BCG vaccination reduces bovine tuberculosis transmission, improving prospects for elimination.

Bacillus Calmette-Guérin (BCG) is a routinely used vaccine for protecting children against Mycobacterium tuberculosis that comprises attenuated Mycobacterium bovis. BCG can also be used to protect livestock against M. bovis; however, its effectiveness has not been quantified for this use. We performed a natural transmission experiment to directly estimate the rate of transmission to and from vaccinated and unvaccinated calves over a 1-year exposure period. The results show a higher indirect efficacy of BCG to reduce transmission from vaccinated animals that subsequently become infected [74%; 95% credible interval (CrI): 46 to 98%] compared with direct protection against infection (58%; 95% CrI: 34 to 73%) and an estimated total efficacy of 89% (95% CrI: 74 to 96%). A mechanistic transmission model of bovine tuberculosis (bTB) spread within the Ethiopian dairy sector was developed and showed how the prospects for elimination may be enabled by routine BCG vaccination of cattle.

Is bovine density and ownership associated with human tuberculosis in India?

Zoonotic tuberculosis in humans is caused by infection with bacteria of the Mycobacterium tuberculosis complex acquired from animals, most commonly cattle. India has the highest burden of human tuberculosis in the world and any zoonotic risk posed by tuberculosis in bovines needs to be managed at the source of infection as a part of efforts to end human tuberculosis. Zoonotic tuberculosis in humans can be severe and is clinically indistinguishable from non-zoonotic tuberculosis. As a consequence, zoonotic tuberculosis remains under-recognised and the significance of its contribution to human tuberculosis is poorly understood. This study aimed to explore any association between bovine density, bovine ownership, and human tuberculosis reporting in India using self-reported tuberculosis data in households and officially reported tuberculosis cases while controlling for common confounders for human tuberculosis. We find an association between human tuberculosis reporting, bovine density and bovine ownership in India. Buffalo density was significantly associated with an increased risk of self-reported tuberculosis in households (odds ratio (OR) = 1.23 (95% credible interval (CI): 1.10-1.39) at household level; incidence rate ratio (IRR) = 1.17 (95% CI: 1.04-1.33) at district level), while cattle density (OR = 0.80, 95% CI: 0.71-0.89; IRR = 0.78, 95% CI: 0.70-0.87) and ownership of bovines in households (OR = 0.94, 95% CI: 0.9-0.99; IRR = 0.67, 95% CI: 0.57-0.79) had a protective association with tuberculosis reporting. It is unclear whether this relates to differences in tuberculosis transmission dynamics, or perhaps an association between bovines and other unexplored confounders for tuberculosis reporting in humans. Our study highlights a need for structured surveillance to estimate the prevalence of tuberculosis in cattle and buffaloes, characterisation of Mycobacterium tuberculosis complex species present in bovines and transmission analyses at the human-animal interface to better assess the burden and risk pathways of zoonotic tuberculosis in India.

Novel Polyherbal Nanocolloids to Control Bovine Mastitis.

Mastitis is a widespread disease in dairy cattle occurring throughout the world. The increased use of antibiotics brings about the development of antibiotic-resistant microbes. The application of antibiotics in dairy farming led to increased antibiotic resistance and represents a major obstacle for the treatment of mastitis. Recent advancements in nanotechnology led to the development of nanocolloids to overcome disadvantages posed by conventional antimicrobial agents. Hence, a novel, environmentally friendly, cost-effective, biocompatible, and long-term antibacterial represents a promising solution for medicine and farming. Hence, polyherbal nanocolloids (PHNc) was formulated by using the extracts of Syzygium aromaticum, Cinnamomum verum, Emblica officinalis, Terminalia belerica, Terminalia chebula, and Cymbopogon citratus and physicochemically characterized. From mastitis milk samples, microorganisms were isolated including Acinetobacter junii, Klebsiella pneumoniae, Pseudomonas stutzeri, and Acinetobacter baumannii and screened for antibiotic susceptibility. All the isolated strains were tested with PHNc and compared with antibiotics. Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and biofilm assays were performed at different concentrations, and antibacterial effects were quantified. In our results, PHNc showed potent bacteriostatic, bactericidal, and antibiofilm activity against all the strains. Our results indicated that PHNc can reduce the virulence factors responsible for infection by different bacterial strains. This study confirmed that PHNc had the potential to inhibit the growth of pathogenic Gram-negative and Gram-positive strains and could be utilized as an alternative to antibiotics to inhibit multidrug-resistant microbial pathogens in cattle.

A Defined Antigen Skin Test for Diagnosis of Bovine Tuberculosis in Domestic Water Buffaloes (Bubalus bubalis).

Bovine tuberculosis (bTB) remains endemic in domestic water buffaloes (Bubalus bubalis) in India and elsewhere, with limited options for control other than testing and slaughter. The prescribed tuberculin skin tests with purified protein derivative (PPD) for diagnosis of bTB preclude the use of Bacille Calmette-Guérin (BCG)-based vaccination because of the antigenic cross-reactivity of vaccine strains with Mycobacterium bovis and related pathogenic members of the M. tuberculosis complex (MTBC). For the diagnosis of bTB in domestic water buffaloes, we here assessed a recently described defined-antigen skin test (DST) that comprises overlapping peptides representing the ESAT-6, CFP-10 and Rv3615c antigens, present in disease-causing members of the MTBC but missing in BCG strains. The performance characteristics of three doses (5, 10 or 20 µg/peptide) of the DST were assessed in natural tuberculin skin test reactor (n = 11) and non-reactor (n = 35) water buffaloes at an organized dairy farm in Hisar, India, and results were compared with the single intradermal skin test (SIT) using standard bovine tuberculin (PPD-B). The results showed a dose-dependent response of DST in natural reactor water buffaloes, although the SIT induced a significantly greater (P < 0.001) skin test response than the highest dose of DST used. However, using a cut-off of 2 mm or greater, the 5, 10, and 20 µg DST cocktail correctly classified eight, 10 and all 11 of the SIT-positive reactors, respectively, suggesting that the 20 µg DST cocktail has a diagnostic sensitivity (Se) of 1.0 (95% CI: 0.72-1.0) identical to that of the SIT. Importantly, none of the tested DST doses induced any measurable skin induration responses in the 35 SIT-negative animals, suggesting a specificity point estimate of 1.0 (95% CI: 0.9-1.0), also identical to that of the SIT and compares favorably with that of the comparative cervical test (Se = 0.85; 95% CI: 0.55-0.98). Overall, the results suggest that similar to tuberculin, the DST enables sensitive and specific diagnosis of bTB in water buffaloes. Future field trials to explore the utility of DST as a defined antigen replacement for tuberculin in routine surveillance programs and to enable BCG vaccination of water buffaloes are warranted.

Corrigendum: A Meta-Analysis of the Effect of Bacillus Calmette-Guérin Vaccination Against Bovine Tuberculosis: Is Perfect the Enemy of Good?

[This corrects the article DOI: 10.3389/fvets.2021.637580.].

A Meta-Analysis of the Effect of Bacillus Calmette-Guérin Vaccination Against Bovine Tuberculosis: Is Perfect the Enemy of Good?

More than 50 million cattle are likely exposed to bovine tuberculosis (bTB) worldwide, highlighting an urgent need for bTB control strategies in low- and middle-income countries (LMICs) and other regions where the disease remains endemic and test-and-slaughter approaches are unfeasible. While Bacillus Calmette-Guérin (BCG) was first developed as a vaccine for use in cattle even before its widespread use in humans, its efficacy against bTB remains poorly understood. To address this important knowledge gap, we conducted a systematic review and meta-analysis to determine the direct efficacy of BCG against bTB challenge in cattle, and performed scenario analyses with transmission dynamic models incorporating direct and indirect vaccinal effects ("herd-immunity") to assess potential impact on herd level disease control. The analysis shows a relative risk of infection of 0.75 (95% CI: 0.68, 0.82) in 1,902 vaccinates as compared with 1,667 controls, corresponding to a direct vaccine efficacy of 25% (95% CI: 18, 32). Importantly, scenario analyses considering both direct and indirect effects suggest that disease prevalence could be driven down close to Officially TB-Free (OTF) status (<0.1%), if BCG were introduced in the next 10-year time period in low to moderate (<15%) prevalence settings, and that 50-95% of cumulative cases may be averted over the next 50 years even in high (20-40%) disease burden settings with immediate implementation of BCG vaccination. Taken together, the analyses suggest that BCG vaccination may help accelerate control of bTB in endemic settings, particularly with early implementation in the face of dairy intensification in regions that currently lack effective bTB control programs.

A Defined Antigen Skin Test That Enables Implementation of BCG Vaccination for Control of Bovine Tuberculosis: Proof of Concept.

In most low- and middle-income countries (LMICs), bovine tuberculosis (bTB) remains endemic due to the absence of control programs. This is because successful bTB control and eradication programs have relied on test-and-slaughter strategies that are socioeconomically unfeasible in LMICs. While Bacillus Calmette-Guérin (BCG) vaccine-induced protection for cattle has long been documented in experimental and field trials, its use in control programs has been precluded by the inability to differentiate BCG-vaccinated from naturally infected animals using the OIE-prescribed purified protein derivative (PPD)-based tuberculin skin tests. In the current study, the diagnostic specificity and capability for differentiating infected from vaccinated animals (DIVA) of a novel defined antigen skin test (DST) in BCG-vaccinated (Bos taurus ssp. taurus x B. t. ssp. indicus) calves were compared with the performance of traditional PPD-tuberculin in both the skin test and in vitro interferon-gamma release assay (IGRA). The IFN-γ production from whole blood cells stimulated with both PPDs increased significantly from the 0 week baseline levels, while DST induced no measurable IFN-γ production in BCG-vaccinated calves. None of the 15 BCG-vaccinated calves were reactive with the DST skin test (100% specificity; one-tailed lower 95% CI: 82). In contrast, 10 of 15 BCG-vaccinated calves were classified as reactors with the PPD-based single intradermal test (SIT) (specificity in vaccinated animals = 33%; 95% CI: 12, 62). Taken together, the results provide strong evidence that the DST is highly specific and enables DIVA capability in both skin and IGRA assay format, thereby enabling the implementation of BCG vaccine-based bTB control, particularly in settings where test and slaughter remain unfeasible.

Reconsidering Mycobacterium bovis as a proxy for zoonotic tuberculosis: a molecular epidemiological surveillance study.

BACKGROUND: Zoonotic tuberculosis is defined as human infection with Mycobacterium bovis. Although globally, India has the largest number of human tuberculosis cases and the largest cattle population, in which bovine tuberculosis is endemic, the burden of zoonotic tuberculosis is unknown. The aim of this study was to obtain estimates of the human prevalence of animal-associated members of the Mycobacterium tuberculosis complex (MTBC) at a large referral hospital in India. METHODS: We did a molecular epidemiological surveillance study of 940 positive mycobacteria growth indicator tube (MGIT) cultures, collected from patients visiting the outpatient department at Christian Medical College (Vellore, India) with suspected tuberculosis between Oct 1, 2018, and March 31, 2019. A PCR-based approach was applied to subspeciate cultures. Isolates identified as MTBC other than M tuberculosis or as inconclusive on PCR were subject to whole-genome sequencing (WGS), and phylogenetically compared with publicly available MTBC sequences from south Asia. Sequences from WGS were deposited in the National Center for Biotechnology Information Sequence Read Archive, accession number SRP226525 (BioProject database number PRJNA575883). FINDINGS: The 940 MGIT cultures were from 548 pulmonary and 392 extrapulmonary samples. A conclusive identification was obtained for all 940 isolates; wild-type M bovis was not identified. The isolates consisted of M tuberculosis (913 [97·1%] isolates), Mycobacterium orygis (seven [0·7%]), M bovis BCG (five [0·5%]), and non-tuberculous mycobacteria (15 [1·6%]). Subspecies were assigned for 25 isolates by WGS, which were analysed against 715 MTBC sequences from south Asia. Among the 715 genomes, no M bovis was identified. Four isolates of cattle origin were dispersed among human sequences within M tuberculosis lineage 1, and the seven M orygis isolates from human MGIT cultures were dispersed among sequences from cattle. INTERPRETATION: M bovis prevalence in humans is an inadequate proxy of zoonotic tuberculosis. The recovery of M orygis from humans highlights the need to use a broadened definition, including MTBC subspecies such as M orygis, to investigate zoonotic tuberculosis. The identification of M tuberculosis in cattle also reinforces the need for One Health investigations in countries with endemic bovine tuberculosis. FUNDING: Bill & Melinda Gates Foundation, Canadian Institutes for Health Research.

A defined antigen skin test for the diagnosis of bovine tuberculosis.

Bovine tuberculosis (bTB) is a major zoonotic disease of cattle that is endemic in much of the world, limiting livestock productivity and representing a global public health threat. Because the standard tuberculin skin test precludes implementation of Bacille Calmette-Guérin (BCG) vaccine-based control programs, we here developed and evaluated a novel peptide-based defined antigen skin test (DST) to diagnose bTB and to differentiate infected from vaccinated animals (DIVA). The results, in laboratory assays and in experimentally or naturally infected animals, demonstrate that the peptide-based DST provides DIVA capability and equal or superior performance over the extant standard tuberculin surveillance test. Together with the ease of chemical synthesis, quality control, and lower burden for regulatory approval compared with recombinant antigens, the results of our studies show that the DST considerably improves a century-old standard and enables the development and implementation of critically needed surveillance and vaccination programs to accelerate bTB control.

Point of Care Tuberculosis Sero-Diagnosis Kit for Wild Animals: Combination of Proteins for Improving the Diagnostic Sensitivity and Specificity.

Tuberculosis is a significant problem globally for domestic animals as well as captive and free ranging wild life. Rapid point of care (POC) serology kits are well suited for the diagnosis of TB in wild animals. However, wild animals are invariably exposed to environmental non-pathogenic mycobacterium species with the development of cross reacting antibodies. In the present study, POC TB diagnosis kit was developed using a combination of pathogenic Mycobacteria specific recombinant antigens and purified protein derivatives of pathogenic and non-pathogenic Mycobacteria. To benchmark the TB antibody detection kit, particularly in respect to specificity which could not be determined in wildlife due to the lack of samples from confirmed uninfected animals, we first tested well-characterized sera from 100 M. bovis infected and 100 uninfected cattle. Then we investigated the kit's performance using sera samples from wildlife, namely Sloth Bears (n = 74), Elephants (n = 9), Cervidae (n = 14), Felidae (n = 21), Cape buffalo (n = 2), Wild bear (n = 1) and Wild dog (n = 1).In cattle, a sensitivity of 81% and a specificity of 90% were obtained. The diagnostic sensitivity of the kit was 94% when the kit was tested using known TB positive sloth bear sera samples. 47.4% of the in-contact sloth bears turned seropositive using the rapid POC TB diagnostic kit. Seropositivity in other wild animals was 25% when the sera samples were tested using the kit. A point of care TB sero-diagnostic kit with the combination of proteins was developed and the kit was validated using the sera samples of wild animals.

Antemortem and postmortem examinations of the cattle calf naturally infected with Mycobacterium avium subsp. paratuberculosis.

A male cattle calf was detected as subclinically and naturally infected with Mycobacterium avium subspecies paratuberculosis (MAP) by a series of antemortem and postmortem tests. The MAP infection was identified by strong antibody and cell-mediated immune (CMI) response by a commercial ELISA kit and an intradermal Johnin test, respectively, in the initial antemortem examination. The antemortem status of the calf was further confirmed by MAP-specific interferon gamma (IFN-γ) response. For detection of IFN-γ response, MAP-specific IFN-γ release assays (IGRAs): (a) immuno capture ELISA (IC-ELISA) and (b) ELISPOT was employed. In addition, the presence of intracellular cytokine IFN-γ was detected by flow cytometry. For all cytokine assays, MAP-specific recombinant antigens HSP65 and 35 kDa were employed to overcome the poor sensitivity and specificity resulting from the use of Johnin, the crude protein purified derivative of MAP. Postmortem examination of the MAP-infected/suspected cattle calf did not reveal any pathognomonic gross lesions in the gastro-intestinal tract. Histopathological examination of multiple organs showed the presence of epithelioid cells/macrophages and edematous lesions in the mesenteric lymph nodes suggestive of MAP; however, no granulomas were observed in the intestinal tract. The necropsy samples of rectum and mesenteric lymph nodes were positive for isolation of MAP by culture in the BACTEC™ MGIT™ 960 system, and acid fast bacilli were demonstrated by fluorescence microscopy confirming the infection. Due to differential and complex expression patterns of MAP antigens reported in literature, a combination of assays such as those based on IGRAs and antibody detection is essential. Therefore, the current experimental evidence confirms the efficacy of the approach adopted. However, further studies will be needed to understand the optimal combination MAP-specific antigens for use in IGRAs or antibody assays that can be used for detecting MAP infection in every stage of the disease.

Multi-antigen print immunoassay for seroepidemiological surveillance of bovine tuberculosis on Indian cattle farms.

Bovine tuberculosis caused by Mycobacterium bovis is a zoonotic disease that is responsible for significant economic losses in many countries. The standard diagnostic method, the tuberculin test (TST) that is used in control programmes has serious shortcomings and, given the complex nature and the economic impact of the disease, a number of other diagnostic methods have been examined. The authors have attempted to characterise antibody response using the multi-antigen print immunoassay (MAPIA). A total of 511 serum samples were collected from farms in India on which bovine tuberculosis was prevalent and on farms with low incidence. These were tested using the MAPIA against a panel of five defined M. bovis recombinant antigens and two purified protein derivatives (bovine PPD and avian PPD) to study the seroprevalence of the disease on Indian cattle farms. Results indicated that the fusion protein of antigen CFP-10:MPB83 showed a positive response in 142 out of 298 serum samples from tuberculosis-prevalent farms, thereby indicating the serological dominance of the proteins post infection. The antigen selected could be used further in the development of a simple, rapid and accurate serological diagnostic test, paired with TST, for use in bovine tuberculosis control programmes.

Use of ESAT-6-CFP-10 fusion protein in the bovine interferon-gamma ELISPOT assay for diagnosis of Mycobacterium bovis infection in cattle.

Bovine tuberculosis (BTB) is a chronic bacterial disease, and a major animal health problem with zoonotic implications. Screening of mycobacterial infections in bovines is traditionally done using the single intradermal tuberculin test. Though the test is widely used, it has its own disadvantages and they include its inability to distinguish between pathogenic and non-pathogenic mycobacterial infections owing to its low specificity. Furthermore, the associated operative difficulties of this test have driven the quest for discovery of new antigens and diagnostic assays leading to the development of the interferon (IFN)- test. Presently, combinatorial testing using the skin test and the interferon gamma assays are being used in the diagnosis of BTB in various control and surveillance programs. In this study, we report the cloning, expression and purification of ESAT-6-CFP-10 fusion protein and its further use in the development of the IFN- gamma ELISPOT assay for accurate diagnosis of BTB in cattle. The BTB diagnosis employing the ELISPOT assay was evaluated using peripheral blood mononuclear cells from culture positive and culture negative cattle. The ELISPOT assay showed higher specificity and sensitivity in detecting BTB when a recombinant ESAT-6-CFP-10 fusion protein was used. The present study indicated that the usefulness of the fusion protein can replace the ESAT-6, CFP-10 or combination of both proteins for detecting BTB in IFN-gamma ELISPOT assay.

Assessment of critical parameters in blood processing for the bovine interferon-gamma ELISPOT assay to detect Mycobacterium bovis infected cattle in India.

In vitro production of bovine interferon gamma (BoIFN-γ) cytokine from bovine peripheral blood mononuclear cells (PBMCs) can be detected using the most sensitive enzyme-linked immunosorbent spot (ELISPOT) assay. ELISPOT assays are dependent on the quantity and quality of PBMC preparations and hence contribute significantly to the performance of this assay. In order to standardise the methods for isolation of PBMCs, we compared two methods for the processing of bovine blood which included aliquots of blood that were stored in a horizontal position without dilution or agitation and aliquots of blood that were immediately diluted 1:1 with complete Rosewell Park Memorial Institute (RPMI) 1640 medium and stored in a horizontal position with gentle agitation. PBMCs were isolated at 2, 4, 6, 8 and 24 h and at 4°C and at 22°C ± 2°C. They were stimulated using tuberculosis-specific antigens, after which the ELISPOT assay was performed. Quantities of spot-forming cells (SFC) created by the release of BoIFN-γ in ELISPOT assays were significantly greater in the samples stored at 22°C ± 2°C than those held +4°C and the intensity of the signals dropped following processing after 6 h. A further drop in SFC was observed in those samples that had been stored undiluted and without agitation. These findings demonstrated that optimisation of PBMC isolation procedures can lead to increased sensitivity in the detection of BoIFN-γ using the ELISPOT assay, thus contributing to an enhanced diagnosis of bovine tuberculosis.

Serotyping of foot-and-mouth disease virus by antigen capture-ELISA using monoclonal antibodies and chicken IgY.

Laboratory detection of specific foot-and-mouth disease virus (FMDV) is routinely carried out by ELISA and RT-PCR. Identification and serotyping of FMDV by ELISA requires polyclonal antibodies raised in rabbits and guinea pigs. The polyclonal antibodies have certain disadvantages such as batch to batch variation, inconsistent yields of antibodies and limited quantity of serum obtained from individual animals. This paper describes a method wherein monoclonal antibodies and chicken IgY were used in an antigen capture-ELISA for serotyping of thirty tongue epithelial samples and sixty tissue culture fluids. The results were compared with the routine antigen detection ELISA. The present study indicated that monoclonal antibodies and chicken IgY can substitute conventional polyclonal antibodies for routine serotyping of FMDV.