Invited Review: Improved control of Johne's disease in dairy cattle through advancements in diagnostics, testing and management of young stock.

Johne's disease (JD; paratuberculosis) control programs have been regionally implemented across the globe, but few have successfully eradicated the pathogen (Mycobacterium avium ssp. paratuberculosis (MAP)) causing this disease. The limited success may partly be attributed to excluding young stock (calves and replacement heifers or bulls) from testing strategies aimed at identifying MAP-infected cattle. Young stock can shed MAP in feces and can have detectable MAP-specific antibodies in blood, as confirmed in experimentally and naturally infected cattle. Furthermore, MAP transmission causes new infections in young stock. Calves and heifers are often included in JD management strategies on dairy farms but excluded from conventional diagnostic tests due to a presumed lag between infection and detection of MAP shedding and/or MAP-specific serum antibodies. We summarize evidence of MAP shedding early in the course of infection and discuss promising diagnostics, testing and management strategies to support inclusion of young stock in JD control programs. Improvements in fecal Polymerase Chain Reaction, interferon-gamma release assay (IGRA), and enzyme-linked immunosorbent assay (ELISA) enable earlier detection of MAP and specific early immune responses. Studies on IGRA and ELISA have focused on evaluation of new antigens and optimal age of testing. There are new diagnostics, including phage-based tests to detect viable MAP, and gene expression patterns and metabolomics to detect MAP-infected young stock. In addition, refinements in testing and management of calves and heifers may enable reductions in MAP prevalence. We provide recommendations for dairy farmers, researchers, veterinarians, and other stakeholders that may improve JD control programs with an objective to control and potentially eradicate JD. Additionally, we have identified the most pressing gaps in knowledge that currently hamper inclusion of young stock in JD prevention and control programs. In summary, transmission among young stock may cause new MAP infections, and appropriate use of new diagnostic tests, testing and management strategies for young stock may improve the efficacy of JD control programs.

Lining the small intestine with mycobacteriophages protects from Mycobacterium avium subsp. paratuberculosis and eliminates fecal shedding.

Johne's disease (JD), a chronic, infectious enteritis caused by Mycobacterium avium subsp. paratuberculosis (MAP), affects wild and domestic ruminants. There is no cure or effective prevention, and current vaccines have substantial limitations, leaving this disease widespread in all substantial dairy industries causing economic, and animal welfare implications. Mycobacteriophages (MPs) have been gaining interest in recent years and are proposed as a promising solution to curtailing MAP infection. Using a well-validated infection model, we have demonstrated the preventative potential of MPs to protect dairy calves against MAP infection. Calves were supplemented daily with a phage cocktail from birth till weaning at 2 m of age and inoculated with MAP at 2 wk of age. Infection status was measured for 4.5 mo through blood, fecal, and postmortem tissue samples. Our findings highlight the remarkable efficacy of orally administered MPs. Notably, fecal shedding of MAP was entirely eliminated within 10 wk, in contrast to the infected control group where shedding continued for the entirety of the trial period. Postmortem tissue culture analysis further supported the effectiveness of MPs, with only 1 out of 6 animals in the phage-treated group testing positive for MAP colonized tissues compared to 6 out of 6 animals in the infected control group. Additionally, plaque assay results demonstrated the ability of phages to persist within the intestinal tract. Collectively, these results underscore the potential of orally administered MP cocktails as a highly effective intervention strategy to combat JD in dairy calves and by extension in the dairy industry.

Evaluation of Mycobacterium avium subsp. paratuberculosis isocitrate lyase (IcL) and ABC transporter (BacA) knockout mutants as vaccine candidates.

There has been little success in controlling Johne's disease, caused by Mycobacterium avium subsp. paratuberculosis, due to suboptimal diagnostics and the ineffectiveness of available vaccines. By knocking out BacA and IcL, genes required for MAP survival in dairy calves, two live-attenuated vaccine candidates were created. This study evaluated the host-specific attenuation of MAP IcL and BacA mutants in mouse and calf models, as well as the elicited immune responses. Deletion mutants were generated in MAP strain A1-157 through specialized transduction and found viable in vitro. First, the mutants' attenuation and elicited cytokine secretion were assessed in a mouse model, 3 weeks after intraperitoneal inoculation with MAP strains. Later, vaccine strains were assessed in a natural host infection model where calves received 109CFU oral dose of MAP wild-type or mutant strains at 2 weeks old. Transcription levels of cytokines in PBMCs were evaluated at 12-, 14-, and 16-weeks post-inoculation (WPI) and MAP colonization in tissue was assessed at 4.5 months after inoculation. Whereas both vaccine candidates colonized mouse tissues similarly to wild-type strain, both failed to persist in calf tissues. In either mouse or calf models, gene deletion did not reduce immunogenicity. Instead, inoculation with ΔBacA induced a greater upregulation of proinflammatory cytokines than ΔIcL and wild-type in both models and a greater expansion of cytotoxic and memory T-cells than uninfected control in calves. ΔBacA and wild-type strains significantly increased secretion of IP-10, MIG, TNFα, and RANTES in mice serum compared to uninfected control. This agreed with upregulation of IL-12, IL-17, and TNFα in calves inoculated with ΔBacA at all time points. The ΔBacA also gave rise to greater populations of CD4+CD45RO+, and CD8+ cells than uninfected control calves at 16 WPI. Low survival rate of MAP in macrophages co-incubated with PBMCs isolated from the ΔBacA group indicated that these cell populations are capable of killing MAP. Overall, the immune response elicited by ΔBacA is stronger compared to ΔIcL and it is maintained over two different models and over time in calves. Further investigation is warranted to evaluate the BacA mutant's protection against MAP infection as a live attenuated vaccine candidate.

Immunogenicity and efficacy of an oral live-attenuated vaccine for bovine Johne's disease.

Mycobacterium avium subsp. paratuberculosis (MAP), the etiological agent of Johne's disease (JD) in ruminants, establishes a prolonged and often lifelong enteric infection. The implementation of control measures for bovine JD has faced obstacles due to the considerable expenses involved in disease surveillance and hindered by unreliable and inadequate diagnostic tests, emphasizing the need for an effective vaccine that can stimulate mucosal immunity in the gastrointestinal tract. Previous investigations have demonstrated that deletion of the BacA gene in MAP produces an attenuated strain that can transiently colonize the calf small intestine while retaining its capacity to stimulate systemic immune responses similar to wildtype MAP strains. This study assessed the efficacy of the BacA gene deletion MAP strain, referred to as the BacA vaccine, when administered orally to young calves. The research aimed to evaluate its effectiveness in controlling MAP intestinal infection and to investigate the immune responses elicited by mucosal vaccination. The study represents the first evaluation of an enteric modified live MAP vaccine in the context of an oral MAP challenge in young calves. Oral immunization with BacA reduced MAP colonization specifically in the ileum and ileocecal valve. This partially protective immune response was associated with an increased frequency of CD4+ and CD8+ T cells with a pro-inflammatory phenotype (IFNγ+/TNFα+) in vaccinated animals. Moreover, re-stimulated PBMCs from vaccinated animals showed increased expression of IFNγ, IP-10, IL-2, and IL-17 at 10- and 12-weeks post challenge. Furthermore, immunophenotyping of blood leukocytes revealed that vaccinated calves had increased levels of T cells expressing cell-surface markers consistent with long-term central memory. Overall, our findings provide new insights into the development and immunogenicity of a modified live MAP vaccine against bovine JD, demonstrating oral vaccination can stimulate host immune responses that can be protective against enteric MAP infection.

Identification of essential genes in Mycobacterium avium subsp. paratuberculosis genome for persistence in dairy calves.

To cause disease Mycobacterium avium subsp. paratuberculosis needs to enter mammalian cells, arrest phagosomal maturation and manipulate the host immune system. The genetic basis of the bacterial capacity to achieve these outcomes remains largely unknown. Identifying these genes would allow us to gain a deeper understanding of MAP's pathogenesis and potentially develop a live attenuated Johne's disease vaccine by knocking out these genes. MAP genes demonstrated to be essential for colonization in the natural host, ruminants, are unknown. Genome-wide transposon mutagenesis and high-throughput sequencing were combined to evaluate the essentiality of each coding region in the bacterial genome to survive in dairy calves. A saturated library of 3,852 MAP Tn mutants, with insertions in 56% of TA sites, interrupting 88% of genes, was created using a MycoMarT7 phagemid containing a mariner transposon. Six calves were inoculated with a high dose of a library of MAP mutants, 1011 CFUs, (input) at 2 weeks of age. Following 2 months of incubation, MAP cells were isolated from the ileum, jejunum, and their associated lymph nodes of calves, resulting in approximately 100,000 colonies grown on solid media across 6 animals (output). Targeted next-generation sequencing was used to identify the disrupted genes in all the mutants in the input pool and the output pool recovered from the tissues to identify in vivo essential genes. Statistical analysis for the determination of essential genes was performed by a Hidden Markov Model (HMM), categorizing genes into essential genes that are devoid of insertions and growth-defect genes whose disruption impairs the growth of the organism. Sequence analysis identified 430 in vivo essential and 260 in vivo growth-defect genes. Gene ontology enrichment analysis of the in vivo essential and growth-defect genes with the highest reduction in the tissues revealed a high representation of genes involved in metabolism and respiration, cell wall and cell processing, virulence, and information pathway processes. This study has systematically identified essential genes for the growth and persistence of MAP in the natural host body.