avium subspecies paratuberculosis (MAP) infection in cases of Johne's disease (JD), but there are no reports on the mucosal antibody response. Faecal immunoglobulin (Ig) G and IgA ELISA responses were examined from sheep experimentally inoculated with MAP for up to 23 months post inoculation (PI). Corresponding serum IgG responses and the presence of viable MAP shed in faeces were also examined. The sheep were divided into three groups: (i) “un-inoculated controls” (n=10), (ii) “clinical cases” (n=8) which were inoculated animals that developed clinical disease and had moderate to high levels of MAP shedding and (iii) “survivors” (n=11) which were inoculated animals from which MAP could not be cultured from tissues at the conclusion of the trial. Serum IgG responses gradually increased in all inoculated animals, peaking at 12–16 months PI. A significant increase in the levels of MAP-specific faecal IgG and IgA was measured in the survivors at 16 and 17 months PI, while levels in the un-inoculated controls and clinical cases remained at baseline levels. The detection of faecal Ig in the survivors coincided with the removal of sheep that developed clinical disease. The data suggest that some sheep produced MAP-specific IgG and IgA in the intestinal mucosa, which was released into their faeces. We hypothesise that the survivors produced faecal Ig as a direct response to ingestion of MAP associated with environmental contamination from clinical cases. Thus MAP specific mucosal antibodies may play a previously unreported role as part of a protective response triggered by environmental exposure.

Dam Mycobacterium avium subspecies paratuberculosis (MAP) infection status does not predetermine calves for future shedding when raised in a contaminated environment: a cohort study.

Uptake of Mycobacterium avium subsp. paratuberculosis (MAP) by calves in the first days of life from colostrum, milk and faeces is regarded an important moment of transmission. The objective of this study was to quantify the association between the MAP status of dams as determined by the presence of MAP DNA and antibody in colostrum and that of DNA in faeces and the environment with subsequent MAP shedding of their daughters. A cohort of 117 dam-daughter pairs giving birth/being born on eight commercial dairy farms with endemic paratuberculosis was followed where colostrum, faecal and environmental samples (dust) were analysed for the presence of MAP using an IS900 real-time PCR. Antibodies in colostrum were measured by ELISA. Analysis of dust samples showed that on all farms environmental MAP exposure occurred continuously. In significantly more colostrum samples (48%) MAP DNA was detected compared to faecal samples (37%). MAP specific antibodies were present in 34% of the colostrum samples. In total MAP DNA was present in faecal samples of 41% of the daughters at least once during the sampling period. The association between faecal shedding in the offspring and the dam MAP status defined by MAP PCR on colostrum, MAP PCR on faeces or ELISA on colostrum was determined by an exact cox regression analysis for discrete data. The model indicated that the hazard for faecal shedding in daughters born to MAP positive dams was not significantly different compared to daughters born to MAP negative dams. When born to a dam with DNA positive faeces the HR was 1.05 (CI 0.6; 1.8) and with DNA positive colostrum the HR was 1.17 (CI 0.6; 2.3). When dam status was defined by a combination of both PCR outcomes (faeces and colostrum) and the ELISA outcome the HR was 1.26 (CI 0.9; 1.9). Therefore, this study indicates that neither the presence of MAP DNA in colostrum, MAP DNA in faeces nor the presence of MAP antibodies in colostrum of the dam significantly influences the hazard of MAP shedding in their subsequent daughters up to the age of two years when raised in a contaminated environment.

Decreased serum protein associated with Mycobacterium avium subspecies paratuberculosis shedding in German Holstein cows

Using well established metabolic parameters, this study aimed to substantiate differences in protein and energy metabolism between Mycobacterium avium subspecies paratuberculosis (MAP) positive and negative dairy cows tested by faecal...

Can early host responses to mycobacterial infection predict eventual disease outcomes?

Diagnostic tests used for Johne's disease in sheep either have poor sensitivity and specificity or only detect disease in later stages of infection. Predicting which of the infected sheep are likely to become infectious later in life is currently not feasible and continues to be a major hindrance in disease control. We conducted this longitudinal study to investigate if a suite of diagnostic tests conducted in Mycobacterium avium subspecies paratuberculosis (MAP) exposed lambs at 4 months post infection can accurately predict their clinical status at 12 months post infection. We tracked cellular and humoral responses and quantity of MAP shedding for up to 12 months post challenge in 20 controls and 37 exposed sheep. Infection was defined at necropsy by tissue culture and disease spectrum by lesion type. Data were analysed using univariable and multivariable logistic regression models and a subset of variables from the earliest period post inoculation (4 months) was selected for predicting disease outcomes later on (12 months). Sensitivity and specificity of tests and their combinations in series and parallel were determined. Early elevation in faecal MAP DNA quantity and a lower interferon gamma (IFNγ) response were significantly associated with sheep becoming infectious as well as progressing to severe disease. Conversely, early low faecal MAP DNA and higher interleukin-10 responses were significantly associated with an exposed animal developing protective immunity. Combination of early elevated faecal MAP DNA or lower IFNγ response had the highest sensitivity (75%) and specificity (81%) for identifying sheep that would become infectious. Collectively, these results highlight the potential for combined test interpretation to aid in the early prediction of sheep susceptibility to MAP infection.

Efficacy of ‘indigenous vaccine’ using native ‘Indian bison type’ genotype of Mycobacterium avium subspecies paratuberculosis for the control of clinical Johne’s disease in an organized goat herd

Therapeutic efficacy of a new 'Indigenous vaccine' prepared from native highly pathogenic 'Indian Bison Type' genotype of Mycobacterium avium subspecies paratuberculosis (MAP) of goat origin has been evaluated with respect to control of clinical Johne's disease in naturally infected Mehsana breed of goat in North Gujarat. Fifty goats from Sheep and Goats Research Station, Sardarkrushinagar Dantiwada Agricultural University, Sardarkrushinagar, were randomly divided into 2 groups viz.,'Vaccinated'(n = 35) and 'Control'(n = 15). After vaccination, goats were monitored for physical condition, morbidity, mortality, body weights, shedding of MAP in feces, internal condition, gross lesions and humoral immune responses up to 120 days (at each interval of 30 days). At the end of 120 days trial, there was marked overall improvement in physical condition and body weights of vaccinated goats as compared to 'Control' goats. Vaccinated goats gained significantly (P < 0.05) higher body weights, hardly exhibited any lesions characteristic of JD, had significantly higher (P < 0.01) antibody titers and shedding of MAP was significantly (P < 0.01) reduced. Few of the vaccinated goats were positive for MAP DNA in faecal PCR and blood PCR before vaccination. However, all were found as negative at 120 days post vaccination (DPV). Overall vaccine exhibited effective in restriction of MAP infection and significant improvement in production parameters and reduction in mortality and morbidity due to JD. The trial in the herd will be continued.

Evaluation of “Indigenous Vaccine” Developed Using “Indian Bison Type” Genotype of &amp;lt;i&amp;gt;Mycobacterium avium subspecies paratuberculosis&amp;lt;/i&amp;gt; Strain “S5” of Goat Origin in a Sheep Flock Endemic for Johne’s Disease: A Three Years Trial in India

“Indigenous vaccine” developed from native “Indian Bison Type” strain (“S5”) of Mycobacterium avium subspecies paratuberculosis (MAP) of “goat origin” was first time evaluated in a sheep flock of Bharat Merino breed located in Mannavanur town of Tamil Nadu in South India. Therapeutic efficacy of the vaccine was evaluated for 3 years between 2008 and 2010, on the basis of improvements in productivity (body weights, reproductive efficiency and survivability), physical condition, clinical symptoms (weakness, diarrhea, wool quality), immune response (sero-conversion) and infection load in feces (shedding). After immunization of the flock in 2008, the successive progenies of 112 and 53 lambs born in 2009 and 2010, respectively were vaccinated. Whereas, 40 lambs born to control animals were kept as unvaccinated controls. Though gain in body weights in vaccinated versus controls were not significant in 2008, growth rates were distinctly superior in Ist and IInd generations of vaccinated lambs. Reproductive performance (tupping percent) and survivability of lambs and adult sheep improved significantly. There was overall reduction in yearly morbidity (diarrhea) and mortality rates of the flock in post vaccination years. Shedding of MAP in feces was reduced in vaccinated sheep by 6.2%, 14.3% and 27.3% in 2008, 2009 and 2010 respectively, whereas shedding increased in the control sheep. Seromonitoring of the animals by “indigenous ELISA kit” showed enhanced “flock immunity” in successive generations. “Indigenous vaccine” reduced clinical disease and shedding and improved immunity and productivity of Bharat Merino flock, endemic for Johne’s disease.

Early infection dynamics after experimental challenge with Mycobacterium avium subspecies paratuberculosis in calves reveal limited calf-to-calf transmission and no impact of Hsp70 vaccination

Efficient control of bovine paratuberculosis is hampered by lack of a vaccine. The purpose of this study was to evaluate efficacy of a candidate vaccine, consisting of recombinant Mycobacterium avium subspecies paratuberculosis (MAP) Hsp70 with DDA adjuvant, in calves experimentally infected with MAP. Four groups of 14 animals each were used. Animals in group 1 and 2 were all vaccinated with Hsp70/DDA at day 0, 84, 168 and 357, and those in group 3 and 4 were non-vaccinated controls. In each group half (n=7) of the animals were challenged and the remaining half served as contacts. Blood and fecal samples were collected at three week intervals until day 588, and subsequently all animals were subjected to necropsy. The primary outcomes assessed were fecal culture (FC) of MAP, tissue colonization of MAP, and transmission of infection to contact animals. The kinetics of MAP shedding in feces of challenged animals showed a peak around 130 days post-challenge, irrespective of vaccination status. At necropsy no differences in the level of tissue colonization between vaccinated animals and controls were observed in the challenged groups. Only one contact animal (non-vaccinated) was positive at necropsy, indicating limited to no transmission within groups. These findings indicate that Hsp70/DDA vaccination does not influence early infection dynamics after experimental infection. However, early shedding of MAP in calves did not result in efficient transmission of infection to contact animals. The latter implies that introduction of an infected calf in a cohort of susceptibles has limited consequences for spread of infection.

Hsp70 vaccination-induced antibodies recognize B cell epitopes in the cell wall of Mycobacterium avium subspecies paratuberculosis

Mycobacterium avium subspecies paratuberculosis (MAP) causes a chronic intestinal infection of ruminants and has been associated with the etiology of human Crohn's disease. A MAP Hsp70/DDA subunit vaccine previously showed a significant reduction in fecal shedding of MAP in cattle, concomitant with pronounced antibody production against MAP Hsp70, rather than T cell reactivity. Our hypothesis is that if Hsp70-specific antibodies are able to confer protection, the first requisite would be that the Hsp70 molecule is accessible for antibodies in intact MAP bacteria. In the current study monoclonal antibodies identified MAP Hsp70 B cell epitopes. Two linear epitopes were also recognized by antibodies of vaccinated calves and goats. These epitopes showed to be accessible by antibodies in the bacterial cell wall and in intestinal lesional tissue during natural infection. These results indicate that vaccination-induced antibodies can bind intact bacteria and have the potential to contribute to the protective effect of Hsp70/DDA subunit vaccination against bovine paratuberculosis.

Correlation between Herrold Egg Yolk Medium Culture and Real-Time Quantitative Polymerase Chain Reaction Results for Mycobacterium Avium Subspecies Paratuberculosis in Pooled Fecal and Environmental Samples

Real-time quantitative polymerase chain reaction (qPCR) testing for Mycobacterium avium subspecies paratuberculosis (MAP) in fecal samples is a rapid alternative to culture on Herrold egg yolk medium (HEYM), the traditional antemortem reference test for MAP. Although the sensitivity and specificity of these 2 tests have been estimated based on dichotomized test results, the correlation between real-time qPCR threshold cycle (Ct) values and colony-forming units (CFU) on HEYM for fresh and thawed samples has not been evaluated. The objectives of the present study were to estimate the correlation and association between Ct and CFU in fresh and thawed pooled fecal and environmental samples. Results of HEYM culture of 1,997 pooled fecal samples from cows in 14 herds, and 802 environmental samples from 109 dairies nationwide were negatively (inversely) correlated with their respective real-time qPCR results. The Spearman's rank correlation between Ct and CFU was good (-0.66) in fresh and thawed pooled fecal samples, and excellent (-0.76) and good (-0.61) in fresh and thawed environmental samples, respectively. The correlation varied from good (-0.53) to excellent (-0.90) depending on the number of samples in a fecal pool. Truncated regression models indicated a significant negative association between Ct and CFU in fecal pools and environmental samples. The use of real-time qPCR instead of HEYM can yield rapid, quantitative estimates of MAP load and allow for incorporation of real-time qPCR results of pooled and environmental samples in testing strategies to identify dairy cow groups with the highest MAP shedding.

Comparative efficacy of an indigenous ‘inactivated vaccine’ using highly pathogenic field strain of Mycobacterium avium subspecies paratuberculosis ‘Bison type’ with a commercial vaccine for the control of Capri-paratuberculosis in India

Johne's disease (JD) is endemic in goatherds located at Central Institute for Research on Goats, Makhdoom, since 1979 and lately it has been reported from farmer's herds in equal frequencies. Despite using test and slaughter method for the control of JD for more than 25 years in these herds, incidence of JD has not been reduced. Efficacy of ‘indigenous vaccine’ containing native ‘Bison type’ genotype of Mycobacterium avium subspecies paratuberculosis (MAP) was compared with commercial vaccine using challenge studies with homologous strain of MAP. Goat kids (85) were randomly divided in to three groups. Kids were vaccinated with 1 ml of vaccine subcutaneously and Sham-immunized with 1 ml of sterile PBS. All kids except 3 in each group were challenged twice at 75- and 275-day post-vaccination (DPV). Four goats each from three groups were sacrificed at 200-day post-challenge to evaluate carcass and histopathologically for vaccine and challenge response in kids of different groups. Samples (blood, serum and fecal) were screened for LTT, ELISA and shedding of bacilli and data on live animal traits, mortality and experimental sacrifice were compared. Average body weights gained by goats in three groups at different stages of trials (0, 1–75, 76–275, 276–425 DPV) showed marked improvements in performance of vaccinated groups over ‘Sham-immunized’ group. Effect of vaccines against challenge became visible in terms of body weights gained at 276–425 DPV (‘Bison’ group gained significantly higher body weights than ‘Sham-immunized’). Mortality was significantly less in two vaccinated as compared to ‘Sham-immunized’. Vaccinated groups also had significant stimulation and sero-conversion for cell mediated and humoral immune response, respectively as compared to ‘Sham-immunized’. Results of post-challenged fecal culture showed significant reduction in shedding of MAP in both vaccinated groups than in ‘Sham-immunized’. There was significant improvement in external and internal body traits and histological lesions in case of vaccinated than ‘Sham-immunized’ group.

Efficacy of spheroplastic and cell-wall competent vaccines for Mycobacterium avium subsp. paratuberculosis in experimentally-challenged baby goats

A Mycobacterium avium subspecies paratuberculosis (MAP) vaccine that reduced the incidence of clinical disease or reduced fecal shedding of MAP would aid control of Johne's disease (JD). The objective of the present study was to evaluate the efficacy of four MAP vaccine combinations, including cell-wall competent (CWC) alum adjuvant, CWC-QS21 adjuvant, cell-wall deficient (CWD) alum adjuvant and CWD-QS21 adjuvant vaccines. Eighty baby goats were vaccinated at 1 and 4 weeks of age with one of these vaccines or a sham control vaccine consisting of alum adjuvant. Kids were challenged orally with approximately 6.0 × 10 9 organisms in four divided doses of 1.5 × 10 9 organisms using a goat isolate of MAP. Vaccinated challenged and challenged control groups had 10 and 6 kids per group, respectively. Half of the kids within each group were necropsied at either 6 or 9 months post-challenge. Gross and microscopic lesions and relative number of acid-fast bacilli were evaluated and scored at necropsy. Results indicated all challenged kids had some lesions compatible with JD suggesting none of the vaccines prevented infection. Three vaccines (CWC-alum, CWC-QS21 and CWD-QS21) reduced lesion scores by 46–51% at 9 months. CWD-alum vaccine resulted in a more severe (+33.5%) lesion score than sham-vaccinated challenged control. Lesion scores were greater at 9 months than at 6 months post-challenge in the sham-vaccinated challenged group and CWD-alum vaccinated group, while lesion scores were generally stable with remaining vaccines. Mean fecal CFU/g were significantly different across time from challenge to 9-month necropsy ( p = 0.043) and the CWC-QS21 vaccine group had a marked reduction in fecal CFU/g at all time points post-challenge. A reduction in MAP CFU/g was also detected in necropsy tissues from kids given the CWC-alum, CWC-QS21 and CWD-QS21 vaccines, and increased CFU/g were detected in tissues from kids given the CWD-alum vaccine. Immunological tests evaluated included, humoral response evaluation by AGID, ELISA and Western blot, and cell mediated response by comparative PPD skin testing ( M. avium, Old Johnin, M. bovis and Lot 2 Johnin PPD's), and production of MAP induced γ-interferon. Vaccination also resulted in false-positive PPD skin test reactions for M. avium PPD, Old Johnin PPD and γ-interferon tests. When a 2-mm cutoff above normal skin thickness was used to define positive skin test reactions, false-positive reactions for M. bovis were detected in only 2 of 32 kids given a vaccine with QS21 adjuvant.