Onchocercosis in red deer (Cervus elaphus) from Spain.

Onchocercosis, caused by Onchocerca flexuosa, was observed in red deer (Cervus elaphus) from Spain for the first time. Adult specimens of P. flexuosa were found in nodules in subcutaneous tissues in 42 of 125 (33%) red deer between October 1994 and September 1995; intensity of infection +/- SD was 3.93 +/- 5.26 nodules per infected host. A clear seasonal pattern in the distribution of nodules was observed, with higher values of prevalence and intensity in fall and winter in contrast to spring and summer. Significant differences were found among age groups in prevalence, but not in the mean intensity. No differences in infection were apparent between male and female adult red deer.     

Neutrophil accumulation around Onchocerca worms and chemotaxis of neutrophils are dependent on Wolbachia endobacteria

Unlike in many other helminth infections, neutrophilic granulocytes are major cellular components in the hosts immune response against filarial worms. The pathways that drive the immune response involving neutrophils are unclear. This study shows that Wolbachia endobacteria (detectable by polyclonal antibodies against endobacterial heat shock protein 60 and catalase and by polymerase chain reaction being sensitive to doxycycline treatment) are direct and indirect sources of signals accounting for neutrophil accumulation around adult Onchocerca volvulus filariae. Worm nodules from untreated onchocerciasis patients displayed a strong neutrophil infiltrate adjacent to the live adult worms. In contrast, in patients treated with doxycycline to eliminate the endobacteria from O. volvulus and to render the worms sterile, the neutrophil accumulation around live adult filariae was drastically reduced. Neutrophils were absent in worm nodules from the deer filaria Onchocerca flexuosa, a species which does not contain endobacteria. Extracts of O. volvulus extirpated from untreated patients showed neutrophil chemotactic activity and in addition, induced strong TNF-alpha and IL-8 production in human monocytes, in contrast to filarial extracts obtained after doxycycline treatment. Thus, neutrophil chemotaxis and activation are induced directly by endobacterial products and also indirectly via chemokine induction by monocytes. These results show that the neutrophil response is a characteristic of endobacteria-containing filariae.     

Onchocerca eberhardi n. sp. (Nematoda: Filarioidea) from sika deer in Japan; relationships between species parasitic in cervids and bovids in the Holarctic region

Onchocerca eberhardi n. sp. from the sika deer, Cervus nippon, in Japan is described. Adult worms lived in the carpal ligament; infection reached high levels (up to 25 female and 16 male worms in a single carpal limb). Skin dwelling microfilariae were mainly found in the ears. Prevalence of infection was 81% at the type locality, Mt. Sobo, in Kyushu. The new material was compared to the 31 species of Onchocerca presently known. Onchocerca eberhardi n. sp. females were characterized by a long slender anterior end and a thin esophagus < or =1 mm long with no or only a slight glandular region. The vulva was located near the level of the mid-esophagus and the cuticle had transverse external ridges and internal striae (two striae between adjoining ridges). The most similar species were O. stilesi (re-examined), O. lienalis, and to a lesser extent O. gutturosa, all from bovids (cattle). Two main lineages of Onchocerca are recognized in cervids with either primitive or with derived characteristics (as exemplified by the new species). The species in both lineages are not restricted to cervids but are also found in bovids in the Holarctic region, suggesting that the species diversified in the two host groups simultaneously, when these host groups lived in the some geographic area.     

Onchocerca sweetae (Nematoda: Filarioidea): notes on the intermediate host.

Microfilariae of Onchocerca sweetae are broadly distributed in the superficial layers of the dermis of the water buffalo (Bubalus bubalis). A total of 2855 insects representing 20 species were collected from O. sweetae-infected bait buffaloes. Only one species, Culicoides sp. "M", ingested microfilariae from buffalo skin. Larval development of O. sweetae was observed in the thorax of this species. Atotal of 829 insects, representing 7 species and including 749 parous Culicoides spp. were collected from light and Manitoba traps. Developing filarioid larvae were observed only in Culicoides sp. "M". It is concluded that Culicoides sp. "M" is a natural intermediate host of O. sweetae in the Northern Territory of Australia.     

Identification and characterization of onchoastacin, an astacin-like metalloproteinase from the filaria Onchocerca volvulus

The tissue-invasive nematode Onchocerca volvulus causes skin and eye pathology in human onchocerciasis. While the adult females reside sessile in subcutaneous nodules, the microfilariae are abundantly released from the nodules, males and juvenile worms migrate through the host tissue. Matrix-degrading metallo- and serine proteinases have been detected in excretory-secretory worm products that may be essential for migration of the mobile stages. In this study, a 1713bp long cDNA encoding for a putative proteinase of O. volvulus has been isolated. The predicted protein sequence includes a signal peptide indicating secretion to the extracellular space, a propeptide, an astacin-like protease domain, an EGF-like and a CUB-domain, thereby identifying the protein as a member of the astacin family of zinc endopeptidases. Onchoastacin, Ov-AST-1, is most closely related to a subfamily comprising nematode astacins including Caenorhabditis and Ancylostoma. Ov-AST-1 was expressed as a recombinant protein in baculovirus-infected insect cells and exhibited enzymatic activity. The exposure of onchoastacin to the host immune system is indicated by demonstration of IgG reacting with the recombinant Ov-AST-1 and with two peptides of the protein. Since a homologous metalloproteinase is part of a promising hookworm vaccine, Ov-AST-1 may be a candidate for intervention strategies in filarial infections.     

Infective larvae of five Onchocerca species from experimentally infected Simulium species in an area of zoonotic onchocerciasis in Japan

Microfilariae of five Onchocerca species, O. dewittei japonica (the causative agent of zoonotic onchocerciasis in Oita, Kyushu, Japan) from wild boar (Sus scrofa), O. skrjabini and O. eberhardi from sika deer (Cenus nippon), O. tienalis from cattle, and an as yet unnamed Onchocerca sp. from wild boar, were injected intrathoracically into newly-emerged black flies of several species from Oita to search the potential vector(s) of these parasites and identify their infective larvae. Development of O. dewittei japonica microfilariae to the infective larvae occurred in Simulium aokii, S. arakowae, S. bidentatum, S. japonicum, S. quinquestriatum, and S. rufibasis while development of infective larvae of O. skrjabini, O. eberhardi, and the unnamed Onchocerca sp. was observed in S. aokii, S. arakawae, and S. bidentatum. Development of O. lienalis microfilaria to infective larvae occurred in S. arakawae. Based on the morphology of infective larvae obtained, we proposed a key of identification of Onchocerca infective larvae found in Oita. We also reconsider the identification of three types of infective larvae previously recovered from Simulium species captured at cattle sheds: the large type I larvae that may be an undescribed species; the small type III identified as O. lienalis may include O. skrjabini too; the intermediary type II that may be O. gutturosa, or O. dewittei japonica, or the unnamed Onchocerca sp. of wild boar.     

Extracellular proteases of Onchocerca

Two important events in infection by Onchocerca parasites involve cutaneous tissue migration by larval stages. L3 larvae migrate from the blackfly bite site to subcutaneous locations for adult development, and microfilariae from subcutaneous nodules to distant regions of the skin and sometimes the eye. By analogy to other tissue-invasive helminth larvae, it has been proposed that migration of Onchocerca larvae through cutaneous tissue is facilitated by secretion of proteolytic enzymes. To test this hypothesis, neutral protease activity capable of degrading a model of cutaneous extracellular matrix was assayed using live L3 larvae of O. lienalis and microfilariae of O. cervicalis and O. cervipedis. Five hundred L3 larvae degraded most of the matrix within 24 hr of incubation. Substrate gel electrophoresis and other protease assays showed a 43-kDa serine elastase was secreted by O. lienalis L3 larvae. Larvae and adults of the free-living nematode, Caenorhobditis elegans, by contrast, did not secrete neutral proteases and large numbers of motile C. elegans juveniles and adults produced no degradation of the extracellular matrix. Expression of Onchocerca neutral protease activity was stage specific. No protease activity corresponding to that seen in L3 larvae was found in adult worms. Microfilariae of O. cervicalis and O. cervipedis produced both a serine and a metalloprotease, but the level of protease activity of these microfilariae was substantially lower than that of L3 larvae, and no significant protease activity was detected in extracts of O. lienalis microfilariae. Uterine microfilariae of O. cervicalis had different protease species than skin microfilariae, suggesting that changes in protease expression parallel other morphologic and biochemical changes in the development of skin microfilariae. The serine protease of L3 larvae probably plays an important parasitic function, facilitating L3 migration from the blackfly bite site to distant regions of the body where adults will develop and form nodules. The protease activity of microfilariae, while individually considerably less than that of L3 larvae, may still contribute to the tissue destruction seen with heavy skin densities of microfilariae.     

Lymphatic Vascularisation and Involvement of Lyve-1+ Macrophages in the Human Onchocerca Nodule

Onchocerciasis, caused by the filarial nematode Onchocerca volvulus, is a parasitic disease leading to debilitating skin disease and blindness, with major economic and social consequences. The pathology of onchocerciasis is principally considered to be a consequence of long-standing host inflammatory responses. In onchocerciasis a subcutaneous nodule is formed around the female worms, the core of which is a dense infiltrate of inflammatory cells in which microfilariae are released. It has been established that the formation of nodules is associated with angiogenesis. In this study, we show using specific markers of endothelium (CD31) and lymphatic endothelial cells (Lyve-1, Podoplanin) that not only angiogenesis but also lymphangiogenesis occurs within the nodule. 7% of the microfilariae could be found within the lymphatics, but none within blood vessels in these nodules, suggesting a possible route of migration for the larvae. The neovascularisation was associated with a particular pattern of angio/lymphangiogenic factors in nodules of onchocerciasis patients, characterized by the expression of CXCL12, CXCR4, VEGF-C, Angiopoietin-1 and Angiopoietin-2. Interestingly, a proportion of macrophages were found to be positive for Lyve-1 and some were integrated into the endothelium of the lymphatic vessels, revealing their plasticity in the nodular micro-environment. These results indicate that lymphatic as well as blood vascularization is induced around O. volvulus worms, either by the parasite itself, e.g. by the release of angiogenic and lymphangiogenic factors, or by consecutive host immune responses.     

Nematocysts and Taxonomy in Laomedea, Gonothyraea and Obelia (Hydrozoa, Campanulariidae)

The cnidoms of Laomedea flexuosa, Gonothyraea loveni, Obelia geniculata, O. longissima and O. dichotoma were studied by light and scanning electron microscopy to find out whether the nematocysts could be used as taxonomic characters. Three b-rhabdoid heteroneme nematocysts (microbasic b-mastigophore) and three isorhizous haploneme nematocysts (atrichous isorhiza) were distinguished. A small b-rhabdoid nematocyst with spindle-shaped capsule occurred in all the species examined. In the polyp and planula of G. loveni , and the planula of L. flexuosa it was the only nematocyst present. Specific for L. flexuosa was a b-rhabdoid with curved capsule. In the polyp and newly-liberated medusa of O. longissima another b-rhabdoid appeared with bean-shaped capsule and markedly long spines at the distal tube. The polyp and newly-liberated medusa of O. dichotoma were characterized by two different isorhizas. A special type of isorhiza occurred in the polyp of O. geniculata . The curved capsules of the different isorhizas varied somewhat in shape and size. Differences in nematocyst structure and occurrence are presumed to provide characters for taxonomy. Thus, O. dichotoma and O. Iongissima are regarded as separate species due to their distinctly different nematocysts, in conjunction with other morphological and ecological differences.     

Cloning of a species-specific DNA probe from Onchocerca gibsoni

A genomic library of Onchocerca gibsoni has been prepared in the vector lambda-gt10 and has been screened for specific DNA sequences by hybridization with radiolabelled total genomic DNA from a number of Onchocerca species. A clone--fOGI--has been isolated which does not interact with DNA prepared from O. gutturosa, O. lienalis, O. ochengi, O. cervicalis or O. volvulus (both Liberian and Mexican isolates). In addition, no hybridization is observed with host (cattle) DNA. fOGI can detect as little as 100-200 pg of O. gibsoni DNA. It is thus concluded that fOGI has the sensitivity to detect microfilariae of O. gibsoni found in the skin of cattle and the specificity to differentiate them from closely related species living in the same environment.     

COMPARATIVE POPULATION GENETIC STRUCTURE OF A PARASITE (FASCIOLOIDES MAGNA) AND ITS DEFINITIVE HOST

The population genetic structure of the American liver fluke, Fascioloides magna, and its definitive host the white-tailed deer, Odocoileus virginianus, was examined in South Carolina. Flukes were significantly more common in deer from river-swamp habitat than upland areas and prevalence increased with host age. The distribution of flukes among deer occurred as a negative binomial with the mean dispersion parameter, k, equal to 0.17 and the range from 0.10 to 1.11 within local areas. Significant spatial genetic differentiation was observed for flukes and deer. Patterns of genetic distance in flukes were not concordant with those of the definitive host nor were they related to geographic distance between sample locations. Spatial genetic differentiation among flukes reflected the tendency for individual hosts to harbor multiple individuals from a limited number of parasite clones. The large population size of the parasite and movements of the definitive host tend to counteract factors that lead to spatial differentiation.     

Rumen Ciliates of White-tailed Deer (Odocoileus virginianus), Axis Deer (Axis axis), Sika Deer (Cervus nippon) and Fallow Deer (Dama dama) from Texas

Samples of rumen contents from 33 white-tailed deer (Odocoileus virginianus), 31 axis deer (Axis axis), 26 sika deer (Cervus nippon), and 25 fallow deer (Dama dama) were collected from four study areas in central Texas. The geometric mean concentration of total protozoa was 50.2 x 10(4) per ml, with no differences between species (P > 0.36). White-tailed deer had a higher percentage of Entodinium and lower percentage of Diplodiniinae (P < 0.01) than the other deer species, which were not different from each other. Occurrence of Epidinium, Isotricha, and Dasytricha was sporadic and did not differ among deer species. Numerous new host records of protozoan species were observed: white-tailed deer--four; axis deer--five; sika deer--five; fallow deer--four. This brings the total number of protozoan species identified in each deer species to: white-tailed--eight; axis--12; sika--15; fallow--16. For all species combined, protozoan concentration were 7.5 to 11-fold higher (P < 0.01) from Area 4, which differed from the other three areas by having a stream that allowed deer to have free access to water. Criteria used for identification of medium-size Eudiplodinium species were evaluated.     

Onchocerciasis in camels (Camelus dromedarius) in Saudi Arabia

During a survey in 1980-81, 125 of 478 (26.2%) camels in Saudi Arabia were found infected with onchocerciasis. The prevalence rates in local and imported camels were 93/272 (34.2%) and 32/206 (15.5%), respectively. The disease was characterized by hard nodules in the connective tissue around the nuchal ligaments and in the subcutis. The nodules consisted of cavities containing live, degenerate or dead Onchocerca fasciata, inflammatory cells, fibrosis and calcification. The microfilariae were concentrated in the skin over the head and neck regions and often caused mild non-suppurative dermatitis.     

Maxillary lymphosarcoma in a white-tailed deer (Odocoileus virginianus)

In 1996, lymphosarcoma was observed in a captive adult female white-tailed deer (Odocoileus virginianus) from northeastern Kansas (USA). A subcutaneous mass on the deer's left cheek was surgically removed and lymphosarcoma was diagnosed. The mass recurred within 3 wk. A second surgical removal was attempted but the tumor had grown much larger, had become intimately involved with the buccal mucosa, and was beginning to interfere with mastication. For these reasons, the deer was euthanized. At postmortem examination the only abnormal findings were the primary tumor and enlarged ipsilateral parotid and mandibular lymph nodes. Histologically these tissues demonstrated changes characteristic of lymphosarcoma but no other organs had evidence of neoplastic disease. A diagnosis of focal lymphosarcoma with local metastasis was made. The organ distribution of lymphosarcoma in this deer differs from previously described cases of lymphosarcoma in cervids.     

Pathology and preliminary characterization of a parapoxvirus isolated from a California sea lion (Zalophus californianus)

Cutaneous pox-like lesions are a common complication in the rehabilitation of pinnipeds. However, the exact identity, taxonomy, and host range of pinniped parapoxviruses remain unknown. During a poxvirus outbreak in May 2003 in California sea lions (Zalophus californianus) at a marine mammal rehabilitation facility, multiple raised, firm, 1-3-cm skin nodules from the head, neck, and thorax of one sea lion weanling pup that spontaneously died were collected. Histologically, the nodules were characterized by inflammation and necrosis of the dermis and epidermis, acanthosis, and ballooning degeneration of the stratum spinosum. Large, coalescing eosinophilic cytoplasmic inclusions were observed in the ballooned cells. A parapoxvirus (sea lion poxvirus 1, SLPV-1) was isolated on early passage California sea lion kidney cells inoculated with a tissue homogenate of a skin nodule. The morphology of the virions on electron microscopy was consistent with that of parapoxviruses. Partial sequencing of the genomic region encoding the putative major virion envelope antigen p42K confirmed the assignment of the sea lion poxvirus to the genus Parapoxvirus. Although SLPV-1 is most closely related to the poxvirus of harbor seals of the European North Sea, it is significantly different from orf virus, bovine papular stomatitis virus, pseudocowpox virus and the parapoxvirus of New Zealand red deer.     

Experimental infection of white-tailed deer (Odocoileus virginianus) with Ehrlichia chaffeensis by different inoculation routes

The infection dynamics of the tick-transmitted organism Ehrlichia chaffeensis were investigated in white-tailed deer (Odocoileus virginianus) using different routes of inoculation. Six deer were each inoculated with 5.4 x 10(6) DH82 cells infected with E. chaffeensis (Arkansas strain) by three different routes: intravenous (n = 2), subcutaneous (n = 2), and intradermal (n = 2). Two control deer were inoculated with uninfected cells. Infections were monitored for 54 days and were continued in one deer from each E. chaffeensis inoculated group for an additional 31 days. All deer inoculated with E. chaffeensis seroconverted (> or = 1: 64) and became 16S rDNA polymerase chain reaction and/or cell culture positive by post-inoculation day 15. There was no apparent (difference in susceptibility to infection between deer inoculated by different routes for the first 50 days based on detection of E. chaffeensis infection by PCR assay of blood or culture isolation. These results demonstrate infection of (deer by intradermal and subcutaneous routes for the first time.     

Observations on the routes of infection of Oswaldocruzia filiformis (Nematoda: Trichostrongylidae) in amphibia

The oral, percutaneous and subcutaneous routes of infection of Oswaldocruzia filiformis were investigated in amphibia. Tadpoles of Bufo bufo and Rana temporaria can be infected with O. filiformis when kept temporarily in a suspension of infective larvae in water. Larval stages and subadults were found in tadpoles. All stages of the parasite, including egg-producing females, were found after metamorphosis of the host. However, under natural circumstances infection of tadpoles seems unlikely. Oral infections in metamorphosed hosts of both species were successful in 97.5% of the host animals used. The first eggs appeared 29 days after infection in the faeces. The oral route seems to be normal for O. filiformis in amphibia. Experiments on percutaneous infections did not reveal actual penetration of larvae in or through the skin nor a subsequent migration through host tissues. Sometimes a few larvae were found in the stomach and intestine, but in these particular cases the experimental conditions did not totally exclude the possibility of oral infections. Consequently, the percutaneous route of infection is not plausible for O. filiformis. Subcutaneous inoculation of infective larvae seems to be a possible way of establishing experimental infections. Erratic localisation of the parasite in the enlarged gall bladder of the host was observed.     

Worldwide phylogeny and biogeography of Cardamine flexuosa (Brassicaceae) and its relatives

Phylogenetic relationships, biogeography, and taxonomy of a group of taxa putatively related to the tetraploid Cardamine flexuosa were explored using sequences of the internal transcribed spacer region of nrDNA (ITS) and the trnL-trnF region of cpDNA. Taxon sampling focused on eastern Asia, North America, and Europe, and included 19 taxa represented by 177 and 182 accessions for each data set, respectively. Our analyses provided unequivocal evidence that Asian weedy populations traditionally assigned to C. flexuosa form an independent evolutionary lineage and represent a distinct taxon from European C. flexuosa. The allopolyploid origin of this common weed in paddy fields, its origin, and/or spread associated with the establishment of suitable man-made habitats are suggested. It is also found as an introduced weed in Australia and North America. Phylogenetic relationships and the associated taxonomic implications are presented and discussed for the group as a whole. Contrasting patterns of genetic variation (particularly in cpDNA) among different species were revealed. While very little haplotype diversity was found in widespread C. hirsuta and C. flexuosa, greater variation, showing phylogeographic structure, was observed in the tetraploid C. scutata within a relatively small area of Japan.     

The filarial endosymbiont Wolbachia sp. is absent from Setaria equina

Wolbachia sp. was first reported in filarial nematodes over 25 yr ago. Today, much research is focused on the role of these bacteria in filarial worm biology. The filarial symbionts are closely related to arthropod symbionts, which are known to modify host reproduction and biology through various mechanisms. Similarly, it has been suggested that Wolbachia sp. is essential for long-term survival and reproduction of filariae. We report that Wolbachia sp. 16S rDNA was not found in the equine filarial nematode Setaria equina, using either polymerase chain reaction (PCR) or DNA hybridization. In addition, ultrastructural analysis of adult worms did not reveal the presence of Wolbachia sp. in hypodermal cords or reproductive tissues. These data suggest that like Onchocerca flexuosa and Acanthocheilonema vitae, S. equina may not be dependent on Wolbachia sp. for survival.