Establishment a real-time reverse transcription PCR based on host biomarkers for the detection of the subclinical cases of Mycobacterium avium subsp. paratuberculosis

Abstract

Bovine paratuberculosis (PTB) is a chronic enteric inflammatory disease of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP) that causes large economic losses in the dairy industry. Spread of PTB is mainly provoked by a long subclinical stage during which MAP is shed into the environment with feces; accordingly, detection of subclinical animals is very important to its control. However, current diagnostic methods are not suitable for detection of subclinical animals. Therefore, the current study was conducted to develop a diagnostic method for analysis of the expression of genes of prognostic potential biomarker candidates in the whole blood of cattle naturally infected with MAP. Real-time PCR with nine potential biomarker candidates was developed for the diagnosis of MAP subclinical infection. Animals were divided into four groups based on fecal MAP PCR and serum ELISA. Eight genes (Timp1, Hp, Serpine1, Tfrc, Mmp9, Defb1, Defb10, and S100a8) were up-regulated in MAP-infected cattle (p <0.05). Moreover, ROC analysis revealed that eight genes (Timp1, Hp, Serpine1, Tfrc, Mmp9, Defb1, Defb10, and S100a8) showed fair diagnostic performance (AUC≥0.8). Four biomarkers (Timp1, S100a8, Defb1, and Defb10) showed the highest diagnostic accuracy in the PCR positive and ELISA negative group (PN group) and three biomarkers (Tfrc, Hp, and Serpine1) showed the highest diagnostic accuracy in the PCR negative and ELISA positive group (NP group). Moreover, three biomarkers (S100a8, Hp, and Defb10) were considered the most reliable for the PCR positive and ELISA positive group (PP group). Taken together, our data suggest that real-time PCR based on eight biomarkers (Timp1, Hp, Serpine1, Tfrc, Mmp9, Defb1, Defb10, and S100a8) might be useful for diagnosis of JD, including subclinical stage cases.

Mycobacterium avium Subspecies paratuberculosis DNA and Antibodies in Dairy Goat Colostrum and Milk

“…The transmission of MAP from dam to offspring by horizontal as well as vertical routes in MAP non-vaccinated cattle (major role: [36][37][38], minor role: [39]), goats, and sheep [31] has been researched in a number of studies. A meta-analysis in cattle quantifying age-and dose-dependence of early and late shedding of MAP indicated that challenging older calves or using multiple-exposure experimental systems resulted in a smaller proportion and shorter duration of early shedding as well as slower transition to late shedding from latent compartments [40].…”

Gene Expression Profiles of Th1-type Chemokines in Whole Blood of Mycobacterium avium subsp. paratuberculosis-Infected Cattle

“…Th1 response decreases as the disease progresses, but the antibody response (Th2 response) to MAP increases (13). Thus, since the Th1 / Th2 shift is related to disease progression and bacterial excretion, many studies have determined the MAP infection stage based on detection of MAP and MAP-specific antibody in specimen such as feces, milk, and sera (12,14,15). DNA-detected with ELISA-negative, and was expected to be detectable at the early state of MAP infection.…”

Biomarkers for Detecting Resilience against Mycobacterial Disease in Animals

“…Among the differentially regulated genes with potential as diagnostic biomarkers in mycobacterial infections are Tfrc, which encodes the transferrin receptor, and LTF, which regulates lactoferrin; they are often attributed to the pathogen's metabolism of host iron via the action of mycobactins (74)(75)(76). Similarly, S100a8 and S100a9 are differentially regulated and have been proposed as biomarkers for comparable inflammatory bowel diseases (75,76).…”

“…This response is thought to be a result of the host's immune system limiting the harmful immunopathology of MAP infection. Matrix metalloproteinase 9 (MMP9) and its inhibitor TIMP1, are both upregulated during paratuberculosis and TB and are documented as consistently up-regulated genes in TB (74,75,79). Two β-defensin genes have also recently been shown to be up-regulated in MAP, Defb1 and Defb10, indicating that their antimicrobial and immunomodulatory role may be indicative of host responses to bacterial infection (74).…”

“…Matrix metalloproteinase 9 (MMP9) and its inhibitor TIMP1, are both upregulated during paratuberculosis and TB and are documented as consistently up-regulated genes in TB (74,75,79). Two β-defensin genes have also recently been shown to be up-regulated in MAP, Defb1 and Defb10, indicating that their antimicrobial and immunomodulatory role may be indicative of host responses to bacterial infection (74). Along with this gene subset, Th1 chemokine genes such as CCL4, CCL5, CXCL9, CXCL10 and genes related to metabolism including IGF1 and TCF7L2, are up and down regulated respectively in paratuberculosis (76).…”