Molecular evidence of spotted fever group rickettsiae ...

Parasitol Res DOI 10.1007/s00436-015-4819-y

ORIGINAL PAPER

Molecular evidence of spotted fever group rickettsiae and Anaplasmataceae from ticks and stray dogs in Bangladesh Yongjin Qiu 1 & Ryo Nakao 2,3 & May June Thu 2 & Shirin Akter 3,4 & Mohammad Zahangir Alam 4 & Satomi Kato 1 & Ken Katakura 3 & Chihiro Sugimoto 1,5

Received: 23 July 2015 / Accepted: 3 November 2015 # Springer-Verlag Berlin Heidelberg 2015

Abstract Emerging tick-borne diseases (TBDs) are important foci for human and animal health worldwide. However, these diseases are sometimes over looked, especially in countries with limited resources to perform molecular-based surveys. The aim of this study was to detect and characterize spotted fever group (SFG) rickettsiae and Anaplasmataceae in Bangladesh, which are important tick-borne pathogens for humans and animals worldwide. A total of 50 canine blood samples, 15 ticks collected from dogs, and 154 ticks collected from cattle were screened for the presence of SFG rickettsiae and Anaplasmataceae using molecular-based methods such as PCR and real-time PCR. The sequence analysis of the amplified products detected two different genotypes of SFG rickettsiae in ticks from cattle. The genotype detected in

Rhipicephalus microplus was closely related to Rickettsia monacensis, while the genotype detected in Haemaphysalis bispinosa was closely related to Rickettsia sp. found in Korea and Japan. Anaplasma bovis was detected in canine b l o o d a n d t i c k s (R h i p i ce p h a l u s s a n gu i n e u s a n d H. bispinosa). Unexpectedly, the partial genome sequence of Wolbachia sp., presumably associated with the nematode Dirofilaria immitis, was identified in canine blood. The present study provides the first molecular evidence of SFG rickettsiae and A. bovis in Bangladesh, indicating the possible emergence of previously unrecognized TBDs in this country. Keywords Anaplasmataceae . Rickettsia . Tick . Bangladesh . Tick-borne diseases

Electronic supplementary material The online version of this article (doi:10.1007/s00436-015-4819-y) contains supplementary material, which is available to authorized users. * Chihiro Sugimoto [email protected]

1

Division of Collaboration and Education, Hokkaido University Research Center for Zoonosis Control, Kita 20, Nishi 10, Kita-ku, Sapporo, Hokkaido 001-0020, Japan

2

Unit of Risk Analysis and Management, Hokkaido University Research Center for Zoonosis Control, Kita 20, Nishi 10, Kita-ku, Sapporo 001-0020, Hokkaido, Japan

3

Laboratory of Parasitology, Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Kita 18, Nishi 9, Kita-ku, Sapporo 060-0818, Hokkaido, Japan

4

Department of Parasitology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

5

Global Station for Zoonosis Control, Global Institution for Collaborative Research and Education, Hokkaido University, Kita 20, Nishi 10, Kita-ku, Sapporo 001-0020, Hokkaido, Japan

Yongjin Qiu [email protected] Ryo Nakao [email protected] May June Thu [email protected] Shirin Akter [email protected] Mohammad Zahangir Alam [email protected] Satomi Kato [email protected] Ken Katakura [email protected]

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Introduction The spotted fever group (SFG) rickettsiae are obligate intracellular Gram-negative bacteria, recognized as the causative agents of important emerging tick-borne human infectious diseases (Raoult and Roux 1997; Parola and Raoult 2001; Renvoisé et al. 2009). They mainly invade endothelial cells and cause acute flu-like symptoms, chills, high fever, headache, skin rash, and photophobia (Renvoisé et al. 2009). Eight different species of human pathogenic SFG rickettsiae have been definitively characterized throughout the world. These are Rickettsia rickettsii in the USA; Rickettsia slovaca in Europe; Rickettsia conorii in Europe, Asia, and Africa; Rickettsia japonica and Rickettsia sibirica in Asia; Rickettsia australis and Rickettsia honei in Australia; and Rickettsia africae in Africa and the West Indies (Parola et al. 2013; Renvoisé et al. 2009). In addition, several SFG rickettsiae, including Rickettsia helvetica, Rickettsia aeschlimannii, Rickettsia heilongjiangensis, Rickettsia monacensis, and Rickettsia mongolotimonae, have been recently isolated from febrile patients and recognized as causative agents of emerging human rickettsioses (Fournier et al. 2000a, b; Jado et al. 2007; Raoult et al. 2002; Zhang et al. 2000. Anaplasma spp. are obligate intracellular bacteria transmitted by ticks. They cause potentially fatal infectious diseases in humans and economically devastating diseases in animals (Dumler et al. 2001; Parola et al. 2005). In humans, anaplasmosis is mainly caused by Anaplasma phagocytophilum, although there has been one reported case of Anaplasma ovis infection in Spain (Dumler et al. 2001; Chochlakis et al. 2010). In addition, in China, Anaplasma capra was newly recognized as a pathogenic agent in humans (Li et al. 2015). In sheep and cattle, Anaplasma marginale and Anaplasma centrale infect erythrocytes and cause several symptoms such as anemia, fever, lymphadenopathy, depression, and loss of conditioning (Inokuma 2007). On the other hand, Anaplasma bovis infects monocytes in ruminants and induces mild symptoms or subclinical infection (Dumler et al. 2001; Inokuma 2007). In canines, Anaplasma platys is known to cause cyclic thrombocytopenia (Harvey et al. 1978). Although some information is available on the diversity of ticks in Bangladesh (Islam et al. 2006; Ghosh et al. 2007), little attention has been paid to the pathogens they transmit. Bangladesh is located between India and South East Asia, where different tick-borne pathogens are expected to circulate. For example, R. conorii subsp. indica, an agent of Indian tick typhus, and Candidatus Rickettsia kellyi are the only known SFG rickettsial agents in India (Rovery and Raoult 2007; Rolain et al. 2006). Meanwhile, several other rickettsial agents, including R. japonica, R. heilongjiangensis, and R. honei, have been reported from east neighboring areas of Bangladesh such as the Thai-Myanmar border (Parola et al. 2003) and Thailand (Jiang et al. 2005; Takada et al. 2009).

Thus, a molecular survey on tick-borne pathogens in Bangladesh may provide useful information for understanding the distribution of tick-borne diseases (TBDs) in Asia. In this paper, we demonstrate the presence of Rickettsia and Anaplasma bacteria in Bangladesh at a molecular level.

Materials and methods Sample collection and DNA extraction A total of 50 stray dogs were captured as part of a surveillance program of Babesia infections in the Mymensingh district in Bangladesh, as described elsewhere (Terao et al. 2015). Peripheral blood samples were collected from dogs and used for DNA extraction, as described (Terao et al. 2015). Ticks were collected from dogs and cattle in the same study area. Tick species were identified using morphological taxonomic keys under a stereomicroscope. Total DNA was extracted from individual ticks using DNAzol (Invitrogen, Carlsbad, CA) according to a previous study (Nakao et al. 2010). PCR, real-time PCR, and sequencing The DNA samples were initially subjected to screening for SFG and typhus group (TG) rickettsiae infections using the citrate synthase gene (gltA) real-time PCR. When gltA realtime PCR yielded a positive result, the samples were further tested by semi-nested gltA PCR and ompA PCR, as described previously (Nakao et al., 2013). Real-time PCR was performed using the primers CS-F (5′TCGCAAATGTTCACGGTACTTT-3′) and CS-R (5′TCGTGCATTTCTTTCCATTGTG-3′) and the probe CS-P (5′-FAM-TGCAATAGCAAGAACCGTAGGCTGGATGBHQ1-3′) (Stenos et al. 2005), using a THUNDERBIRD Probe qPCR mix (Toyobo, Osaka, Japan). The semi-nested gltA PCR and ompA PCR were conducted with primers RpCS.780p (5′-GACCATGAGCAGAATGCTTCT-3′), RpCS.877p (5′-GGGGACCTGCTCACGGCGG-3′), and RpCS.1273r (5′-CATAACCAGTGTAAAGCTG-3′) (Ishikura et al. 2003), and the primers Rr.190.70p (5′ATGGCGAATATTTCTCCAAAA-3′), Rr.190.701n (5′G T T C C G T TA AT G G C A G C AT C T- 3 ′ ) , a n d Rr.190.602n (5′-AGTGCAGCATTCGCTCCCCCT-3′) (Roux et al. 1996), respectively, using the Amplitaq Gold 360 kit (Applied Biosystems, Foster City, CA). To detect Anaplasmataceae, the PCR was performed using the primers EHR16SD (5′-GGTACCYACAGAAGAAGTCC-3′) and EHR16SR (5′-TAGCACTCATCGTTTACAGC-3′), which amplify a 345-bp fragment of the 16S ribosomal DNA

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(rDNA) of the family of Anaplasmataceae (Parola et al. 2000). When the samples tested positive, a semi-nested PCR was performed using primers fD1 (5′AGAGTTTGATCCTGGCTCAG-3′), EHR16SR, and GA1UR (5′-GAGTTTGCCGGGACTTCTTCT-3′) (Warner and Jacqueline 1996). A nearly complete sequence of 16S rDNA (approx. 1400 bp) was obtained by two semi-nested PCR reactions using primers fD1, Rp2 (5′ACGGCTACCTTGTTACGACTT-3′), EHR16SR, and EHR16SD, as previously reported (Ybañez et al. 2012). The PCR products were purified using ExoSap-IT (USB Corporation, Cleveland, OH) according to the manufacturer’s instructions. Cycle sequencing was performed using the BigDye Terminator version 3.1 chemistry (Applied Biosystems). Sequencing products were run on a 3130xl Genetic Analyzer (Applied Biosystems). The DDBJ/EMBL/ GenBank accession numbers obtained are as follows: ompA of Rickettsia, LC064749131 and LC064750; 16S rDNA of Anaplasma, LC066136 to LC066139; and 16S rDNA of Wolbachia, LC063848. Phylogenetic analysis Sanger sequencing data were analyzed using GENETYX version 9.1 (GENETYX Corporation, Tokyo, Japan). The phylogenetic analysis was conducted using MEGA version 6.05 (Tamura et al. 2013). The sequences were aligned with closely related bacterial sequences deposited in the database using

Table 1

ClustalW, and a maximum likelihood phylogram was applied to draw phylogenetic trees.

Results Out of 50 canine blood samples tested, 29 samples were positive by Anaplasmataceae-PCR (EHR16SD/EHR16SR) (Table 1). Subsequent PCR and sequence analyses of 16S rDNA using the primers fD1 and GA1UR gave two different sequences in 14 samples. One sequence (423 bp) obtained from 13 samples showed 99.7 % identity with a Wolbachia endosymbiont of Dirofilaria immitis (GenBank no. AF088187) and was clustered together with Wolbachia spp. found in nematodes (Fig. 1). The other sequence, which was recovered from one dog, showed 99.5 % similarity with A. bovis (GenBank no. U03775). Further amplification of the longer sequence was only successful using the primer set fD1 and EHR16SR (approx. 770 bp), still showing 99.6 % identity with the same A. bovis strain. This sequence showed 99.5 % similarity with the sequence detected from Tick 9 as mentioned below. A total of seven samples tested positive for SFG/TG rickettsiae by gltA real-time PCR (Table 1); however, none of these samples were amplified by gltA and ompA PCR. Fifteen ticks were recovered from dogs. Further morphological investigation identified them as Rhipicephalus sanguineus. Out of 15 samples tested, ten samples were amplified by conventional Anaplasmataceae-PCR. Subsequent analysis of a

Detection of SFG rickettsiae and Anaplasmataceae by PCR and real-time PCR

No. of samples

Canine blood Dog

Cattle

Rhipicephalus sanguineus

Rhipicephalus microplus Haemaphysalis bispinosa Hyalomma anatolicum

50 7 3 5

gltA real-time PCR positive 7 1 0 0

gltA PCR positivea 0 0 nd nd

ompA PCR positivea 0 0 nd nd

AnaplasmataceaePCR positive 29 5 2 3

fD1-GA1UR PCR positiveb 14 4 0 2

Subtotal Male Female Nymph Male Female Nymph Male

15 6 57 7 23 58 1 2

1 0 10 0 3 13 0 1

0 nd 6 nd 1 10 nd 1

0 nd 3 nd 0 3 nd 0

10 0 0 0 1 2 0 0

6 nd nd nd 1 0 nd nd

Subtotal

154

27

18

6

3

1

Sex/stage

Tested

Male Female Nymph

nd not done a

Only tested for the samples positive by gltA real-time PCR

b

Only tested for the samples positive by Anaplasmataceae-PCR

Parasitol Res Fig. 1 Phylogenetic analysis of Wolbachia sp. detected in canine blood based on partial sequences of 16S rDNA using a maximum likelihood method. The tree is rooted with Anaplasma marginale. All bootstrap values from 1000 replications are shown on the interior branch nodes

nearly complete sequence of 16S rDNA gave two different sequence types (Tick 6 and Tick 9). These sequences showed the highest similarities with A. bovis detected in China (99.2 %) (GenBank no. HQ913646) and in South Africa (99.4 %) (GenBank no. U03775), and were located in a clade of A. bovis in a phylogenetic tree (Fig. 2). Although one sample tested positive by gltA real-time PCR, subsequent gltA and ompA PCR were negative. A total of 154 ticks were collected from cattle and identified as Rhipicephalus microplus (n=70), Haemaphysalis bispinosa (n = 82), and Hyalomma anatolicum (n = 2). Three samples of H. bispinosa were positive by Anaplasmataceae-PCR (Table 1). Subsequent PCR using the primers fD1 and GA1UR was successful in one of the samples. Sequence analysis of the amplicon showed 99.5 % similarity with A. bovis (GenBank no. AB983376) detected from the Haemaphysalis longicornis in the southern area of Japan (Tateno et al. 2015). The sequence was located in a clade of A. bovis in a phylogenetic tree (Supplemental Figure 1). A total of 27 samples were positive for SFG/TG rickettsiae by gltA real-time PCR. Subsequent PCR assays amplified gltA and ompA genes in 18 and 6 samples, respectively. Sequence analysis of the ompA products gave two different sequence types. Sequence type 1, recovered from three individual R. microplus, was clustered with R. monacensis in a phylogenetic tree with the highest sequence similarity of 99.7 % to R. monacensis strain MT34 (GenBank no.

Fig. 2 Phylogenetic analysis of Anaplasma spp. detected in Rhipicephalus sanguineus based on almost the full length sequences of 16S rDNAs using a maximum likelihood method. The tree is rooted with Rickettsia rickettsii. All bootstrap values from 1000 replications are shown on the interior branch nodes

JX972178) (Fig. 3). Sequence type 2, recovered from three individual H. bispinosa showed the highest similarity to Rickettsia spp. found in H. longicornis in South Korea (Kim et al. 2006) and in Ixodes nipponensis in Japan (Ishikura et al. 2003).

Discussion The aim of this study was to detect and characterize SFG rickettsiae and Anaplasmataceae in Bangladesh, where very little attention has been paid to TBDs, due to a lack of resources to perform molecular-based surveys. This is the first report of the molecular detection of SFG rickettsiae and Anaplasmataceae in Bangladesh. Sequence analysis of Anaplasma sp. found in canine blood showed 99.5 % similarity (766/770) with A. bovis or a closely related species, detected in the R. sanguineus (Tick9) (Fig. 2). The presence of A. bovis in dogs has been reported elsewhere (Sakamoto et al. 2010), although its pathogenicity to dogs is totally unknown. Several reports described a high positive rate of anaplasmosis in cattle in Bangladesh (Chowdhury et al. 2006; Alim et al. 2012; Belal et al. 2014). In these studies, only A. marginale and A. centrale have been identified by microscopic examination. A lack of information on A. bovis may be attributed to the differences in the infection sites in mammalian hosts; A. marginale and A. centrale infect erythrocytes, while A. bovis parasitizes monocytes

Parasitol Res Fig. 3 Phylogenetic analysis of Rickettsia spp. detected in Rhipicephalus microplus and Haemaphysalis bispinosa based on partial sequences of ompA using a maximum likelihood method. The tree is rooted with Rickettsia australis. All bootstrap values from 1000 replications are shown on the interior branch nodes

(Sreekumar et al. 1996; Yang et al. 2015). Thus, it is possible that infection with A. bovis has been overlooked in this country. Wolbachia sp. was detected in 13 canine blood samples (Table 1). This bacterial genus, a member of alphaproteobacteria such as Anaplasma, is well known as an essential symbiont of arthropods and nematodes (Casiraghi et al. 2001; Saint André et al. 2002). A phylogenetic analysis indicated that the detected sequence is closely related to the Wolbachia endosymbiont of D. immitis (heartworm) (Fig. 1). Detection of Wolbachia sequences in canine blood has been previously reported in Japan, where filarial nematodes are endemic (Sakamoto et al. 2010). The authors found that all Wolbachia-positive dogs were also positive for D. immitis infection (Sakamoto et al. 2010). Only one case report is available so far on D. immitis infection in Bangladesh (Fuehrer et al. 2013). Our results indirectly suggest that D. immitis infection might be common, at least among stray dogs, in the study area in Bangladesh. Further studies should be carried out to clarify the endemic status of D. immitis infection in dogs by means of parasite-specific methods. An increasing amount of evidence supports human infections with this filarial roundworm (Orihel and Eberhard 1998; Pampiglione et al. 2009; Foissac et al. 2013). Therefore, clinicians should be aware of the infection as a case of pulmonary illness. Two different sequence types of rickettsial ompA were detected in ticks collected from cattle (Fig. 3). Sequence type 1 from R. microplus showed the highest similarity with R. monacensis, previously reported in European countries such as Spain, Germany, and Hungary, as well as in two Far East Asian countries, Korea and Japan (Simser et al. 2002; Sréter-Lancz et al. 2005; Lee et al. 2013; Ishikura et al. 2002). The other sequence type

(type 2), detected from H. bispinosa, was located in the same clade with Rickettsia spp. reported from Korea and Japan. It is of great importance to accumulate such information in order to understand the geographic distribution of each rickettsial species/genotypes. Of note, the presence of R. monacensis, which was reported from two human cases in Spain (Jado et al. 2007), indicates that human SFG rickettsial diseases may exist in this country. In conclusion, this study is the first to provide genetic evidence for the presence of SFG rickettsiae and Anaplasma spp. in Bangladesh. Further studies, targeting a wide range of tick species and mammalian hosts, are required to capture the whole picture of the diversity of tick-borne pathogens in this country.

Acknowledgments This work was supported in part by the Global Center of Excellence Program for International Collaboration Centers for Zoonosis Control and JSPS KAKENHI Grant nos. 22405037 and 24380163 from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT). We thank all veterinarians, technicians, and dairy farmers in the region for their assistance in sample collection.

Compliance with ethical standards Competing interests The authors declare that they have no competing interests. Author’s contributions RN, KK, SA, and MZA collected samples. YQ and RN carried out tick identification, molecular genetic studies, and the preparation of this manuscript. SK participated in tick identification. MJT participated in the molecular genetic studies of Anaplasma. CS designed the experiments and helped to draft the manuscript. All authors read and approved the final manuscript. Ethical approval Approval to capture stray dogs and collect ticks was obtained from the Mymensingh Municipality Bureau.

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