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Molecular Characterization of Orientia tsutsugamushi in Domestic Rodents In Kolar Region, Karnataka



Gangannagari Vijay Kumar and Parvangada Madappa Beena
 
ABSTRACT
Background and Objective: Orientia tsutsugamushi (O. tsutsugamushi) is the causative agent of scrub typhus, which is an acute febrile illness seen in human beings. Rodents were implicated as the natural reservoirs for Orientia tsutsugamushi and they served as hosts for chigger mite. This study was undertaken to investigate the molecular prevalence and characterization of Orientia tsutsugamushi in domestic rodents in Kolar region. Methodology: Blood samples were collected from 177 rodents (138 Bandicota bengalensis, 16 Rattus rattus, 9 Bandicota indica, Rattus norvegicus and 2 Mus musculus). Conventional PCR, targeting the 56 kDa type specific gene was performed and DNA sequenced. Results: O. tsutsugamushi DNA was detected by conventional PCR in 10 (5.6 %) rodents. Two PCR purified samples were sequenced, phylogenetic analyses showed that one strain was closely related to Orientia chuto Dubai strain and another was closely related to Boryang strain. Conclusion: This suggests a high incidence of scrub typhus in this region. Further research on the characterization of O. tsutsugamushi from rodents as well as vectors throughout this district is needed to study the prevalence of O. tsutsugamushi in reservoir hosts.
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Gangannagari Vijay Kumar and Parvangada Madappa Beena, 2017. Molecular Characterization of Orientia tsutsugamushi in Domestic Rodents In Kolar Region, Karnataka. Asian Journal of Epidemiology, 10: 123-127.

DOI: 10.3923/aje.2017.123.127

URL: http://scialert.net/abstract/?doi=aje.2017.123.127
 
Received: September 22, 2017; Accepted: November 16, 2017; Published: November 30, 2017

INTRODUCTION

Orientia tsutsugamushi is the causative agent of scrub typhus, which is an acute febrile illness seen in human beings. Rodents were implicated as the natural reservoirs for O. tsutsugamushi and they serve as hosts for chigger mite which in turn transmits the bacteria to man following the bite1. Scrub typhus is endemic in the Asia-Pacific region and report an annual incidence of 1 million cases/year2. Mortality rates in untreated cases range from 0-30%3. Scrub typhus was also known to occur all over India and have been reported in states like Haryana4, Himachal pradesh5, Jammu and Kashmir6, Karnataka7, Assam8, Tamil Nadu3, Andhra Pradesh9, Kerala10 and Utharakand11. Antigenic variations among the strains of O. tsutsugamushi were reported and also interstrain variability associated with the virulence of the disease resulting in mild disease or fatal disease among humans as well as rodents12. Till the date more than 20 antigenic variants of O. tsutsugamushi were identified by immunological and molecular methods, which included previously identified prototype strains Karp, Gilliam and Kato13. 56 kDa type specific antigen is the one of the major immunogen of O. tsutsugamushi14 and used as a target gene in methods like genetic sequence analysis15. However, there were no studies regarding molecular characterization of O. tsutsugamushi in the rodents in India. This study was undertaken to investigate the molecular prevalence and characterization of Orientia tsutsugamushi in domestic rodents in Kolar region.

MATERIALS AND METHODS

The study was done in the Department of Microbiology, Sri Devaraj Urs Medical College attached to R.L Jalappa Hospital and Research Centre, Kolar. From December, 2014-November, 2015, 177 domestic rodents were trapped from the residences of farmers in rural areas, chicken market and the tomato market in the Kolar region (Table 1). Rodents included in this study were not an endangered or protected species. Captured rodents were euthanized and species identified16. Two milliliter of whole blood was collected by cardiac puncture and placed in tubes containing EDTA17 and stored at -80°C before testing. This study was approved by the Institutional Ethical Committee.

DNA amplification: The DNA was extracted from the Blood sample of 177 rodents using the QIAamp Blood DNA Mini Kit (QIAGEN GmbH, Hilden, Germany) according to the manufacturer’s instructions. Conventional PCR, targeting the 56 kDa type specific gene was standardized and performed with slight modifications; initial denaturation at 94°C for 5 min, followed by 40 cycles, each consisting of denaturation at 94°C for 30 sec, 58°C for 30 sec, 72°C for 1 min and the final extension at 72°C for 10 min using the primers of 56 kDa F: 5-AATTGCTAGTGCAATGTCTG-3' and of 56 kDa R: 5-GGCATTATAGTAGGCTGAG-3' (Sigma Aldrich, Bangalore, India)18. This particular region encompasses 410 bp and contains the VD I-III hyper variable regions19. The PCR products were visualized by electrophoresis on a 1.5% agarose gel, stained with ethidium bromide and were purified by using the Gene JET Gel Extraction Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Purified products were subjected to a sequencing reaction using the BigDye Terminator Mix (Applied Biosystems, Foster City, CA, USA) and subsequent automatic sequencing using the ABI 3500 Genetic Analyzer (Applied Biosystems).The sequence obtained was submitted to GenBank (accession nos. KY497019, MF140631). A phylogenetic and molecular evolutionary analysis was conducted by using Mega 7 software. A phylogenetic tree with 1,000 bootstrap replications was constructed by using the neighbour-joining methods with distances calculated by the maximum composite likelihood20.

RESULTS AND DISCUSSION

Rodents were recognized as reservoirs of various zoonotic diseases. Scrub typhus is a zoonotic disease caused by O. tsutsugamushi, where rodents act as reservoir hosts.

Table 1:Domestic rodents trapped in Kolar region
Arh: Arahalli, Bmn: Bommidi nagar, Ckm: Chicken Market, Elm: Eelam, Hrl: Horhalli, Kk: Karanji Katte, Khl: Kuppahalli, Mbr: Mulbagal road, Prh: Prasanth nagar, Rhn: Rahmath nagar, Shn: Saheed nagar, Tmk: Tomato market, San: Sangondahalli

Fig. 1:Agarose-gel electrophoresis of domestic rodents PCR products
  Lane 1 and 16: PC, Lane 6 and 18: 100 bp DNA marker, Lane 7, 8 and 17: PCR positives

Fig. 2:Phylogenetic relationships of Orientia tsutsugamushi detected in domestic rodents in Kolar region, from December, 2014-November, 2015
 
Relationships were determined on the basis of the partial 56 kDa type-specific antigen gene of O. tsutsugamushi by the minimum-evolution method with the maximum composite likelihood. Bootstrap values >50% were showed at the branches. Location and GenBank accession numbers are indicated for each sequence. Solid diamonds indicate sequences determined in this study

The present study helped to understand the prevalence of O. tsutsugamushi among domestic rodents in Kolar region. Among 177 rodents trapped, 138 (78.4%) were identified as Bandicota bengalensis, 16 (9.09%) Rattus rattus, 9 (5.11%) Bandicota indica, 12 (6.7%) Rattus norvegicus and 2 (1.12%) Mus musculus (Table 1), which were captured during the months of December, February and March. O. tsutsugamushi DNA was detected by conventional PCR in 10 (5.6%) rodents (Fig. 1), 7 from Bandicota bengalensis, 2 from Rattus rattus and 1 from Rattus norvergicus; O. tsutsugamushi DNA was detected in the rodents captured from Arahalli (4), Bommidi nagar (3), Karanji katte (2) and Sangondahalli (1) villages situated in Kolar. This indicates high risk of acquiring scrub typhus infection in this area. Zhang et al.21, reported 2.6% positivity for O. tsutsugamushi DNA by semi nested PCR among 385 domestic rodents from Northern China, which was slightly lower than the results reported in this study. The predominant rodent species in this study was Bandicota bengalensis as the reservoir host and the study by Zhang et al.21, reported Mus musculus as the predominant species.

Among 10 purified DNA recovered from the rodents blood sample, 2 were sequenced and analyzed. One strain was phylogeneticaly closely related to novel Orientia species O. chuto Dubai strain, which was isolated from Humans22 in Dubai and another strain was closely related to Boryang strain which was reported in wild rodents from South Korea23 (Fig. 2).

Studies from different parts of China, reported multiple genotypes of O. tsutsugamushi in rodents which were phylogeneticaly related to Kawasaki, Fuji, SDM related types, Karp, Gilliam, Kato, TA763 strains and Sdu-1, similar to Japan Kawasaki strain21,24,25 indicating different genotypes of Orientia tsutsugamushi exists in the environmental ecology.

CONCLUSION

Scrub typhus is one of the important cause of acute febrile illness in Karnataka. This present study was done in the Kolar region in Karnataka state from where a large number of clinical cases of scrub typhus were reported in recent years in humans. To the best of researchers knowledge this is the first molecular characterization of O. tsutsugamushi in domestic rodents in India. The strain characterized in this study was phylogeneticaly closely related to novel Orientia chuto Dubai strain isolated from human and Boryang strain from wild rodents. These strains were circulating in reservoir rodents suggesting a high incidence of scrub typhus in this region. Further research on the characterization of O. tsutsugamushi from rodents as well as vectors throughout this district are needed to study the prevalence of O. tsutsugamushi in reservoir hosts.

SIGNIFICANCE STATEMENT

This study highlights the prevalence and molecular characterization of Orientia tsutsugamushi in domestic rodents in the Kolar region, Karnataka, India. This study will helps the researchers to uncover the circulating strains of Orientia tsutsugamushi in domestic rodents and increases the knowledge in the community about epidemiological role of rodents in scrub typhus in this region.

ACKNOWLEDGMENT

The authors acknowledge Mr. vaigundan, Research Assistant and Mrs. Divya Bose, Research Assistant in Cell Biology and Molecular Genetics, Sri Devraj Urs Academic Higher Education and Research, Kolar, India for the assistance in DNA sequencing. Researchers used the equipment and materials provided by the institution for research work.

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