Co-Infection Of Rickettsiale Bacteria In Hedgehogs From China
Background
Bacteria of the order Rickettsiales (Alphaproteobacteria) are obligate intracellular parasites of eukaryotes. At present, the order contains three established families (Rickettsiaceae, Holosporaceae, and Anaplasmataceae) as well as one proposed family (Candidatus Midichloriaceae). Most described species of Rickettsiales are well known as zoonotic emerging or reemerging pathogens that may cause life-threatening diseases, including rickettsioses, ehrlichiosis, anaplasmosis and scrub typhus in humans and also be linked with devastating agricultural losses by infecting meat and milk producing animals. The number of newly discovered organisms in the Rickettsiales has markedly increased over the last 20 years and even bacteria that had previously been considered nonpathogenic are now associated with human disease. Rickettsiales are associated with a diverse host range, including diverse protists, annelids, arthropods, mammals and birds. Although numerous studies conducted worldwide have focused on the ecology of Rickettsiales, investigation related to vector-pathogen interaction and the role of vertebrate hosts in the maintenance and dissemination of Rickettsiales in nature remain scarce. Previous investigations showed that ectoparasites in hedgehogs carried several rickettsial agents. Rickettsia helvetica, Candidatus Neoehrlichia mikurensis and Anaplasma phagocytophilum were also detected in European hedgehogs (Erinaceus europaeus) in Europe.
Erinaceus europaeus has also been suggested as reservoir hosts for Anaplasma phagocytophilum and different genospecies of the Borrelia burgdorferi sensu lato complex. Thus, hedgehog may play a role in the transmission cycle of Rickettsiale species as well as acting as a reservoir. The aim of this study was to investigate the prevalence of Rickettsia, Anaplasma and Ehrlichia in hedgehogs from central China.
Methods
This study was approved by the Ethics Committee of Wuhan University (2018010). Hedgehogs were handled in accordance with good animal practices required by the Animal Ethics Procedures and Guidelines of the People’s Republic of China.
Hedgehog samples
Hedgehogs (n=52) were captured using traps baited with meat from April to August in 2018 from Wuhan and Xianning cities, Hubei Province of China. The hedgehogs were morphologically identified as Erinaceus amurensis and were further confirmed by sequencing PCR amplified cytochrome b (cytb) gene as described previously. Animals were classified in two age groups: young and adults. Age was estimated from the appearance of the animal, following the criteria set out by Robinson, gender was identified and recorded. Captured hedgehogs were anesthetized by an intramuscular injection (20 mg/kg) of Ketamine. One of the front feet was sterilized immediately after sedation and blood samples were obtained by clipping a nail 3mm short. After sampling all individuals were released back to the wild.
Tick samples
Ticks were also collected from infested hedgehogs and later morphologically identified in our laboratory. Ticks (n=27) collected from hedgehogs were pooled (6 pools in total) and screened for Rickettsia, Anaplasma and Ehrlichia using the same method as described below.
PCR amplification of Rickettsia, Anaplasma and Ehrlichia
Hedgehog blood DNA was extracted with the Qiagen DNA Kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. Blood DNA samples were used as templates for PCR amplification of Ehrlichia, Rickettsia and Anaplasma DNA with primers. Anaplasma 16S rDNA (rrs) gene was amplified with a nested PCR by using the primers Ana1-out and Ana1-in in the first-round reaction, and Ana2-out and Ana2-in in the second-round reaction as described previously.
For Ehrlichia, nested PCR amplification of 16S rRNA, heat sock protein gene (groEL) and citrate synthase gene (gltA) was performed. For detection of Rickettsia, nested PCR amplification of Rickettsia 16S rRNA, citrate synthase gene (gltA), outer membrane protein B gene (ompB) was performed. To avoid contamination, all steps were performed in separate rooms. Negative control with distilled water was run for each reaction.
PCR products were separated with 1. 2% agarose gel electrophoresis and visualized with UV light after ethidium bromide staining. PCR products with expected sizes were excised from gels and extracted using a Gel Extraction Kit (Promega, Madison, WI, USA), which were then cloned into the pMD19-T vector (TaKaRa) for sequencing.
Phylogenetic analysis
All sequences were searched using BLAST in the GenBank database. After alignment by ClustalW with MEGA 7. 0, phylogenetic trees were constructed using the Maximum Likelihood method with the Tamura-Nei model in MEGA7. 0, and the robustness of the trees was tested with 1, 000 bootstrap replications.
Results
Tick index of hedgehogs
A total of 52 hedgehogs were captured and sampled. Ticks were found on 34. 6% (18/52) of hedgehogs with 17. 3% (9/52) infested with Ixodes sinensis, 32. 7% (17/52) with Haemaphysalis longicornis, 15. 4%(8/52) with both tick species. The tick index of hedgehogs was 2-9 per animal, indicating the hedgehogs were highly infested with ticks. One pool (1/6) was tested positive for rrs gene of A. bovis and none was positive for target gene of Ehrlichia and Rickettsia.
Anaplasma in hedgehogs
PCR amplification with Anaplasma rrs primers showed that 13 (25%) hedgehogs were positive. DNA sequences analysis revealed that the sequences were 98. 6 -100% homologous to each other and they were 98. 6-99. 4% similarity with A. bovis (GenBank: KM114612) obtained from monkey (Macaca fascicularis) in Malaysia. Phylogenetic result also showed all sequences from hedgehogs were in the same cluster with A. bovis in Malaysia. No significant differences in A. bovis infection rates were observed between hedgehogs infested with I. sinensis or H. longicornis and those not infested.
Ehrlichia in hedgehogs
PCR amplification with Ehrlichia rrs primers showed that 11 hedgehogs were positive. The 11 rrs positive hedgehogs were further amplified for the groEL and gltA genes. All 11 hedgehogs were positive to the groEL and 2 were gltA positive. BLAST analysis showed that the rrs sequences from hedgehogs were 100% identical to a sequence (KT886409) from ticks in Zhejiang Province in the East China and a sequence (KJ410252) from ticks in Xinjiang Province in the West China. The sequences from hedgehogs were 99. 3% homologous to E. ewingii (U96436). The gltA sequences detected from two hedgehogs were 99. 5% homologous and the groEL sequences detected from 11 hedgehogs were 99. 1-100% to each other. In consistence with the rrs sequences, the gltA and groEL were highly homologous to Ehrlichia detected in ticks from variety places in China including Xinjiang, Zhejiang, and Hubei in China and E. ewingii (92% -100% for groEL and 88. 9%-99. 5% for gltA). Phylogenetic analysis based on the rrs showed that Ehrlichia sequences detected from hedgehogs formed a clade together with uncultured Ehrlichia species from ticks in Zhejiang and Xinjiang provinces and E. ewingii. Phylogenetic analysis of groEL and gltA genes also showed that Ehrlichia detected in hedgehogs clustered together with uncultured Ehrlichia that was previously documented in ticks in Xinjiang, Hubei, and Zhejiang provinces of China and E. ewingii. The results indicated that the Ehrlichia from hedgehogs appeared to be a member of E. ewingii.
The infection rates of hedgehogs
The infection rate of A. bovis and Ehrlichia in hedgehogs was 25% and 21. 2%, respectively. Co-infections were found in 6 hedgehogs (11. 3%). No genomic sequence of Rickettsia was detected in our survey. Discussion: To our knowledge, this is the first of identification of A. bovis, E. ewingii in hedgehogs. These results suggest that hedgehogs could be reservoir hosts for A. bovis and E ewingii.
Economically devastating disease in livestock was usually caused by A. bovis and phylogenetic analysis shows great similarity between A. phagocytophilum and A. bovis. The major clinical symptoms of A. bovis infection include fever, anemia, drowsiness, convulsions, weight loss, and enlargement of lymph nodes. There are many reports of ruminants, raccoon, cats and deer infected with A. bovis worldwide, but information about the epidemiology of this agent is scarce in China, especially among wildlife. Our study revealed that A. bovis infection is common in wild hedgehogs and may be subclinical since all of the molecularly detected agents were found in apparently healthy animals.
The maintenance of Ehrlichia involve complex zoonotic systems including ticks and reservoir hosts. White-tailed deer serve as the natural reservoir host for E. chaffeensis. Little is known about the natural system of E. ewingii, although it is prevalent in dogs throughout the central and southeastern United States and white-tailed deer may also be important reservoirs. Human infections have increasingly reported since the disease became reportable in 2008. Ehrlichia ewingii has not been reported previously in China. Other wildlife, such as raccoons, coyotes, and domestic dogs, have also been implicated in the natural cycles of Ehrlichia. Ehrlichia canis, E. chaffeensis and A. phagocytophilum have been detected in ticks, animals or humans in China. We did not find these pathogens in hedgehogs. Hedgehogs are confirmed host of several tick species in China including Dermacentor sinicus and H. longicornis. In this study we demonstrated that hedgehogs are infested with I. sinensis and H. longicornis ticks. In traditional Chinese medicine, the skin of hedgehogs has long been used in treatments for hemorrhoids, thus this species is frequently taken from the wild and eaten across central China. Also, hedgehogs frequently forage long distances and hibernate near areas where humans and domestic animals live. Hedgehogs may spread ticks and tick bore diseases to humans and animals around and in long distance. We did no found Rickettsia species in hedgehogs, which may be caused by small sample size and need to be further investigated.
Conclusions
We detected A. bovis and E. ewingii in hedgehogs collected from central China, suggesting that hedgehogs may be important in the ecology of these tick borne intracellular bacteria.
