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Table of Contents
Year : 2022  |  Volume : 59  |  Issue : 2  |  Page : 182-185

Molecular epidemiology of Orientia tsutsugamushi from outbreak regions, Madhya Pradesh, central India

1 Division of In-Vivo Research, ICMR-National Institute of Research in Tribal Health, Jabalpur, Madhya Pradesh, India
2 Integrated Disease Surveillance Program (IDSP), Govt. of India, Madhya Pradesh, India

Date of Submission27-Feb-2020
Date of Acceptance08-Mar-2022
Date of Web Publication08-Sep-2022

Correspondence Address:
H V Manjunathachar
Division of In-vivo Research, ICMR- National Institute of Research in Tribal Health (NIRTH), Jabalpur-482003, Madhya Pradesh
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0972-9062.345176

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Scrub typhus is a seriously neglected life threatening disease in Asia and the islands of the Pacific and Indian oceans. The causative agent is Orientia tsutsugamushi, an obligate intra-cytosolic bacterium transmitted to vertebrate animals such as human or rodent by larval trombiculid mites (chiggers). Here, we conducted an outbreak investigation in Madhya Pradesh state in central India after sudden upsurge in scrub typhus cases and mortality in humans. Sixty-five live traps were placed for capturing the rodents. The captured rodent species were identified and the samples were subjected to nested PCR targeting 56kDa TSA gene. Analysis revealed for the first-time predominant circulation of Karp genotype in this region. The vector species involved in transmission is Leptotrobidium deliense. In conclusion, we reported the circulation of Karp strain in outbreak regions of Madhya Pradesh and suggest that public health monitoring and surveillance needs to be improved. Studies on antigenic diversity, association of strains with clinical spectrum and pathogenicity in the local setup will be essential for development of region-specific diagnostics and vaccines.

Keywords: Central India; Leptotrobidium spp; Oreintia tsutsugamushi; Rodents; Polymerase chain reaction; Scrub typhus

How to cite this article:
Manjunathachar H V, Tiwari P, Raut C G, Singh SK, Das A. Molecular epidemiology of Orientia tsutsugamushi from outbreak regions, Madhya Pradesh, central India. J Vector Borne Dis 2022;59:182-5

How to cite this URL:
Manjunathachar H V, Tiwari P, Raut C G, Singh SK, Das A. Molecular epidemiology of Orientia tsutsugamushi from outbreak regions, Madhya Pradesh, central India. J Vector Borne Dis [serial online] 2022 [cited 2022 Oct 5];59:182-5. Available from: https://www.jvbd.org/text.asp?2022/59/2/182/345176

  Background Top

Scrub typhus is a systemic life threatening zoonotic disease caused by Orientia tsutsugamushi, an obligate intracellular gram-negative bacterium. The pathogen is transmitted to humans or rodents through bite of infected chiggers or mite larvae belonging to the genus Leptotrombium, where both human and rodents are incidental hosts[1]. The clinical signs are largely nonspecific, represent a spectrum of severity ranging from mild illness to frequently fatal disease in both humans and laboratory animals[2],[3],[4]. Originally, scrub typhus was endemic to Asia-Pacific region, but recently, reports from South America and Middle East region have surfaced, making scrub typhus a major public health concern globally[4],[5],[6]. In Asia, approximately one million cases are reported annually and fatality rate is upto 70% depending on virulence of O. tsutsugamushi[1],[7]. Currently the epidemic foci of scrub typhus is tremendously spreading as a re-emerging disease in several regions of India, Micronesia, and the Maldives where the disease had been remarkably neglected[1].

In the past, prevalence and outbreaks of scrub typhus have been reported from southern, northern and north-east regions of India, in humans and rodents[4],[8],[9],[10],[11]. However, central India have scanty data on scrub typhus. In recent days, upsurge in scrub typhus cases and outbreaks with fatality was reported from Ratlam and Mandsaur districts of Madhya Pradesh, central India due to heavy rain fall and flood situations (www.patrika.com/mandsaur-news/ scrub-typhus-fever-spreading-rapidly-in-mandsaur-region-5167929/). Owing to lack of molecular characterization of O. tsutsugamushi from Madhya Pradesh, the outbreak investigation has been conducted to investigate the circulating strains of O. tsutsugamushi in the rodents.

  Material & Methods Top

Study area and rodents ' trappings

After high positivity of human cases of scrub typhus, the outbreak investigation was carried out in Ratlam (23.3315° N, 75.0367° E) and Mandsaur (24.0768° N, 75.0693° E) districts of Madhya Pradesh during August and September 2018. Sixty-five live traps (wire cages and wonder traps) were placed which trapped rodents. The captured rodent, vector species were identified from each rodent species; different organs and ear pinna samples were collected and stored in -20°C until use [Figure 1].
Figure 1: Outbreak investigation areas, rodent dissection and sampling A. Map of India and outbreak districts in Madhya Pradesh (circled). B. & C. depict the presence of chigger mite and adult Leptotrobidium spp. in ear pinna of rodents.

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Extraction of DNA and PCR amplification of 56 kDa gene

Genomic DNA was extracted from each specimen (pooled tissue samples of spleen, heart, liver and blood clots) depending on the quantity of the samples by using DNA Sure Mini Kit (GENETIX, India) and then stored at -20°C for further use. Amplification of 56 kDa gene was carried out by using two set of primers and following Polymerase Chain Reaction (PCR) cycling conditions as shown in [Table 1]. The secondary PCR was carried out with the same conditions along with 5μL of 1:10 diluted primary PCR product. After amplification, nested PCR products were electrophoresed in 1.5% agarose gel and visualized under UV light.
Table 1: PCR primers and cycling conditions for amplification of 56 kDa gene

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DNA sequencing and phylogenetic analysis by maximum likelihood method

The positive amplicons were sequenced by using Big Dye Terminator v3.1 Cycle Sequencing Kit and ABI PRISM 3130XL Genetic Analyser (Applied Biosystems, USA) as per manufacturer’s instructions at the in-house sequencing facility. The obtained sequences were compared with available sequences in GenBank (www.blast. ncbi.nlm.nih.gov./Blast.cgi). Phylogenetic analysis was performed by MEGA X software and phylogenetic tree was constructed by using a neighbor-joining method and Tamura-Nei model after 1000 bootstrapped replicates[12].

Ethical statement: Not applicable

  Results Top

Acute febrile illness (AFI) patient blood samples were subjected to IgM ELISA (Inbios, International, Inc.) for scrub typhus diagnosis and early treatment at All India Institute of Medical Sciences (AIIMS), Bhopal. From the same regions, rodents belonging to different species were trapped and the trapping percentage was 30.7 % (20/65). The trapped rodent species are Rattus rattus (n-7), Banidicota bengalensis (n-4), Mus musculus (n- 4), Rattus norvegicus (n-2), and Tatera indica (n-3). The vector species involved in transmission is Leptotrobidium deliense. Nested PCR targeting 56 kDa-TSA gene of O. tsutsugamushi was performed on all rodent DNA samples. The district wise and species wise positivity rate of O. tsutsugamushi are represented in [Figure 2]. Ten sequences were determined from different rodent species and submitted to GenBank (Accession numbers: MN579497 to MN579506). The sequences showed 91.77–92.48% sequence identity with the reference Karp strain (Accession number: M33004.1). The nucleotide sequences showed 98.93–99.79% identity between the strains. Phylogenetic analysis revealed that all ten circulating strains of O. tsutsugamushi in Ratlam and Mandsaur belong to Karp genotype [Figure 3]. BLASTn search results further showed that Karp isolates of Ratlam and Mandsaur share maximum similarity (99.02–99.75%) with Vietnam Karp isolate (GU128876.1). Genetic homology of Ratlam and Mandsaur isolates with other Indian isolates and reference strains are represented in [supplementary Table 1] [Additional file 1] & [supplementary Table 2] [Additional file 2] .
Figure 2: Status of O. tsutsugamushi infection in captured rodents of Ratlam and Mandsaur districts of Madhya Pradesh, central India. A. O. tsutsugamushi positivity in captured rodents of both districts. B. O. tsutsugamushi infection rate in different species of captured rodents from Ratlam. C. O. tsutsugamushi infection rate in different species of captured rodents from Mandsaur.

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Figure 3: Phylogenetic tree showing the genetic relatedness among different isolates of O. tsutsugamushi revealed by maximum likelihood method and Tamura-Nei model based on 56 kDa gene sequence.

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  Discussion Top

Globally, zoonotic pathogens are attributed to number of outbreaks and they account for more than 1 billion human illness cases annually[13],[14]. Scrub typhus is a re-emerging zoonotic disease in India where rodents act as a potential natural reservoir cum maintenance hosts for O. tsutsugamushi. The family of Muridae (rats and mice) play a crucial role in transmission of scrub typhus by hosting chigger mites. Hence, understanding the epidemiology as well as potential impact on human health depends on the geographical distribution of vectors and reservoir hosts[14]. Over the last few years many outbreaks of scrub typhus in humans have been reported from different parts of India with case fatality[4],[11]. Nonetheless, there is a scarcity of reports/underdiagnoses of scrub typhus from Madhya Pradesh state, located at the Basin of Narmada river and is among the richest repositories of biological diversity in India. In 2018 after heavy rainfall and flood in the state, upsurge in scrub typhus cases and fatalities were encountered and health authorities alerted scrub typhus outbreak situation in Ratlam and Mandsaur districts of Madhya Pradesh.

In this study we identified the involvement of different rodent species and vector in scrub typhus outbreak regions by detecting O. tsutsugamushi in rodents via PCR. Rattus rattus is the predominant rodent species collected which accounts for high rate of positivity for O. tsutsugamushi among other species of rodents. The vector involved in this region is Leptotrobidium deliense. The re-emergence of this neglected disease might be due to change in climatic conditions and increase in the rodent and vector populations above the threshold level[1]. The heavy rainfall and associated flood in the state altered the rodent habitation and subsequently infested rodents cohabited with human dwellings for shelter and food. It is possible that this might be a reason for sudden upsurge and outbreak of scrub typhus in these regions.

This first report revealed circulation of Karp genotype in two outbreak districts of Madhya Pradesh. Sequence analyses revealed that the identified strains are 98–99.75% identical with Karp isolate from febrile patients, rodents and mites of Vietnam, Japan, China, Sri Lanka and Taiwan. Conversely, our sequences are 94% and 71–72% similar with southern (Karnataka) and northern Indian (Uttar Pradesh) Karp isolates from rodents, respectively. The variation of strains may be due to geographical variation in O. tsutsugamushi and plasticity of Orientia genome[15]. Studies have reported that Karp is the highly virulent strains of O. tsutsugamushi[16]. The reported mortality in the outbreak region may be due to the circulation of highly virulent Karp strain, which was determined in all the samples in our study. Similarly, in human samples phylogenic studies revealed the predominance circulation of Karp followed by Gilliam and Ikeda strains in the Madhya Pradesh region (unpublished data). Rodent sampling and gene sequence analysis from Nagpur region of Maharashtra state revealed Karp-like strain of O. tsutsugamushi[17]. Screening of animal hosts in the Gorakhpur region of Uttar Pradesh state reported high prevalence of acute encephalitis syndrome scrub typhus patients and revealed circulation of Karp and Gilliam strains[18].

  Conclusion Top

We have reported the circulation of Karp strain in outbreak regions of Madhya Pradesh state of central India and it is suggested that public health monitoring and surveillance needs to be improved. Studies on antigenic diversity, association of strains with clinical spectrum and pathogenicity in the local setup will be essential for development of region-specific diagnostics and vaccines.

Key message

Scrub typhus has reemerged as an important cause of acute febrile illness in India. The predominant circulating pathogenic genotype in central India is Karp strain. The vector species involved in transmission cycle is Leptotrobidium deliense. Re-emergence of this disease along with mortality and evolving strain diversity is alarming.

Conflict of interest: None

  References Top

Walker DH. Scrub typhus—scientific neglect, ever-widening impact. N Engl 2016; 375(10): 913–915.  Back to cited text no. 1
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Candasamy S, Ayyanar E, Paily K, Karthikeyan PA, Sundararajan A, Purushothaman J. Abundance & distribution of trombiculid mites &Orientiatsutsugamushi, the vectors & pathogen of scrub typhus in rodents & shrews collected from Puducherry & Tamil Nadu, India. IJMR 2016; 144(6): 893.  Back to cited text no. 10
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Kim DM, Yun NR, Neupane GP, Shin SH, Ryu SY, Yoon HJ, et al. Differences in clinical features according to Boryoung and Karp genotypes of Orientiatsutsugamushi. PloS one 2011; 6(8): e22731.  Back to cited text no. 15
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Sadanandane C, Jambulingam P, Paily KP, Kumar NP, Elango A, Mary KA, et al. Occurrence of Orientia tsutsugamushi, the etiological agent of scrub typhus in animal hosts and mite vectors in areas reporting human cases of acute encephalitis syndrome in the Gorakhpur region of Uttar Pradesh, India. Vector- Borne and Zoonotic Dis 2018; 18(10): 539–547.  Back to cited text no. 18


  [Figure 1], [Figure 2], [Figure 3]

  [Table 1]


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