Elsevier

Vaccine

Volume 39, Issue 23, 27 May 2021, Pages 3131-3140
Vaccine

Reduced tissue colonization of Mycobacterium avium subsp. paratuberculosis in neonatal calves vaccinated with a cocktail of recombinant proteins

https://doi.org/10.1016/j.vaccine.2021.04.051Get rights and content

Highlights

  • Vaccination reduced colonization of intestinal tissues in calves.

  • Higher dose of the vaccine provided further protection.

  • Vaccination increased numbers of CD4+ T cells and B cells.

  • Vaccination increased expression of IFN-γ, IL-12 and IL-17.

Abstract

An increasing prevalence of paratuberculosis supports the need for new efficacious vaccines as an essential management tool. Two separate studies were performed in neonatal calves to evaluate the effectiveness of pooled recombinant Mycobacterium avium subsp. paratuberculosis (MAP) proteins (MAP1087, MAP1204, MAP1272c, MAP2077c) as a potential vaccine. In the first study vaccinated calves were immunized with 400 µg protein cocktail per dose, whereas the second study compared doses of 400 µg and 800 µg of protein cocktail, followed by challenge with live MAP for both vaccinated and nonvaccinated control calves 28 days post-vaccination. At the end of 12 months, tissue colonization with MAP was significantly reduced for the vaccinated calves compared to control animals. A higher dose of vaccine improved protection, with further reductions of MAP burden. Antigen-specific IFN-γ responses and serum antibody responses were similar regardless of vaccination, indicating exposure to MAP invoked conventional host immune responses. Host immunity differed due to vaccination, resulting in increased percentages of CD4+ T cells and B cells after stimulation of PBMCs with antigen. Interestingly, gene expression in PBMCs was similar for both control and vaccinated calves except for significant increases in IFN-γ, IL-12, and IL-17 expression observed in vaccinated calves. Vaccination with a cocktail of immunogenic recombinant MAP proteins was efficacious in reducing the level of infection and fecal shedding of neonatal calves and may be a potential tool for curtailing the spread of Johne’s disease.

Introduction

Johne’s disease, a chronic enteritis associated with ruminants, is caused by the intracellular pathogen, Mycobacterium avium subsp. paratuberculosis (MAP). The incidence of paratuberculosis is worldwide, with prevalence in large and small ruminant species, particularly domestic livestock species such as cattle, sheep, and goats. In the US, the dairy industry has the highest incidence of paratuberculosis, with infection observed in over 90% of dairy herds [1]. The conundrum with paratuberculosis control lies in the ability to detect the presence of MAP infection within a herd, with further difficulty in assessing infection within an individual animal. Animals generally become infected as neonates due to ingestion of MAP in colostrum and milk and exposure to MAP in manure from infected dams [2]. The primary infection is followed by a protracted asymptomatic period that is mainly dependent upon measures of cell-mediated immunity such as the antigen-specific bovine interferon-gamma assay for detection [3], [4], a test that is not yet approved in the US or abroad for detection of Johne’s disease. Most herd screening is done by either fecal detection of MAP by culture or PCR, or by serum ELISA for MAP antibodies [5]. Both of these methods are fairly rapid and cost-effective, but their efficacy is wholly dependent upon the status of infection within the host. Although asymptomatic hosts can shed MAP intermittently and at low levels in their feces and may also have a low level of serum antibody, the success of detection is minimal. Therefore, animals may be well into advanced disease, shedding MAP into the milk and feces and transmitting it to herd mates before infection is properly diagnosed.

Vaccination has long been proven to be an effective intervention method to prevent infection in livestock. Administering a vaccine to neonates can effectively halt the cycle of disease, or at a minimum reduce the incidence. For Johne’s disease, a vaccination program may be tantamount to allaying the spread of infection throughout the herd. The three major commercial vaccines for Johne’s disease are Mycopar (Boehringer Ingelheim), Gudair (CZ Veterinaria), and Silirium (Pfizer). Other vaccines for paratuberculosis are available, but accessibility may differ depending upon licensing agreements within the country of origin [6]. Current research on development of new vaccines encompasses live attenuated vaccines [7], [8], [9], as well as a more directed approach utilizing specific MAP proteins as the antigen [10]. In the US, Mycopar is the only vaccine licensed for use and it is restricted to cattle only [11]. Administration of Mycopar has been shown to be protective by reducing fecal shedding of MAP and clinical signs of disease, but there are also shortcomings associated with the use of this vaccine. The Mycopar vaccine is noted for severe granulomatous lesions at the site of injection, a disadvantage for meat livestock. Another major disadvantage of most commercial vaccines comprised of the whole cell organism, either killed or live attenuated, is interference with sero-diagnostics of paratuberculosis and bovine tuberculosis. The development of new vaccines is critical to eliminate the aforementioned tissue injury and serologic interference, whilst adequately reducing tissue colonization of the host and fecal shedding of MAP.

In the present study, the selection of 4 recombinant proteins of MAP (MAP1087, MAP1204, MAP1272c, MAP2077c) combined into a protein cocktail was evaluated as a potential vaccine for paratuberculosis. These four proteins showed promising results when administered in mice, as evidenced by reduced colonization of tissues [12]. Furthermore, these proteins have been shown to be strong antigens in cattle [13], [14], [15]. In two separate studies reported here, calves were vaccinated with the protein cocktail and then challenged with live MAP to ascertain protection. Protection was assessed by tissue colonization and fecal shedding, with concurrent assessment of host immune responses due to vaccination.

Section snippets

Animals

Neonatal dairy calves were obtained from a local herd in Iowa at less than one week of age. Preliminary screening was performed to confirm negative status of dams prior to acceptance of neonates. An acclimation period of one week was allowed prior to initiation of the study. Standard commercial milk replacer (Land O’Lakes, Shoreview, MN) was fed to calves twice per day at 10 hr intervals during the acclimation and vaccination periods. After challenge with live MAP, animals were weaned onto calf

Infection status of calves

Measures of antigen-specific whole blood IFN-γ and MAP-specific antibody were performed to assess robustness of diagnostic immune responses to live MAP, with and without vaccination. Antigen-specific IFN-γ responses were observed by day 90 post-inoculation, significantly (P < 0.05) greater than previous sampling dates, with further increases throughout the study (Fig. 1A). Calves had robust IFN-γ responses to both ConA and PWM on all sampling dates (data not shown). Vaccination of calves did

Discussion

Vaccination is a management tool that is widely used internationally as a control measure for paratuberculosis, but less so in the US. The only vaccine that was approved for use within the US is Mycopar (no longer for sale), a heat-killed whole cell organism-based vaccine in oil. Although Mycopar vaccination results in reduced fecal shedding by infected cattle and lessens the incidence of advanced clinical disease, it does not prevent infection [11]. These results conform with those of other

Funding

This work was supported by the US Department of Agriculture, Agricultural Research Service Project (CRIS#5030–32000-221). USDA is an equal opportunity employer.

CRediT authorship contribution statement

J.R. Stabel: Investigation, Data curation, Formal analysis, Writing - original draft, Writing - review & editing. J.P. Bannantine: Investigation, Resources, Formal analysis, Writing - review & editing.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

We thank Amy Turner and Margaret Walker for their excellent technical support and Brian Conrad and his animal care staff at the NADC for excellent animal care and handling.

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