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 ~  Abstract
 ~ Introduction
 ~  Materials and Me...
 ~ Results
 ~ Discussion
 ~ Acknowledgements
 ~  References
 ~  Article Figures

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  Table of Contents  
BRIEF COMMUNICATION
Year : 2015  |  Volume : 33  |  Issue : 3  |  Page : 422-425
 

Detection of flea-borne Rickettsia species in the Western Himalayan region of India


1 Department of Veterinary Microbiology, Dr. GC Negi College of Veterinary and Animal Sciences, Chaudhary Sarwan Kumar Himachal Pradesh Krishi Vishvavidyalaya, Palampur, Himachal Pradesh, India
2 Department of Veterinary Public Health and Epidemiology, Dr. GC Negi College of Veterinary and Animal Sciences, Chaudhary Sarwan Kumar Himachal Pradesh Krishi Vishvavidyalaya, Palampur, Himachal Pradesh, India
3 Department of Veterinary Parasitology, Dr. GC Negi College of Veterinary and Animal Sciences, Chaudhary Sarwan Kumar Himachal Pradesh Krishi Vishvavidyalaya, Palampur, Himachal Pradesh, India

Date of Submission02-Nov-2013
Date of Acceptance07-Apr-2014
Date of Web Publication12-Jun-2015

Correspondence Address:
R Chahota
Department of Veterinary Microbiology, Dr. GC Negi College of Veterinary and Animal Sciences, Chaudhary Sarwan Kumar Himachal Pradesh Krishi Vishvavidyalaya, Palampur, Himachal Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0255-0857.158572

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 ~ Abstract 

Human infections by various rickettsial species are frequently reported globally. We investigated a flea-borne rickettsial outbreak infecting 300 people in Western Himalayan region of India. Arthropod vectors (ticks and fleas) and animal and human blood samples from affected households were analysed by gltA and ompB genes based polymerase chain reaction (PCR). Rat flea (Ceratophyllus fasciatus) samples were found harbouring a Rickettsia sp. Phylogenetic analysis based on gltA gene using PHYLIP revealed that the detected Rickettsia sp. has 100% identity with SE313 and RF2125 strains of Rickettsia sp. of flea origin from Egypt and Thai-Myanmar border, respectively and cf1 and 5 strains from fleas and lice from the USA. But, the nucleotide sequence of genetically variable gene ompB of R14 strain was found closely related to cf9 strain, reported from Ctenocephalides felis fleas. These results highlight the public health importance of such newly discovered or less recognised Rickettsia species/strains, harboured by arthropod vectors like fleas.


Keywords: Epidemiology, polymerase chain reaction, Rattus Rickettsia, vector-borne


How to cite this article:
Chahota R, Thakur S D, Sharma M, Mittra S. Detection of flea-borne Rickettsia species in the Western Himalayan region of India. Indian J Med Microbiol 2015;33:422-5

How to cite this URL:
Chahota R, Thakur S D, Sharma M, Mittra S. Detection of flea-borne Rickettsia species in the Western Himalayan region of India. Indian J Med Microbiol [serial online] 2015 [cited 2019 Nov 22];33:422-5. Available from: http://www.ijmm.org/text.asp?2015/33/3/422/158572



 ~ Introduction Top


Genus Rickettsia has three groups. Ancestral group (AG) has Rickettsia bellii and R. canadensis harboured by various types of ticks. Typhus group (TG), consisting of R. prowazekii and R. typhi, is associated with lice and fleas. The spotted fever group (SFG) contains more than 20 species and mostly found in ticks but mites and fleas can also act as vectors. [1],[2],[3]

Human rickettsiosis is reported globally. [2] In the Indian subcontinent rickettsial species such as O. tsusugamushi (Scrub typhus) and Rickettsia spp. (Indian tick typhus) are reported to be prevalent. [4],[5] The role of livestock and arthropods infesting them has been established in transmitting the rickettsial infection to humans. We describe an outbreak of rickettsial febrile illness in the sub-Himalayan region of Himachal Pradesh, India. Outbreaks of human rickettsiosis have been reported from this region in the past. [6],[7] Citrate synthtase (gltA) and outer membrane protein B (ompB) DNA sequence based polymerase chain reaction (PCR) tests confirmed the role of a Rickettsia sp. spread by rat fleas in human population. [8],[9] This report suggests public health significance of such rarely reported flea-borne rickettsial species outside TG cluster.


 ~ Materials and Methods Top


Symptoms of high fever (up to 39.9°C) persisting for 3 to 8 weeks, without skin lesions (rashes/eschar) were reported by approximately 23% (300/1300) people (109 households) from a cluster of three villages to Department of Health, Himachal Pradesh. Patients responded to empirical treatment with doxycycline. The Weil-Felix test with OX19, OX2 and OXK antigens performed on select human serum samples (titer >320 was considered positive) indicated the possible involvement of rickettsial agent without conclusive diagnosis. [2]

For identification of rickettsial species, blood samples from humans (n = 7), (5 from convalescent human patients and 2 from persons with fever) and animals (n = 18), tested positive by Weil-Felix test were analysed by PCR. The arthropod samples analysed by PCR included 18 ticks [Boophilus microplus (n = 11) and Haemephysalis bispinosa (n = 7)] from the livestock [cows (n = 12) and buffaloes (n = 6)], 9 flea samples, [Ceratophyllus fasciatus, rat flea (n = 6); Ctenocephalides canis (n = 2) and Pulex irritans (n = 1)] from trapped rats (Rattus rattus) and mice (R. norvegicus), human beings and animal houses.

DNA was extracted from all human and animal blood samples as well as arthropod samples using QIAamp DNA mini kit (Qiagen, Hilden, Germany) as per manufacturer's instructions. PCR tests competent to detect all rickettsial species, were performed by protocols as reported previously with primers Rp877p and Rp1258n for gltA (382 bp amplicon) and 120-M59 and 120-807 primers for ompB (856-bp amplicon) for DNA amplification as reported, previously. [8],[9] DNA of R. parkeri and R. rhipicephali were used as positive controls (Kindly provided by Dr. Marcelo B. Labruna, University of São Paulo, São Paoulo, SP, Brazil).

Direct nucleotide sequencing of PCR products was done and the sequences were compared by BLAST search. Phylogenetic analysis was done using Phylogeny Inference Package software (PHYLIP) [Version. 3.69; (http://evolution.genetics.washington.edu/phylip.html)] as reported before. [10]


 ~ Results Top


PCR amplification of both the targeted genes, glt and ompB, were observed in 7 out of 9 flea samples. The National Centre for Biotechnology-Basic Local Alignment Search Tool (NCBI-BLAST) (http://www.ncbi.nlm.nih.gov) search revealed that the detected Rickettsia species (R14 strain) has 100% gltA gene identity with SE313 (GenBank accession no. DQ166938) and RF2125 (AF516333) strains of Rickettsia spp. of flea origin from Egypt and Thai-Myanmar border, respectively and cf1and 5 strains from fleas and lice from the USA. But, the ompB gene sequence of R14 strain was found closely related to cf9 strain (DQ379483) reported from Ctenocephalides felis fleas by the Centers for Disease Control and Prevention, Atlanta, Georgia, USA with 99% DNA sequence identity. The R14 strain was found 92% identical to R. felis (AY394854). The nucleotide sequences of gltA and ompB genes of R14 strain were deposited in GenBank under accession no. HM370112 and HM370113, respectively.

Phylogenetic analysis showed that rickettsial strains of flea origin including R14 strain are forming a distinct cluster within SFG largely reported from ticks. The R14 strain was also different from highly pathogenic TG that has fleas and lice as arthropod vectors [Figure 1]a. Genetically variable ompB gene based clustering of R14 strain vis-à-vis ticks, fleas, and lice transmitted rickettsiae and resultant human disease conditions are shown in [Figure 1]b.
Figure 1: Phylogenic position of R14 strain (bold-faced) determined by neighbour-joining method. Bootstrap percentages calculated by generating 1000 data sets using SEQBOOT of PHYLIP programmes are shown at respective nods of the trees. The accession numbers are shown in parentheses. Clustering of flea-borne rickettsial species is well evident in both trees. (a) R14 strain vis-a-vis rickettsial species of TG, SPG and AG clusters based on nucleotides of gltA. Genetic distance is shown in the 0.01 scale; (b) tree based on ompB nucleotides as compared to other rickettsial species with known human pathologies. Genetic distance is shown in the 0.02 scale

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 ~ Discussion Top


We report an outbreak of human rickettsiosis by still unreported Rickettsia spp. (R14) harboured by rat flea, C. fasciatus in the Himalayan region. The identification of this Rickettsia spp. was done by determining DNA sequences of ompB, a highly genetically variable gene and gltA, a highly conserved gene, which are established methods for the detection of rickettsial spp. [8],[9] The C. fasciatus, arthropod vector of detected Rickettsial spp. (R14) were observed feeding on captured rat and mice (their natural reservoirs) and also human dwellings and animal sheds. Analysis of epidemiological data, revealed higher prevalence of febrile illness among people who stayed at home (women and children compared to men going out for work) and in people residing in mud houses (data not shown). These findings can be attributed to the exposure of the former group to repeated flea-bites, leading to higher doses of pathogen inoculation and higher rodent population in mud houses.

Flea-borne (murine) typhus caused by R. typhi, is considered globally endemic attributed to the distribution of rat flea (Xenopsilla cheopis). But, in recent years, other rickettsial species of flea origin like R. felis are considered as emergent threat to the human health. [11],[12] Our findings and other studies substantially indicate that the flea- borne rickettsial species other than TG may be noteworthy pathogens of public health significance. [8],[9] Such febrile illness without typical skin lesions of rashes or eschar may remain undiagnosed many times if not tested specifically for rickettsiae. Further studies are warranted to screen large arthropod vector populations, especially fleas, for detecting the novel rickettsial species of public health consequences.

In this investigation, we could not detect rickettsial DNA in small number of tested human blood samples, as the infections must have cleared to undetectable level in patients under treatment. The effective containment of the outbreak by controlling flea and rat population, establish the disease and causative agent relationship.


 ~ Acknowledgements Top


We thank University authorities for financial and infrastructure support and R.K Sood for helping in identification and selection of studied households based on his preliminary screening of human patients, and the Department of Animal Husbandry, Himachal Pradesh, staff for logistics.

We are thankful to Dr. Marcelo B. Labruna, Faculty of Veterinary Medicine, University of São Paulo, São Paoulo, SP, Brazil for providing DNA of R. parkeri and R. rhipicephali, which was used as positive controls in this study.

RC is an Associate Professor in the Department of Veterinary Microbiology, DGCN COVAS, Palampur. His research focuses on chlamydial and rickettsial diseases.

 
 ~ References Top

1.
Weisburg WG, Dobson ME, Samuel JE, Dasch GA, Mallavia LP, Baca O, et al. Phylogenetic diversity of the Rickettsiae. J Bacteriol 1989;171:4202-6.  Back to cited text no. 1
    
2.
Raoult D, Roux V. Rickettsioses as paradigms of new or emerging infectious diseases. Clin Microbiol Rev 1997;10:694-719.  Back to cited text no. 2
    
3.
Parola P, Paddock CD, Raoult D. Tick-borne rickettsioses around the world: Emerging diseases challenging old concepts. Clin Microbiol Rev 2005;18:719-56.  Back to cited text no. 3
    
4.
Kumar S, Yadav MP, Singh VB, Padbidri VS. Rickettsioses surveillance in animals and man in Uttar Pradesh. Indian J Med Res 1982;76:179-84.  Back to cited text no. 4
    
5.
Mathai E, Lloyd G, Cherian T, Abraham OC, Cherian AM. Serological evidence for the continued presence of human rickettsioses in southern India. Ann Trop Med Parasitol 2001;95:395-8.  Back to cited text no. 5
    
6.
Mahajan SK, Rolain JM, Kashyap R, Bakshi D, Sharma V, Prasher BS, et al. Scrub typhus in Himalayas. Emerg Infect Dis 2006;12:1590-2.  Back to cited text no. 6
    
7.
Mahajan SK, Kashyap R, Sankhyan N, Sharma, V, Rolain JM, Prasher BS, et al. Spotted fever group rickettsioses in Himachal Pradesh. J Assoc Physicians India 2007;55:868-70.  Back to cited text no. 7
    
8.
Regnery RL, Spruill CL, Plikaytis BD. Genotypic identification of rickettsiae and estimation of intraspecies sequence divergence for portions of two rickettsial genes. J Bacteriol 1991;173:1576-89.  Back to cited text no. 8
    
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Roux V, Raoult D. Phylogenetic analysis of members of the genus Rickettsia using the gene encoding the outer-membrane protein rOmpB (ompB). Int J Syst Evol Microbiol 2000;50:1449-55.  Back to cited text no. 9
    
10.
Chahota R, Ogawa H, Mitsuhashi Y, Ohya K, Yamaguchi T, Fukushi H. Genetic diversity and epizootiology of Chlamydophila psittaci prevalent among the captive and feral avian species based on VD2 region of ompA gene. Microbiol Immunol 2006;50:663-78.  Back to cited text no. 10
    
11.
Gilles J, Just FT, Silaghi C, Pradel I, Passos LM, Lengauer H, et al. Rickettsia felis in fleas, Germany. Emerg Infect Dis 2008;14:1294-6.  Back to cited text no. 11
    
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Perez-Osorio CE, Zavala-Velazquez JE, Arias Leon JJ, Zavala-Castro JE. Rickettsia felis as emergent global threat for humans. Emerg Infect Dis 2008;14:1019-23.  Back to cited text no. 12
    


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