Exp Appl Acarol DOI 10.1007/s10493-015-9896-1

Identification of intestinal bacterial flora in Rhipicephalus microplus ticks by conventional methods and PCR– DGGE analysis Xing-Li Xu1,2 • Tian-Yin Cheng1 • Hu Yang2 Fen Yan1

•

Received: 19 May 2014 / Accepted: 5 March 2015 Ó Springer International Publishing Switzerland 2015

Abstract In this study, we have analyzed the intestinal microbial flora associated with Rhipicephalus microplus ticks using both culture-dependent and independent methods based on PCR and denaturing gradient gel electrophoresis (PCR–DGGE). The R. microplus ticks were collected from cattle and goats in Jiangxi, Hunan and Guizhou Provinces of China. Three distinct strains of bacteria were isolated using culture-dependent methods: Staphylococcus simulans, Bacillus subtilis and Bacillus flexus strain. Nineteen distinct DGGE bands were found using PCR–DGGE analysis, and their search for identity shows that they belonged to Rickettsiaceae, Xanthomonadaceae, Coxiella sp., Ehrlichia sp., Pseudomonas sp., Ehrlichia sp., Orphnebius sp., Rickettsia peacockii, Bacillus flexus. Rickettsia peacockii and Coxiella genus were the dominant strain of the R. microplus ticks from cattle, Pseudomonas sp. and B. flexus strain were the most common species in all tick samples from goats. Ehrlichia canis were detected only in R. microplus ticks from Yongshun area in Hunan Province. The results indicate that the intestinal microbial diversity of R. microplus ticks was influenced by tick hosts and local differences in the sampling location and these two aspects may affect transmission of pathogen to humans and animals.

& Tian-Yin Cheng [email protected] Xing-Li Xu [email protected] Hu Yang [email protected] Fen Yan [email protected] 1

College of Veterinary Medicine, Hunan Agricultural University, Changsha City, Hunan Province, China

2

College of Life Science and Resource Environment, Jiangxi Yichun University, Yichun City, Jiangxi Province, China

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Keywords

Endosymbionts  Intestinal microbiota  DGGE  Diversity

Introduction Ticks are associated with a variety of bacteria, viruses, and protozoa, and they are regarded as important vectors of zoonose disease. Rhipicephalus microplus (Acari: Ixodida), known as the cattle tick, is the main blood-sucking ectoparasite of bovines in many countries around the world. The tick causes major economic losses on account of the transmission of diseases and reducing animal production. Ticks harbour a variety of endosymbionts such as Coxiella-, Francisella-, Rickettsiaand Arsenophonus-like symbionts as well as Candidatus Midichloria mitochondrii and Wolbachia, which are important in determining the transmission of pathogens (Ahantarig et al. 2013). In addition to bacterial endosymbionts, ticks are known to transmit many pathogenic bacteria that contain species in the genera Borrelia, Ehrlichia, Rickettsia, Anaplasma, Francisella, Coxiella and so on. These pathogen can cause tick-borne zoonoses, endangering human and animal health. For example, in Europe, ticks are known to transmit diseases including mediterranean spotted fever and tick-borne lymphadenopathy, caused by Rickettsia sp. (Oteo and Portillo 2012). Therefore, it is important to investigate the composition of the microbial population associated with tick and their relationship and understanding of the host pathogen-interactions would lead to effective prevention of the disease transmission. Methods for analysis of tick microbiota include molecular tools and culture-dependent methodology. Next-generation sequencing (NGS) as an efficient approach has been successfully used to identify the bacterial communities in ticks (Williams-Newkirk et al. 2012; Vayssier-Taussat et al. 2013; Zhang et al. 2014). Considering the high cost associated with NGS, DGGE profiling has proven to be an economical and effective tool to analyze the intestinal microbial diversity in many animals (Ariefdjohan et al. 2010; Li et al. 2012a, b). Temporal temperature gradient gel electrophoresis (TTGE) is a variation of DGGE, which has also been used to detect the microbial populations of ticks (Moreno et al. 2006; Halos et al. 2006). It was suggested that single molecular method were not sufficient to analyze the gut bacterial diversity, because they will not be identified owing to PCR bias when there are too few bacteria (Li et al. 2012a, b). Culture-dependent techniques typically can find a minor portion of the bacteria. A total of 151 bacterial strains were isolated using several kinds of culture media from Ixodes ricinus, Dermacentor reticulatus and Haemaphysalis concinna ticks (Rudolf et al. 2009). Up until now, the bacteria of several ticks were investigated by using DGGE technique (Schabereiter et al. 2003; Van Overbeek et al. 2008; Tveten 2013; Tveten et al. 2011, 2013) or culture-dependent method (Rudolf et al. 2009; Murrell et al. 2003; Egyed and Makrai 2014), but information on the microbial diversity of R. microplus ticks is scanty. In this study, we investigate the bacterial communities of R. microplus ticks using a combination of the culture-dependent and the culture-independent method of PCR–DGGE for the first time. There is evidence that different habitats (Van Overbeek et al. 2008) and animals (Kittelmann and Janssen 2011) can influence the distribution of pathogens in tick. The arthropod gut is considered to be a pivotal microbial entry point (Narasimhan et al. 2014). In the present work, the intestinal flora of R. microplus ticks from different hosts (goat and cattle) and different habitats in China were compared and analyzed, in order to

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investigate to what extent these two factors can influence bacterial composition in R. microplus. In addition, we identified some well-known tick-infecting bacteria, such as members of genus Coxiella, Ehrlichia, Pseudomonas, which highlights the need for further investigation considering the medical and veterinary significance of controling R. microplus ticks.

Materials and methods Collection of ticks A total of 145 adult female R. microplus ticks were collected from Jiangxi, Hunan and Guizhou Province of China. Feeding ticks were individually picked from cattle and goats using sterile tweezers. After all ticks were rinsed by 70 % ethanol for about 2 min, placed individually into sterile tubes. In this study, we distinguish between feeding tick from cattle and goats (Table 1), and ticks collected from five different areas: Changsha (C), Xiangxi Yongshun (X-y), Xiangxi Huaheng (X-h) of Hunan Province; Guizhou Province (G); Jiangxi Province (J). (as showed in Fig. 1).

Dissection of tick intestine The abdominal cuticula was rived, and the intestinal sample was immediately placed in microcentrifuge tubes with 1 ml sterile double-distilled (dd) H2O, for bacterial culture and DNA extraction. Total 35 guts were used to culture bacteria, and 110 guts to extract genome DNA. All works were completed under stringent conditions in a biohazard cabinet.

Analysis of the intestinal flora by culturing bacteria Each intestine sample was ground in microcentrifuge tube containing 1 ml sterilized water. Ten-fold serial dilutions of the suspension were prepared, and appropriate dilution of 10-4 was plated on Luria–Bertani (LB) agar media for culturing bacteria. Plates were incubated for 24–48 h at 30 °C. The isolates were identified by their colonial morphology,

Table 1 Grouping of Rhipicephalus microplus ticks in present study Origin (area)

Host

Ticks number

DGGE profiles obtained

Changsha in Hunan Province (C)

Cattle

15

8

Goat

16

5

Cattle

14

8

Goat

12

5

Cattle

15

8

Goat

13

6

Cattle

18

7

Goat

14

5

Xiangxi Yongshun in Hunan Province (X-y) Xiangxi Huaheng in Hunan Province (X-h) Jiangxi Province (J) Guizhou Province (G)

Cattle

15

6

Goat

13

5

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Fig. 1 Sampling distribution of Rhipicephalus microplus ticks from five different geographical areas of three Province (Guizhou, Hunan, Jiangxi) (the left). The right map is the map of China. Hunan located in the Central China region. It borders Jiangxi to the east and Guizhou to the west. Yongshun (X-y) and Huaheng (X-h) located in western area of Hunan Province and the capital is Changsha Table 2 List of primer in 50 –30 orientation Primera

Primer sequence

References

341FGCb

50 -CGCCCGCCGCGCGCGGCGGGCGGGGGGGGGGCACG GGGGCTCCTACGGGAGGCAGCAG-30

Schabereiter et al. (2003)

341F

50 -CCTACGGGAGGCAGCAG-30

Schabereiter et al. (2003)

518R

50 -ATTACCGCGGCTGCTGG-30

Schabereiter et al. (2003)

27F

50 -AGAGTTTGATCCTGGCTCAG-30

Yu et al. (2013)

1492R

50 -GGTTACCTTGTTACGACTT-30

Yu et al. (2013)

a

F, Forward primer; R, Reverse primer

b

The GC clamp was attached to the 50 -end of the primer

biochemical tests and microscope examination (Gram staining). DNA was extracted from every strain using DNeasy bacteria kit according to manufacturers’ protocol (TransGen Corporation, Beijing, China). PCR amplification were performed using 16S rDNA universal primers 27F and 1492R (Table 2). Sequencing of the amplicons carried out by Shanghai Biological Engineering Company. Sequences were compared to those in the GenBank (http://www.ncbi.nlm.nih.gov/blast/) databases using online tools.

Extraction of genomic DNA from ticks DNA was extracted from each gut sample using DNeasy bacteria kit according to manufacturers’ protocol (TransGen Corporation, Beijing, China). The DNA was eluted with 100 ll elution buffer. DNA was analyzed by 10 g L-1 agarose gels with a molecular weight standard and stored at -20 °C until further use.

PCR amplification Primer GC-F341 and R518 (Table 2) were used to amplify V3 regions of gene fragments of 16S rDNA. Reaction mixtures of 50 ll contained 4 ll of genomic DNA, 2 ll of each primer, 25 ll of 2 9 Taq mix, 110 DNA samples were amplified with the following steps:

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initial denaturation for 4 min at 94 °C, denaturation 30 cycles for 30 s at 94 °C, annealing for 30 s at 55 °C, primer extension for 15 s at 72 °C and final extension for 7 min at 72 °C. A negative control was included in all PCR–DGGE experiments. Presence of PCR products were confirmed by electrophoresis on 1.5 % agarose gels stained with ethidiumbromide in 1 9 TAE buffer using a 100 bp DNA mass ladder. Gels were visualized and photographed by UV transillumination.

Denaturing gradient gel electrophoresis The PCR amplicons were subjected to DGGE with a 30–60 % linear denaturing gradient of urea and formamide in a 6 % acrylamide gels according to the manufacturer’s instructions (JunYi, Beijing, China). 5 ll of PCR product along with 1 ll of 6 9 loading buffer was loaded in each lane. Electrophoresis was performed in 1 9 TAE buffer (40 mM Tris, 20 mM acetic acid, 1 mM EDTA, PH8.0 with NaOH) for 4 h at a constant voltage of 120 V at 60 °C. The gels stained with argentation before they photographed by UV transillumination, and then DGGE band recoverd.

Second amplification The selected dominant bands were excised from the gel and eluted in 20 ll sterile water at 4 °C overnight and then frozen at -20 °C. 3 ll of DNA was used as a template and reamplified with the forward primer 341F without GC clamp (Table 2) and 518R by following the program described previously. Each PCR product was also subjected to DGGE analysis to confirm where the bands have been eluted.

Clone library construction for sequencing Purified PCR products were then cloned into the PMD18-T vector (Takara, Dalian, China) for sequencing and introduced into Escherichia coli DH5a by transformation, according to the protocol provided by the manufacturer. The transformed cells were plated onto LB medium (1.0 % Bacto-Tryptone, 0.5 % Bacto-yeast extract, 1.0 % NaCl, 1.5 % Bacto agar, pH 7.0) containing ampicillin and X-Gal (5-bromo-4-chloro-3-indolyl-b-D-galgactopyrano-side: 0.1 mM) to identify white-colored recombinant colonies. The clones were selected for sequencing. All sequencing was conducted at the Shanghai Sangon Biological Engineering Technology and Service Co., Ltd. in China.

DGGE profile analysis Each sequence was compared to sequences of known bacterial species in the BLAST database. The fringerprints of the DGGE profile were analyzed using the Quantity One analysis software version 4.6.2. Comparisons between different animals and different regions were performed. The number of bands in every DGGE profile was determined as an indicator of richness.

Results Analysis of Rhipicephalus microplus ticks in culture Three different bacterial strains were isolated by using culture-dependent method, combined with partial amplification of 16 s DNA with specific primers. The sequence of the

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amplified products were blast-searched against the Genbank which showed that they belonged to Staphylococcus simulans, Bacillus subtilis and Bacillus flexus strain. The corresponding results of identity analyses were 99, 97, and 99 % respectively. These three bacteria were found from different areas of cattle- and goat-collected R. microplus ticks. Bacteria S. simulans were detected in tick samples from J, C, X-y and X-h. The bacteria B. subtilis was isolated from ticks samples collected from cattle. Gram staining revealed that they were Gram-positive, and three different colony morphology isolates were observed under microscope (Table 3). Ten different biochemical tests were developed to aid identification of these strains, the results are shown in Table 3.

Bacteria diversity determined using PCR–DGGE analysis DGGE pattern of intestinal flora in Rhipicephalus microplus ticks from different hosts PCR amplification was performed on the total DNA extracted from gut samples of R. microplus ticks by using primer GC-F341 and R518. Presence of PCR products were confirmed by electrophoresis on 1.5 % agarose gels. A total of 19 distinct bands were identified from all PCR–DGGE profiles, and the bands were present at different positions in the Fig. 2. Figure 2a shows the DGGE fringerprint of tick samples collected from cattle. Pseudomonas sp., Coxiella endosymbiont, Rickettsia peacockii and Xanthomonadaceae were occurred in all tick samples collected from cattle. Coxiella sp. (Band 4) and Rickettsia peacockii (Band 7) were the dominant strain of these tick samples. Figure 2b shows the DGGE fingerprint of tick samples collected from goats. Uncultured bacterium, Pseudomonas sp. and B. flexus strain were the most common species in tick samples collected from goats. Prominent bands in

Table 3 Bacterial morphology, colony characteristics and the biochemical tests of intestinal bacteria in Rhipicephalus microplus ticks The biochemical tests; bacterial modality; colony characteristics

Strain name Staphylococcus simulans

Bacillus subtilis

Bacillus flexus

Nitrate reduction

?

?

-

L-pectinose

-

?

-

Oxidase

-

?

?

Glucose

?

?

?

Gelatin liquefaction

ND

?

?

Catalase

?

ND

ND

Amylohhydrolysis

-

?

?

Mannitol

?

?

?

VP test

-w

?

-

Gram stain

G?

G?

G?

Arginine double hydrolysis enzyme

?

-

-

Mycelium form

Bulbiform

Rod-shaped

Rod-shaped

Colony characteristics

Milk white, round, regular edge, raise,smooth, moist

Ough, opacity, incanus or yellowish

Round, creamy yellow, irregular edge, opaque

?, Positive; -, negative; ND, undetermined; -w, from negative to faintly reaction

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Fig. 2 PCR–DGGE profile of intestinal flora in Rhipicephalus microplus ticks from different hosts. a The sample collected from cattle; b The sample collected from goats. Jiangxi Province (J), Guizhou Province (G), Changsha in Hunan Province (C), Xiangxi Yongshun in Hunan Province (X-y), Xiangxi Huaheng in Hunan Province (X-h)

these tick samples were related to Uncultured bacterium (band12, 19) and Pseudomonas sp. (Band15). The results of the percentage abundance of different classes of bacteria in R. microplus ticks from different hosts are shown in Fig. 3. Figure 3a shows the percentage abundance of bacteria in tick samples collected from cattle. The identified bacteria in these samples were mainly grouped into nine different classes: Xanthomonadaceae, Rickettsiaceae, Ehrlichia sp., Pseudomonas sp., Orphnebius sp., Coxiella sp., Rickettsia peacockii, Ehrlichia canis and Uncultured bacterium. Figure 3b shows the percentage abundance of different classes of bacteria in tick samples collected from goats. The Phyla of the identified bacteria in samples from goats were less than those of the samples from cattle. The identified bacteria in samples from goats were mainly assigned to four different phyla: Uncultured bacterium, Rickettsiaceae, Pseudomonas sp. and B. flexus. Rickettsiaceae was

Xanthomonadaceae

90% 80% 70% 60% 50% 40% 30% 20% 10% 0%

Orphnebius Ehrlichia Rickettsia peacockii pseudomonas Ehrlichia canis coxiella Rickettsiaceae Uncultured bacterium

C

X-y

X-h

cattle

J

G

B 100% Relative abundance

Relative abundance

A100%

90% 80% 70% 60% 50% 40% 30% 20% 10% 0%

Bacillus flexus Pseudomonas Rickettsiaceae Unculture bacterium

C

X-y

X-h

J

G

goat

Fig. 3 Percentage abundance diagram of different classes of bacteria in Rhipicephalus microplus ticks from different hosts. a The sample collected from cattle; b the sample collected from goat. Jiangxi Province (J), Guizhou Province (G), Changsha in Hunan Province (C), Xiangxi Yongshun in Hunan Province (X-y), Xiangxi Huaheng in Hunan Province (X-h). Percentage distribution was calculated on the basis of relative abundance in the total of PCR amplification

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mostly found in samples from J and G. These results demonstrated that the diversity in the intestine of R. microplus ticks from different hosts were significantly different.

DGGE pattern of intestinal flora in Rhipicephalus microplus ticks from different areas Bacterial diversity differed significantly among different areas. The core microbiome was detemined by comparing all samples from different regions, and the Venn diagram (Fig. 4) was constructed based on Fig. 2. According to different areas, the bands were analyzed, the results show that the bacterial diversity in the sample from Xiangxi Huaheng was the highest in all tick samples. From Fig. 4, bands 4, 6, 7, 10, 11, 12, 15, 17 and 19 were the most common microorganisms, found in all five regions. Many bands were only present in one area, for example, Bands 5 was detected in samples from Yongshun area of Xiangxi only, meanwhile there were some bands that present in two or three regions, such as bands 16, 2, 3, 9 and 14. These results demonstrated that the diversity in the intestine of R. microplus ticks from different areas were significantly different.

Identification of DGGE bands from Rhipicephalus microplus ticks samples To identify the intestinal flora in R. microplus ticks, the bands cut from DGGE gel profiles were sequenced and the results are displayed in Table 4. The majority of the bands (except for Band 19) showed more than 97 % similarity with known sequences in the BLAST database. Among the detected DGGE bands, four bands were not cloned or sequenced (Band 1, 13, 16, 18) and fifteen bands were successfully sequenced. To sum up, some bands (such as Band 5, 7) were identified at the species level, others bands (such as Band 6, 9) were only identified at the genus level. Their sequences blasting analysis shows that they

Fig. 4 Venn diagram of the intestinal core microbiomes according to the different areas. 1–19: band number labeled in DGGE gel

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Exp Appl Acarol Table 4 Nearest match of bacterial 16S rDNA sequences isolated from midguts of Rhipicephalus microplus ticks in GenBank Areasc

Band no.

Sequence size

Closest related sequencea

Similarity (%)

Accession numberb

2

156

Uncultured bacterium

98

GQ351496.1

J, C, X-y

Cattle

3

164

Rickettsiaceae bacterium

99

JQ700915.1

J, C, X-h, X-y

Cattle

4

167

Coxiella endosymbiont

99

JQ480818.1

J, C, X-y, X-h, G

Cattle

5

156

Ehrlichia canis strain 222

97

KC479024.1

X-y

Cattle

Host

6

155

Pseudomonas sp.

99

FR870455.1

J, C, X-y, X-h, G

Cattle

7

156

Rickettsia peacockii

99

U55820.1

J, C, X-y, X-h, G

Cattle

8

155

Ehrlichia sp. strain Jena

98

AJ312939.2

X-h

Cattle

9

150

Orphnebius sp.

97

JN581837.1

X-h, G

Cattle

10

153

Xanthomonadaceae

99

EU520432.1

J, C, X-y, X-h, G

Cattle

11

155

Pseudomonas sp.

98

FR870455.1

J, C, X-y, X-h, G

Cattle

12

166

Uncultured bacterium

98

GQ289435.1

J, C, X-y, X-h, G

Goat

14

164

Rickettsiaceae bacterium

99

JQ700915.1

J,G

Goat

15

163

Pseudomonas sp.

99

FR870455.1

J, C, X-y, X-h, G

Goat

17

168

Bacillus flexus strain

99

HM451429.1

J, C, X-y, X-h, G

Goat

19

165

Uncultured bacterium

94

GQ289422.1

J, C, X-y, X-h, G

Goat

a

Closest related sequence indentified by BLAST search

b

GenBank accession number of closest identified strain

c

Jiangxi Province (J), Guizhou Province (G), Changsha in Hunan Province (C), Xiangxi Yongshun in Hunan Province (X-y), Xiangxi Huaheng in Hunan Province (X-h)

belonged to Rickettsiaceae, Xanthomonadaceae, Coxiella endosymbiont, Pseudomonas sp., Ehrlichia sp., Orphnebius sp., Ehrlichia canis, Rickettsia peacockii, B. flexus and Uncultured bacterium. Band 6, 11 and 15 were related to Pseudomonas sp., and Bands 8 was assigned to Ehrlichia. sp.

Discussion In our study, a combination of the conventional culture and DGGE analysis was used to compare the intestinal microbial diversity of R. microplus ticks from different areas, different hosts. The results show that Rickettsiaceae, Coxiella sp., Ehrlichia sp., Rickettsia peacockii were found in the intestine of R. microplus ticks. Common bacteria identified in R. microplus ticks have been described in other studies of tick-associated bacterial communities (Murrell et al. 2003; Tveten and Sja˚stad 2011; Maia et al. 2014; Schabereiter et al. 2003). Staphylococcus and Xanthomonas detected in this study have been isolated from I. ricinus, D. reticulatus and H. concinna ticks (Rudolf et al. 2009). Pseudomonas sp. was identified in all samples, it has also been found in a study of microbial communities in I. ricinus ticks from Northwest Norway (Tveten et al. 2013). Our findings are thus in agreement with these previous reports. To our knowledge, the presence of Orphnebius sp. in the present paper have not been detected in R. microplus ticks before. The results show that S. simulans strains only appeared in the culture-dependent method, and they were not detected by DGGE methods, indicating that there were less

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amounts of S. simulans strains in R. microplus ticks, so that they were not found by DGGE analysis. In addition, some bacteria (such as Coxiella sp. and Pseudomonas sp.) only appeared in the DGGE profile, however, these strains were not isolated by the culturedependent method. Only three bacterial strains (S. simulans, B. subtilis and B. flexus strain) were isolated using culture-dependent method. These may be due to the low diversity of bacteria isolated by culture-dependent method, it is associated with the culture media used, which may affect the culturability of bacteria. The optimization of culture conditions and medium composition is very important for the growth of bacteria. In this study, since the nutrients of LB agar media for intestinal flora may be other than common bacteria, so few bacteria were detected in culture. The differences between culture-dependent method and PCR–DGGE technology may result in the fact that only B. flexus strain was detected by the two techniques. These results are consistent with the findings reported by others. Xu et al. (2013) showing that there were differences for detection of heterogeneity of Borrelia burgdorferi in Ixodes ticks by culture-Dependent and culture-independent methods. A single LB agar media was used for culturing bacteria, which could decrease the quantity of same bacteria. It is necessary that using kinds of culture mediums to identify bacteria, and then comparing the result of the two methods. The traditional culture method and DGGE method have their own pros and cons in analyzing microbial diversity. DGGE analysis has the advantages such as reproducibility, rapidity, reliability (Kusˇar and Avgusˇtin 2012) and allows screening of multiple samples, but shortcomings include: requiring specialized equipment, choice of primers, effect of variable DNA extraction efficiency on DGGE profiles (Theron and Cloete 2000). While traditional culturing technique has superiority in some aspects, for example, allowing the recovery of potentially relevant strains, there are some disadvantages associated with the methods as well. It is a time-consuming, complex process and many microorganisms that exist in nature are not amenable to culturing. The use of these two methods synthetically may provide a better overview of the microbial communities in ticks. In this study, the effect of host species on intestinal flora of R. microplus ticks were investigated. Rickettsia peacockii and Coxiella sp. were the dominant strains of the R. microplus ticks from cattle (Fig. 3a), while Uncultured bacterium and Pseudomonas were the most abundant in all samples from goats (Fig. 3b). This may indicate that the bacterial communities of ticks changes with different hosts, which agrees with previously published data reported by others (Kittelmann and Janssen 2011), who examined potential effects of ruminant species on the ciliate community structure in sheep, red deer, and cattle. It turned out that sheep were more similar to deer than they were to cattle. In a study by Alessandra and Santo (2012), ruminants samples were tested in several Italian regions from 2004 to 2009, Anaplasma ovis was the main etiological agent of anaplasmosis with a different prevalence for different hosts (82.9 % for sheep and 74.9 % for goats). In general, our results show that DGGE fingerprints from ticks collected from goats included fewer bands than that of the samples from cattle (Fig. 2a, b). These changes may be connected with host diet and physiological environment. We also examined the effect of natural habitats on intestinal flora of R. microplus ticks. It turned out that geographic differences in regions might be one of the factors producing bacterial diversity in these ticks. Different geographic environments lead to the diversity of vegetation type in five areas. Variation in vegetation determines the occurrence of local tick hosts, which has a big impact on tick-associated bacterial communities. Our findings are consistent with previous study (Van Overbeek et al. 2008). In a study on I. ricinus in four different areas in the Netherlands in the period from 2000 to 2004, Borrelia-,

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Anaplasma- and Ehrlichia-species infection rates in the ticks varied substantially for the four areas and there existed obvious space–time difference (Wielinga et al. 2006). In this study, because distances between locations in five areas are very close, there was little variation in tick bacterial composition, except that bacterial diversity in R. microplus ticks collected from region X-h were more abundant than that of other regions. In summary, culture-dependent method and DGGE profiling were used to identify microorganisms associated with R. microplus ticks. The bacterial diversity in R. microplus ticks collected from five regions and two hosts were significantly different. The bacterial content of ticks may be somewhat defined by the different environmental conditions, so that the microbial diversity in ticks are dissimilar per habitat. Moreover, variation in animal host species composition could also affect the bacterial communities of ticks. The present study has presented an overview of the microbial community of R. microplus ticks. Further studies will be necessary to assess the effects of bacterial flora on the biology of R. microplus ticks and disease transmission. Acknowledgments This research was financially supported by grant from the National Natural Science Foundation of China (No. 31372431). The authors also thank Ya Yang and Ling-Xuan Qu for their help in sampling.

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