Creating a Marked Mycobacterium avium subsp. paratuberculosis Vaccine Strain and Detecting Marker-Specific Immune Responses in Calves

Luo, Yi Yi

doi:10.11575/prism/31814

Cited by 2 publications

(4 citation statements)

References 164 publications

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“…Bacteria growth conditions were previously explained ( Luo, 2018 ). Shortly, the Escherichia coli strains DH5α (New England BioLabs (NEB)), HB101 (Bio-Rad, Hercules, CA, USA) and ClearColi® BL21 (Lucigen, Middleton, WI, USA) and fast-growing mycobacteria Mycobacterium smegmatis mc ( Pavlik et al., 2000 )155 (ATCC, Manassas, VA, USA), were grown in Luria-Bertani (LB) Miller broth (Invitrogen, Carlsbad, CA, USA) at 37°C.…”

Section: Methodsmentioning

confidence: 99%

“…Cosmid pYUB854 (kindly supplied by the Jacobs’ lab, Albert Einstein College of Medicine) was employed to generate the allelic exchange substrates (AESs), as previously described ( Luo, 2018 ). This cosmid possess a selectable resistance cassette (resolvase-hygromycin-resolvase) surrounded by multiple cloning sites.…”

Section: Methodsmentioning

confidence: 99%

“…Temperature-sensitive shuttle phasmid phAE159 (45 kb), which contain an ampicillin resistance cassette and replicates as a lytic phage at 30°C, was employed to generate the specialized transducing mycobacteriophage ( Luo, 2018 ). The shuttle phasmid and allelic exchange substrates DNA were concentrated three times by lypholization, resulting in concentration of 150 ng/μl and 500 ng/μl, respectively.…”

Section: Methodsmentioning

confidence: 99%

“…Before proceeding with in vitro and in vivo experiments, the gene knockouts were confirmed. by screening for homologous recombination using PCR, as previously explained ( Luo, 2018 ). The old school boiling method was employed to extract MAP genomic DNA as follows: single colonies from the plate were collected and dissolved in 800 μl pure ethanol and spun, and the resulting pellet was washed twice with 900 μl sterile Dulbecco’s Phosphate Buffered Saline (DPBS; Gibco), then simmer in 100 μl of sterile UltraPure DNase/RNase-Free Distilled Water (Gibco) for 30 minutes.…”

Section: Methodsmentioning

confidence: 99%

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

Eshraghisamani

Arrazuria

Luo

et al. 2023

Front. Cell. Infect. Microbiol.

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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.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

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

Eshraghisamani

Arrazuria

Luo

et al. 2023

Front. Cell. Infect. Microbiol.

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Oral vaccination stimulates neutrophil functionality and exerts protection in a Mycobacterium avium subsp. paratuberculosis infection model

et al. 2021

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Mycobacterium avium subsp. paratuberculosis (Map) causes paratuberculosis (PTB), a granulomatous enteritis in ruminants that exerts high economic impact on the dairy industry worldwide. Current vaccines have shown to be cost-effective against Map and in some cases confer beneficial non-specific effects against other pathogens suggesting the existence of trained immunity. Although Map infection is mainly transmitted by the fecal-oral route, oral vaccination has not been deeply studied. Therefore, the aim of this study was to compare the oral route with a set of mycobacterial and non-mycobacterial vaccines with a subcutaneously administered commercially available vaccine. Training effects on polymorphonuclear neutrophils (PMNs) and homologous and heterologous in vivo protection against Map were investigated in the rabbit infection model. Oral vaccination with inactivated or live vaccines was able to activate mucosal immunity as seen by elevation of serum IgA and the expression of IL4 in peripheral blood mononuclear cells (PBMCs). In addition, peripheral PMN phagocytosis against Map was enhanced by vaccination and extracellular trap release against Map and non-related pathogens was modified by both, vaccination and Map-challenge, indicating trained immunity. Finally, PBMCs from vaccinated animals stimulated in vitro with Map antigens showed a rapid innate activation cytokine profile. In conclusion, our data show that oral vaccination against PTB can stimulate neutrophil activity and both innate and adaptive immune responses that correlate with protection.

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Creating a Marked Mycobacterium avium subsp. paratuberculosis Vaccine Strain and Detecting Marker-Specific Immune Responses in Calves

Cited by 2 publications

References 164 publications

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

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

Oral vaccination stimulates neutrophil functionality and exerts protection in a Mycobacterium avium subsp. paratuberculosis infection model

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