Investigations On Natural Foci And Pathogenic Characteristics Of Two Vector-borne Diseases

Emerging and re-emerging infectious diseases are an important issue in global health.The well-known SARS and highly pathogenic avian influenza cause great hazards insome regions, while the huge hazards from emerging and re-emerging vector-borneinfectious diseases can not be ignored. A disease that is transmitted to humans or otheranimals by an insect or other arthropod is called a vector-borne disease. Manyvector-borne diseases are natural focus diseases, and the etiologic pathogens includeviruses, rickettsiae, bacteria, parasites and so on. Vector-borne diseases are easilyprevalent in nature due to the widely distribution of vectors and reservoir hosts. Theoccurrence of natural disasters and the destruction of the natural environment willaffect the occurrence and prevalence of vector-borne diseases.Because of the complex natural and geographical conditions, diverseenvironment, as well as rich flora and fauna, there are many kinds of vector-borneinfectious diseases in China. Recently, the emergence and prevalence of emergingtick-borne infectious diseases poses a serious threat to public health in China, such asthe emergence of anaplasmosis and the outbreak of severe fever withthrombocytopenia syndrome. Therefore, to find out the natural foci and epidemiologicfeatures, and to understand the pathogenic characteristics and the interaction betweenhosts and vectors, in-depth investigations on emerging and re-emerging vector-borneinfectious diseases should be performed. The findings from these researches willprovide a scientific basis and theoretical support for prevention and control ofdiseases.“Anaplasmosis” is a generic name for infections caused by obligate intracellularbacteria in the family Anaplasmataceae, which is a kind of tick-borne disease andmainly includes human granulocytic anaplasmosis (HGA), human monocyticehrlichiosis and human ewingii ehrlichiosis. An outbreak of HGA including10caseswere reported in Anhui province in2006, and this study suggested human-to-humantransmission of HGA. Subsequently, no confirmed cases have been reported in China.Our previous surveys detected various tick-borne agents in ticks and animal hosts from forest areas of northeastern China, such as Anaplasma, Borrelia and rickettsia.In addition, our laboratory successfully isolated A. phagocytophilum from rodents andsheep in northeastern China, suggesting the potential existence of human cases ofHGA in this area.In the current study, we initiated a surveillance project in Mudanjiang ForestryCentral Hospital, one of the largest hospitals treating patients with tick bites innortheastern China, and found7cases infected with Candidatus Neoehrlichiamikurensis. Field investigation on rodents and ticks confirmed the existence of localnatural foci.When we used rhesus macaques for isolation of Candidatus Neoehrlichiamikurensis, Bartonella quintana infection in the macaques were unexpectedlyobserved. B. quintana was firstly recognized as the pathogenic bacteria of trenchfever. In China, few studies focus on B. quintana, which is recognized as are-emerging vector-borne pathogen now. The study was then aimed to identify theanimal hosts and vectors of B. quintana, and to investigate the genetic characteristicsof this agent.The main results:1Investigations on human infection with Candidatus Neoehrlichiamikurensis in eastern ChinaDuring May-July in2010, a total of628febrile patients who had a history ofrecent tick bites and sought treatment at Mudanjiang Forestry Central Hospital ofHeilongjiang Province were enrolled into the study. The peripheral blood sampleswere collected from the patients on acute phase of the illness. A board-range PCRtargeting rrs gene and a species-specific PCR targeting groEL gene were used todetect the infections of tick-borne agents. As a result,7patients were found to beinfected with Candidatus Neoehrlichia mikurensis through the amplifications of bothrrs and groEL. The amplified nearly entire rrs (1501bp) and partial groEL (914bp)nucleotide sequences from the7patients were identical, and same to those ofCandidatus Neoehrlichia mikurensis detected from I. persulctus ticks and field micein the Asian part of Russia.Clinical symptoms of fever, headache and malaise were present in all7patients.Other major manifestations included nausea (5/7), vomiting (5/7), myalgia (4/7) andstiff neck (4/7). The less common symptoms were arthralgias (2/7) and cough (2/7),diarrhea (1/7), confusion (1/7) and erythema (1/7). Laboratory test results showed leucopenia in one patient, leukocytosis in one patient, thrombocytopenia in twopatients and anemia in two patients; serum levels of alanine aminotransferase andaspartate aminotransferase within reference ranges in all patients. Unlike previouslyreported cases with old age or immunocompromised conditions who developedoverwhelming disease, all7patients in the current study presented with relativelymild disease. The clinical manifestations might be typical of Candidatus Neoehrlichiamikurensis infection in otherwise healthy population.A total of516host-seeking ticks, including316Ixodes persulcatus,187Haemaphysalis concinna and13Dermacentor silvarum, were collected on vegetationand individually examined. Candidatus Neoehrlichia mikurensis DNA was detected in6I. persulctus and2H. conicinna with an overall infection rate of1.6%, whereas noDNA from the agent was detected in D. silvarum. A total of211wild rodents from6species were captured and tested. Eight rodents from3species, includingClethrionomys rufocanus, Rattus norvegicus and Tamias sibiricus, were foundnaturally infected, with an overall infection rate of3.8%. These results suggest theexistence of natural foci of Candidatus Neoehrlichia mikurensis in Mudanjiang Cityof Heilongjiang Province.2Prevalence and genetic diversity of Candidatus Neoehrlichia mikurensis inrodents from ChinaTotally,841rodents of22species from10genera were collected at15study sitesin11regions at local peak seasons of tick activities. The predominant species wereApodemus agrarius (14.6%) and the other species accounted for0.8%-10.3%. Eachregion had3-9species of rodents sampled. Candidatus Neoehrlichia mikurensis wasdetected in34(4.0%) of the841rodents by amplifications of both groEL and rrs. The34rodents with positive results represented9species, with the prevalence in eachspecies ranging from1.1%to25.0%. When each rodent species was compared withall others, the infection rates in Eothenomys custos (25.0%), Apodemus agrarius(12.0%) and A. sylvaticus (12.5%) were significantly higher. Candidatus Neoehrlichiamikurensis was detected in10of the15study sites, representing Heilongjiang, Jilin,Henan, Zhejiang, Fujian, Yunnan Provinces and Inner Mongolia Autonomous Region.The prevalence of Candidatus Neoehrlichia mikurensis in the7regions ranged from1.5%to13.8%and showed significant difference. Rodents collected from JilinProvince were at significantly higher risk for infection in contrast to other regions(P<0.001). Sequence comparisons revealed three different Candidatus Neoehrlichiamikurensis variants that were consistent with the three geographic regions, NortheastChina, Southwest China and Southeast China. Phylogenetic analysis based on allavailable and comparably long groEL nucleotide sequences (874bp) revealed fourdistinct clusters of Candidatus Neoehrlichia mikurensis strains that related togeographic origins. Sequences obtained from Northeast China were identical to thosefrom the Asia part of Russia and included in Cluster I. Sequences obtained fromSouthwest China and Southeast China were separately clustered in Cluster III andCluster IV. No sequence of the present study was identified in Cluster II, which wasexclusively restricted to Europe. Similar to the phylogenetic analysis of groELsequences, neighbor-joining tree based on all available and nearly entire rrs (1303bp)also separated these sequences into four different clusters.3Investigations on infection and transmission of Bartonella quintana inmacaquesFour of10one-year old male macaques housed in the Laboratory Animal Centerof the Academy of Military Medical Sciences were found to be infected with B.quintana by PCR, blood smear staining, transmission electron microscopy and bloodculture. Then, we performed an active surveillance of five primate centers acrossmainland China to identify prevalence and distribution of B. quintana infection incaptive monkeys. Blood samples were collected from328rhesus macaques and308cynomolgus macaques. A total of96(15.1%) macaques were positive for B. quintanaby PCR assay, including59rhesus macaques and37cynomolgus macaques; B.quintana isolates were successfully cultured from the blood of8.3%(53/636) of themacaques tested, including37rhesus macaques and16cynomolgus macaques.Prevalence of B. quintana in rhesus macaques was significantly higher than that incynomolgus macaques. B. quintana was detected in the macaques from each region,but the monkey colony from Nanyang City was at significantly higher risk (P<0.001).In both rhesus macaques and cynomolgus macaques, the B. quintana infection rates injuvenile (≤3years) and young animals (4~6y) were significantly higher than those inadult animals (≥7y)(P<0.05). No significant association was observed between theinfection and gender of the monkeys (P>0.05).In three consecutive screening tests of a group of10monkeys, an increasedprevalence of B. quintana was observed with4of them being found positive at day1,7positive at day15, and all ten positive at day35during the period of observation. All10monkeys were found to be infested with lice at the last day of the observation(day36). Lice from each infested monkey were positive for detection of B. quintanaDNA, suggesting the lice might act as efficient vectors. By phylogenetic analysis andmorphologic features, the lice were identified as Pedicinus obtusus.After intravenous inoculation of the Bartonella-negative macaques with B.quintana strain RM-11, the macaques presented a long-lasting chronic bacteremia, butdid not show abnormal clinical features. In addition, we found4workers involvedwith the macaques care showed seroconversion to antigens derived from our strain,suggesting that macaques could be a source for B. quintana human infection.4Clonal and phylogenetic analysis of B. quintanaThe current study identified44new nucleotide variable sites,25newly allelesand14novel sequence types (STs8-21) among53B. quintana isolates by MLSTanalysis. In contrast with the relatively low level of sequence divergence of B.quintana reported in humans, our investigation revealed much higher genetic diversityin non-human primates. To display the relationships between related STs, all macaqueisolates were analysed by eBURST v3in combination with the data from humanisolates. Two novel clonal complexes and three new singletons were found.Phylogenetic analysis based on the concatenated MLST sequences revealed3distinctclusters. Sequences (STs1–7) from B. quintana isolates recovered from humans wereincluded in Group1. Sequences (STs8–14) from16isolates cultured fromcynomolgus macaques belonged to Group2. Sequences (STs15–21) from37isolatescultured from rhesus macaques were found exclusively in Group3, which displayed ahigher homology to Group2than to Group1. The clustering of concatenatedsequences resembles the relationship between STs and host species, suggesting thegenetic diversity and host-cospeciation among the B. quintana populations. Inaddition, splits decomposition analysis and phi test were performed using Splitstree4,and revealed no apparent recombination among B. quintana isolates.To further investigate genetic characteristics, the complete genome of B.quintana strain RM-11, which was isolated from rhesus macaques, was obtained by acombined strategy involving Solexa and Roche/454. A total of1204predicted proteincoding genes,42tRNA genes, and two copies each of the5S,16S, and23S rRNAgenes were identified. The genome sequence and annotation information areaccessible in the GenBank database with accession number CP003784. Using areciprocal best BLAST hit strategy, we identified a total of1096common genes between strains RM-11and Toulouse, a human B. quintana isolate whose genome hasbeen sequenced.The main conclusions:This study is the first report on human infection of the Candidatus Neoehrlichiamikurensis variant in the world other than Europe. Field survey on ticks and rodentssuggested that the existence of natural foci of Candidatus Neoehrlichia mikurensis innortheastern China. We detected Candidatus Neoehrlichia mikurensis in diversespecies of rodents from various regions, indicating the agent is widely distributed inChina. We also found that the Candidatus N. mikurensis gene clusters correlated withdistinct geographic origins.The study demonstrated the prevalence of B. quintana infection was common inmacaques from main primate centers in mainland China. We firstly reported thetransmission of B. quintana in macaques, and identified Pedicinus obtusus lice as thevectors. These results indicated that macaques may serve as reservoir hosts for B.quintana. Serologic test suggested that macaques could be a source for B. quintanahuman infection. MLST analysis of the B. quintana isolates revealed14novelsequence types, suggesting much higher genetic diversity in non-human primates thanin humans. Phylogenetic analysis provided the evidence of host-cospeciation of B.quintana. These findings suggested that trench fever may primarily be a zoonoticdisease with macaques as the natural hosts. In addition, we sequenced the genome of aB. quintana strain, which was isolated from the blood of a rhesus macaque.