Infectious diseases caused by pathogens including viruses, bacteria, fungi, and parasites are ranked as the second leading cause of death worldwide by the World Health Organization. Despite tremendous improvements in global public health since 1950, a number of challenges remain to either prevent or eradicate infectious diseases. Many pathogens can cause acute infections that are effectively cleared by the host immunity, but a subcategory of these pathogens called “intracellular pathogens” can establish persistent and sometimes lifelong infections. Several of these intracellular pathogens manage to evade the host immune monitoring and cause disease by replicating inside the host cells. These pathogens have evolved diverse immune escape strategies and overcome immune responses by residing and multiplying inside host immune cells, primarily macrophages. While these intracellular pathogens that cause persistent infections are phylogenetically diverse and engage in diverse immune evasion and persistence strategies, they share common pathogen type-specific mechanisms during host-pathogen interaction inside host cells. Likewise, the host immune system is also equipped with a diverse range of effector functions to fight against the establishment of pathogen persistence and subsequent host damage. This article provides an overview of the immune effector functions used by the host to counter pathogens and various persistence strategies used by intracellular pathogens to counter host immunity, which enables their extended period of colonization in the host. The improved understanding of persistent intracellular pathogen-derived infections will contribute to develop improved disease diagnostics, therapeutics, and prophylactics.

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The quantitative detection of viable pathogen load is an important tool in determining the degree of infection in animals and contamination of foodstuffs. Current conventional culture methods are limited in their ability to determine these levels in Mycobacterium avium subspecies paratuberculosis (MAP) due to slow growth, clumping and low recoverability issues. The principle goal of this study was to evaluate a novel culturing process (TiKa) with unique ability to stimulate MAP growth from low sample loads and dilutions. We demonstrate it was able to stimulate a mean 29-fold increase in recoverability and an improved sensitivity of up to three logs when compared with conventional culture. Using TiKa culture, MAP clumping was minimal and produced visible colonies in half the time required by standard culture methods. Parallel quantitative evaluation of the TiKa culture approach and qPCR on MAP loads in tissue and gut mucosal samples from a MAP vaccine-challenge study, showed good correlations between colony counts (cfu) and qPCR derived genome equivalents (Geq) over a large range of loads with a 30% greater sensitivity for TiKa culture approach at low loads (two logs). Furthermore, the relative fold changes in Geq and cfu from the TiKa culture approach suggests that non-mucosal tissue loads from MAP infected animals contained a reduced proportion of non-viable MAP (mean 19-fold) which was reduced significantly further (mean 190-fold) in vaccinated “reactor” calves. This study shows TiKa culture equates well with qPCR and provides important evidence that accuracy in estimating viable MAP load using DNA tests alone may vary significantly between samples of mucosal and lymphatic origin.

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This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.Page 1 Relatively few of the described antigens were evaluated for their use in CMI based diagnostic 30 assays and so far, no obvious candidate has been identified for this application. Most of the novel 31 diagnostic candidates were evaluated in few animals and it is recommended that an appropriate 32 sample size is included for evaluation of antigen candidates in future studies. 33 34