珠江上游地区蚊虫乙型脑炎病毒核酸检测及PrM和E基因序列分析

试验旨在了解珠江上游地区乙型脑炎病毒（JEV）主要传播媒介蚊虫的种类、分布及携带JEV基因型和分子特征。2013年7月在珠江上游地区师宗县采集蚊虫和库蠓标本,并对蚊虫进行分类鉴定;同时采用黄病毒属引物对采集到的蚊虫和库蠓标本进行检测,阳性标本用JEV PrM和E基因特异引物进行扩增、测序,然后采用生物信息学软件进行序列分析。在4个采集点共采集蚊虫3 705只,分别属于库蚊、按蚊、伊蚊和阿蚊4个属的7种蚊虫,牛圈以中华按蚊、骚扰阿蚊为优势蚊种,羊圈以中华按蚊、骚扰阿蚊为优势蚊种,猪圈以三带喙库蚊、中华按蚊和骚扰阿蚊为优势蚊种。采用JEV PrM和E基因特异引物对采集到蚊虫和库蠓标本分162批进行扩增、测序,采用生物信息学软件进行序列分析。结果显示,4株蚊虫携带的病毒均为基因Ⅰ型JEV,与疫苗株SA14-14-2 E蛋白存在15个氨基酸差异位点,在8个影响病毒毒力的重要位点上未发生突变。以上结果提示珠江上游地区存在多种蚊虫种类,三带喙库蚊和中华按蚊是该地区的优势蚊种,三带喙库蚊是当地JEV的主要传播媒介,在当地的媒介中存在基因Ⅰ型JEV流行,而流行株的毒力并未降低。     

Nucleotide Detection of Mosquito Japanese Encephalitis Virus and Sequence Analysis of PrM and E Genes in Upper Pearl River Region

This experiment was conducted to understand mosquito species, distribution of the main vector of Japanese encephalitis virus (JEV), JEV genotype and molecular characteristics in upper Pearl River region. We collected mosquito and Culicoides specimen in Shizong county, upper Pearl River region in July 2013, and these specimens were classified and identified. Meanwhile, JEV nucleotide were detected using PrM and E genes specific primers in 162 pools of mosquito and Culicoides specimen, and the positive specimens were sequencd and analyzed by bioinformatics software. Total 3 705 mosquitoes were collected from 4 collection spots, and the mosquitoes belong to 7 species in 4 generas: Culex, Anopheles, Aedes and Armigeres. In cattle pens, Anopheles Sinensis and Armigeres subalbatus were the dominant mosquito species. In sheep pens, Anopheles Sinensis and Armigeres subalbatus were the dominant mosquito species. And in pig pens, Culex tritaeniorhynchus, Anopheles Sinensis and Armigeres subalbatus were the dominant mosquito species. 4 of 162 pools mosquito and Culicoides were positive detecting by using PrM and E genes specific primers. Sequence analysis of JEV PrM and E genes results showed that 4 strains virus from Culex tritaeniorhynchus samples belonged to genotype Ⅰ JEV, and existing 15 amino acid difference sites between vaccine strain SA14-14-2 in the E gene protein, and 8 important sites which affected virus virulence did not mutant. These results suggested that there were many mosquito species in upper Pearl River region, and Culex tritaeniorhynchus, Anopheles Sinensis were dominant mosquito species in this region, and Culex tritaeniorhynchus was the main media of JEV in local region, genotypeⅠ JEV was popular in local media, and virulence of the pandemic strain did not reduce.     

云南省乙型脑炎病毒宿主和媒介研究

于１９７８年至１９９７年,从云南猪血液和乳猪脑组织中分离到乙型脑炎（ＪＥ）病毒５株,从蝙蝠脑组织中分得ＪＥ病毒１０株,从鸟类脑组织中分得ＪＥ病毒５株,从１５种蚊虫体内分离到ＪＥ病毒６３株,从２种蠓类中分得ＪＥ病毒２株。分析认为,猪是ＪＥ病毒的主要扩散宿主,蝙蝠和鸟类在该病毒保存和传播中起重要作用;三带喙库蚊是该病毒的主要传播媒介,伪杂鳞库蚊、霜背库蚊、蚊腿库蚊、白蚊伊和刺扰伊蚊为重要媒介,蠓类亦可     

Isolation of Ross River virus from mosquitoes and from horses with signs of musculo-skeletal disease

OBJECTIVE: To report clinical and clinicopathological findings in horses naturally infected with Ross River virus (RRV) and identify likely mosquito arbovirus vector species. PROCEDURES: Veterinarians submitted serum samples from 750 horses because they suspected Ross River virus (RRV) infection. The samples were tested for the presence of IgM and IgG antibody to RRV and for the presence of virus. Mosquitoes were trapped, differentiated to species level and tested for the presence of RRV by virus isolation. RESULTS: RRV was isolated from six species of mosquitoes (Ochlerotatus camptorhyncus, Culex globocoxitus, Cx. australicus, Cx. annulirostris, Cx. quinquefasciatus, Anopheles annulipes) and from 13 horses with clinical signs of musculo-skeletal disease. Antibody to RRV was detected in 420 of the 750 serum samples; 307 contained IgG only; 76 contained both IgM and IgG and 37 contained only IgM antibody to RRV. Virus was isolated from horses with IgM antibody only. CONCLUSIONS: RRV can be isolated from infected horses during the short time period when there is an overlap of clinical signs, positive IgM serology and viraemia. Early spring infections of horses may occur if RRV infected mosquito vectors are present. RRV has not been shown to cause clinical disease in horses. This is the first report of isolation of RRV from Oc. camptorhyncus in the Murray region and indicates a potential for infection of humans and animals in autumn as well as in spring.     

Detection and molecular characterization of avian Plasmodium from mosquitoes in central Turkey

Assessing vector-parasite relationship is important in understanding the emergence of vector-borne diseases and the evolution of parasite diversity. This study investigates avian Plasmodium parasites in mosquitoes collected from Kayseri province in Central Anatolian, Turkey and determines the haemosporidian parasite lineages from these mosquito species. A total of 6153 female mosquitos from 6 species were collected from 46 sites during June-August of 2008 and 2009. Each mosquito's head-thorax and abdomen were separated, categorized with respect to species and collection area and pooled for DNA extraction. A total of 1198 genomic DNA pools (599 thorax-head, 599 abdomen) were constituted of which 128 pools (59 thorax-head, 69 abdomen) were positive for avian haemosporidian parasites (Plasmodium and Haemoproteus) by Nested-PCR analysis. Culex pipens, Aedes vexans, Culex theileri and Culiseta annulata were positive with minimum infection rates (MIRs) of 16.22 and 18.15, 4.72 and 5.98, 5.18 and 10.36, 10.64 and 10.64 in their thorax-head and abdomen parts, respectively. No avian haemosporidian DNA was detected from Culex hortensis and Anopheles maculipennis. Phylogenetic analyses of the partial cytb gene of avian haemosporidian mt-DNA from 13 positive pools revealed that 11 lineages in four phylogenic groups were Plasmodium and the other two were Haemoproteus. Our results suggest that Cx. pipiens could probably be the major vector of avian Plasmodium in Central Turkey. This is the first report of molecular detection and characterization of avian Plasmodium lineages from mosquitoes in Turkey.     

Aedes vexans and Culex pipiens as the potential vectors of Dirofilaria immitis in Central Turkey

This study was carried out to investigate the potential vectors and relative mosquito infection rates of Dirofilaria immitis throughout two mosquito seasons (2008-2009) in Kayseri province where is located in Central Anatolian part of Turkey. For this aim, totally 1198 genomic DNA pools, extracted and grouped according to the species and collection site (1-17 specimens/pool) from 6153 mosquito specimens, were examined by PCR using species-specific primers for D. immitis. The captured mosquitoes from 46 focuses were survived under in vitro conditions for 7 days to allow the development of larval stages of D. immitis. DNA extraction was performed individually to both thorax-head and abdomens in order to determine infective and infected mosquito specimens, respectively. The most abundant mosquito species in the study area was determined as Ae. vexans (51.7%) and this was followed by Cx. pipiens (42.1%), Cx. theileri (3.1%), Cs. annulata (1.5%), An. maculipennis (1.0%) and Cx. hortensis (0.6%). The PCR results indicated that 9/312 and 12/312 pools from Ae. vexans abdomens and thorax-heads were positive for filarial DNAs, respectively where as 3/241 pools of each abdomens and thorax-heads from Cx. pipiens were positive for D. immitis DNAs. The minimum infection rates (MIRs) for Ae. vexans and Cx. pipiens were calculated as 0.41 and 0.12, respectively. Although D. immitis DNA's were found in both pools from Ae. vexans and Cx. pipiens, the calculated MIRs provide evidence that Ae. vexans could be the main potential vector of D. immitis in Kayseri.     

First record of avian Plasmodium DNA from mosquitoes collected in the Yaeyama Archipelago, southwestern border of Japan

We studied the prevalence of avian Plasmodium in 509 mosquitoes of 9 species collected from the Ishigaki and Iriomote islands in the Yaeyama Archipelago, located southwest from the mainland of Japan. Two identical avian Plasmodium lineages were detected from Culex (Culiciomyia) nigropunctatus. Detected lineages were phylogenetically classified into different clade to avian Plasmodium lineages from birds and mosquitoes in the mainland of Japan but identical to a lineage detected from a resident bird, White-breasted Waterken (Amaurornis phoenicurus). This is the first detection of avian Plasmodium DNA from mosquitoes in the Yaeyama Archipelago and suggested that resident birds might have been infected with an avian Plasmodium lineage specific to the studied area and C. nigropunctatus could be the candidate vector mosquito species.     

Research Progress on Rapid Serological Assays for Diagnosis of Japanese Encephalitis B

Japanese encephalitis B (JE) is a zoonosis caused by Japanese encephalitis virus which invades the central nervous system,infected by mosquito.Various animals could be the sower and reservoir of JE including swine,horse,cattle and avian.JE could cause a major public health trouble and a horrible crisis of the farm all in Asia and the Western Pacific region.Therefore,it is necessary to diagnose it rapidly and accurately.This review article made a sketch of approaches for the rapid serological assays of JE,including traditional method,emerging technologies and specific improvements of the assay,so as to provide references for the study of rapid,simple and accurate JE diagnose methods.     

流行性乙型脑炎快速血清学诊断方法研究进展

流行性乙型脑炎(简称乙脑)是由日本脑炎病毒引起的,以中枢神经系统受损病变为主的一种急性蚊媒人畜共患传染病。由于乙脑病毒的增殖宿主和贮存宿主涉及猪、马、牛和禽等多种家畜,且主要在亚洲和西太平洋地区广泛流行,不仅造成畜牧业严重经济损失,而且危害公共卫生安全。因此,对乙脑进行快速而准确的诊断具有实际意义。文章对乙脑的快速血清学诊断方法包括传统方法、新兴技术和试验特异性改良等进行较全面的综述,意在为快速、简单、准确的乙脑诊断方法研究提供参考。     

The inhibitory effect of a combination of imidacloprid and permethrin on blood feeding by mosquitoes in dogs raised under outdoor conditions

Combinations of imidacloprid and permethrin were frequently used to control harmful arthropod of companion animals. The inhibitory effects on blood-feeding activity of mosquitoes in dogs raised under outdoor conditions were evaluated by using combination of 10% (w/v) of imidacloprid and 50% (w/v) of permethrin as spot-on form. Dogs in the treated group received the combination imidacloprid/permethrin spot-on. After treatment, dogs in the control and treated groups were kept separately from the evening (17:00) to the morning of the following day (09:00) in two different kennels installed outdoors to mimic realistic dog-raising conditions. Mosquitoes in the kennels were collected by light traps placed in the kennels and a sweep net to determine evidence of blood feeding, and for species identification. Mosquitoes were collected at Days 5, 3 and 1 before agent treatment, and the Day of treatment, and Days 3, 7, 14, 21, 28, 35 and 42 after treatment. The percentages of blood-fed mosquitoes measured at Days 0, 3, 21, 28 and 42 after treatment were statistically significantly lower (p<0.01) in the treated group than in the control group. The most commonly collected mosquito, Culex tritaeniorhynchus, revealed statistically significant lower percentages (p<0.01) of blood-fed mosquitoes in the treated group than in the control group at the Day of treatment, and Days 3, 7, 21, 28 and 42 after treatment. It appeared that the test agent was effective in inhibiting blood feeding by adult female mosquitoes, and the efficacy lasts for 42 days after treatment under outdoor conditions.     

Mosquito vectors of dog heartworm in the United States: vector status and factors influencing transmission efficiency

Dog heartworm (Dirofilaria immitis) is dependent on mosquito vectors for its maintenance and transmission among vertebrate hosts. Consequently, D. immitis abundance and distribution are closely linked with mosquito vector biology and ecology. Information on the important dog heartworm vectors in the United States is limited and no comprehensive surveillance of dog heartworm in US mosquitoes has been undertaken to date. Here, we review information gleaned from a number of field surveys documenting heartworm presence in wild mosquito populations as well as laboratory assessments of mosquito vector capacity. Various biological and ecological factors likely contribute to the relative importance of different vector species. We describe some of these factors, rank the leading criteria for efficient vectors, and present the most likely vector species found across the United States. Considering the recent emergence of drug resistance among D. immitis strains, practical knowledge of heartworm vector biology and control should be incorporated into heartworm disease management programs. We conclude by proposing that heartworm control would benefit by targeting mosquito vectors, and we suggest ways in which veterinarians can incorporate the recognition of vector importance into heartworm prevention recommendations imparted to clients.     

Isolation of Western Equine Encephalomyelitis (Wee) Virus From Mosquitoes In Saskatchewan, 1962

Saskatchewan, in the summer of 1962, was the scene of an extensive outbreak of western equine encephalomyelitis (WEE) in horses. The results of mosquito survey work showed Culiseta inornata and Culex tarsalis respectively to be the two most abundant mosquito species during midsummer. These species are those reported to be most commonly associated with outbreaks of WEE. Five hundred and sixty-four pools of mosquitoes were examined for the presence of WEE virus. Six pools, three of C. tarsalis and one each of C. inornata, Aedes flavescens and Aedes dorsalis, yielded WEE virus. Positive mosquitoes were from St. Walburg (C. inornata), Saskatoon (C. tarsalis — two, A. dorsalis — one), Outlook (C. tarsalis) and Kisbey (A. flavescens).     

A long-term field study on mosquito vectors of avian malaria parasites in Japan

Avian malaria is a mosquito-borne disease of birds caused by avian Plasmodium spp. in worldwide scale. Some naïve birds show serious symptoms which can result in death. Surveillance of vectors and parasites are important to understand and control this disease. Although avian malaria has been found in Japan, detailed prevalence and dynamics remained understudied. We aimed to observe annual changes in the abundance of mosquitoes and the prevalence of avian Plasmodium parasites in Japan. Mosquitoes were collected using dry ice traps over a 10-year period, at a fixed research area located in Kanagawa prefecture. Collected mosquitoes were investigated for the species composition, population size and prevalence of avian Plasmodium by PCR. Mosquitoes belonging to 13 species in 7 genera were collected (n=8,965). The dominant species were Aedes (Ae.) albopictus and Culex (Cx.) pipiens group (gr.). Seven avian Plasmodium lineages, all of which were previously known, were detected from Cx. pipiens gr., Ae. albopictus, and Tripteroides bambusa. Three genetic lineages were dominant and were probably transmitted by Cx. pipiens gr. whose could be the primary vector of these parasites. Annual variations in the seasonal prevalence of mosquitoes and avian Plasmodium were revealed for the first time during recent 10 years in Japan. Namely, avian Plasmodium occurrence in the vector population peaked often in June to July and September to October when the density of the vector population was presumably high enough for the transmission of avian Plasmodium upon appearance of infected birds.     

Antibodies to Akabane virus in horses,sheep and goats in Japan

High prevalences of neutralizing (NT) antibody to Akabane virus were obtained with horse (72%), sheep (38%) and goat (67%) serum samples collected in Chiba Prefecture, where outbreaks of abortion and congenital deformities caused by Akabane virus occurred among cattle. In these animal sera, titers of hemagglutination-inhibiting (HI) antibody to Akabane virus and of NT antibody were closely correlated.     

Laboratory Studies on the Transmission of Western Equine Encephalitis Virus by Saskatchewan Mosquitoes I. Culex tarsalis

A Saskatchewan strain of the mosquito Culex tarsalis, transmitted a local strain of western equine encephalitis virus from chick to chick, between four and 44 days after an infective blood meal. At incubation temperatures of 69 and 75°F, 120 transmissions occurred out of a possible 141, and all but seven of these were by single infected mosquitoes. At 75°F virus titers in individual mosquitoes were more uniform and transmission was more efficient, than at 69°F, although infection rates were similar at both temperatures. The minimum concentration of virus required to infect 50% of C.tarsalis was 10^2.5 intracerebral three-week old mouse LD₅₀ per 0.03 ml of donor blood. These findings provide direct evidence that C. tarsalis of Saskatchewan is a highly efficient vector of western equine encephalitis virus.     

Ongoing Activity of Toscana Virus Genotype A and West Nile Virus Lineage 1 Strains in Turkey: A Clinical and Field Survey

Toscana virus (TOSV), West Nile virus (WNV) and tickborne encephalitis virus (TBEV) are among major viral pathogens causing febrile disease and meningitis/encephalitis. The impact of these viruses was investigated at a referral centre in Ankara Province, Central Anatolia in 2012, where previous reports suggested virus circulation but with scarce information on clinical cases and vector activity. Serum and/or cerebrospinal fluid samples from 94 individuals were evaluated, in addition to field‐collected arthropod specimens that included 767 sandflies and 239 mosquitoes. Viral nucleic acids in clinical samples and arthropods were sought via specific and generic nested/real‐time PCRs, and antibody responses in clinical samples were investigated via commercial indirect immunofluorescence tests (IIFTs) and virus neutralization. A WNV antigen assay was also employed for mosquitoes. WNV neuroinvasive disease has been identified in a 63‐year‐old male via RNA detection, and the WNV strain was characterized as lineage 1. TOSV infections were diagnosed in six individuals (6.3%) via RNA or IgM detection. Partial sequences in a 23‐year‐old female, presented with fever and transient pancytopenia, were characterized as TOSV genotype A. Febrile disease with arthralgia and/or peripheral cranial nerve involvement was noted in cases with TOSV infections. Previous WNV and TOSV exposures have been observed in 5.3% and 2.1% of the subjects, respectively. No confirmed TBEV exposure could be identified. Morphological identification of the field‐collected mosquitoes revealed Culex pipiens sensu lato (74.4%), Anopheles maculipennis (20.9%), An. claviger (2.1%) and others. Sandfly species were determined as Phlebotomus papatasi (36.2%), P. halepensis (27.3%), P. major s. l. (19.3%), P. sergenti (8.9%), P. perfiliewi (4.4%), P. simici (2.6%) and others. Viral infections in arthropods could not be demonstrated. TOSV genotype A and WNV lineage 1 activity have been demonstrated as well as serologically proven exposure in patients. Presence of sandfly and mosquito species capable of virus transmission has also been revealed.     

Incidence of abortions caused by leptospirosis in sheep and goats in Spain

From 1970 to 1985, 973 outbreaks of abortion in sheep and 262 outbreaks of abortion in goats from southern Spain were studied. Of these, 1.7 and 2.6% respectively were caused by leptospiras, chiefly by the serovar pomona (11 flocks of sheep, 64.7%, and six herds of goats, 75.0%) and in a lower proportion the serovars sejroe (three flocks of sheep 17.6%, and one herd of goats, 12.5%), icterohaemorrhagiae (two and one outbreaks respectively) and grippotyphosa in one ovine flock (5.8%). The mean rates of attach (X + SD) of abortions by leptospirosis have been estimated as 16.9 + 6.5% in ovine outbreaks and 20.7 + 8.3% among goats.     

Potential vectors of Dirofilaria immitis in Macon County, Alabama

A study was conducted in Macon County, Alabama (east-central) during the mosquito breeding seasons of 1978 and 1979 to determine the potential vector(s) of Dirofilaria immitis. Live mosquitoes were collected with CO2-baited, miniature light traps, and small hand aspirators. These mosquitoes were subsequently dissected to determine whether immature stages of the heartworm were present. Specific communities were selected based on their having at least 4 known cases of D immitis infection present in the local dog population. All positive collections were made between the months of May and September. Of 326 Anopheles punctipennis females dissected, 34 were positive, and of 84 Aedes vexans 6 were positive for infective (L3) larvae. Of 520 Culex quinquefasciatus females tested, 14 contained 2nd-stage larvae only. Immature stages of D immitis were collected during most of the mosquito breeding season. Data collected during the survey indicate that An punctipennis and to a lesser extent, Ae vexans are probably the principal vectors of D immitis in Macon County, Ala.     

蝙蝠作为流行性乙型脑炎病毒宿主的研究

1986、1988、1990和1997年的7－8月，在云南省共捕获653只蝙蝠，其中棕果蝠（Rousettus leschenaulti)593只，金管鼻蝠（Murina aurata)60只，采集血清及剖取脑组织作病毒分离和抗体检查。用细胞法和乳鼠法分离到6株病毒，带毒率为0．96％，其中从捕自景洪的棕果蝠脑组织中分离到3株，从河口的棕果蝠血清中分离到2株，从捕自耿马的金管鼻蝠脑组织中分离出1株。棕果蝠和金管鼻蝠的带毒率分别为0．84％和1．67％。这6株病毒能引起C6／36和BHK21细胞病变和导致乳小白鼠或3－4周龄小白鼠发病和死亡，并具有典型的嗜神经病毒感染症状，经血凝抑制，补体结合，免疫荧光和中和试验鉴定。新分离的6株病毒均为乙脑病毒（JE virus)。同期用血凝抑制试验对采获的425份棕果蝠血清作乙脑病毒抗体检查。阳性153－份，阳性率为36％，人有较高的感染率，。本次从棕果蝠和金管鼻蝠体内分离出乙脑病毒属具有重要流行病学意义。