Phylogenetic comparison of Leucocytozoon spp. from wild birds of Japan

Eight species of Japanese birds were found to be infected with Leucocytozoon species using microscopic analysis. We used PCR and sequence analysis of the mitochondrial cytochrome b gene (cyt b) to compare the genetic background among these detected protozoa species. In 20 individuals of 22 samples, a single amplified band was detected from 6 of 8 bird species; 9 Japanese rock ptarmigans (Lagopus mutus japonicus), 4 large-billed crows (Corvus macrorhynchos), 2 carrion crows (C. corone), 2 scops owls (Otus scops), 1 Japanese grosbeak (Eophona personata), and 2 brown-eared bulbuls (Hypsipetes amaurotis), respectively. Phylogenetic analysis based on the partial cyt b sequences revealed that all Leucocytozoon isolates in Japan closely grouped with other Leucocytozoon species previously reported in the literature. Among the Japanese isolates, the phylogenetic tree suggested that L. lovati from the Japanese rock ptarmigan may be basal to the parasites found in other bird species. Our study is the first to identify the molecular relationships among Leucocytozoon parasites in the avifauna of Japan.     

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).     

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.     

The prevalence of blood parasites in helmeted guineafowls, Numida meleagris, in the Kruger National Park

Bloodsmears were taken from separate groups of five helmeted guineafowls, Numida meleagris, shot at approximately monthly intervals at Skukuza and near Lower Sabie in the Kruger National Park during the period August 1988 to August 1990. Ninety-eight (86%) of 114 guineafowls had single or multiple infections of Aegyptianella sp., Haemoproteus pratasi, Hepatozoon sp., Leucocytozoon neavei, Plasmodium circumflexum and Trypanosoma numidae. The apparent seasonal prevalence of Aegyptianella sp., H. pratasi and L. neavei, the three most commonly occurring parasites (42%, 49% and 56% of birds infected respectively), is probably dependent on the presence of their respective vectors.     

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.     

Ultrastructural and histochemical properties of the olfactory system in the japanese jungle crow, Corvus macrorhynchos

Although it has been commonly believed that birds are more dependent on the vision and audition than the olfaction, recent studies indicate that the olfaction of birds is related to the reproductive, homing, and predatory behaviors. In an attempt to reveal the dependence on the olfactory system in crows, we examined the olfactory system of the Japanese jungle crow (Corvus macrorhynchos) by histological, ultrastructural, and lectin histochemical methods. The olfactory epithelium (OE) of the crow occupied remarkably a small area of the nasal cavity (NC) and had the histological and ultrastructural features like other birds. The olfactory bulb (OB) of the crow was remarkably small and did not possess the olfactory ventricle. The left and right halves of the OB were fused in many cases. In the lectin histochemistry, soybean agglutinin (SBA) and Vicia villosa agglutinin (VVA) stained a small number of the receptor cells (RCs) in the OE and the olfactory nerve layer (ONL) and glomerular layer (GL) on the dorsocaudal region of the OB. Phaseolus vulgaris agglutinin-E (PHA-E) stained several RCs in the OE and the ONL and GL on the ventral region of the OB. These results suggest that 1) the crow has less-developed olfactory system than other birds, and 2) the dedicated olfactory receptor cells project their axons to the specific regions of the OB in the crow.     

Blood parasites of imported psittacine birds.

Of 117 imported psittacine birds examined for the presence of blood parasites, 18 (15-3 per cent) were found to be infected. The most common parasites were microfilaria and Haemoproteus, but Aegyptianella and a Trypanosoma sp were also observed. Haemoproteus handai was diagnosed in several birds and for the first time in lesser sulphur-crested cockatoos. The significance of the parasites observed is discussed and also that of other haematozoa recorded from psittacines but not found during the present survey.     

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.     

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。     

Rhipicephalus sanguineus (Ixodida, Ixodidae) as intermediate host of a canine neglected filarial species with dermal microfilariae

The life cycles of filarioids of dogs presenting dermal microfilariae have been little studied. Following the recent retrieval of dermal microfilariae identified as Cercopithifilaria sp. in a dog from Sicily (Italy), this study was designed to assess the role of the brown dog tick Rhipicephalus sanguineus as an intermediate host of this filarial species. An experimental tick infestation was performed on an infected dog using 300 nymphs of R. sanguineus. Engorged nymphs were collected and examined by both microscopic dissection and molecular analysis at five time points (i.e., the same day of tick detachment and 10, 20, 30 and 50 days post-detachment) to detect the presence and developmental stage of filariae in the ticks. A total of 270 engorged nymphs were collected from the dog and developing filarioid larvae detected in 10 (5%) out of 200 ticks dissected. Infective third-stage larvae were observed in 4 (2%) of the all dissected ticks, 30 days post-detachment. Twelve (6.6%) out of 181 samples molecularly tested were positive for Cercopithifilaria sp. This study demonstrates that nymphs of R. sanguineus feeding on a dog naturally infected by Cercopithifilaria sp. can ingest microfilariae, which develop up to the third infective stage thus suggesting that this tick species might act as an intermediate host of this little known canine filarioid.     

Geographical distribution and seasonality of the prevalence of Leucocytozoon lovati in Japanese rock ptarmigans (Lagopus mutus japonicus) found in the alpine regions of Japan

In this study, we investigated the geographical distribution and seasonality of Leucocytozoon lovati infection in the Japanese rock ptarmigan (Lagopus mutus japonicus); this bird is one of the special natural monuments of Japan that inhabits the Japanese alpine regions. We examined blood samples from birds captured in the Kubiki, Hida, and Akaishi mountain ranges for three years from 2002 to 2005. Seventy-three blood samples from 42 males, 19 females, and 12 birds of unknown sex were used for this study. The rate of infection with L. lovati was 78.1% in the 73 birds examined. We demonstrated that the L. lovati infection was distributed across wide ranges of ptarmigan populations from the northern to the southern alpine zones. There was no sex bias in the prevalence ratio. The prevalence of L. lovati and the level of parasitization of the blood cells tended to increase from spring through summer; in contrast, a decrease was observed from summer through autumn. Although L. lovati infection was observed in a number of local populations inhabiting three mountainous regions, no infected birds were found in Mt. Johnen-dake and Mt. Maejohnen-dake. It is necessary to continue surveying the relationship between the population dynamics of the ptarmigan and the density of the arthropod vector from the perspective of in situ conservation of this endangered species.     

Toxoplasma gondii prevalence in Israeli crows and Griffon vultures

A cross-sectional Toxoplasma gondii seroprevalence study was performed on free ranging crows (Corvus cornis, Corvus monedula, Corvus splendens) and Griffon vultures (Gyps fulvus) from Israel in order to assess exposure to this pathogen in scavenger birds that feed on animal carcasses and their possible role in the epidemiology of toxoplasmosis. Using the modified agglutination test (MAT) with a cutoff titer of 1:25, 52 of 122 crows (42.6%) and 40 of 101 Griffon vultures (39.6%) were found to be T. gondii seropositive. Crow T. gondii seroprevalence was significantly higher in northern areas of Israel (p = 0.007) where annual precipitation is higher and annual summer maximum temperatures are lower than in the drier and warmer south. Seroprevalence in crows was positively associated with higher human population densities possibly related to the increased cat population in these areas. PCR analysis of brain extracts from crows resulted in the detection of T. gondii DNA in 1 seropositive crow from northern Israel. Genetic analysis of DNA from the positive crow brain confirmed infection with T. gondii type 2 using a multiplex multilocus nested PCR-RFLP (Mn-PCR-RFLP) of the SAG1, 5–3′ SAG2, alt.SAG2, SAG3, BTUB, GRA6, C22-8, c29-2, L358, PK1 and Apico loci. The high T. gondii seroprevalence in these bird species suggests that infected carrion may be responsible for widespread infection of carcass scavenger birds which may further transmit infection to other carnivorous intermediate hosts or feline definitive hosts when consumed post-mortally.     

Fatal hemoprotozoal infections in multiple avian species in a zoological park.

Over a 3-yr span, two juvenile lesser flamingos (Phoeniconaias minor), two green jays (Cyanocorax yncas glaucescens), and two Montezuma oropendolas (Psarocolius montezuma) died peracutely with no premonitory signs at a zoological park in the southern United States. At necropsy, the birds were in excellent body condition. Except for one green jay, the coelomic cavities were filled with a dark serosanguineous fluid. Splenomegaly and hepatomegaly were present. The livers were tan to purple with numerous, randomly distributed red-to-black foci, ranging in size from 1 to 4 mm. The predominant histopathologic finding, except in one green jay, was large protozoal cysts in the hepatic parenchyma. Histologically, the protozoal cysts were restricted to the liver, and none were identified in the skeletal muscle, spleen, or other tissues. Frozen tissue samples harvested at necropsy had a nested polymerase chain reaction assay performed to amplify the mitochondrial cytochrome B gene of the protozoa. The amplified gene sequences were compared with reference cytochrome B gene sequences for avian Plasmodium spp., Haemoproteus spp., and Leucocytozoon spp. The protozoal parasite within the hepatic parenchyma from the Montezuma oropendolas and the lesser flamingos was identified as Haemoproteus spp. Both green jays had Plasmodium spp. isolated from the submitted tissue samples. The peracute nature of the infections precluded any successful medical intervention, making prevention by exclusion the principal means to control hemoprotozoal transmission. There are no reports in the literature documenting identified fatal hemoprotozoal infections in oropendolas, green jays, or lesser flamingos.     

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.     

Role of birds in transmission of classical swine fever virus

Active transmission of classical swine fever virus (CSFV) was studied in six birds (five ravens, one hooded crow) and two laying hens. Cloacal swabs, blood and organs of birds and hens as well as blood and organ samples of pigs which had been fed with faeces derived from CSFV infected birds or which had come in contact with faeces of infected hens were negative for CSFV. None of the animals seroconverted during the study. This result demonstrates that active virus transmission by these animals is unlikely. Dissemination of CSFV from wild boar to domestic pigs is discussed.     

First molecular identification and subtype distribution of Blastocystis sp. isolated from hooded crows (Corvus cornix) and pigeons (Columba livia) in Tehran Province,Iran

Blastocystis is a common intestinal parasite among humans and animals such as non-human primates, pigs, cattle, birds, amphibians, and less frequently, rats, reptiles and insects. Since Blastocystis is a widely transmissible parasite between humans and mammals or birds, it is prominent to determine whether newly secluded non-human isolates are zoonotic. There are no comprehensive studies in Iran assessing the prevalence and molecular identification of Blastocystis infection in birds, especially in pigeons and crows. So, the aim of this study was to identify Blastocystis subtypes (STs) in crows and pigeons in Tehran province, Iran, using Nested PCR-RFLP and sequencing. Overall, 300 Blastocystis isolates from birds (156 pigeons and 144 crows) were subtyped by PCR, and the homology among isolates was then confirmed by RFLP analysis of the 18S rRNA gene. The prevalence of Blastocystis infection was detected 42.9% in pigeons and 44.4% in crows. All positive pigeons were owned by ST13 (100%). Among crows, 46 samples (71.8%) like pigeons were ST13, and 13 samples (20.3%) were ST14. Five samples (7.9%) remained unknown. This study was the first report of ST13 and ST14 of Blastocystis from birds. In the present study, our data revealed a high prevalence of Blastocystis sp. in pigeon’s and crow’s samples and the isolates from these birds were classified into two genetically distinct STs. Therefore, birds appear to be infected with various STs. It is important to determine the phylogenetic relationships between unknown STs from these birds and the multiple STs of Blastocystis.     

DNA vaccination of the American crow (Corvus brachyrhynchos) provides partial protection against lethal challenge with West Nile virus

The New York 1999 strain of West Nile virus (WNV) is nearly 100% fatal in the American crow (Corvus brachyrhynchos). We evaluated four WNV vaccine formulations in American crows, including intramuscular (i.m.) DNA vaccine, i.m. DNA vaccine with adjuvant, orally administered microencapsulated DNA vaccine, and i.m. killed vaccine. Neutralizing antibodies developed in approximately 80% of crows that received the DNA vaccine i.m. (with or without adjuvant), and in 44% that received the killed vaccine. However, no crows that received the oral microencapsulated DNA vaccine or the placebo developed WNV antibodies. All crows were challenged 10 wk after initial vaccination. No unvaccinated crows survived challenge, and survival rates were 44% (i.m. DNA vaccine), 60% (i.m. DNA vaccine with adjuvant), 0% (oral microencapsulated DNA vaccine), and 11% (killed vaccine). Peak viremia titers in the birds that survived were significantly lower as compared to titers in birds that died. Parenteral administration of a WNV DNA vaccine was associated with reduced mortality but did not provide sterile immunity.     

Serologic evidence of West Nile virus and Usutu virus infections in Eurasian coots in the Netherlands

West Nile virus (WNV) and Usutu virus (USUV) are arboviruses that are maintained in enzootic transmission cycles between mosquitoes and birds and are occasionally transmitted to mammals. As arboviruses are currently expanding their geographic range and emerging in often unpredictable locations, surveillance is considered an important element of preparedness. To determine whether sera collected from resident and migratory birds in the Netherlands as part of avian influenza surveillance would also represent an effective source for proactive arbovirus surveillance, a random selection of such sera was screened for WNV antibodies using a commercial ELISA. In addition, sera of jackdaws and carrion crows captured for previous experimental infection studies were added to the selection. Of the 265 screened serum samples, 27 were found to be WNV–antibody‐positive, and subsequent cross‐neutralization experiments using WNV and USUV confirmed that five serum samples were positive for only WNV‐neutralizing antibodies and seven for only USUV. The positive birds consisted of four Eurasian coots (Fulica atra) and one carrion crow (Corvus corone) for WNV, of which the latter may suggest local presence of the virus, and only Eurasian coots for USUV. As a result, the screening of a small selection of serum samples originally collected for avian influenza surveillance demonstrated a seroprevalence of 1.6% for WNV and 2.8% for USUV, suggesting that this sustained infrastructure could serve as a useful source for future surveillance of arboviruses such as WNV and USUV in the Netherlands.     

Neuroangiostrongyliasis and other parasites in tawny frogmouths (Podargus strigoides) in south-eastern Queensland

The neurological, clinical and pathological findings and endoparasites in 10 wild tawny frogmouths (Podargus strigoides) presented to the Australian Wildlife Hospital in Beerwah, Queensland during a 28-day period in May 2009 are reported. Affected birds had a history of being found in poor body condition on the ground and unable to fly. Clinical examination revealed paresis with variable but generally weakened deep pain responses, withdrawal reflexes and an inability to perch. Severely affected birds that failed to respond to anti-inflammatory, antibiotic and anthelmintic treatments all had larval Angiostrongylus cantonensis in the brain and or spinal cord, with occasional larvae found in the visceral organs. Other parasites detected included the liver fluke, Brachylecithum podargi, the intestinal nematode, Allodapa suctoria, an unidentified species of Trichostrongylus and unidentified cestodes. Gametocytes of Leucocytozoon sp. were found in peripheral blood smears and low numbers of microfilariae were found in histological sections of various blood vessels of several birds. However, no adult filarioids were recovered. Unidentified subcutaneous mites were identified in the connective tissue of the thoracic inlet. Attempts to treat two birds with ivermectin or oxfendazole-praziquantel were unsuccessful, but a third bird treated with a combination of steroidal and non-steroidal anti-inflammatory therapy followed by moxidectin steadily recovered, such that by 6 weeks post presentation its ability to perch, grasp, fly and judge distances was considered normal and it was subsequently released.