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RESEARCH ARTICLE
Year : 2022 | Volume
: 59
| Issue : 4 | Page : 348-355
Vector and rodent surveillance for Orientia tsutsugamushi in north India
Taruna Kaura1, Jasleen Kaur2, Kamlesh Bisht2, Shriya Goel2, PVM Lakshmi3, Gagandeep Singh Grover4, Abhishek Mewara1, Manisha Biswal2
1 Department of Medical Parasitology; Postgraduate Institute of Medical Education and Research, Chandigarh, India
2 Department of Medical Microbiology; Postgraduate Institute of Medical Education and Research, Chandigarh, India
3 Department of Community Medicine and School of Public Health, Postgraduate Institute of Medical Education and Research, Chandigarh, India
4 Department of Health and Family Welfare, Punjab, Parivar Kalyan Bhawan, Chandigarh, India
Correspondence Address:
Dr. Manisha Biswal
Professor, Department of Medical Microbiology, Postgraduate Institute of Medical Education and Research, Chandigarh-160012
India
Source of Support: None, Conflict of Interest: None
CheckDOI: 10.4103/0972-9062.355958
Background & objectives: Scrub typhus or chigger borne typhus, caused by Orientia tsutsugamushi is an emerging vector-borne disease as large numbers of cases have been reported in various tropical countries. It is transmitted to humans through bites of infected chiggers (larval mites). The knowledge about the vector, its distribution, density and habitat are important so as to understand the epidemiology of scrub typhus in a given area. To control rickettsial infections, regular rodent-vector surveillance should be planned in areas where the disease transmission is occurring and it will also help to strengthen the existing entomological data related to the vector of scrub typhus in northern India.
Methods: In the present study, rodent-vector surveillance was planned for one whole year, covering both mite active and non-active seasons (October 2019-December 2020) in selected areas of Chandigarh and Punjab in north India. Rodent tissues and mites were also examined for the presence of O. tsutsugamushi by nested PCR for 56 kDa gene and real-time PCR for 47 kDa outer membrane protein gene. 18S gene PCR was performed for molecular identification of mites.
Results: In the surveillance, three types of ectoparasite, viz. mites, fleas and ticks were obtained in rodents. All mites found were of Laelapidae family. None of the pooled rodent tissue samples as well as mite samples were found positive for O. tsutsugamushi by nested PCR for rickettsial DNA.
Interpretation & conclusion: In the present study, we did not get any evidence of carriage of O. tsutsugamushi in either mites or rodents collected and sampled in selected regions in Chandigarh and Punjab. We need to strengthen the entomological surveillance over a broader region and increase the frequency of trapping rodents to increase clarity on vector-reservoir dynamics in this geographical region.
Keywords: Scrub typhus; O. tsutsugamushi; rodent vector; chigger mite; surveillance; north India
Scrub typhus is a vector-borne zoonotic rickettsial disease caused by Orientia tsutsugamushi through bites of trombiculid mites and rodents as common host for them[1],[2]. The studies from India have reported that the case fatality rate (CFR) ranges from 1.3% to 33.5% for scrub typhus depending upon the severity of this disease in the patient[3],[4]. Since 1999, the World Health Organization (WHO) states that scrub typhus is probably one of the most under-diagnosed and under-reported febrile illnesses[5]. In north India this disease has been increasingly reported in recent years[6],[7]. Around 40% of the patients presenting with acute febrile illness (AFI) in the monsoon months are diagnosed to have scrub typhus at our tertiary care center, the Postgraduate Institute of Medical Education and Research (PGIMER), Chandigarh, in north India. The rate of transmission of this disease is influenced by the rodent population as well as the contact frequency between rodents harboring mites infected with O. tsutsugamushi and humans[8],[9].
The establishment of a rodent-vector surveillance strategy, to assess the density of rodent population and the type of ectoparasites in scrub typhus affected area is a prerequisite to understand the potential risk of this disease. This ensures timely diagnosis and implementation of serological surveillance of scrub typhus in the at-risk population. Such pre-emptive efforts help to reduce the morbidity and mortality caused by this rodent-chigger borne disease in endemic seasons. Despite the public health importance of ectoparasite chiggers, the studies in India on the epidemiology of this vector remain neglected. Though, few studies have been carried out to identify the trombiculid mite vectors and their rodent host from the areas where scrub typhus cases has been reported[10],[11],[12],[13] while no such study has been done in Punjab and Chandigarh, from where cases of scrub typhus have been reported. Therefore, the current study was planned to initiate entomological surveillance to enable control of scrub typhus in this region.
Material & MethodsSurveillance and trapping of rodents
The study was jointly conducted by Departments of Medical Microbiology, Medical Parasitology and Community Medicine, Chandigarh, India. An entomological surveillance was planned in affected areas based on monthly data of notified scrub typhus cases from 2017-2020 to ascertain the prevalence of rodent-vector mite association. The rodent surveys were undertaken from October 2019 to December 2020 in seven areas in District S.A.S. Nagar, Punjab (Village Chajju Majra; Village Raipur Kalan; Village Saidpura; Village Sambhalki; Village Harlalpur; Village Baroli; Gobind Nagar, Nayagaon) and eight areas of Chandigarh (Sector-12, PGIMER, Sector-41 B, Sector-41 D, Adarsh Colony, Sector-54, Village Kaimbwala; Daddu Majra; Industrial area; Village Mauli Jagran). The rodent traps (26.5 × 10 × 8.5 cm) were set-up at various points in a particular area from where a case had been reported previously, viz. garbage areas, student hostel, cafeteria, forest areas inside the house, store room, and slum areas. The numbered and tagged baited traps were laid in suspected areas before dusk and collected the following morning for investigation. The captured animals were transported back to the animal house, PGIMER, anaesthetized and euthanized by sodium pentothal I.P. (40 mg/kg body weight). The genus, species, and gender of the rodent was recorded by following the standard morphological keys[14]. Tissues (liver, spleen, kidney and lung) were collected from all the rodents and stored at −80°C until further processing.
Collection and processing of ectoparasites
For ectoparasite collection, the fur of the animal was combed thoroughly over a white A4-size plain white sheet paper using a fine-toothed comb[15]. The parasites that fell on the paper from the fur were collected and transferred into a bottle containing 70% ethanol for preservation. Separate containers were used for each animal. The mites, fleas and ticks that were difficult to comb were dislodged using forceps and preserved as above before morphological identification[16]. The chigger index was calculated by using the formula: Number of chigger collected/No. of host examined.
DNA extraction, amplification and sequencing for mite species identification
DNA was extracted from the individual mite specimens. The DNA extraction was done by Qiagen DNAeasy blood and tissue extraction kit. The following modifications were added to the manufacturers' protocol: after the addition of lysis buffer and proteinase K the sample was incubated for 6 hours at 56°C and overnight at 37°C. In the last step, the DNA was eluted in 20 μl AE buffer.
The 18S gene was amplified by using genomic DNA of individual mite with the following reaction conditions: initial denaturation at 94°C for 5 min, 35 cycles of denaturation at 94°C for 1 min, annealing at 56.1°C for 1 min, extension at 70°C for 1 min, and final extension at 72°C for 10 min [Table 1][17]. The PCR products were visualized on 1.5% agarose gel. Subsequently, the amplicons were sequenced (Eurofins Genomics Pvt. Ltd., Bengaluru, India) and the sequences were submitted to GenBank.
Nested PCR and real-time PCR for detection of Orientia tsutsugamushi in chiggers and tissues collected from rodents
For the detection of Orientia from rodents and chiggers, the tissues collected from each rodent were pooled (lung, liver and spleen of individual rodent was considered as single pool sample), whereas the mites belonging to the same species and same host were pooled (n=4). The DNA was extracted using the Qiagen DNeasy blood and tissue kit as per the manufacturer' s instructions.
Nested-PCR
A nested PCR targeting 56 kDa gene for O. tsutsugamushi in all the DNA samples extracted from mouse tissue and mites was performed. The nested PCR protocol consisted of 35 cycles of primary PCR with initial denaturation at 94°C for 10 min, denaturation for 20 sec, annealing at 57°C for 1 min, extension at 70°C for 1 min, and final extension at 72°C for 10 min followed by 35 cycles of nested PCR with initial denaturation at 94°C for 10 min, denaturation for 20 sec, annealing at 57°C for 1 min, extension at 70°C for 1 min, and final extension at 72°C for 10 min as described previously[18]. The primers used are summarized in [Table 1].
Real-time PCR
The 47 kDa outer membrane protein gene was targeted to detect O. tsutsugamushi in rodent and mite samples[19]. The total volume reaction was 10 μl, including 5 μl of Taqman master mix, 0.8 μl of the primer-probe mix, 3.2 μl nuclease-free water and 1 μl of DNA template. The details of primers used are given in [Table 1]. The probe was labeled at the 5'-end with 6-carboxyfluorescein (FAM) reporter dye and at the 3'- end with 6-carboxytetramethylrhodamine (TAMRA) quencher dye. The cycling condition for the 47kDa outer membrane protein was Uracil-DNA glycosylases (UNG) incubation at 50°C for 2 min, polymerase activation at 95°C for 10 min followed by 40 cycles of 95°C for 15 sec, 60°C for 1 min as described previously[19].
Ethical statement: Not applicable
ResultsA total 645 traps were placed in the selected areas of Chandigarh and Punjab to trap the rodents [Figure 1]. The overall trap positivity rate was 37.5%. A total of 242 rodents comprising of five species, viz., Mus musculus (45.4%), Rattus rattus (33.4%), Bandicota indica (10%), Suncus murinus (7%), and Rattus norvegicus (4.1%) were trapped. Of these, 24.7% rodents were found to be infested with ectoparasites. A total of 237 ectoparasites comprising 138 chiggers, 64 flea and 35 tick specimens were retrieved from the trapped rodents, thus giving an overall ectoparasite index of 5.5. The ectoparasites identified were oriental rat flea (Xenopsylla cheopis), two species of mites (the spiny rat mite Laelaps echidninus, and Laelaps nutalli), and a species of hard tick (Rhipicephalus sanguineus) [Figure 2].
Figure 2: Types of ectoparasites collected during the surveillance.The ectoparasite abundancy in each host was also calculated and it was highest in Bandicota indica followed by Rattus rattus and Mus musculus, whereas no ectoparasite was found from Suncus murinus and Rattus norvegicus [Table 2]. The chigger infestation (chigger index) of areas selected was 0.6 and 0.63 in Chandigarh and Punjab, respectively. The chigger index for rodent species positive for ectoparasite was 3.6 in Bandicota indica, 0.24 in Mus musculus and 0.3 in Rattus rattus [Table 2].
18SPCR-sequencing for mite species identification
18S PCR was performed for mite identification. Lelapidae echidninus was morphologically identified and was the commoner species and used for molecular study, while the samples of the other species Lelapidae nutalli were not sufficient to process further. The 471 bp size product of 18S PCR of Laelaps echidninus [Figure 3] was sequenced and showed a 97.65% similarity to Ondatralaelaps multispinosus (Accession no. FJ911843.1). Ondatralaelaps is a genus of mites which belong to family Laelaps. Laelaps echidninus, Lelapidae nutalli and Laelaps multispinosus belong to the same family, Laelaps. The sequence was submitted to GenBank and the following accession number was obtained: MZ723920.
Figure 3: Gel image of 18S PCR for the identification of Laelaps echidninus.Nested PCR and real-time PCR for Orientia tsutsugamushi in chiggers and rodent tissues
Rodent tissues and mites collected in this study were screened for O. tsutsugamushi by 56 kDa gene nested PCR assay and 47 kDa outer membrane protein gene by real-time PCR. None of the 242 pooled tissues (liver, lung and spleen) of individual rodents, and 23 pools (n=4 each pool) of chiggers were found positive.
DiscussionThe ectoparasites found on rodents have an important role as vectors of pathogenic microorganisms that cause diseases in humans[20]. The present study was planned to understand the distribution of species of rodents and ectoparasites prevalent in this region in north India from where scrub typhus cases have been reported in past few years. In the present surveillance, among the trapped rodents, the prevalence rate of mites was 57%. We did not get any Leptotrombidium mites, the commonest vector for O. tsutsugamushi in the 242 rodents trapped. In the present study, we did not find evidence of O. tsutsugamushi infection in pooled samples of Laelaps by PCR which possibly indicates that a larger sample size of mites must be screened and that the rodent trapping must be more intense in time and more extensive in targeted areas.
All the chiggers found in the surveillance belonged to the genus Laelaps. Recent studies have shown that the mites belonging to Laelaps genus are common ectoparasite of rodents although studies on their role as disease vectors remains largely unknown[21]. In a study carried out in Kuala Selangor Nature Park, Malaysia, six more species of mites in addition to Leptotrombidium deliense were found. Among these six species, there were two species of mesostigmatid mites, viz., Laelaps nuttalli and Laelaps echidninus. It was further reported that in Malaysia, murine typhus is known to occur and L. deliense is a known vector species, however, the role of L. echidninus as a vector is yet not studied. The two species, L. echidninus and L. nuttalli, are haematophagous and known to bite and cause irritation in humans[22],[23]. Similarly, in a three-year study carried out in Kuala Lumpur and Pulau Pinang, the ectoparasites collected from wild rats were mainly spiny rat mites, i.e., Laelaps spp. although none were found positive for rickettsiae by gltA PCR assay[24]. Recently a study from Taiwan was carried out in which rickettsiae were detected in Laelaps mites. In this study, L. nuttalli was found to be the most abundant mite species (51.7% of total mites), followed by L. echidninus (24.2%)[25]. The rickettsia detected were similar to spotted fever group (SFG) rickettsiae.
In areas of South west Slovakia the infestation rates of rodents was investigated and their role as possible vectors of pathogenic rickettsiae. In this two-year study, they collected 2821 mites from 615 rodents, and found three species from Laelapide and six from Trombiculidae family. Rickettsial DNA was found in 32.5% of the 345 pooled samples of mites, and 9.4% of the 487 rodent blood samples. In the above study the most common species of Laelaps mite found was L. agilis[26],[27]. We have also detected spotted fever and murine typhus rickettsia in patients presenting with fever to our hospital. From the screening of patients for ompA gene of Rickettsia in northern India, Rickettsia conorii and R. typhi were reported after confirmation with sequencing[28],[29]. Hence, the role of Laelaps species in transmission of rickettsial infections prevalent in our region needs to be further investigated.
In the present study, the chigger index in both Chandigarh and Punjab was found to be high, which indicates that vector control measures must be adopted in this region[30]. In addition, the present surveillance clearly indicates that Laelaps were the most common mites found, although in the current sampling their role as vector of O. tsutsugamushi was not evident. In the scrub typhus endemic regions of Chandigarh and Punjab, the next logical step is to broaden the scope of this surveillance so as to accumulate more data on rodent-borne ectoparasites, characterization of the diversity and distribution of these ectoparasites in rodents using conventional and molecular tools, regular surveillance of the pathogens borne by these vectors and assessment of the risk of the rodent borne zoonoses to humans.
Ectoparasite surveillance studies conducted so far in scrub typus endemic areas from different parts of India have been summarized in [Table 3]. From south India, in a study carried out in districts of Andhra Pradesh and Tamil Nadu, found Leptotrombidium deliense as the dominant species of mites, however, the rodent and chiggers were not screened for O. tsutsugamushi[31]. From Puducherry and Tamil Nadu, L. deliense was recorded as dominant species of mite and 50 rodents were screened for the pathogen, of which only 28 samples were positive for OX-K Weil-Felix test and only two samples were positive only for GroEL gene of O. tsutsugamushi[11]. From central India (Nagpur and Raipur), Ornithonyssus bacoti was reported as the most common species of mite. In this study, 59 blood samples from rodents were screened for both 56kDa and 47kDa gene sequences, of which ten samples were positive for 56kDa and four gene PCR[12],[32]. Thus, O. bacoti was reported as the dominant species, and their abundance was found more in post-monsoon season as compared to pre-monsoon[33]. From eastern India, Schoengastiella ligula was found as the dominant species in Darjeeling, West Bengal. However, the blood samples collected from rodents in this study could not be processed for Weil-Felix test and PCR, as the samples were hemolysed[34]. In a study carried by Cossan et al., 2015 a total of1334 rodents were trapped from areas of France, Senegal and Thailand and spleens were screened for the presence of bacteria using universal primers targeting the hyper variable region V4 of the 16S rRNA gene. 110 samples were found to positive for Orientia and further by sequence analyses it was confirmed to be 100-94.4% identical to already published Orientia sequences in GenBank[35]. During a rodent survey done in north-west of Nairobi in 2017, a total of 54 small rodents were captured during five trap-nights and after molecular analysis found lung tissue and a pool of chigger mites from one of rodent M. natalensis which were positive for Orientia by PCR[36]. However, in a recent surveillance done in two distinct sites in Gwangju Metropolitan City, Republic of Korea, a total of47 rodents were captured and spleen, kidneys and blood collected from rodents were screened by PCR but none were found positive for Orientia[37]. Thus, there are many lines of evidence that Orientia may chronically infect rodents but the persistence of the bacteria in organs, and spleen in particular is currently poorly known suggesting more studies should be carried out.
Table 3: Studies from India focused on ectoparasite surveillance in scrub endemic areasFrom northern India, ectoparasite surveillance has been carried out in Delhi and few endemic districts of Himachal Pradesh, wherein, L. deliense has been found as the most abundant species, however, they were not screened for the presence of pathogens[38],[39]. Thus, despite the proven endemicity of scrub typhus all across India by reported human cases as well as rodent seropositivity for O. tsutusgamushi there is still a gap in our understanding of the host-pathogen-vector transmission dynamics in the country which needs to be focused in future in well-designed studies on patients as well as their immediate environment.
ConclusionThus, in this rodent-vector surveillance in scrub typhus endemic regions, ectoparasites such as mites, fleas and ticks were found to commonly infest rodents. The most common mites were of the Laelapidae family. However, none of the rodent or chiggers showed any evidence of carriage of O. tsutsugamushi by serology and molecular assays. This calls for enhanced rodent-vector surveillance since scrub typhus is prevalent in the region and it is vital to identify the vector and mode of transmission of the disease. The focus on chigger research along with preparedness for timely serological surveillance in scrub typhus endemic season in humans and rodents, timely investigation of cases and clusters, and prompt vector control measures will help to curtail the rodent-borne emerging diseases.
LimitationsDue to logistic issues, sometimes we could not do trapping as soon as we got the addresses of the patients. Thus, a more regular and rigorous approach should be utilized while carrying out surveillance for scrub typhus as the epidemiology of this disease still has gaps. In some instances, the patient's family in members did not cooperate for the trapping. There were some disturbances in our schedule because of COVID-19 pandemic. All of these factors may have affected results that we obtained.
Conflict of interest: None
AcknowledgementsThe authors thank Mr. Kamaldeep Singh, Field Worker, Department of Department of Community Medicine and School of Public Health, PGIMER, Chandigarh, India who helped carry out the surveillance activities. This study is funded by DST, Chandigarh vide letter No. Sanc/02/2019/336-343 dated 28.02.2019.
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