Original Article J Vet Sci 2017, 18(S1), 343-349ㆍhttps://doi.org/10.4142/jvs.2017.18.S1.343 JVS Potential biomarkers as an indicator of vertical transmission of Johne’s disease in a Korean native cattle farm Hong-Tae Park1,†, Hyun-Eui Park1,†, Yong-Il Cho2, Eui-Hyung Kim3, Myunghwan Jung1, Seung Won Shin1, Su-Hyung Lee4, Dae-Yong Kim4, Han Sang Yoo1,* Departments of 1Infectious Diseases and 4Veterinary Pathology, College of Veterinary Medicine, Seoul National University, Seoul 08826, Korea 2Department of Animal Science & Technology, Suncheon National University, Suncheon 57922, Korea 3National Institute of Animal Science, Rural Development Administration, Pyeongchang 25340, Korea Paratuberculosis (PTB) is caused by Mycobacterium avium subsp. paratuberculosis (MAP) and is one of the most widespread and economically important diseases in cattle. After birth, calves are raised with natural breast feeding without separation from their mothers in most Korean native cattle (Hanwoo breed) farms. Vertical transmission of PTB has been reported, but the exact PTB infection route has not been revealed in Hanwoo farms. Calves of MAP seropositive dams were tested for MAP presence and MAP antibodies in feces and tissues. MAP was detected in calf tissues by using polymerase chain reaction. Expressions of genes reported to be prognostic biomarkers of MAP infection changed in both calves and cows (p ＜ 0.05). Expression of two genes (HGF and SERPINE1) were significantly decreased in MAP-infected cattle and their offspring (p ＜ 0.01). The results suggest that biomarker gene expression profiles can be useful in detecting early stage MAP infection. Based on the results, complete eradication of MAP may be possible if accurate diagnostic methods to detect infected calves are added to the current PTB eradication strategy, which, because infected individuals are likely to develop into fecal MAP shedders at any time, includes isolation of new born calves and feeding sterilized colostrum. Keywords: Korean native cattle, Mycobacterium avium subsp. paratuberculosis, biomarker, subclinical infection, vertical transmission Introduction through their feces [20]. Although this strategy has reduced the prevalence of MAP infection, it has not generally eliminated Johne’s disease, also called paratuberculosis (PTB), is caused MAP infection. Moreover, general diagnostic methods such as by Mycobacterium avium subsp. paratuberculosis (MAP) and enzyme-linked immunosorbent assay (ELISA), fecal is one of the most widespread and economically important polymerase chain reaction (PCR), and fecal culture often fail to diseases in cattle [23]. Because MAP is a slow-growing bacteria identify the subclinical stage of infection due to variations in with a very long incubation period, the clinical signs of PTB PTB progression and MAP shedding, resulting in intermittent may not be observed for more than two years after initial detection [21]. infection [32]. Therefore, MAP-infected animals usually have a Vertical transmission is another cause of difficulty in the prolonged subclinical stage of infection. However, such eradication of PTB. Vertical transmission is the acquisition of animals can be a source of infection because they can infection by new born calves from dams and can be divided into contaminate the surrounding environment by shedding MAP prenatal and postnatal infections. MAP is reported to be capable bacteria through their feces during their subclinical stage of of producing a prenatal infection with evidence supporting that infection. Animals can be easily infected by ingestion of capability including the presence of MAP in placenta or sex MAP-contaminated environmental components such as feed, organs [33]. Postnatal infection occurs mainly by exposure to water, and bedding [33]. Therefore, the primary PTB control bacteria present in colostrum or fecal material. In the calf, the strategy is reduction of the number of cattle shedding MAP highest susceptibility to MAP occurs during the first day of life Received 25 May 2016, Revised 19 Jan. 2017, Accepted 7 Feb. 2017 pISSN 1229-845X *Corresponding author: Tel: +82-2-880-1263; Fax: +82-2-874-2738; E-mail: [email protected] eISSN 1976-555X †The first two authors contributed equally to this work. Journal of Veterinary Scienceㆍⓒ 2017 The Korean Society of Veterinary Science. All Rights Reserved. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 344 Hong-Tae Park et al. (within 24 h after birth) [32,40]. Therefore, a key element of PCR testing performed 4 times within a 6-month interval. Age PTB eradication strategies, so far, has been periodic MAP of the Hanwoo cows varied from 2 to 10 years and their parity screening of farms, isolation of calves from dams right after ranged between 0 and 7. None of the seropositive cows parturition, and the provision of sterilized colostrum [30,33]. exhibited any of the typical clinical signs of MAP infection. The However, in the Korean native cattle (Hanwoo breed) calves were raised at the same farm with natural breast feeding industry, most farms use a conventional breeding system, which from their mothers until the age of 4 to 5 months. involves feeding calves through natural breast milking without dam separation for a period of 3 to 4 months. Thus, the calves Analysis of infection status live in same herd as their mother until weaning. Many previous Infection status of cows and calves were analyzed by using studies have warned that rearing the calf with the dam serves as commercial ELISA and fecal PCR assays even though the cows an intervening factor in infection [3,26,30,36]. Current rearing were identified as seropositive. In addition, detection of MAP systems have had these problems pointed out in previous was carried out in mesenteric lymph node, ileum, and Peyer’s studies; regardless, there is a need to investigate the risk of patch of calves by using PCR and acid-fast staining. For fecal vertical transmission in the current Korean Hanwoo rearing PCR, fecal samples were collected individually from rectum. system. In addition, studies on the transmission of MAP Collected samples were subjected to DNA extraction carried infections in Korean Hanwoo farms have not been conducted in out by using the mGITC/SC method [25]. For downstream Korea. analysis, IS900 real-time PCR and ISMAP02 nested PCR were A biomarker can be used as an indicator of normal biological conducted to identify specific genomic materials of MAP. Both states, pathogenic conditions, or therapeutic responses to of those PCR methods were performed by using previously treatment [31]. In that regard, biomarkers have been used for the described methods [16,27], with a slight modification in the diagnosis of diseases such as cancers, metabolic diseases, nested PCR. The PCR reaction mixture for nested PCR was set autoimmune diseases, and infectious diseases [10,15]. In up in a volume of 20 L, using 1 L of template DNA, particular, host biomarkers such as transcripts, microRNA nuclease-free water, 2 L of i-Taq 10× PCR buffer (Intron (miRNA), and metabolites have been proposed as diagnostic Biotechnology, Korea), 2.5 mM MgCl2, 0.25 mM indicators of chronic infectious diseases in humans and animals deoxyribonucleotide triphosphates, 500 nM of each primer, and [9,11]. In recent years, transcriptomics and proteomics have 2.5 U i-Taq DNA polymerase. Samples were run according to been used to analyze the expression of genes and proteins in the following protocol: initial denaturation at 94oC for 5 min, 35 attempts to identify biomarkers of early stage MAP infection cycles of 94oC for 15 sec, 58oC for 15 sec, and 72oC for 20 sec, [13,18,19]. In our previous microarray method-based studies of followed by final extension at 72oC for 7 min. For the second cells and animal models, several biomarkers for diagnosis of step of the nested PCR, 1 L of amplicon was used as the early stage of PTB were proposed [24,28,29]. template and the reaction run according to the same conditions In this study, we used several diagnostic tools to confirm the as the first step PCR, but with the number of cycles reduced MAP infection status of calves immediately after weaning in from 35 to 30. Fresh tissues (mesenteric lymph node, Peyer’s order to determine the degree of infection transmitted to calves patch, and ileum) were obtained during necropsy of the eight born from dams that were positive for MAP in the conventional calves. One gram of each tissue sample was prepared for DNA Korean Hanwoo rearing system. To that end, we analyzed extraction. The samples were homogenized by mortar and differences in expression levels of several biomarkers selected pestle and, except for large particles, suspended in 1× phosphate from our previous study to identify whether MAP-infected buffered saline. Suspended samples were then vortexed and calves could be successfully diagnosed. centrifuged at 13,000 × g for 2 min. Pellets were re-suspended in L6 lysis buffer followed by vortexing and incubation in a heat Materials and Methods block at 95oC for 15 min. The remaining steps were the same as described in the mGITC/SC method [25]. Tissue DNA was then Animals and farm analyzed by performing IS900 real-time PCR and ISMAP02 About 1,000 Hanwoo cattle were raised in a Korean native nested PCR. Histopathological examination and acid-fast cattle farm located in a mountainous area at an approximate 800 staining were performed as previously described [28]. m altitude. The cattle were put out to pasture except during the winter season. Fifteen Hanwoo cows previously diagnosed as Analysis of gene expression of prognostic biomarkers positive for PTB by using a commercial ELISA kit (IDEXX Seven genes identified as prognostic biomarkers for MAP Laboratories, USA) and 8 calves borne from them were infection in previous research [5,24,28,29] were selected for included in the study (Table 1).