重庆快乐十分

Genetic diversity of Bartonella quintana in macaques suggests zoonotic origin of trench fever

基因多样性

Bartonella quintana has been isolated from a cynomolgus monkey imported to the USA from South-East Asia(O’Rourke et al。2005)and more recently from2captive rhesus macaques in China (Huang et al。2011)。Previous experimental studies involving inoculation of rhesus macaques with B。quintana isolated from human patients could result in chronic bacteremia in the animals(Mooser&Weyer 1953;Zhang et al。2004)。Thesefindings suggested a possibility that nonhuman primates might serve as reservoir hosts for B。quintana。However,there has been no other documented evidence of B。quintana in monkey populations,including infection prevalence and distribution。

Multilocus sequence typing(MLST)is a powerful approach for studying the evolution,epidemiology and population/landscape genetics of microorganisms based on comparison of nucleotide sequences derived from the internal fragments of housekeeping genes (Maiden2006;Margosa et al.2008;Unemo&Dillon 2011;Achtman et al.2012).For the genus Bartonella, MLST wasfirst applied to the study of B.henselae pop-ulation structure,revealing a considerable genetic diversity among isolates from the feline reservoir,but a relative homogeneity among isolates from humans(Ire-dell et al.2003;Arvand et al.2007;Yanagihara et al. 2010).In2010,Arvand et al.developed a MLST scheme for B.quintana based on the nucleotide sequences of9 genetic loci and a wide spectrum of B.quintana isolates, mostly recovered from human patients sampled from3 continents over60years(Arvand et al.2010).This analysis revealed7relatively similar sequence types (STs)(Arvand et al.2010),thus suggesting a relatively low level of sequence divergence comparing with B.henselae.

For other Bartonella species,animal reservoir hosts, acting as healthy carriers,usually play a more impor-tant role in the contemporary expansion and homogeni-zation of the bacteria than humans.Hence,we expect that a broadened MLST analysis of B.quintana isolates from nonhuman primates can help to enhance our understanding of population structure of the bacteria and their evolutionary relationships with hosts.As B.quintana is recognized as a genomic derivative of B.henselae(Alsmark et al.2004),we presumed that the genetic diversity of B.quintana might be higher in its animal reservoirs,an observation that would be similar to ones made for B.henselae.

In this study,we performed an active surveillance of major primate centres across mainland China to identify prevalence and distribution of B.quintana infection in captive monkeys and used MLST analysis to investigate the genetic diversity of obtained bacte-rial isolates.Materials and methods

Ethics statement

All animals were handled in strict accordance with ani-mal welfare regulations,and all animal use procedures were taken in accordance with the recommendations of the Weatherall Report on the use of nonhuman pri-mates in research(SDWF2006).All studies and proce-dures were reviewed and approved by the Institutional Animal Care and Use Committee(IACUC)of the Labo-ratory Animal Center at Academy of Military Medical Sciences(IACUC-2012-003).

Sample collection

A total of636blood samples were obtained from2Old World monkey species,the rhesus macaque(Macaca mulatta)and cynomolgus macaque(Macaca fascicularis), that are commonly used for modelling human diseases. In brief,328blood samples were collected from rhesus macaques from4primate centres,which were located in4cities(Nanyang,Nanning,Yibin and Putian Cities) (Fig.1).The other308blood samples were collected from cynomolgus macaques from another primate centre in Haikou City.These primate centres represent major facilities across mainland China that house mon-keys.Approximately2mL of EDTA-anticoagulated blood was collected from each monkey by venipuncture and immediately deep-frozen atÀ80°C until used for subsequent laboratory tests.

重庆快乐十分Bacterial culture

For bacterial isolation,frozen blood samples were thawed at37°C,and subsequently,100l L of the EDTA-anticoag-ulated blood was inoculated onto chocolate agar plates. The plates were incubated at35°C in a5%CO2and water-saturated atmosphere up to5weeks until the cul-ture was deemed negative in the absence of observed bac-teria growth.Each blood sample was plated in duplicate, and colony forming units(CFU)were counted after the five-week observation period.A strain of B.grahamii was cultured as a positive control.Any suspicious colonies were subjected to PCR assay and subsequently confirmed by sequencing.Subcultures of Bartonella colonies from the original agar plates were streaked onto secondary agar plates and incubated with the same conditions until sufficient growth was observed,usually after5–7days. Bartonella DNA detection

Genomic DNA was extracted from200l L of the freeze-thawed EDTA-anticoagulated blood using the

©2013Blackwell Publishing Ltd

2H。L I E T A L。

Genetic diversity of Bartonella quintana in macaques suggests zoonotic origin of trench fever的相关文档搜索

相关文档
甘肃快3 山东11选5开奖 青海福彩网 天津体育彩票网 上海福彩网 上海体彩网 博乐彩票计划群 浙江福彩 上海快3走势图 上海11选5