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ORIGINAL ARTICLE
Year : 2020  |  Volume : 64  |  Issue : 1  |  Page : 27-31  

Evidence of natural infection of Orientia tsutsugamushi in vectors and animal hosts – Risk of scrub typhus transmission to humans in Puducherry, South India


1 Scientist B, Division of Vector Biology and Control, ICMR-Vector Control Research Centre, Puducherry, India
2 MSc, Public Health Entomology Graduates, ICMR-VCRC, Puducherry, India
3 Technical Officer, Division of Microbiology and Molecular Biology, ICMR-VCRC, Puducherry, India
4 Scientist G and Head, Division of Vector Biology and Control, ICMR-VCRC, Puducherry, India
5 Scientist G and Director, ICMR-VCRC, Puducherry, India

Date of Submission19-Mar-2019
Date of Decision04-Jun-2019
Date of Acceptance10-Feb-2020
Date of Web Publication16-Mar-2020

Correspondence Address:
Panneer Devaraju
Division of Vector Biology and Control, ICMR-Vector Control Research Centre, Medical Complex, Indira Nagar, Puducherry - 605 006
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijph.IJPH_130_19

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   Abstract 


Background: Scrub typhus infection is endemic in India and reported to be the major cause for acute encephalitis syndrome (AES) in humans. Periodic occurrence of scrub typhus cases and presence of pathogen in rodents were also reported in areas with human cases of scrub typhus in Puducherry. Objectives: This study was carried out to screen Orientia tsutsugamushi in rodent/shrew reservoirs and vectors in villages of Puducherry with no reports of human scrub typhus cases. Methods: This study was conducted during October 2017 to January 2018 in ten randomly selected villages in Puducherry. Rodents/shrews in the peridomestic area were trapped using Sherman traps. Screening of O. tsutsugamushi in rodents/shrews and mite vectors was done by polymerase chain reaction (PCR). Weil-Felix test was done to screen antibodies against O. tsutsugamushi in rodent serum samples. Results: Among the 54 rodents trapped, Suncus murinus was the major small animal and Leptotrombidium deliense was the major mite species retrieved. PCR screening revealed pathogen positivity in 8 rodent blood and 3 pooled mite samples. Phylogenetic analysis has shown that Kato was the circulating serotype of O. tsutsugamushi. None of the rodent serum samples was tested positive for antibodies against O. tsutsugamushi by Weil-Felix test. Conclusions: The presence of pathogen in both vectors and reservoir animal hosts imposes a risk for scrub typhus transmission to the inhabitants; hence, initiation of vector control measures before the start of winter is recommended in the study area. It is also recommended to screen scrub typhus in patients with undifferentiated acute febrile illness and AES.

Keywords: 56 KDa, acute encephalitis syndrome, Leptotrombidium deliense, Orientia tsutsugamushi, scrub typhus


How to cite this article:
Devaraju P, Arumugam B, Mohan I, Paraman M, Ashokkumar M, Kasinathan G, Purushothaman J. Evidence of natural infection of Orientia tsutsugamushi in vectors and animal hosts – Risk of scrub typhus transmission to humans in Puducherry, South India. Indian J Public Health 2020;64:27-31

How to cite this URL:
Devaraju P, Arumugam B, Mohan I, Paraman M, Ashokkumar M, Kasinathan G, Purushothaman J. Evidence of natural infection of Orientia tsutsugamushi in vectors and animal hosts – Risk of scrub typhus transmission to humans in Puducherry, South India. Indian J Public Health [serial online] 2020 [cited 2020 Apr 8];64:27-31. Available from: http://www.ijph.in/text.asp?2020/64/1/27/280763

Bhuvaneswari Arumugam, Iswaryalakshmi Mohan, Mariselvam Paraman contributed equally as first authors





   Introduction Top


Scrub typhus is an acute febrile illness endemic in wider areas of South and South East Asia, the Asian Pacific Rim, and Northern Australia (“tsutsugamushi triangle”). Over one billion people are under the risk of acquiring the infection, and an average of one million cases are being reported every year from these regions.[1] The scrub typhus pathogen, Orientia tsutsugamushi, is an obligate intracellular rickettsial organism transmitted by the bite of trombiculid mites (chiggers). The mites serve as a reservoir, and the infection in them is maintained by transovarial and transstadial transmission. Humans and rodents acquire the infection by the bite of the mites. Humans are the accidental host, whereas rodents are the natural as well as the reservoir host for O. tsutsugamushi.[2] In humans, the infection may manifest as a self-limiting disease to a fatal illness in 30%–70% of cases, with multiorgan dysfunction.[3] Despite being least diagnosed, several human cases of scrub typhus were reported from many states in India.[4],[5],[6],[7],[8] In the recent past, acute encephalitis syndrome (AES) with high case fatality rate (20%–25%) is a major public health issue in Gorakhpur district, Uttar Pradesh, India, during cooler season of the year (October to December), and during this season, the incidence of scrub typhus was also reported to be higher. Murhekar et al., 2016, reported serological evidence of scrub typhus infection in 63% of patients with AES and 54% of patients with acute febrile illness in Gorakhpur.[9] Khan et al., 2017, reported that scrub typhus contributed to 20% of AES cases in Assam.[10] Sadanandane et al., 2018, demonstrated the presence of scrub typhus pathogen in rodents and mites collected from the AES-reported villages of Gorakhpur.[11] Human scrub typhus cases were reported in Pondicherry union territory (UT) in the year 2010.[12] Subsequently, a preliminary study carried out in the scrub typhus reported villages of Puducherry, and the adjoining regions of Tamil Nadu recorded the abundance of vector mite, Leptotrombidium deliense, and the pathogen, O. tsutsugamushi in rodents.[13] In this context, the present study was undertaken to screen O. tsutsugamushi in rodent/shrew reservoirs and vectors in villages of Puducherry with no reports of human scrub typhus cases.

This study confirmed the presence of scrub typhus pathogen, O. tsutsugamushi, in rodents/shrews and vectors in villages of Puducherry with no reports of human scrub typhus cases.


   Materials and Methods Top


This exploratory study to investigate the presence of animal hosts and vector for scrub typhus was carried out for a period of 4 months (October 2017 to January 2018) in ten randomly selected villages [Figure 1] of UT of Puducherry, with no reports of human scrub typhus cases. The Institutional Animal Ethics Committee (IAEC) approved the study (ICMR-VCRC/IAEC/2017/NP-2 dated November 29, 2017). Trapping and identification of rodents/shrews, collection of blood samples from rodents, retrieval, storage, and identification of ectoparasites were done as per the protocol by Sadanandane et al., 2018.[11] Weil-Felix test was performed using a commercial kit (Tulip Diagnostics, Pune, India) to detect the presence of antibodies in rodent sera against O. tsutsugamushi and other rickettsial pathogens based on the agglutination of OX-K and OX-19 and OX-2 antigens, respectively. DNA from the rodent blood samples and the pooled abdominal contents of L. deliense mites (30 mites constituted a pool) was extracted using the commercial DNA isolation kits (DNeasy Blood and Tissue Kit, Invitrogen, Germany). The samples were screened for O. tsutsugamushi by amplification of 56 KDa outer membrane by nested polymerase chain reaction (PCR) as per the protocol of Lee et al., 2006,[14] and the PCR-positive samples were (nucleotide) sequenced (Genetic Analyzer 3130XL, Applied Biosystems, USA). The sequences were edited using BioEdit software 7.0.0 and explored using BLAST for the identification of serotype. A phylogenetic tree was constructed with 1000 bootstrap replicates using neighbor-joining method, and the distances between the strains were analyzed using maximum likelihood method using the MEGA version 10 [Figure 1].
Figure 1: Location of study sites in Puducherry union territory, India.

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   Results Top


A total of 54 rodents were trapped showing a trap positivity rate of 13.7%. The rodents mainly belonged to two orders, namely Rodentia (Rattus rattus [17.9%] and Mus booduga [1.8]) and Soricomorpha (Suncus murinus [79%]). The details on rodents trapped and the ectoparasites retrieved are given in [Table 1]. S. murinus was the predominant shrew species trapped (79%). In total, 555 mites were retrieved from the rodents, among which L. deliense was the most prevalent mite species (79.62%). None of the sera samples collected from the rodents were tested positive by Weil-Felix test for the presence of antibodies against O. tsutsugamushi. PCR screening revealed that six shrews (S. murinus), two rodents (R. rattus) and three pooled mite samples collected from the villages, Thuthipet, Sooramangalam, and Ramanathapuram, were tested positive for O. tsutsugamushi. While none of the rodents trapped from the village Sooramangalam was tested positive, the mites retrieved from them were tested positive for O. tsutsugamushi by PCR. BLAST and phylogenetic analysis of all the PCR positive samples revealed that the pathogen belonged to the major serotype, Kato [Figure 2].
Table 1: Details on the study villages, rodents trapped, mites retrieved, and polymerase chain reaction positivity in rodents and vectors

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Figure 2: Phylogenetic distribution of Orientia tsutsugamushi isolated in animal reservoirs and vectors from Puducherry. The isolates from Puducherry are mentioned as consensus (S5A, S47, S36, S25, S24, S19, S26, and S53). Phylogenetic tree was constructed with 1000 bootstrap replicates using neighbor-joining method, and evolutionary distances between the strains were analyzed using maximum likelihood method.

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   Discussion Top


Scrub typhus is an endemic, re-emerging vector-borne illness contributing to 50% of the acute undifferentiated febrile illness reported from many parts of India.[4],[5],[6],[7],[8] The nonspecific clinical symptoms and the lack of adequate point of care diagnostic tools make it difficult to diagnose the disease at the bedside. More attention is paid to scrub typhus, as it was found to be a major etiology for AES reported from Gorakhpur, UP (63%), and Assam (20%).[9],[10] The proportion of scrub typhus among the total incidence and the case fatality reported due to AES/undifferentiated febrile illness remains to be explored throughout the country. Furthermore, there are only a very few reports on the epidemiology of maintenance and transmission of the pathogen from rodents/shrews and vectors to humans. The variable climatic conditions across the country have been reported to favor the pathogen maintenance in the vectors and its transmission to humans.[15] The current study was carried out to explore the presence of pathogen in the vectors and in rodents from the villages in Puducherry with no reports on human cases of scrub typhus. The shrew, S. murinus, constituted 79% of the total rodents captured and harbored 79.6% of trombiculid mite vectors. The chigger index in S. murinus was 10.2. Our findings are concurrent with the earlier reports from Gorakhpur and Puducherry.[11],[13] L. deliense was the major trombiculid mite observed in the current study that matches with the earlier reports in India, Taiwan, Thailand, and Malaysia.[11],[13],[15],[16],[17]

None of the sera samples collected from the animal reservoirs were tested positive by Weil-Felix test for the presence of antibodies against O. tsutsugamushi. The nonreactivity to Weil-Felix test observed might be due to the lack of development antibodies during the earlier phase of O. tsutsugamushi infection in rodents/shrews or these reservoirs, which might not have acquired the infection.

PCR screening of DNA samples showed that six shrews, two domestic rats, and three L. deliense pools were tested positive for O. tsutsugamushi. PCR positivity in shrews and rodents indicated the acquisition of infection from mites and pathogen amplification in these animals. If these rickettsemic rodents are bitten by uninfected mites, they may acquire and pass on the infection to its progeny through transovarial and transstadial transmission,[15] and thus, the infection is being maintained in nature. PCR positivity in mites confirmed that L. deliense was the major vector for scrub typhus in the study area. Similar findings have been reported elsewhere in India[11],[13] and also in other Asian countries, namely Taiwan, Thailand, and Malaysia.[15],[16],[17] The eight sequences of our isolates together with 42 near similar homologous sequences, which included the major serotypes (Gilliam, Karp, Kato, Boryong), were aligned, and a distance phylogeny was constructed. Nucleotide sequence analysis revealed the presence of a pathogenic strain Kato, which was reported to be the predominant circulating strain in South India.[18] Overall, the mean distance and the mean diversity among all the analyzed sequences were 1.04 and 1.46, respectively. The Puducherry isolates from the current study showed a mean distance of 2.68 and a mean diversity of 0.00, implying that there was no diversity among the Puducherry isolates but exists with the other isolates from India. The diversity within the type strains can be attributed to the higher plasticity of O. tsutsugamushi genome.[19]

Candasamy et al., 2016, demonstrated the presence of O. tsutsugamushi only in rodents/shrews but not in vectors from the areas with the reports of human scrub typhus cases in Puducherry and adjoining regions of Tamil Nadu.[13] In this study, we have demonstrated the vector potential and the enzootic maintenance of the pathogen in small animal hosts in ten randomly selected villages of Puducherry, with no reports of human scrub typhus cases. In addition, these infected animal hosts were trapped from the peridomestic area, which is in closer proximity with the human dwellings. Furthermore, we demonstrated the presence of pathogen in both vectors and animal hosts during the cooler season,[20] which coincides with the earlier reports of scrub typhus outbreaks in cooler seasons in Gorakhpur, Uttar Pradesh, India.[9] Data on the incidence of scrub typhus in Puducherry for the last 2 years, i.e., 2017 and 2018, were obtained from IDSP, Puducherry. It was observed that no human cases of scrub typhus were reported from the villages included in the current study. The lack of report of human cases from these study sites could be due to the underdiagnosis of scrub typhus due to its overlapping symptoms with other diseases reported during cooler season, empirical use of doxycycline to treat febrile illness, and/or the minimal/no human-vector contact.[1] Therefore, considering the risk on the presence of pathogen in animal hosts and mite vectors, it is suggested to include scrub typhus in the differential diagnosis in patients with undifferentiated acute febrile illness and AES and to implement vector control measures, before the winter onset.

The major limitation of Weil-Felix test in diagnosis of scrub typhus is its lower sensitivity and its utility after the postinfection to detect the presence of antibodies. Hence, Weil-Felix test has a limited scope to detect the pathogen at the time of infection or rickettsemia in the animal reservoirs. However, it may be used for serosurveillance in reservoir animal hosts in view of its lower cost and requirement of less expertise to perform the test. Nevertheless, the results of Weil-Felix test will also give a hint on the enzootic maintenance of other rickettsial pathogens in the reservoir animal hosts. Although the PCR assay for scrub typhus is very sensitive for the detection of scrub typhus during prevailing infection/rickettsemia, its application is limited after the development of antibodies against the pathogen. Hence, the use of both Weil-Felix test and PCR will aid in the detection of antibody and infection, prevailing serotype, respectively. However, the limitations of both these tests necessitate the development of a newer diagnostic test for the detection of antigen and antibodies.



The evidence on the presence of O. tsutsugamushi in both the vectors and small animal hosts suggests a potential risk of disease transmission to humans in the study area. Although no human scrub typhus cases were reported from the study area, in view of the vector potential and enzootic maintenance of the pathogen in small animal reservoirs, human–vector contact would enhance the risk for infection in humans. Based on our findings, it is suggested to include scrub typhus in the differential diagnosis in patients with acute febrile illness and AES and to implement vector control measures before the start of winter. It is recommended to use both Weil-Felix test and PCR protocol for scrub typhus surveillance in animal reservoirs.

Acknowledgments

The funds and facilities provided by ICMR to carry out the study and the technical assistance extended by Dr. S. Lakshmy, Mr. S. Rajkumar, and Mrs. S. Pushpa are gratefully acknowledged.

Financial support and sponsorship

This study is supported by the extramural funds received from Indian Council of Medical research (ICMR) F. No. 8/1/14/2019-RMC dated 03.06.2019.

Conflicts of interest

There are no conflicts of interest.



 
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