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.     

Aedes albopictus is a natural vector of Dirofilaria immitis in Italy

Investigations were carried out in Padova town (Veneto region, NE Italy) to define the actual role of Aedes albopictus in the natural transmission of Dirofilaria nematodes, and to assess the risk that its presence might represent for veterinary and medical health. During summer 2000-2002 daytime captures of human-attracted mosquitoes were carried out in three areas of the town. The presence of filarial parasites in mosquitoes was evaluated by PCR, and sequencing confirmed species assessment. DNA extraction was performed separately on pools of the insect abdomen and thorax-head, to discriminate between Dirofilaria infected/infective specimens. A total of 2721 mosquitoes were caught and A. albopictus was the most abundant species (2534). Filarial DNA was found in 27.5% (19/69) of the abdomen pools formed with mosquitoes collected in summer 2000, and in 11.1% (16/144) and 4.9% (6/123) thorax-head pools coming from samplings 2001 and 2002, respectively. Filarial DNA was belonging to D. immitis and all studied areas harboured infective specimens. These results prove A. albopictus as natural vector of D. immitis in Italy. Moreover, they support the hypothesis that the presence of the mosquito could affect the transmission pattern of canine heartworm disease in urban environment and, considering the aggressive anthropophylic behaviour of the species (30-48 bites/h) proven in Padova town, could enhance the circulation of filarial nematodes from animals to humans.     

Haemosporidian infection in captive masked bobwhite quail (Colinus virginianus ridgwayi), an endangered subspecies of the northern bobwhite quail

The avian haemosporidian parasites (phylum Apicomplexa) are taxonomically diverse and cosmopolitan in distribution; infecting most bird families. Sources of concern are reports of clinical haemosporidian infections in birds kept as part of zoo and aviary collections. Recently, severe and acute mortality episodes have been reported in masked bobwhite quail (Colinus virginianus ridgwayi), an endangered subspecies from the American Southwest. Two hundred and five eggs of the captive flock held in Arivaca, Arizona, were hatched at a zoo in the American Southwest. Thirty-four sub-adult or adult animals had lesions associated with tissue phases of haemoparasites, especially vasculitis, ventricular leiomyositis and ulcerative pododermatitis. Molecular techniques applied to blood collected from the zoo's last twelve remaining animals resulted in the detection of a Plasmodium juxtanucleare-like and Haemoproteus sp. parasites. A Raven (Corvus corax), in a contiguous exhibit, was positive for the same P. juxtanucleare-like parasite, but remained asymptomatic for three years following detection. These findings indicate that other birds in the exhibit within the zoo premises could act as reservoirs. We conclude that haemosporidian infections could be a factor in the demise of the captive masked bobwhite quails housed at the zoo. We suggest that active surveillance for haemoporidian parasites should be incorporated as a precaution to ex situ conservation efforts of susceptible endangered species.     

Avian haemosporidian parasites infection in wild birds inhabiting Minami-daito Island of the Northwest Pacific, Japan

Haemosporidian parasites infection among wild birds inhabiting Minami-daito Island was studied. Blood films from 183 birds representing 4 species of 4 families were examined microscopically. Avian haemosporidian parasites were detected in 3 species with an overall prevalence of 59.6%. None of the 30 Daito scops owls (Otus scops interpositus) examined were infected. Either Haemoproteus sp. or Plasmodium sp. infection was found in 14 of 31 (45.2%) Borodino islands white-eyes (Zosterops japonicus daitoensis). Plasmodium spp. were found in 94 of 102 (92.2%) bull-headed shrikes (Lanius bucephalus) and 1 of 20 (5%) tree sparrows (Passer montanus).     

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.     

Haemoproteus columbae in domestic pigeons in Sebele, Gaborone, Botswana.

Mature and immature stages of Haemoproteus columbae gametocytes, an avian haemosporidian protozoan parasite were found in 75% of blood smears prepared from 30 healthy domestic pigeons in Sebele location, Gaborone, Botswana. Parenteral administration of an immuno-suppressive dose of dexamethasone, lowered the level of parasitaemia, the packed cell volume and the heterophil:lymphocyte ratio in the test pigeons. However, both the dexamethasone-treated and the control pigeons remained clinically normal.     

Avian malaria (Plasmodium spp) in yellow-eyed penguins: investigating the cause of high seroprevalence but low observed infection

AIM: To investigate the cause of a high seroprevalence of antibodies to Plasmodium spp known to cause avian malaria, but extremely low levels of observed infection, in yellow-eyed penguins, Megadyptes antipodes. METHODS: A polymerase chain reaction (PCR) test specific for malarial parasites was applied to DNA extracted from blood samples collected from 143 yellow-eyed penguins from an area where seroprevalence for malarial antibodies was known to be high but no parasites were observed in blood smears. RESULTS: None of the samples tested positive for malarial parasite DNA using the PCR test. Assuming a sensitivity of 90% for this test, this means that prevalence of infection was 95% likely to be <2.3% in this population during this sampling period. CONCLUSIONS: Serological studies of a population of adult yellow-eyed penguins indicated a high level of exposure to avian malaria parasites, but a correspondingly high level of infection was not observed and no evidence of malarial parasite DNA was found in the current study. Discrepancies between these findings and historical records of Plasmodium spp found in blood smears and post mortem may be explained either by inaccuracy of the serological test used, or by infection occurring in juveniles which is subsequently cleared in surviving adults.     

Plasmodium (Bennettinia) juxtanucleare infection in a captive white eared-pheasant (Crossoptilon crossoptilon) at a Japanese zoo

An adult male white eared-pheasant (Crossoptilon crossoptilon) at a Japanese zoo exhibited lethargy and emaciation. Microscopic examination of a blood smear revealed a haemosporidian parasitic infection. Based on the morphological characteristics and molecular analysis of the parasite, it was identified as Plasmodium (Bennettinia) juxtanucleare. This is the first report of P. juxtanucleare infection in bird species belonging to the genus Crossoptilon. Caution against avian malaria infection is required for the conservation of endangered bird species in zoos.     

幼丹顶鹤感染血孢子虫的PCR鉴定

2006～2008年,北京动物园繁殖的17只1.5～3.5月龄丹顶鹤幼鹤出现厌食、精神倦怠、呼吸困难等症状。采用PCR方法鉴定病原,使用特异性引物对线粒体细胞色素b、细胞色素氧化酶Ⅰ、18S rRNA及线粒体部分非编码区基因进行扩增、克隆、测序,结合GenBank中已知疟原虫序列,经DNAMAN软件处理分析,并对Cytb序列用软件MEGA 4.0建NJ树。序列分析表明丹顶鹤血孢子虫靶序列与鸟疟原虫序列相似性达到98%以上,进化关系分析表明与残疟原虫各株属同一个种群,证明该病病原为残疟原虫。但该血孢子虫与残疟原虫已报道各株形态有一定差异,可能为寄生于丹顶鹤的一个新基因型。     

Prevalence of haematozoa in birds of prey in Catalonia, north-east Spain.

Blood samples from birds of prey, 95 Strigiformes and 190 Falconiformes, were examined for the presence of haematozoan parasites. The birds had been admitted to a raptor recovery centre in Catalonia, north-east Spain. Parasites were counted in positive smears. A second blood sample was obtained from 99 birds at least seven days after their arrival at the centre. Haematozoa were detected in all seven species of Strigiformes and in nine of 19 species of Falconiformes. The overall prevalences in the two groups were significantly different, 30.5 per cent and 46.3 per cent in the nocturnal and diurnal raptors, respectively. Eleven species of haematozoan parasites were identified. The genus most commonly detected in members of the nocturnal Strigiformes was Leucocytozoon, followed by Haemoproteus, Plasmodium and Trypanosoma. In the diurnal Falconiformes only Leucocytozoon and Haemoproteus were detected. The highest infection rates were found in Accipiter nisus (sparrow hawks), Accipiter gentilis (goshawks) and Athene noctua (little owls). Relapses were detected in 9 per cent of the birds sampled twice. The highest intensity of infection (6.2 per cent) was observed in a Buteo buteo (buzzard) infected with Haemoproteus.     

Blood parasites of some West African rainforest birds

A total of 969 birds representing 121 species of 21 families from the West African nations of Cameroon, Equatorial Guinea and Ivory Coast were examined for haematozoa using thin blood smears; 277 individuals (28.6%) harbored blood parasites. The parasites identified included species of Haemoproteus (7.7% prevalence), Plasmodium (10.7%), Leucocytozoon (4.6%), and Trypanosoma (7.3%). In addition, microfilariae of filariid nematodes were present in 3.6% of the individuals examined. The birds were collected over a period of 12 years, from 1989-2001, from rainforest and ecotone habitats. We report a relatively high prevalence of parasites in colonial nesting birds, and two species of ground nesting birds. In addition, we compared data from bird species collected at a site identical to a previously published study, and did not find significant differences in parasite prevalence between the two years constituting two different seasons. Our results are also compared to other studies in Africa that implement similar and different methodologies.     

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.     

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。     

Prevalence of avian haematozoa in wild birds in a high-altitude forest in Japan

The infection dynamics of avian haematozoa, which includes the genera Plasmodium, Haemoproteus, and Leucocytozoon, are complicated by a variety of environmental factors and host-parasite interactions. In Japan, the prevalence of haematozoa in wild birds has recently been determined in several local areas. However, no information on the annual prevalence of avian haematozoa in a single study site has been reported. Here, we investigated the long-term infection dynamics of haematozoa in wild birds inhabiting a mountain forest of Japan. Blood samples were collected from 415 wild birds captured in the Chichibu mountains in Saitama Prefecture at an altitude of 1650 m between 2007 and 2010. All obtained samples were examined for haematozoan infection using nested polymerase chain reaction (PCR) of the cytochrome b (cytb) genes of haematozoa. A total of 62 out of 415 (14.9%) forest birds were PCR positive for haematozoa. Relatively high infection rates of Leucocytozoon were found among several bird species (Parus ater, 64.3%; Parus montanus, 81.8%) and may be due to the host preference of vector black flies and host nestling pattern in this forest. Phylogenetic analysis of amplified cytb sequences revealed for the first time that a variety of lineages of avian haematozoa are distributed among wild bird hosts in a high-altitude forest stand in Japan. Notably, significant seasonal changes of the prevalence of avian haematozoa were not observed; however, continuous investigation will likely provide detailed information on host-parasite interactions, including local environmental factors, that influence the dynamics of avian haematozoan infections.     

Unidentified Plasmodium species in Australian black swans (Cygnus atratus) hatched and raised in North America

A pair of Australian black swans (Cygnus atratus) with origins in Wakefield, Virginia, USA, was admitted to the quarantine area at the Baltimore Zoo for general health assessments before housing in the collections. During the quarantine period, no clinical signs of disease were manifest; however, upon examination of a blood smear, intraerythrocytic parasites were detected and initially determined to be Haemoproteus species. Diagnostic polymerase chain reaction (PCR) and sequencing results, however, indicated that the parasites were within the genus Plasmodium. Subclinical infections with Plasmodium species in birds may affect collection management, and transmission from refractory hosts to susceptible hosts should be considered when multispecies exhibits are used. In addition, changes in the dynamics of host-vector-parasite interactions might have significant impacts on wild or domesticated populations of birds.     

Preliminary results of an anticircumsporozoite DNA vaccine trial for protection against avian malaria in captive African black-footed penguins (Spheniscus demersus).

Captive juvenile African black-footed penguins (Spheniscus demersus) housed in an outdoor enclosure at the Baltimore Zoo have an average 50% mortality from avian malarial (Plasmodium sp.) infection each year without intense monitoring for disease and chemotherapeutic intervention. During the 1996 malaria transmission season, the safety and efficacy of an anti-circumsporozoite (CSP) DNA vaccine encoding the Plasmodium gallinaceum CSP protein against P. relictum were studied. The goal was to reduce clinical disease and death without initiating sterile immunity after release into an area with stable, endemic avian malaria. The birds were monitored for adverse clinical signs associated with vaccination, the stimulation of an anti-CSP antibody response, and protection afforded by the vaccine. The presence of P. relictum in trapped culicine mosquitoes within the penguin enclosure was monitored to assess parasite pressure. Among the vaccinated penguins, the parasitemia rate dropped from approximately 50% to approximately 17% despite intense parasite pressure, as determined by mosquito infection rate. During the year of the vaccine trial, no mortalities due to malaria occurred and no undesirable vaccination side effects occurred. This is the first trial of an antimalarial vaccine in a captive penguin colony.     

Hepatic hemorrhage, hemocoelom, and sudden death due to Haemoproteus infection in passerine birds: eleven cases.

Haemoproteus spp. are ancient apicomplexan hemoparasites that have undergone extensive coevolution with their natural hosts and are typically species specific, with inapparent or minimal pathogenicity. A promiscuous genotype of Haemoproteus capable of undergoing host switching on a familial level was identified. This protozoan caused severe disease with high mortality in 6 species of exotic passerine birds housed in California at the San Diego Zoo's Wild Animal Park: Surinam crested oropendola (Psarocolius decumanus decumanus), Guianan turquoise tanager (Tangara mexicana mexicana), blue-necked tanager (Tangara cyanicollis caeruleocephala, Guianan red-capped cardinal (Paroaria gularis gularis), magnificent bird of paradise (Diphyllodes magnificus hunsteini), and superb bird of paradise (Lophorina superba). The birds had few or no clinical signs. Necropsy findings consisted of hemocoelom and irregularly scattered areas of hemorrhage and hepatocellular necrosis. Affected areas of liver contained solitary protozoal megaloschizonts in varied states of degeneration and peripheral nonsuppurative inflammation. No other parasite life stages were found in parenchymal organs or blood smears. Polymerase chain reaction using consensus primers for an avian malarial mitochondrial cytochrome B gene segment was positive in all cases. Sequencing and BLAST analysis identified the protozoan as a Haemoproteus sp. related to Haemoproteus spp. found in asymptomatic passerine birds native to North America. In situ hybridization was performed in 3 animals with a mitochondrial cytochrome B probe and was positive only in megaloschizonts. These findings suggest the recognition of a genotype of Haemoproteus that exhibits high levels of host infidelity and causes severe disease in captive birds exotic to North America.     

Attraction of mosquitoes to domestic cats in a heartworm enzootic region

Heartworm disease is caused by a mosquito-borne parasite that can affect many different mammalian species and has worldwide distribution. The agent, Dirofilaria immitis (Leidy 1856), infect mainly dogs but feline infection have been frequently reported in the last decade. Feline heartworm infection is difficult to detect, therefore, low reported prevalence could reflect true low prevalence or poor diagnostic efficiency. As mosquitoes are known to be attracted differently by different mammalian species, mosquitoes were collected from both a cattery and a contiguous home located in a canine heartworm enzootic area in Niterói, state of Rio de Janeiro, Brazil. For 14 months, mosquitoes were collected weekly for genus identification, speciation when possible, and for individual blood meal identification. Culex species mosquitoes were the most captured and those most frequently found with feline blood meal, followed by Aedes species that, although captured in lower numbers, also fed on feline blood. While Culex species mosquitoes have been reported as potential secondary heartworm vectors for dogs and primary vectors for cats, the present results suggest that Aedes species mosquitoes may also be involved in feline heartworm transmission in a larger proportion than previously thought.     

Malaria in an eastern screech owl (Otus asio)

Owls are frequent carriers of blood parasites but clinical malaria infections are rare. Various stages of Plasmodium subpraecox were seen in 90% of the erythrocytes of an Eastern screech owl (Otus asio) showing symptoms consistent with malaria 1 wk after admission for traumatic injuries. An additional unidentified blood parasite, either a Plasmodium or a Haemoproteus spp. was found in small numbers of red blood cells on blood films examined at admission and at day 7 postadmission. Combined infestation, trauma-induced stress, and iatrogenic corticosteroid administration are possible factors that could have induced disease. Oral treatment with mefloquine at 30 mg/kg, repeated after 12, 24, and 48 hr, proved successful in eliminating both organisms and signs of clinical disease.