Detection of Mycobacterium avium subsp. paratuberculosis in Kerman Province’s Dairy Cows using Microbial Culture, PCR and Nested PCR Methods

Document Type : Genetics & breeding

Author

• Mahdi Soltani

Graduate University of Advanced Technology, Kerman

10.22067/ijasr.v10i2.65920

Abstract

Introduction Mycobacterium avium subsp. paratuberculosis (Map) is a very slow growing bacterium that solely infect the digestive tract and leads to Johne’s disease (JD) that is characterized by immedicable diarrhea. Considering innate resistance and different transmission modes of Map and its possible role in development or progression of some human diseases (mainly Crohn’s disease), strong zoonotic potential has been proposed for Map. Diagnosis of JD depends on isolation of viable Map (culture), tracking of its consequences on host immune system (ELISA) or amplification of Map genome (PCR). Fecal culture considered as gold standard for Map detection but obstacles like very slow growth rate of Map, decontamination issues and its high expenses led to development of alternatives like PCR based approaches to gain PCR advantages like high sensitivity, specificity and rapidity for diagnosis of Map. Since no published report was found on Map infection status in Kerman, the current study aimed to investigate the presence of Map in bovine feces samples using 3 different detection methods including culture, IS900 PCR and IS900 nested PCR. Also IS1311 PCR-REA was used for strain typing of Map isolates.
Materials and Methods 212 fecal samples were taken from 4 dairy cattle farms located in Kerman province, southeast Iran. The fecal fractions for culture and PCR were prepared separately to inhibit potential cross-contaminations. Per each sample, 300 µl of prepared inoculum was cultured on slope of Herrold’s egg yolk medium (HEYM) alone, and two HEYM + 2.0 mg/L of mycobactin J slopes. Inoculated slopes were incubated at 37 °C for 4 months and monitored at biweekly intervals. After extraction of DNA from fecal samples, PCR reaction performed in 25 µl volume and products were analyzed by electrophoresis on 2% agarose gels. The samples were considered as positive if 413 bp amplified band was present. To avoid false positive results, a REA approach using AlwI restriction enzyme was adopted. For IS900 nested PCR reaction, 3 ml from IS900 PCR products were used as template. Resultant products were screened as before. To evaluate the limit of detection of PCR reactions, the DNA extracted from confirmed MAP field strain culture was 10 fold serially diluted (1 µg – 1 fg) and the minimum detection level of PCR and nested PCR were observed. Strain typing of Map isolates was based on IS1311 PCR-REA.
Results and Discussion It takes 3-4 months of incubation for appearance of MAP positive colonies. Map was detected in 32/212 (15.1%) of cultured samples. All colonies were verified as MAP using IS900 PCR and REA with AlwI. 44 positive samples were detected by IS900 PCR (20.7%) while nested PCR was able to find 52 infected samples (24.5%). All isolates detected by PCR based methods were also verified as MAP by IS900 PCR-REA. According to expectations, nPCR offered higher sensitivity than conventional PCR. However, comparing the methods used in the present study showed that as a simple and cheap assay, nested PCR is inherently successful in amplification even with rare amount of starting template and has the better ability to detect the Map infection in fecal samples and can be used as a routine method in diagnostic processes. Although this study was the first attempt to access the Map infection status in Kerman province, as summarized in table 3, many studies were conducted all around Iran that can be compared with. Altogether, Map incidence in other central Iran provinces (Isfahan, Fars and Chaharmahal and Bakhtiari) were between 14.1 to 31.8 percent of tested populations based on examination of fecal samples. Considering that most of reported values came from clinically infected or suspected animals, the estimates obtained in this study showed the possibility that the Map incidence in Kerman province may be higher than other parts of central Iran, but it more investigations needed to create a realistic scheme of Map infection status in Kerman province. Remembering that a copy of genome was considered as equivalent to 5 fg of DNA, sensitivity analysis using serially diluted DNA preparations revealed that the minimum detection level were 1 pg (200 genomes) and 10 fg (2 genomes) for PCR and nested PCR, respectively. According to results, compared to nested PCR, conventional PCR is not sensitive enough for diagnostic tests on field samples. Analysis of fragments produced after IS1311 REA showed the presence of explicit patterns of four bands for all obtained isolates that is the indication of MAP C type. Obtained DNA sequences from the amplified IS1311 locus was exactly consistent with previously published sequence for MAP C type. S type was not detected in current study that was consistent with results of researches performed in Razavi Khorasan province. Failure to find S Map strain was not surprising, since its global prevalence in cow population is very low and hard to find. Also, there are few papers dealing with Map strain typing in Iran, so it is conceivable to find S Map type in Iran’s cattle population if more research conducted in this respect.
Conclusion Although paratuberculosis is a global neglected disease, the situation is worse in developing countries like Iran. Contrary to bovine tuberculosis, due to absence of a national program to control paratuberculosis infection in Iran’s dairy herds, no reliable data is available for arrangement of tools for efficient restriction of disease which leads to inadequate attention to JD and eventually receive the least priority to control. Unfortunately, paratuberculosis as an overlooked disease, is a severe hazard for cows’ health and also for economic activities related to dairy cattle industry. On the other hand, Map is still considered as an important suspicious zoonotic agent which cause serious health problems for humans. Meanwhile, considering aforementioned warnings, enactment of crucial health standards is vital to minimize the chance of infection transmission to non-infected populations and alleviating health problems and economic losses due to Map infection.

Materials and Methods 212 fecal samples were taken from 4 dairy cattle farms located in Kerman province, southeast Iran. The fecal fractions for culture and PCR were prepared separately to inhibit potential cross-contaminations. Per each sample, 300 µl of prepared inoculum was cultured on slope of Herrold’s egg yolk medium (HEYM) alone, and two HEYM + 2.0 mg/L of mycobactin J slopes. Inoculated slopes were incubated at 37 °C for 4 months and monitored at biweekly intervals. After extraction of DNA from fecal samples, PCR reaction performed in 25 µl volume and products were analyzed by electrophoresis on 2% agarose gels. The samples were considered as positive if 413 bp amplified band was present. To avoid false positive results, a REA approach using AlwI restriction enzyme was adopted. For IS900 nested PCR reaction, 3 ml from IS900 PCR products were used as template. Resultant products were screened as before. To evaluate the limit of detection of PCR reactions, the DNA extracted from confirmed MAP field strain culture was 10 fold serially diluted (1 µg – 1 fg) and the minimum detection level of PCR and nested PCR were observed. Strain typing of Map isolates was based on IS1311 PCR-REA.
Results and Discussion It takes 3-4 months of incubation for appearance of MAP positive colonies. Map was detected in 32/212 (15.1%) of cultured samples. All colonies were verified as MAP using IS900 PCR and REA with AlwI. 44 positive samples were detected by IS900 PCR (20.7%) while nested PCR was able to find 52 infected samples (24.5%). All isolates detected by PCR based methods were also verified as MAP by IS900 PCR-REA. According to expectations, nPCR offered higher sensitivity than conventional PCR. However, comparing the methods used in the present study showed that as a simple and cheap assay, nested PCR is inherently successful in amplification even with rare amount of starting template and has the better ability to detect the Map infection in fecal samples and can be used as a routine method in diagnostic processes. Although this study was the first attempt to access the Map infection status in Kerman province, as summarized in table 3, many studies were conducted all around Iran that can be compared with. Altogether, Map incidence in other central Iran provinces like Isfahan, Fars and Chaharmahal and Bakhtiari were between 14.1 to 31.8 percent of tested populations based on examination of fecal samples. Considering that most of reported values came from clinically infected or suspected animals, the estimates obtained in this study showed the possibility that the Map incidence in Kerman province may be higher than other parts of central Iran, but it more investigations needed to create a realistic scheme of Map infection status in Kerman province. Remembering that a copy of genome was considered as equivalent to 5 fg of DNA, sensitivity analysis using serially diluted DNA preparations revealed that the minimum detection level were 1 pg (200 genomes) and 10 fg (2 genomes) for PCR and nested PCR, respectively. According to results, compared to nested PCR, conventional PCR is not sensitive enough for diagnostic tests on field samples. Analysis of fragments produced after IS1311 REA showed the presence of explicit patterns of four bands for all obtained isolates that is the indication of MAP C type. Obtained DNA sequences from the amplified IS1311 locus was exactly consistent with previously published sequence for MAP C type. S type was not detected in current study that was consistent with results of researches performed in Razavi Khorasan province. Failure to find S Map strain was not surprising, since its global prevalence in cow population is very low and hard to find. Also, there are few papers dealing with Map strain typing in Iran, so it is conceivable to find S Map type in Iran’s cattle population if more research conducted in this respect.
Conclusion Although paratuberculosis is a global neglected disease, the situation is worse in developing countries like Iran. Contrary to bovine tuberculosis, due to absence of a national program to control paratuberculosis infection in Iran’s dairy herds, no reliable data is available for arrangement of tools for efficient restriction of disease which leads to inadequate attention to JD and eventually receive the least priority to control. Unfortunately, paratuberculosis as an overlooked disease, is a severe hazard for cows’ health and also for economic activities related to dairy cattle industry. On the other hand, Map is still considered as an important suspicious zoonotic agent which cause serious health problems for humans. Meanwhile, considering aforementioned warnings, enactment of crucial health standards is vital to minimize the chance of infection transmission to non-infected populations and alleviating health problems and economic losses due to Map infection.

Keywords

• Culture
• Detection
• Johne’s disease
• Mycobacterium avium subsp. Paratuberculosis
• Nested PCR
• PCR

20.1001.1.20083106.1397.10.2.9.4

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