Molecular Detection of Mycobacterium avium paratuberculosis by Large Sequence Polymorphisms (LSPs)

Johne’s disease (JD), a chronic granulomatous enteritis that affects ruminants worldwide and has a severe negative impact on productivity and results in major economic loss, is caused by Mycobacterium avium subsp. paratuberculosis (MAP). It is an intestinal disease affecting both domestic and wild ruminants (Singh et al., 2014). It is contagious, persistent and frequently fatal. Clinically affected animals exhibit wasting and emaciation symptoms. The most typical route for M. paratuberculosis transmission is through the fecal-oral pathway. The quantity of organisms in the exposure and the animal’s age has an effect on the intensity and rate at which the disease develops. When substantial faecal shedding is observed in late stages of disease, other mechanisms of transmission, such as transplacental and direct excretion in milk and colostrum, should not be disregarded and should be of special concern (Roller et al., 2020). PCR techniques that target the IS900 insertion element are the most common approach for MAP detection in faecal samples. This method is frequently used as a diagnostic marker for M. avium subsp. paratuberculosis. With a frequency of 14 to 18 copies per genome seen in the genomes of M. avium subsp. paratuberculosis, multicopy insertion elements known as IS900 elements offer increased sensitivity for their detection (Bull et al., 2000).
     
Numerous polymorphic genomic regions have been identified through genome sequencing research that may be specific to the diagnosis of M. avium subsp. paratuberculosis known as Large Sequence Polymorphisms (LSPs). These Large Sequence Polymorphisms (LSPs), which are regions that are specific to M. avium subsp. paratuberculosis strain K10 that have been found so far, range in size from 2.9 to 66 kb and can be employed in nucleic acid-based diagnostic assays. A 38-kb fragment is defined as a M. avium subsp. paratuberculosis-specific putative pathogenicity island and contains a 19-kb sequence dubbed LSPP 12 as well as a 98-kb stretch of the genome that includes elements called LSPP 14 and LSPP 15 (Stratmann et al., 2004) and these can be used in diagnosis of MAP.

The experiment was conducted in year 2021-2022 in department of veterinary microbiology, GADVASU, Ludhiana. Fecal samples (n=218) were taken from cattle (n=180) and buffaloes (n=38) from organized dairy farms in the state of Punjab that had a history of or occurrence of cases of chronic intermittent diarrhea. Following the collection of faecal samples, all of the samples were stained using the Ziehl Neelsen (acid fast) staining method for microscopic analysis. The samples were categorized positive or negative after staining based on the number of bacilli per High power (HP) field on the smears: 1+ (1-5 bacilli per HP field), 2+ (6–10 bacilli per HP field), 3+ (11-15 bacilli per HP field) and 4+ (>15 bacilli per HP field).
 
Isolation of MAP from fecal samples
 
After the initial screening of fecal samples with Ziehl Neelsen (acid fast) staining, the highly positive acid-fast bacilli (AFB) positive fecal samples were treated with 0.95% Hexadecyl pyridinium chloride (HPC) for decontamination and then were inoculated on Middlebrook 7H10 media slants supplemented with mycobactin J (1 μg/ml) (OIE Terrestrial Manual, 2014). After inoculation, the slants were incubated at 37°C and were periodically observed for a period of 6 months (Quinn and Carter, 1994). The observed growth if any, was stained with acid fast stain and DNA was extracted from the samples as well as isolates using a commercially available DNA extraction kit (Qiagen).
 
Polymerase chain reaction
 
Large sequence polymorphism (LSP)-targeting primer sets, including LSP^P 2 (MAP0284), LSP^P 4 (MAP0865), LSP^P 11 (MAP2154), LSP^P 12 (MAP2182c), LSP^P 12 (MAP2188c), LSP^P 15 (MAP3774), LSP^P 16 (MAP3815) (Table 1) and IS900 sequence (Table 2), were used to amplify the extracted DNA from faecal samples (n=218) (Semret et al., 2005 and Vary et al., 1990). Each PCR experiment employed a 25 µl volume that contained 12.5 µl of GoTaq® Green Master Mix (2X), 1 µl of forward primers (25 pmol), 1 µl of reverse primers (25 pmol), 8 µl of DNA template and 2.5 µl of nuclease-free water. Along with all of the test samples, a positive control Mycobacterium avium subsp. paratuberculosis (K10) (IMTECH, Chandigarh) DNA was also amplified.




     
LSP amplification started with a denaturation stage at 94°C for 5 minutes, followed by 30 cycles of denaturation at 94°C for 30 seconds, annealing at 58°C for 45 seconds and extension at 72°C for 2 minutes (Semret et al., 2005). The cyclic conditions for IS900 amplification were as follows: an initial denaturation at 95°C for 5 minutes, 40 cycles of denaturation at 95°C for 45 seconds, primer annealing at 62°C for 45 seconds, extension at 72°C for 45 seconds and final extension at 72°C for 10 minutes were performed after (Vary et al., 1990). On a 1.5% agarose gel, the amplified PCR products were separated and the Gel Documentation System was used to display them (Alpha Innotech). PCR amplicons of 600 bp, 597 bp, 375 bp, 430 bp, 719 bp, 621 bp and 611 bp were considered positive for LSP^P 2 (MAP0284), LSP^P 4 (MAP0865), LSP^P 11 (MAP2154), LSP^P 12 (MAP2182c), LSP^P 12 (MAP2188c), LSP^P 15 (MAP3774), LSP^P 16 (MAP3815) and PCR amplicons of 229 bp were considered positive for IS900.

In this experiment a total of 218 fecal samples from cattle and buffaloes with a history or incidence of cases of chronic intermittent diarrhea were screened out of which 90 animals were detected positive by acid-fast staining and findings were interpreted based on the number of acid-fast bacilli per HP field. These 90 ZN staining positive fecal samples were subjected to isolation of MAP, growths were seen in two fecal samples after 16 weeks of incubation and the colonies were diffused greyish and off white. The isolates from these tubes were further subjected to acid fast staining which showed acid-fast bacilli under 100x oil emulsion.
 
Molecular detection of MAP by large sequence polymorphism (LSPs) and IS900 PCR
 
DNA extracted from fecal samples as well as isolates using QIAmp DNA Purification kit was amplified using different sets of primers targeting large sequence polymorphism (LSP^Ps) viz; LSP^P2 (MAP0284), LSP^P4 (MAP0865), LSP^P11 (MAP2154) LSP^P12 (MAP2182c), LSP^P12 (MAP2188c), LSP^P15 (MAP3774), LSP^P16 (MAP3815) and IS900 sequence (Semret et al., 2005 and Vary et al., 1990) with an amplicon size of 600, 597, 375, 430, 719, 621, 611 and 229 bp.  DNA extracted from the standard culture of MAP was used as positive control in all the PCR reactions and the primers were initially standardized using standard culture DNA (Fig 1). Out of 218 fecal samples tested, 7 samples were positive by both LSP PCR (Fig 2, 3, 4, 5, 6, 7 and 8) and IS900 PCR (Fig 10). DNA was also extracted from the isolates (n=2) were confirmed by PCR targeting Large Sequence Polymorphism (LSPs) (Fig 9) and IS900 (Fig 10).




















     
Various methods, such as fecal smears, fecal culture and PCR, can be used to diagnose paratuberculosis in feces samples. Goal of present study was to use culture and molecular approaches to detect Mycobacterium avium subsp. paratuberculosis (MAP). In the chronic stage of infection, MAP organisms are shed in feces, hence fecal samples can be examined to diagnose paratuberculosis. For the initial screening of MAP, Ziehl–Neelsen staining can be used (Rathnaiah et al., 2017). The number of acid-fast bacteria present in the sample determines the success of microscopic screening of feces (Ris et al., 1988). Although acid-fast staining is the simplest, fastest and most cost-effective way of diagnosis, its specificity and sensitivity are limited because it is difficult to distinguish between different members of the family Mycobacteriaceae and other acid-fast bacteria (Manning and Collins, 2001). Although acid-fast staining is of limited and benefits in the early stages of infection, it can be helpful in diagnosing the disease as it progresses. According to Whitlock et al., (2000), the majority of Johne’s disease-affected animals were moderate shedders. The “gold standard” for JD diagnosis is the isolation of MAP by culturing (OIE, 1996). The fact that MAP requires mycobactin J to grow in Middle brook media can be used to distinguish it from other acid-fast bacteria. Because, MAP grows slowly (12-16 weeks for colony formation on solid media), culture-based diagnosis takes a long period but it is considered more sensitive than most other detection procedures (Ellingson et al., 2000). But the decontamination technique has been found to be damaging to MAP growth. The decontamination procedures followed during the sample processing for isolation also decreases the number of viable mycobacteria (Whittington and Sergeant, 2001). Decontamination of samples with low concentrations of MAP may result in a negative culture result (Clark et al., 2008). Overgrowth of contaminating bacteria and fungus pose a problem, while culturing the samples. All of these variables make isolation of MAP colonies using culture techniques problematic, resulting in lower viable MAP recovery and, as a result, fewer culture positive animals. According to reports, fecal culture can only detect upto 15 to 25% of sub-clinically infected animals (Stabel, 1998). As a result, a highly sensitive and quick PCR-based test was employed to identify MAP in clinical samples. PCR IS900 insertion element is specific to MAP (Chaubey et al., 2016). With fecal samples, PCR tests are approximately 70% sensitive and 85% specific (Clark et al., 2008).
     
In a comparative genome study conducted by Semret et al., (2005) to detect the genomic differences between M. avium subsp. paratuberculosis and its close relative M. avium subsp. avium, two types of large sequence polymorphisms (LSPs): those present in the M. avium subsp. avium but missing in the M. avium subsp. paratuberculosis (LS) and those only present in the M. avium subsp. paratuberculosis (LS) were found. Distribution of 3 LS and 17 LS across a panel of 383 M. avium complex isolates was examined for their potential use for accurate diagnostic test development. Of the 17 LS, only 7 (LSP^P2 (MAP0284), LSP^P4 (MAP0865), LSP^P11(MAP2154) LSP^P12 (MAP2182c), LSP^P12 (MAP2188c), LSP^P15 (MAP3774), LSP^P16 (MAP3815) were seen highly specific (>98%) and in some cases highly sensitive (up to 95%) for M. avium subsp. paratuberculosis indicate the LSPs best suited for M. avium subsp. paratuberculosis diagnostics. Additionally, Paustian et al., (2005) discovered genetic differences between M. avium subsp. paratuberculosis and other M. avium complex organism and described seven regions unique to only M. avium subsp. paratuberculosis (LSP^P2 (MAP0284), LSP^P4 (MAP0865), LSP^P11(MAP2154) LSP^P12 (MAP2182c), LSP^P12 (MAP2188c), LSP^P15 (MAP3774), LSP^P16 (MAP3815). Sohal et al., (2009) conducted a study in which different markers such as IS900, LSPs, were used to characterize MAP ‘Bison type’ S5 and this study showed the polymorphic profile for LSP^P 4 was also seen in MAP ‘Bison type’ S5. LSP^P 2, LSP^P 4, LSP^P 12 and LSP^P 15 amplified showing their presence in MAP S5.
     
Early detection of paratuberculosis can be aided by molecular detection technologies. The development of PCR technologies has increased the specificity and sensitivity of laboratory diagnosis (Vary et al., 1990). The insertion IS900 is thought to be exclusive to Mycobacterium avium subsp. paratuberculosis and can be used for detection of MAP in fecal samples. Vary et al., (1990) also reported the presence of mycobacterial insertion sequence IS900 in multiple copies and the DNA probes that hybridize to this sequence were found to be highly specific for MAP and synthesized primers for IS900 DNA sequences. This resulted in highly specific direct detection of MAP DNA in feces from cattle with Johne’s disease. The results of bacterial culture and IS900-specific PCR were also compared by Sivakumar et al., (2005) to detect M. avium subsp. paratuberculosis (MAP) from the intestinal and mesenteric lymph node tissues of water buffaloes with paratuberculosis (JD) lesions and found PCR to be more sensitive than bacterial culture in detecting subclinical paratuberculosis. Efficacy of fecal culture and IS900 Polymerase chain reaction (PCR) assay was compared by Soumya et al., (2009) in 40 fecal samples of dairy cattle and found the PCR assay to be more sensitive 90% (36/40) than fecal culture 52.5% (21/40).

Detection of MAP in all the 7 fecal samples both by IS900 and by 7 LS indicates the detection potential of selected 7 LS (LSP^P2, LSP^P4, LSP^P11, LSP^P 12 (MAP2182c), LSP^P 12 (MAP2188c), LSP^P15, LSP^P16) in fecal samples of cattle and buffaloes suspected for JD. They can also have a potential use for the development of an accurate diagnostic test. 

The authors are thankful to Department of Biotechnology (DBT) for providing the necessary funds under the scheme “Mycobacterial diseases in animals network (MyDAN) Programme” and the Director of Research, GADVASU, Ludhiana for providing the necessary facilities.

None.