Acta Tropica 131 (2014) 117–123
Contents lists available at ScienceDirect
Acta Tropica journal homepage: www.elsevier.com/locate/actatropica
Field assessment of Orientia tsutsugamushi infection in small mammals and its association with the occurrence of human scrub typhus in Taiwan Pey-Ru Lin, Hui-Ping Tsai, Ming-Hui Weng, Hung-Chi Lin, Kuo-Ching Chen, Ming-Der Kuo, Pei-Yi Tsui, Yao-Wen Hung, Hui-Ling Hsu, Wen-Tssann Liu ∗ Institute of Preventive Medicine, National Defense Medical Center, Taipei, Taiwan
a r t i c l e
i n f o
Article history: Received 13 August 2013 Received in revised form 29 November 2013 Accepted 30 November 2013 Available online 17 December 2013 Keywords: Orientia tsutsugamushi Prevalence Rodent Scrub typhus
a b s t r a c t We conducted an extensive study in Taiwan of Orientia tsutsugamushi (OT) infection in small wild mammals. Field trapping was carried out at six districts in eastern and western Taiwan as well as various offshore islands during the period 2006–2010. A total of 1061 specimens representing 11 rodent species were captured. The presence of OT infection was assessed by indirect immunofluorescence assay and polymerase chain reaction assays of 56-kDa type-specific antigen gene. The chigger infestation rate among the animals was 35% (371/1061). Among these, OT was detected in 64% (238/371) of the chiggers from the infested animals and in the spleens from 273 (34.3%) of 797 animals. Excluding animals in the Suncus murinus group, the antibody positive rate of scrub typhus was 69.1% (477 of 690 of serum samples). The prevalence of OT infection in animals from areas with a low incidence of human cases of scrub typhus was significantly lower than that in rodents obtained from regions with a high incidence of human cases of the disease (44.4% ± 4.0% vs. 71.2% ± 9.7%, p < 0.001). In Taiwan, the prevalence of OT infection in wild rodents is considerably high and appears to correlate positively with the occurrence of scrub typhus in humans. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Scrub typhus is a mite-borne disease that is endemic in the AsiaPacific region (Kelly et al., 2009; Lee et al., 2006; Watt and Parola, 2003). The disease in humans is caused by bites from larval trombiculid mites (Leptotrobidium spp known as chiggers) that are infected with Orientia tsutsugamushi (OT), a gram-negative, obligate, intracellular bacterium (Ewing et al., 1978; Frances et al., 1999; Kuo et al., 2011a). The mites are believed to be the only reservoir of OT, from which the bacteria can be transovarially transmitted to the next generation in egg cells (Takahashi et al., 1994; Traub and Wisseman, 1974; Walker et al., 1975). Rodents, on the other hand, play pivotal roles as hosts for vector mites (ectoparasites) in scrub typhus transmission, providing blood meals for the mites at various stages of the life cycle (Coleman et al., 2003; Traub et al., 1975). Although the clinical symptoms of OT infection in rodents are usually unapparent (Allen and Spitz, 1945), antibody responses to the pathogen are readily detectable using serological methods, such
∗ Corresponding author. Tel.: +886 28177703819841; fax: +886 226733025. E-mail address: [email protected] (W.-T. Liu). 0001-706X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.actatropica.2013.11.029
as Weil–Felix assays and indirect immunofluorescence assays (IFA) (Bourgeois et al., 1977; O’Guinn et al., 2010; Shirai et al., 1982). Alternatively, PCR-based diagnostic techniques that target specific genes are sensitive and cost-effective methods for detecting the presence of OT (Furuya et al., 1993; Jiang et al., 2004; Kee et al., 1994). Outbreaks of scrub typhus depend on several factors, including the prevalence of OT, the abundance of the chigger vectors, prevalence of infected wild rodents, and socioeconomic development of the geographic region or country (Takahashi et al., 1994). Thus, the prevalence of OT infection in rodent hosts epidemiologically describes the risk of scrub typhus. The 56-kDa type-specific antigen gene codes a dominant outer membrane protein that is specific to OT (Ohashi et al., 1992). Phylogenetic analysis of OT strains, based on the 56-kDa gene, isolated from patients in Taiwan revealed that there are different genetic variants (Lu et al., 2010). The DNA sequences of the 56-kDa type-specific antigen gene of OT isolated from wild rodents are highly homologous to those of clinical isolates (Lin et al., 2011). This study was conducted to determine the OT prevalence in small mammals trapped in different environments from 2006 to 2010 in Taiwan and the relationship of OT prevalence to the occurrence of scrub typhus in humans.
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P.-R. Lin et al. / Acta Tropica 131 (2014) 117–123
Fig. 1. Map of the geographic areas in Taiwan showing population density and incidence (IR) of human scrub typhus for each of the study sites. IR expressed as the mean number of annual cases (2006–2010)/population in log10 is indicated in parentheses.
2. Materials and methods
2.2. DNA extraction
2.1. Study sites and sample collection
DNA from each mite that was obtained from each rodent was extracted and processed as described previously (Tsui et al., 2007). DNA samples from mites and from splenic tissue were extracted using the QIAamp DNA minikit (Qiagen, Hilden, Germany), according to the protocol provided by the manufacturer.
Field trapping of rodents was conducted from April 2006 to September 2010 in six regions in Taiwan (latitude: 22◦ 3 N to 26◦ 13 N; longitude: 119◦ 59 E to 121◦ 32 E) (Fig. 1). Tainan, located in the south-central region of western Taiwan is the largest city in the largest plain (Chianan Plain) on the island. The cities of Taitung and Hualien are located in mountainous areas in eastern Taiwan; Kinmen and Matsu islands are located off the coast of China’s Fujian province. Penghu is a large archipelago located off the western coast of Taiwan. Approximately 15 sites were selected in each study district, with general coverage of nonresidential field sites as well as suburbs, villages, towns, and residential sites. Rodents were captured in randomly set traps. Twenty traps were set at night at each site. The captured animals were identified to the species level. During the study period, a total of 10 rodent species and one shrew species were captured. Mites collected from the captured rodents were placed in Eppendorf tubes for later use. The captured animals were anesthetized with an intraperitoneal dose of tiletamine/zolazepam (Zoletil 50, Virbac SA, Carros, France) 30 mg/kg and the blood samples were collected by cardiac puncture. The animals were killed, and their spleens were removed. Each spleen sample was placed in a cryogenic tube, and stored using dry ice for delivery to the laboratory.
2.3. Antibody assay IFA was performed to determine the titers of OT-specific antibodies in the rodent serum samples as described previously (Robinson et al., 1976). Serum samples were diluted in phosphate buffered saline (PBS, 1:40) and then applied to antigencoated slides (pooled antigens of Karp, Kato, and Gilliam OT strains). The slides were incubated in a humidified chamber at 37 ◦ C for 30 min. A secondary antibody mixture of fluorescein isothiocyanate-conjugated goat-anti-rat IgG + A + M (H+L) antibodies (Zymed Laboratories, Inc., San Francisco, CA, USA) diluted 1:100 in PBS were then added to the slides. The reaction was scored as positive when bright green fluorescence was observed using a fluorescence microscope (Olympus IX71, Tokyo, Japan). 2.4. Primers and PCR amplification Nested PCR was performed to detect OT DNA in the DNA extracts. The primer sequences were designed based on the DNA
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Table 1 Rodent species captured by gender in Taiwan during the period 2006–2010. Rodent species
Rattus flavipectus R. norvegicus R. losea R. rattus mindanensis Mus musculus Mus caroli Apodemus agrarius Bandicota indica R. exulans R. rattus Suncus murinus Total
Rodent numbers
Male
Female
67 79 91 51 2 8 2 24 18 1 211 554
67 69 103 63 2 5 2 26 9 1 160 507
Total
Percentage of total (%)
134 148 194 114 4 13 4 50 27 2 371 1061
12.6 14 18.3 10.8 0.4 1.2 0.4 4.7 2.5 0.2 35
sequence from the Karp strain (GenBank accession no. AY956315), which amplified the 56-kDa type-specific antigen, as described previously (Furuya et al., 1993; Horinouchi et al., 1996). The two pairs of primers used were as follows: pair 1, comprising F6 (5 -GTTGGAGGAATGAATTACTGG-3 ) (nucleotides 406–425) and R6 (5 -AGCGCTAGGTTTATTAGCAT-3 ) (nucleotides 1059–1040), and pair 2, comprising F7 (5 -AGGATTAGAGTGTGGTCCTT-3 ) (nucleotides 369–388) and R7 (5 -ACAGATGCACTATTGGCAA-3 ) (nucleotides 1175–1156). DNA with a predicted length of approximately 0.7 kb was visualized with a UV transilluminator after agarose gel electrophoresis and staining with ethidium bromide (Lin et al., 2011; Fig. S1). 2.5. Human cases of scrub typhus in Taiwan Reported human cases of scrub typhus during the period 2006–2010 in Taiwan were anonymously retrieved from the Database of the Centers for Disease Control, 2011, Taiwan (http://nidss.cdc.gov.tw/NIDSS). The population data was retrieved from the website of the Department of Statistics, Ministry of the Interior, Taiwan (http://sowf.moi.gov.tw/stat/month/list.htm) (Fig. 1). 2.6. Statistical analysis The results are presented as means ± standard errors of means (SEM). Student’s t tests were conducted using Microsoft Excel 2007. P values less than 0.05 were considered statistically significant. Pearson correlation analysis was used to evaluate the relationships between the variables. 2.7. Ethics statement This research was approved by the Institutional Animal Care and Use Committee of the Institute of Preventive Medicine, permission number IPM-98-A1. The animal experiments were performed according to the guidelines of the Council of Agriculture, Executive Yuan, Taiwan. The data regarding human cases of scrub typhus was retrospectively and anonymously analyzed. 3. Results 3.1. Collection of field rodents During the study period, we captured 1061 small mammals including 10 species of rodents and one species of shrew in the six study districts (Table 1). Suncus murinus accounted for 35% (n = 371) of the captures and was the predominant shrew species.
Fig. 2. Geographic distribution of prevalence of Orientia tsutsugamushi infection in rodents and incidence (IR) of human scrub typhus. Line graph indicating the prevalence of OT infection in samples collected from the small mammals, as determined by the presence of OT-specific antibody in sera and bacterial DNA in spleen and chiggers, respectively, using IFA and nested PCR. Gray bar chart indicates the incidence (IR) of human scrub typhus for each study sites. a IR is expressed as annual scrub typhus cases (mean values of 2006–2010)/population in log10 .
Rattus losea (n = 194, 18.3%) was the most common and abundant rodent species found in this study. Rattus flavipectus (n = 134, 12.6%) and Rattus rattus mindanensis (n = 114, 10.8%) were the predominant rodent species exclusively present on Kinmen and Lan-yu islands, respectively. Overall, there was no significant difference in sex (approximately 1.1; 554/507) between male (48.9%) and female (49.8%) animals, respectively.
3.2. OT infection in chiggers and field rodents Results of the nested PCR studies to detect OT in the rodent chiggers and spleen samples are shown in Tables 2 and 3 respectively. Approximately 35% (371/1061) of animals were infested with chiggers. A heavy infestation in R. rattus mindanensis (91.2%; 104/114) and R. flavipectus (76.9%; 103/134), and moderate infestation in R. losea (40.2%; 78/194) were seen. No chigger positive rodents contained fewer than 10 chigger mites. As no characterization was carried out, detailed mite species were not known. In addition to chigger mites, other ectoparasites such as ticks, and fleas, alone or in combination, were collected from rodents. Tick infestation of rodents ranged from 10 to 24%, and flea infestation was
Contents lists available at ScienceDirect
Acta Tropica journal homepage: www.elsevier.com/locate/actatropica
Field assessment of Orientia tsutsugamushi infection in small mammals and its association with the occurrence of human scrub typhus in Taiwan Pey-Ru Lin, Hui-Ping Tsai, Ming-Hui Weng, Hung-Chi Lin, Kuo-Ching Chen, Ming-Der Kuo, Pei-Yi Tsui, Yao-Wen Hung, Hui-Ling Hsu, Wen-Tssann Liu ∗ Institute of Preventive Medicine, National Defense Medical Center, Taipei, Taiwan
a r t i c l e
i n f o
Article history: Received 13 August 2013 Received in revised form 29 November 2013 Accepted 30 November 2013 Available online 17 December 2013 Keywords: Orientia tsutsugamushi Prevalence Rodent Scrub typhus
a b s t r a c t We conducted an extensive study in Taiwan of Orientia tsutsugamushi (OT) infection in small wild mammals. Field trapping was carried out at six districts in eastern and western Taiwan as well as various offshore islands during the period 2006–2010. A total of 1061 specimens representing 11 rodent species were captured. The presence of OT infection was assessed by indirect immunofluorescence assay and polymerase chain reaction assays of 56-kDa type-specific antigen gene. The chigger infestation rate among the animals was 35% (371/1061). Among these, OT was detected in 64% (238/371) of the chiggers from the infested animals and in the spleens from 273 (34.3%) of 797 animals. Excluding animals in the Suncus murinus group, the antibody positive rate of scrub typhus was 69.1% (477 of 690 of serum samples). The prevalence of OT infection in animals from areas with a low incidence of human cases of scrub typhus was significantly lower than that in rodents obtained from regions with a high incidence of human cases of the disease (44.4% ± 4.0% vs. 71.2% ± 9.7%, p < 0.001). In Taiwan, the prevalence of OT infection in wild rodents is considerably high and appears to correlate positively with the occurrence of scrub typhus in humans. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Scrub typhus is a mite-borne disease that is endemic in the AsiaPacific region (Kelly et al., 2009; Lee et al., 2006; Watt and Parola, 2003). The disease in humans is caused by bites from larval trombiculid mites (Leptotrobidium spp known as chiggers) that are infected with Orientia tsutsugamushi (OT), a gram-negative, obligate, intracellular bacterium (Ewing et al., 1978; Frances et al., 1999; Kuo et al., 2011a). The mites are believed to be the only reservoir of OT, from which the bacteria can be transovarially transmitted to the next generation in egg cells (Takahashi et al., 1994; Traub and Wisseman, 1974; Walker et al., 1975). Rodents, on the other hand, play pivotal roles as hosts for vector mites (ectoparasites) in scrub typhus transmission, providing blood meals for the mites at various stages of the life cycle (Coleman et al., 2003; Traub et al., 1975). Although the clinical symptoms of OT infection in rodents are usually unapparent (Allen and Spitz, 1945), antibody responses to the pathogen are readily detectable using serological methods, such
∗ Corresponding author. Tel.: +886 28177703819841; fax: +886 226733025. E-mail address: [email protected] (W.-T. Liu). 0001-706X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.actatropica.2013.11.029
as Weil–Felix assays and indirect immunofluorescence assays (IFA) (Bourgeois et al., 1977; O’Guinn et al., 2010; Shirai et al., 1982). Alternatively, PCR-based diagnostic techniques that target specific genes are sensitive and cost-effective methods for detecting the presence of OT (Furuya et al., 1993; Jiang et al., 2004; Kee et al., 1994). Outbreaks of scrub typhus depend on several factors, including the prevalence of OT, the abundance of the chigger vectors, prevalence of infected wild rodents, and socioeconomic development of the geographic region or country (Takahashi et al., 1994). Thus, the prevalence of OT infection in rodent hosts epidemiologically describes the risk of scrub typhus. The 56-kDa type-specific antigen gene codes a dominant outer membrane protein that is specific to OT (Ohashi et al., 1992). Phylogenetic analysis of OT strains, based on the 56-kDa gene, isolated from patients in Taiwan revealed that there are different genetic variants (Lu et al., 2010). The DNA sequences of the 56-kDa type-specific antigen gene of OT isolated from wild rodents are highly homologous to those of clinical isolates (Lin et al., 2011). This study was conducted to determine the OT prevalence in small mammals trapped in different environments from 2006 to 2010 in Taiwan and the relationship of OT prevalence to the occurrence of scrub typhus in humans.
118
P.-R. Lin et al. / Acta Tropica 131 (2014) 117–123
Fig. 1. Map of the geographic areas in Taiwan showing population density and incidence (IR) of human scrub typhus for each of the study sites. IR expressed as the mean number of annual cases (2006–2010)/population in log10 is indicated in parentheses.
2. Materials and methods
2.2. DNA extraction
2.1. Study sites and sample collection
DNA from each mite that was obtained from each rodent was extracted and processed as described previously (Tsui et al., 2007). DNA samples from mites and from splenic tissue were extracted using the QIAamp DNA minikit (Qiagen, Hilden, Germany), according to the protocol provided by the manufacturer.
Field trapping of rodents was conducted from April 2006 to September 2010 in six regions in Taiwan (latitude: 22◦ 3 N to 26◦ 13 N; longitude: 119◦ 59 E to 121◦ 32 E) (Fig. 1). Tainan, located in the south-central region of western Taiwan is the largest city in the largest plain (Chianan Plain) on the island. The cities of Taitung and Hualien are located in mountainous areas in eastern Taiwan; Kinmen and Matsu islands are located off the coast of China’s Fujian province. Penghu is a large archipelago located off the western coast of Taiwan. Approximately 15 sites were selected in each study district, with general coverage of nonresidential field sites as well as suburbs, villages, towns, and residential sites. Rodents were captured in randomly set traps. Twenty traps were set at night at each site. The captured animals were identified to the species level. During the study period, a total of 10 rodent species and one shrew species were captured. Mites collected from the captured rodents were placed in Eppendorf tubes for later use. The captured animals were anesthetized with an intraperitoneal dose of tiletamine/zolazepam (Zoletil 50, Virbac SA, Carros, France) 30 mg/kg and the blood samples were collected by cardiac puncture. The animals were killed, and their spleens were removed. Each spleen sample was placed in a cryogenic tube, and stored using dry ice for delivery to the laboratory.
2.3. Antibody assay IFA was performed to determine the titers of OT-specific antibodies in the rodent serum samples as described previously (Robinson et al., 1976). Serum samples were diluted in phosphate buffered saline (PBS, 1:40) and then applied to antigencoated slides (pooled antigens of Karp, Kato, and Gilliam OT strains). The slides were incubated in a humidified chamber at 37 ◦ C for 30 min. A secondary antibody mixture of fluorescein isothiocyanate-conjugated goat-anti-rat IgG + A + M (H+L) antibodies (Zymed Laboratories, Inc., San Francisco, CA, USA) diluted 1:100 in PBS were then added to the slides. The reaction was scored as positive when bright green fluorescence was observed using a fluorescence microscope (Olympus IX71, Tokyo, Japan). 2.4. Primers and PCR amplification Nested PCR was performed to detect OT DNA in the DNA extracts. The primer sequences were designed based on the DNA
P.-R. Lin et al. / Acta Tropica 131 (2014) 117–123
119
Table 1 Rodent species captured by gender in Taiwan during the period 2006–2010. Rodent species
Rattus flavipectus R. norvegicus R. losea R. rattus mindanensis Mus musculus Mus caroli Apodemus agrarius Bandicota indica R. exulans R. rattus Suncus murinus Total
Rodent numbers
Male
Female
67 79 91 51 2 8 2 24 18 1 211 554
67 69 103 63 2 5 2 26 9 1 160 507
Total
Percentage of total (%)
134 148 194 114 4 13 4 50 27 2 371 1061
12.6 14 18.3 10.8 0.4 1.2 0.4 4.7 2.5 0.2 35
sequence from the Karp strain (GenBank accession no. AY956315), which amplified the 56-kDa type-specific antigen, as described previously (Furuya et al., 1993; Horinouchi et al., 1996). The two pairs of primers used were as follows: pair 1, comprising F6 (5 -GTTGGAGGAATGAATTACTGG-3 ) (nucleotides 406–425) and R6 (5 -AGCGCTAGGTTTATTAGCAT-3 ) (nucleotides 1059–1040), and pair 2, comprising F7 (5 -AGGATTAGAGTGTGGTCCTT-3 ) (nucleotides 369–388) and R7 (5 -ACAGATGCACTATTGGCAA-3 ) (nucleotides 1175–1156). DNA with a predicted length of approximately 0.7 kb was visualized with a UV transilluminator after agarose gel electrophoresis and staining with ethidium bromide (Lin et al., 2011; Fig. S1). 2.5. Human cases of scrub typhus in Taiwan Reported human cases of scrub typhus during the period 2006–2010 in Taiwan were anonymously retrieved from the Database of the Centers for Disease Control, 2011, Taiwan (http://nidss.cdc.gov.tw/NIDSS). The population data was retrieved from the website of the Department of Statistics, Ministry of the Interior, Taiwan (http://sowf.moi.gov.tw/stat/month/list.htm) (Fig. 1). 2.6. Statistical analysis The results are presented as means ± standard errors of means (SEM). Student’s t tests were conducted using Microsoft Excel 2007. P values less than 0.05 were considered statistically significant. Pearson correlation analysis was used to evaluate the relationships between the variables. 2.7. Ethics statement This research was approved by the Institutional Animal Care and Use Committee of the Institute of Preventive Medicine, permission number IPM-98-A1. The animal experiments were performed according to the guidelines of the Council of Agriculture, Executive Yuan, Taiwan. The data regarding human cases of scrub typhus was retrospectively and anonymously analyzed. 3. Results 3.1. Collection of field rodents During the study period, we captured 1061 small mammals including 10 species of rodents and one species of shrew in the six study districts (Table 1). Suncus murinus accounted for 35% (n = 371) of the captures and was the predominant shrew species.
Fig. 2. Geographic distribution of prevalence of Orientia tsutsugamushi infection in rodents and incidence (IR) of human scrub typhus. Line graph indicating the prevalence of OT infection in samples collected from the small mammals, as determined by the presence of OT-specific antibody in sera and bacterial DNA in spleen and chiggers, respectively, using IFA and nested PCR. Gray bar chart indicates the incidence (IR) of human scrub typhus for each study sites. a IR is expressed as annual scrub typhus cases (mean values of 2006–2010)/population in log10 .
Rattus losea (n = 194, 18.3%) was the most common and abundant rodent species found in this study. Rattus flavipectus (n = 134, 12.6%) and Rattus rattus mindanensis (n = 114, 10.8%) were the predominant rodent species exclusively present on Kinmen and Lan-yu islands, respectively. Overall, there was no significant difference in sex (approximately 1.1; 554/507) between male (48.9%) and female (49.8%) animals, respectively.
3.2. OT infection in chiggers and field rodents Results of the nested PCR studies to detect OT in the rodent chiggers and spleen samples are shown in Tables 2 and 3 respectively. Approximately 35% (371/1061) of animals were infested with chiggers. A heavy infestation in R. rattus mindanensis (91.2%; 104/114) and R. flavipectus (76.9%; 103/134), and moderate infestation in R. losea (40.2%; 78/194) were seen. No chigger positive rodents contained fewer than 10 chigger mites. As no characterization was carried out, detailed mite species were not known. In addition to chigger mites, other ectoparasites such as ticks, and fleas, alone or in combination, were collected from rodents. Tick infestation of rodents ranged from 10 to 24%, and flea infestation was
