Evaluation of a rapid immunochromatographic test kit to the gold standard fluorescent antibody test for diagnosis of rabies in animals in Bhutan.

BACKGROUND: Rabies kills approximately 59,000 people each year worldwide. Rapid and accurate diagnosis of rabies is important for instituting rapid containment measures and for advising the exposed people for postexposure treatment. The application of a rapid diagnostic tests in the field can greatly enhance disease surveillance and diagnostic activities, especially in resource poor settings. In this study, a total of 179 brain tissue samples collected from different rabies suspect animal species (113 dogs, 50 cattle, 10 cats, 3 goats, 2 horses, and 1 bear) were selected and tested using both rapid immunochromatographic kit and the reference standard fluorescent antibody test (FAT). We evaluated the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of a rapid antigen detection test kit produced by BioNote, Inc. (Hwaseong-si, Korea) relative to a FAT for its fit-for-purpose for confirmation of clinical cases of rabies for early response and enhancing rabies surveillance. RESULTS: Among 179 samples examined in this study, there was a concordance in results by the rapid test and FAT in 115 positive samples and 54 negative samples. Test results were discordant in 10 samples which were positive by FAT, but negative (false negative) by rapid kit. The rapid test kit showed a sensitivity of 92% (95% CI: 85.9-95.6) and specificity of 100% (95% CI: 93.4-100) using FAT as the reference standard. The positive and negative predictive values were found to be 100% (95% CI:96.7-100) and 84.4% (95% CI: 73.6-91.3), respectively. Overall, there was 94.4% (95% CI: 90-96.9) test agreement between rapid test and FAT (Kappa value = 0.874) with a positive percent agreement and negative percent agreement of 92 and 100%, respectively. CONCLUSIONS: Our finding demonstrated that the rapid test kit (BioNote) can be used for rabies surveillance and confirming clinical case of rabies in animals for making rapid decisions particularly controlling rabies outbreaks in resource poor settings.

Seroprevalence and risk factors associated with bovine tuberculosis in cattle in Eastern Bhutan.

Bovine tuberculosis (bTB) is a chronic zoonotic disease affecting cattle of all age groups including wild animals. It poses a significant threat to public health and high economic losses to dairy farmers. While the disease has been eradicated from most of the developed countries through extensive surveillance, testing and culling strategy, it is endemic in Africa, Asia, and the Middle East countries. Currently, there is limited research regarding the prevalence of bTB in cattle in Bhutan. This study aimed to determine the seroprevalence of bTB in cattle in six districts of eastern Bhutan. A two-stage probability proportional to size (PPS) sampling strategy was used to determine the number of animals from which serum samples needed to be collected in each district and sub-district. All farms and cattle for sampling were randomly selected from the data in the annual livestock census of 2020. The samples were tested using bTB ELISA test kit. The seroprevalence and their 95% confidence intervals were calculated. Logistic regression models were constructed to assess the influence of various individual animal and environmental risk factors (breed, age, sex, source of animal, body condition scores of animals, respiratory system status) associated with sero-positivity in animals. The study revealed an apparent seroprevalence of 2.57% (25/971 cattle; 95% CI:1.58-3.57), with an estimated true seroprevalence of 0.91% (95% CI: 0.0-2.81). However, none of the variables were found to be significantly associated with bTB seroprevalence in cattle. We recommend, further sampling and employment of confirmatory testing to fully ascertain the extent of bTB in the cattle herds in eastern Bhutan for prevention and control.

Antimicrobial Consumption in the Livestock Sector in Bhutan: Volumes, Values, Rates, and Trends for the Period 2017-2021.

Data on the use of antimicrobials in humans and livestock may provide evidence to guide policy changes to mitigate the risk of antimicrobial resistance (AMR). However, there is limited information available about antimicrobial use in livestock in low- and middle-income countries, even though these nations are most vulnerable to the impact of AMR. This study aimed to assess the consumption of veterinary antimicrobials in Bhutan and identify areas for improvement to reduce the use of antimicrobials in livestock. National data on livestock numbers and annual procurement of veterinary antimicrobials over five years (2017-2021) were used to calculate rates of antimicrobial consumption and annual national expenditure on veterinary antimicrobials in Bhutan. The rate of antimicrobial consumption in Bhutan was 3.83 mg per population correction unit, which is lower than most countries in Europe, comparable with the rates of consumption in Iceland and Norway, and approximately 120-fold lower than published rates of antimicrobial consumption in South Asian countries, including Nepal and Pakistan. The low rates of antimicrobial consumption by the animal health sector in Bhutan could be attributable to stronger governance of antimicrobial use in Bhutan, higher levels of compliance with regulation, and better adherence to standard guidelines for antimicrobial treatment of livestock.

The potential of diagnostic point-of-care tests (POCTs) for infectious and zoonotic animal diseases in developing countries: Technical, regulatory and sociocultural considerations.

Remote and rural communities in low- and middle-income countries (LMICs) are disproportionately affected by infectious animal diseases due to their close contact with livestock and limited access to animal health personnel). However, animal disease surveillance and diagnosis in LMICs is often challenging, and turnaround times between sample submission and diagnosis can take days to weeks. This diagnostic gap and subsequent disease under-reporting can allow emerging and transboundary animal pathogens to spread, with potentially serious and far-reaching consequences. Point-of-care tests (POCTs), which allow for rapid diagnosis of infectious diseases in non-laboratory settings, have the potential to significantly disrupt traditional animal health surveillance paradigms in LMICs. This literature review sought to identify POCTs currently available for diagnosing infectious animal diseases and to determine facilitators and barriers to their use and uptake in LMICs. Results indicated that some veterinary POCTs have been used for field-based animal disease diagnosis in LMICs with good results. However, many POCTs target a small number of key agricultural and zoonotic animal diseases, while few exist for other important animal diseases. POCT evaluation is rarely taken beyond the laboratory and into the field where they are predicted to have the greatest impact, and where conditions can greatly affect test performance. A lack of mandated test validation regulations for veterinary POCTs has allowed tests of varying quality to enter the market, presenting challenges for potential customers. The use of substandard, improperly validated or unsuitable POCTs in LMICs can greatly undermine their true potential and can have far-reaching negative impacts on disease control. To successfully implement novel rapid diagnostic pathways for animal disease in LMICs, technical, regulatory, socio-political and economic challenges must be overcome, and further research is urgently needed before the potential of animal disease POCTs can be fully realized.

A knowledge, attitudes, and practices study on ticks and tick-borne diseases in cattle among farmers in a selected area of eastern Bhutan.

Livestock farming plays an important role in supporting the livelihood of resource-poor subsistence farmers in Bhutan. However, ticks and tick-borne diseases (TBDs) are one of the major constraints to livestock farming due to their negative effect on health and production. To date, no study has been conducted in Bhutan to assess farmers' knowledge, attitude, and practices (KAP) about ticks and TBDs in cattle, although such information is essential in ensuring the development and adoption of effective prevention and control measures. Therefore, a KAP survey was conducted among 246 cattle owners in the Samkhar sub-district of eastern Bhutan in June 2019, using a structured questionnaire. Based on our scoring criteria, 52% [95%CI: 45.5-58.4] had adequate knowledge about ticks as potential vectors of diseases. Logistic regression analysis showed that the individuals who practiced a stall-feeding system of cattle rearing were 2.8 times [OR = 2.8 (95%CI: 1.66-4.78)] more likely to have adequate knowledge than others. Sixty-eight percent [95%CI: 62.5-74.4] had a favorable attitude toward tick prevention and control programs. Men were 1.95 times [OR = 1.95 (95%CI: 1.09-3.55)] more likely to have a favorable attitude than women, and the individuals who practiced a stall-feeding system were 2.59 times [OR = 2.59 95%CI: 1.45-4.78)] more likely to have a favorable attitude than others, after adjusting for the effect of other variables in the model. Overall, only 38% [95%CI 32.5-45] of the respondents reported tick infestation as one of the most important animal health problems, but 100% reported using acaricides to control ticks in cattle. Despite a high level of acaricide usage, the level of knowledge was low among the farmers interviewed. Findings from this study underline the importance of considering identified knowledge gaps and initiating education efforts to improve the adoption of effective tick prevention and control measures among farmers.

Identification, Distribution, and Habitat Suitability Models of Ixodid Tick Species in Cattle in Eastern Bhutan.

Tick infestation is the most reported parasitological problem in cattle in Bhutan. In May and June 2019, we collected ticks from 240 cattle in two districts of Eastern Bhutan. Tick presence, diversity, and infestation prevalence were examined by morphological identification of 3600 live adult ticks. The relationships between cattle, geographic factors, and infestation prevalence were assessed using logistic regression analyses. Habitat suitability for the tick species identified was determined using MaxEnt. Four genera and six species of ticks were found. These were Rhipicephalus microplus (Canestrini) (70.2% (95% confidence interval (CI): 68.7-71.7)), Rhipicephalus haemaphysaloides Supino (18.8% (95% CI: 17.5-20.1)), Haemaphysalis bispinosa Neumann (8.2% (95% CI: 7.3-9.1)), Haemaphysalis spinigera Neumann (2.5% (95% CI: 2-3)), Amblyomma testudinarium Koch (0.19% (95% CI: 0.07-0.4)), and a single unidentified Ixodes sp. Logistic regression indicated that the variables associated with infestation were: longitude and cattle age for R. microplus; latitude for R. haemaphysaloides; and altitude and cattle breed for H. bispinosa and H. spinigera. MaxEnt models showed land cover to be an important predictor for the occurrence of all tick species examined. These findings provide information that can be used to initiate and plan enhanced tick surveillance and subsequent prevention and control programs for ticks and tick-borne diseases in cattle in Bhutan.

Occurrence of Echinococcusgranulosussensulato and Other Taeniids in Bhutan.

The present research shows the results of a national study documenting the occurrence and genetic diversity of Echinococcus and Taenia species across Bhutan. Environmental dog faecal samples (n = 953) were collected from 2016 to 2018 in all 20 Bhutanese districts, mainly in urbanised areas. Cystic echinococcosis cysts were isolated from 13 humans and one mithun (Bos frontalis). Isolation of taeniid eggs from faeces was performed by sieving/flotation technique, followed by DNA isolation, PCR and sequence analyses for species identification (gene target: small subunit of ribosomal RNA). Genetic diversity of E. granulosuss.s. was based on the sequence (1609 bp) of the cox1 gene. A total of 67 out of 953 (7%) dog faecal samples were positive for at least one taeniid species. From the 670 free-roaming dog faecal samples, 40 (5.9%) were positive for taeniid DNA, 22 (3.2%) of them were identified as E. granulosuss.s. and four (0.5%) as E. ortleppi (G5). From the 283 faecal samples originating from yak-grazing areas, 27 (9.5%) were taeniid positive, including eight (2.8%) infected with E. granulosuss.s. and four (1.4%) with E. ortleppi. E. granulosuss.s. was identified in all isolates from human and the cyst from mithun. A haplotype network (cox1 gene) from E. granulosuss.s, including isolates from 12 dogs, two human and one mithun, revealed eight different haplotypes. The most common cox1 haplotype was the globally distributed Eg01, followed by Eg40 and Eg37 (previously described in China). Five new cox1 haplotypes (EgBhu1-5) originated from human, dogs, and a mithun were identified. The study indicated the contamination of urban areas and pastures with Echinococcus eggs in seven districts in Bhutan. The molecular characterisation of E. granulosuss.l. revealed different E. granulosuss.s. haplotypes as well as E. ortleppi. The transmission of T. multiceps was documented only in the western part of the country. Considering the zoonotic feature of E. granulosus s.s. and E. ortleppi and the economic impact of coenurosis caused by T. multiceps (also known as gid) in Bhutan, the findings of this study represent a significant contribution towards an epidemiological baseline for the establishment of a national control programme.

Cerebral cysticercosis in a wild Bengal tiger (Panthera tigris tigris) in Bhutan: A first report in non-domestic felids.

The endangered Bengal tiger (Panthera tigris tigris) is a keystone species playing an essential role in ecology as well as in the social and spiritual lives of the Himalayan people. The latest estimate of the Bengal tiger population in Bhutan accounts for 103 individuals. Infectious organisms, including zoonotic parasites causing high burden in human health, have received little attention as a cause of mortality in tigers. Taeniosis/cysticercosis, caused by the cestode Taenia solium, is considered one of the major neglected tropical diseases in Southeast Asia. We present here a case of neurocysticercosis in a Bengal tiger showing advanced neurological disease outside Thimphu, the capital city of Bhutan. After palliative care, the animal died, and necropsy revealed multiple small cysts in the brain. Here we show the presence of two genetic variants of T. solium in the parasite material collected based on PCR and sequencing of the complete cox1 and cytB genes. The sequences form a discrete branch within the Asia plus Madagascar cluster of the parasite. On other hand, tests for feline morbillivirus, feline calicivirus, canine distemper virus, Nipah, rabies, Japanese encephalitis, feline leukaemia and feline immunodeficiency virus were negative. In contrast, PCR for feline herpesvirus was positive and a latex agglutination test revealed an elevated antibody titer against Toxoplasma gondii (titer 1:256). The molecular examination of taeniid eggs isolated from the tiger faeces produced sequences for which the highest homology in GenBank is between 92% and 94% with T. regis and T. hydatigena. This fatal case of T. solium neurocysticercosis, a disease previously unrecorded in tigers or other non-domestic felids, demonstrates an anthropogenically driven transmission of a deadly pathogen which could become a serious threat to the tiger population.

Further development of a reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of foot-and-mouth disease virus and validation in the field with use of an internal positive control.

Foot-and-mouth disease (FMD) is a highly contagious viral disease of cloven-hooved animals. Global outbreaks have highlighted the significant economic, trade, psychosocial and animal welfare impacts that can arise from the detection of disease in previously 'FMD-free' countries. Rapid and early diagnosis provides significant advantages in disease control and minimization of deleterious consequences. We describe the process of further development and validation of a reverse-transcription loop-mediated isothermal amplification foot-and-mouth disease virus (RT-LAMP-FMDV) test, using a published LAMP primer set, for use in the field. An internal positive control (IPC) was designed and introduced for use with the assay to mitigate any intrinsic interference from the unextracted field samples and avoid false negatives. Further modifications were included to improve the speed and operability of the test, for use by non-laboratory trained staff operating under field conditions, with shelf-stable reaction kits which require a minimum of liquid handling skills. Comparison of the assay performance with an established laboratory-based real-time reverse transcriptase PCR (rRT-PCR) test targeting the 3D region of FMD virus (Tetracore Inc) was investigated. LAMP has the potential to complement current laboratory diagnostics, such as rRT-PCR, as a preliminary tool in the investigation of FMD. We describe a strategic approach to validation of the test for use in the field using extracted RNA samples of various serotypes from Thailand and then finally unextracted field samples collected from FMD-suspected animals (primarily oral lesion swabs) from Bhutan and Australia. The statistical approach to validation was performed by Frequentist and Bayesian latent class methods, which both confirmed this new RT-LAMP-FMDV test as fit-for-purpose as a herd diagnostic tool with diagnostic specificity >99% and sensitivity 79% (95% Bayesian credible interval: 65, 90%) on unextracted field samples (oral swabs).

Serological Evidence of Rickettsia, Orientia, and Coxiella in Domestic Animals from Bhutan: Preliminary Findings.

There is no information on rickettsial diseases in domestic animals in Bhutan. This study provides preliminary serological data on exposure of domestic animals to Rickettsia, Orientia, and Coxiella. Animal sera were collected opportunistically from Bhutan and tested in the Australian Rickettsial Reference Laboratory for IgG antibodies against spotted fever group (SFG) and typhus group (TG) Rickettsia, scrub typhus group (STG), and Q fever (QF). Of the 294 animals tested, 136 (46%) showed serological evidence of past exposure to one or more rickettsiae: 106 (36%), 62 (21%), 45 (15%), and 11 (4%) being positive against SFG Rickettsia, Orientia, TG Rickettsia, and Coxiella, respectively. Dogs appeared to exhibit the highest seropositivity against SFG (55%) and TG Rickettsia (45%), horses against STG (91%), while goats were mostly positive for Coxiella (9%). Dogs also appeared to have high risk of being exposed to SFG Rickettsia (odd ratios [OR] 5.71, 95% confidence interval [CI] 3.02-10.80, p < 0.001), TG Rickettsia (OR 48.74, 95% CI 11.29-210.32, p < 0.001), and STG (OR 6.80, 95% CI 3.32-13.95, p < 0.001), but not against QF (OR 1.95, 95% CI 0.42-8.95, p = 0.390). Differences in seropositivity rates between animal species may have been significant for SFG, TG, and STG, but not for QF. The differences in the seropositivity rates of the four infections between districts appeared to be significant for TG and STG, but not for SFG and QF. The seropositivity rates of domestic animals to the four rickettsial infections were consistent with similar studies on the human population in the same areas and appear to demonstrate a high prevalence of exposure to rickettsiae in Bhutan. These preliminary findings constitute baseline data for Bhutan. The findings of this study call for an increased human-livestock sector collaboration in rickettsial diseases research aimed at developing diagnostic and therapeutic guidelines and formulating preventive and control measures through a One Health approach.

A national serosurvey to determine the prevalence of paratuberculosis in cattle in Bhutan following detection of clinical cases.

Johne's disease is an economically important ruminant disease predominantly affecting cattle, sheep and goats. The economic losses are due to early culling, reduced growth rate, progressive weight loss and reduced production. It is caused by Mycobacterium avium subspecies paratuberculosis (MAP). Johne's disease was reported in cattle in Bhutan, based on clinical signs and histopathology; in the late 1990s samples from one mithun that was suspected to have died due to this disease was confirmed by molecular testing at the Faculty of Veterinary Science, University of Sydney, Australia. However, no detailed study on prevalence of JD has been attempted in Bhutan. Objective of this study was to conduct serosurveillance to determine the national prevalence of Johne's disease in cattle for the period 2013-2014 to provide the basis for planning a future control strategy. A national serosurvey was conducted wherein a two-stage sampling procedure was used with 95% confidence and an error level of ±0.05. The sample size required for the survey was calculated using the software-Survey Toolbox for Livestock Diseases, available as Epitools at http://www.ausvet.com.au. A total of 1123 serum samples were collected from an administrative structure of 52 villages, 40 sub-districts and 15 districts. Serum samples were tested using commercially available antibody enzyme linked immunosorbent assay. Statistical analysis was performed using GraphPad Prism 5.0. Illustration such as maps was produced using QGIS version 2.18 'Las Palmas. The mean national apparent prevalence of Johne's disease was found to be 2.31 (26/1123) (95% CI: 0.80-4.50) with an estimated true prevalence was found to be 8.00 (95% CI: 2.00-17.00). Trongsa district had the highest prevalence (12.96) followed by Zhemgang (4.34), Lhuntse (4.25), Sarpang (3.89), Bumthang (3.60), Trashigang (2.67) and Haa (2.63). Prevalence for all other districts was 2.00 or below. Seropositive samples were reported from all over the country with varying levels of sero-positivity. In the recent past many more cattle were imported from India to boost dairy production. Nevertheless, the wide distribution of seroreactive JD cattle all over the country is a concern for future control. Therefore, in future, a detailed study on the impact of cattle import with regard to disease incursion such as Johne's disease and other diseases should be undertaken.

Immunopathological changes and apparent recovery from infection revealed in cattle in an experimental model of Johne's disease using a lyophilised culture of Mycobacterium avium subspecies paratuberculosis.

Johne's disease (JD) or paratuberculosis is an economically significant, chronic enteropathy of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). Experimental models of JD in cattle are logistically challenging due to the need for long term monitoring, because the clinical disease can take years to manifest. Three trials were undertaken, the largest involving 20 cattle exposed orally to a low dose of C strain MAP and 10 controls studied for 4.75 years. Frequent blood and faecal sampling was used to monitor immunological and infection parameters, and intestinal biopsies were performed at two time points during the subclinical disease phase. Although clinical disease was not seen, there was evidence of infection in 35% of the animals and at necropsy 10% had histopathological lesions consistent with JD, similar to the proportions expected in naturally infected herds. Faecal shedding occurred in two distinct phases: firstly there was intermittent shedding <∼9 months post-exposure that did not correlate with disease outcomes; secondly, in a smaller cohort of animals, this was followed by more consistent shedding of increasing quantities of MAP, associated with intestinal pathology. There was evidence of regression of histopathological lesions in the ileum of one animal, which therefore had apparently recovered from the disease. Both cattle with histopathological lesions of paratuberculosis at necropsy had low MAP-specific interferon-gamma responses at 4 months post-exposure and later had consistently shed viable MAP; they also had the highest loads of MAP DNA in faeces 4.75 year s post-exposure. In a trial using a higher dose of MAP, a higher proportion of cattle developed paratuberculosis. The information derived from these trials provides greater understanding of the changes that occur during the course of paratuberculosis in cattle.

Reconstructing the evolutionary history of pandemic foot-and-mouth disease viruses: the impact of recombination within the emerging O/ME-SA/Ind-2001 lineage.

Foot-and-mouth disease (FMD) is a highly contagious disease of livestock affecting animal production and trade throughout Asia and Africa. Understanding FMD virus (FMDV) global movements and evolution can help to reconstruct the disease spread between endemic regions and predict the risks of incursion into FMD-free countries. Global expansion of a single FMDV lineage is rare but can result in severe economic consequences. Using extensive sequence data we have reconstructed the global space-time transmission history of the O/ME-SA/Ind-2001 lineage (which normally circulates in the Indian sub-continent) providing evidence of at least 15 independent escapes during 2013-2017 that have led to outbreaks in North Africa, the Middle East, Southeast Asia, the Far East and the FMD-free islands of Mauritius. We demonstrated that sequence heterogeneity of this emerging FMDV lineage is accommodated within two co-evolving divergent sublineages and that recombination by exchange of capsid-coding sequences can impact upon the reconstructed evolutionary histories. Thus, we recommend that only sequences encoding the outer capsid proteins should be used for broad-scale phylogeographical reconstruction. These data emphasise the importance of the Indian subcontinent as a source of FMDV that can spread across large distances and illustrates the impact of FMDV genome recombination on FMDV molecular epidemiology.

Detection of Echinococcus granulosus and Echinococcus ortleppi in Bhutan.

In this pilot study, fecal samples were collected from community dogs around slaughterhouses and from the city of Thimphu (n=138) as well as from carnivores in the forest area around a farm in Bhutan (n=28). Samples were analyzed microscopically for the presence of taeniid eggs by the floatation and sieving method. Further molecular analyses of 20 samples of community dogs positive for taeniid eggs confirmed 10 Echinococcus granulosus sensu lato and one Taenia hydatigena case. From 14 environmental fecal samples from the forest area positive for taeniid eggs, one contained E. granulosus s.l., six T. hydatigena and one Taenia taeniaeformis DNA. In the remaining samples considered positive for taeniid eggs, no molecular confirmation could be achieved. Additionally, Echinococcus cysts were collected from locally slaughtered cattle and imported cattle organs. Seven Echinococcus cysts (one fertile) from the local animals and 35 (four fertile) from imported cattle organs were confirmed as E. granulosus (G1-3) by PCR/sequencing. One Echinococcus cyst each from a local animal and from an imported cattle organ (both fertile) were confirmed to be Echinococcus ortleppi (G5). Sterile Echinococcus cysts were also collected from local yaks (n=10), and all revealed to be E. granulosus (G1-G3). Hospital records of cystic echinococcosis in humans and the presence of Echinococcus spp. in dogs and ungulates indicate the existence of local transmission for both E. ortleppi and E. granulosus in Bhutan.

Antigenicity in sheep of synthetic peptides derived from stress-regulated Mycobacterium avium subsp. paratuberculosis proteins and comparison with recombinant protein and complex native antigens.

Serum antibody enzyme-linked immunosorbent assay is the most commonly used test for diagnosis of Mycobacterium avium subsp. paratuberculosis infection in ruminants. However, the assay requires serum preabsorption with Mycobacterium phlei proteins to reduce cross reactions potentially contributed by the exposure of livestock to environmental mycobacteria. To trial the discovery of novel antigens which do not require serum absorption, synthetic MAP-specific peptides were selected based on in silico research to identify putative B cell epitopes. Four peptides from previously identified stress-regulated proteins were synthesized and evaluated using enzyme linked immunosorbent assay to detect Mycobacterium avium subsp. paratuberculosis specific antibodies in sheep. Two peptides were from hypothetical MAP proteins (MAP3567 and MAP1168c) and two were from proteins with known function (MAP2698c, an acyl-acyl carrier protein desaturase-DesA2 and MAP2487c a carbonic anhydrase). The ability of each peptide to discriminate between unexposed and MAP exposed (infected and vaccinated) animals was similar to that of the parent recombinant MAP antigen, with area under receiver operating curve values of 0.86-0.93. Assays run with a combination of two peptides showed slightly higher reactivity than those of individual peptides. Peptides evaluated in this study had diagnostic potential similar to corresponding recombinant proteins but not superior to a complex native MAP antigen or a commercial assay. Further study is required to investigate other peptides for their diagnostic potential, and this may be simpler and cheaper than subunit protein-based research.

Cellular and humoral immune responses in sheep vaccinated with candidate antigens MAP2698c and MAP3567 from Mycobacterium avium subspecies paratuberculosis.

Control of Johne's disease, caused by Mycobacterium avium subspecies paratuberculosis (MAP) in ruminants using commercially available vaccine reduces production losses, mortality, fecal shedding and histopathological lesions but does not provide complete protection from infection and interferes with serological diagnosis of Johne's disease and bovine tuberculosis. At this time no recombinant antigens have been found to provide superior protection compared to whole killed or live-attenuated MAP vaccines. Therefore, there is a need to evaluate more candidate MAP antigens. In this study recombinant MAP antigens MAP2698c and MAP3567 were formulated with four different MONTANIDE™ (ISA 50V2, 61VG, 71VG, and 201VG) adjuvants and evaluated for their ability to produce specific immune responses in vaccinated sheep. The cellular immune response was measured with an interferon-gamma (IFN-γ) release assay and the humoral immune response was measured by antibody detection enzyme linked immunosorbent assay. Recombinant vaccine formulation with the antigen MAP2698c and MONTANIDE™ ISA 201VG adjuvant produced strong whole-MAP as well as MAP2698c-specific IFN-γ responses in a high proportion of the vaccinated sheep. The formulation caused less severe injection site lesions in comparison to other formulations. The findings from this study suggest that the MAP2698c + 201VG should be evaluated in a challenge trial to determine the efficacy of this vaccine candidate.

Immunoreactivity of protein tyrosine phosphatase A (PtpA) in sera from sheep infected with Mycobacterium avium subspecies paratuberculosis.

Evasion of host defense mechanisms and survival inside infected host macrophages are features of pathogenic mycobacteria including Mycobacterium avium subspecies paratuberculosis, the causative agent of Johne's disease in ruminants. Protein tyrosine phosphatase A (PtpA) has been identified as a secreted protein critical for survival of mycobacteria within infected macrophages. The host may mount an immune response to such secreted proteins. In this study, the humoral immune response to purified recombinant M. avium subsp. paratuberculosis PtpA was investigated using sera from a cohort of sheep infected with M. avium subsp. paratuberculosis and compared with uninfected healthy controls. A significantly higher level of reactivity to PtpA was observed in sera collected from M. avium subspecies paratuberculosis infected sheep when compared to those from uninfected healthy controls. PtpA could be a potential candidate antigen for detection of humoral immune responses in sheep infected with M. avium subspecies paratuberculosis.

Lymphoproliferative and gamma interferon responses to stress-regulated Mycobacterium avium subsp. paratuberculosis recombinant proteins.

Johne's disease in ruminants is a chronic infection of the intestines caused by Mycobacterium avium subsp. paratuberculosis. An important strategy to control disease is early detection, and a potentially efficient method for early detection is measurement of cell-mediated immune responses developed by the host in response to exposure or infection. One method is to measure lymphoproliferation and cytokine release from the host cells when exposed to the organism or parts of the organism. In this study, 10 recombinant M. avium subsp. paratuberculosis proteins known to be upregulated under in vitro stress conditions were evaluated by examining their ability to evoke memory as a result of exposure by vaccination or oral challenge with live Mycobacterium avium subsp. paratuberculosis. Out of 10 proteins, MAP2698c was found to induce higher cell-mediated immune responses in vaccinated and challenged sheep in comparison to healthy controls. The findings suggest that not all stress-regulated proteins have the diagnostic potential to detect cell-mediated immune responses in ovine paratuberculosis.

Antigenicity of recombinant maltose binding protein-Mycobacterium avium subsp. paratuberculosis fusion proteins with and without factor Xa cleaving.

Mycobacterium avium subsp. paratuberculosis causes Johne's disease (JD) in ruminants. Proteomic studies have shown that M. avium subsp. paratuberculosis expresses certain proteins when exposed to in vitro physiological stress conditions similar to the conditions experienced within a host during natural infection. Such proteins are hypothesized to be expressed in vivo, are recognized by the host immune system, and may be of potential use in the diagnosis of JD. In this study, 50 recombinant maltose binding protein (MBP)-M. avium subsp. paratuberculosis fusion proteins were evaluated using serum samples from sheep infected with M. avium subsp. paratuberculosis, and 29 (58%) were found to be antigenic. Among 50 fusion proteins, 10 were evaluated in MBP fusion and factor Xa-cleaved forms. A total of 31 proteins (62%) were found to be antigenic in either MBP fusion or factor Xa-cleaved forms. Antigenicity after cleavage and removal of the MBP tag was marginally enhanced.