Larva migrans by Baylisascaris transfuga: fatal neurological diseases in Mongolian jirds, but not in mice

Raccoon roundworms (Baylisascaris procyonis) and other Baylisascaris species cause patent or latent larva migrans (LM) in a variety of mammals and birds, including humans. It is not clear whether LM by Baylisascaris transfuga, roundworms of bears, is associated with clinical neurological disorders. To clarify this issue, ICR and BALB/c mice as well as Mongolian jirds (Meriones unguiculatus) were orally inoculated with 2,000-5,000 embryonated eggs of B. transfuga. In mice, the ascarid caused symptomatic LM of limited extent and duration, whereas the infection was fatal in jirds; i.e., they exhibited general signs such as severe depression and emaciation on days 8-11 postinfection (PI) and died, or they developed progressive and fatal neurological disorders after day 14 PI. Histological examination showed B. transfuga larvae in the brain of all mice and jirds examined, and the larvae collected from them developed to a size comparable with that of B. procyonis. There existed, however, critical differences in host reactions against larvae localized in the brain of mice and jirds; B. transfuga larvae found in mice were surrounded by granulomatous reactions and immobilized, whereas larvae found in jirds were free from any host reaction and mobile, causing extensive malacia.     

Complete mitochondrial genomes of Baylisascaris schroederi, Baylisascaris ailuri and Baylisascaris transfuga from giant panda, red panda and polar bear

Roundworms of the genus Baylisascaris are the most common parasitic nematodes of the intestinal tracts of wild mammals, and most of them have significant impacts in veterinary and public health. Mitochondrial (mt) genomes provide a foundation for studying epidemiology and ecology of these parasites and therefore may be used to assist in the control of Baylisascariasis. Here, we determined the complete sequences of mtDNAs for Baylisascaris schroederi, Baylisascaris ailuri and Baylisascaris transfuga, with 14,778 bp, 14,657 bp and 14,898 bp in size, respectively. Each mtDNA encodes 12 protein-coding genes, 22 transfer RNAs and 2 ribosomal RNAs, typical for other chromadorean nematodes. The gene arrangements for the three Baylisascaris species are the same as those of the Ascaridata species, but radically different from those of the Spirurida species. Phylogenetic analysis based on concatenated amino acid sequences of 12 protein-coding genes from nine nematode species indicated that the three Baylisascaris species are more closely related to Ascaris suum than to the three Toxocara species (Toxocara canis, Toxocara cati and Toxocara malaysiensis) and Anisakis simplex, and that B. ailuri is more closely related to B. transfuga than to B. schroeder. The determination of the complete mt genome sequences for these three Baylisascaris species (the first members of the genus Baylisascaris ever sequenced) is of importance in refining the phylogenetic relationships within the order Ascaridida, and provides new molecular data for population genetic, systematic, epidemiological and ecological studies of parasitic nematodes of socio-economic importance in wildlife.     

Seasonal changes in the prevalence of ova of Diphyllobothrium ursi and Baylisascaris transfuga in the feces of the black bear (Ursus americanus)

An analysis of 114 samples of feces from black bears (Ursus americanus) from La Verendrye Park, Quebec revealed that the prevalence of eggs of Diphyllobothrium ursi and Baylisascaris transfuga varied seasonally. D. ursi ova were most prevalent during the summer and fall and at their lowest in spring while the converse was true for B. transfuga. This phenomenon possibly is associated with the cessation of feeding during the winter denning of black bears.     

Biochemical and immunological systematics of some ascaridoid nematodes: genetic divergence between congeners

Vertical starch gel electrophoresis and trefoil immunodiffusion were used to study the systematics of some ascaridoid nematodes. Within the Ascarididae, the time scale of divergence was too great for intergeneric electrophoretic comparisons. Congeneric electrophoretic comparisons of Baylisascaris procyonis (host--raccoon) versus Baylisascaris transfuga (host--black bear), and Toxocara canis (host--domestic dog) versus Toxocara cati (host--domestic cat) yielded Nei genetic distance coefficients of 1.21 and 1.55, respectively. Estimates of times of divergence made from 1 electrophoretic clock calibration suggest that the Baylisascaris species have not shared a common ancestor for 25 million years (Myr), and that the Toxocara species diverged 33 Myr ago. The Baylisascaris divergence estimate corresponds to host-family divergence estimates based on immunological and paleontological evidence, which suggests that cospeciation has occurred. In contrast to this, Ascaris suum (host--pig) and Ascaris lumbricoides (host--human) have a distance coefficient of 0.09. This indicates that these species diverged comparatively recently and may represent a case of host range expansion. Trefoil immunodiffusion comparisons of ascaridoid albumins yielded reactions of identity for A. suum, A. lumbricoides, Parascaris equorum, B. procyonis, B. transfuga, T. canis, and T. cati. This confirms that these taxa are members of a monophyletic group.     

Parasitic helminths of black bear cubs (Ursus americanus) from Florida

Twenty-two Florida black bear (Ursus americanus floridanus) cubs (< or = 12 mo old) from Florida were examined for endoparasites between 1998 and 2003. Eleven species of helminths were found (8 nematodes, 2 trematodes, and 1 acanthocephalan). The most prevalent helminths were Ancylostoma caninum (64%), Macracanthorhynchus ingens (36%), Strongyloides sp. (27%), Baylisascaris transfuga (23%), and A. tubaeforme (14%). New host records include A. tubaeforme and Brachylaima virginianum. This is the first report of B. transfuga in black bears from Florida.     

Physicochemical characterization and monoclonal and polyclonal antibody recognition of Baylisascaris procyonis larval excretory-secretory antigens

Baylisascaris procyonis larval excretory-secretory (ES) antigens consisted of complex glycoproteins ranging from 10 kDa to over 200 kDa as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and lectin binding. Five monoclonal antibodies (Bapr1-Bapr5) produced against B. procyonis ES antigens were assayed by western blotting with larval ES antigens from B. procyonis, Baylisascaris melis, Baylisascaris transfuga, Ascaris suum, and Toxocara canis. Bapr1 and Bapr2 recognized periodate-sensitive epitopes on 14-kDa ES components of B. procyonis, B. melis, and B. transfuga, whereas Bapr4 and Bapr5 recognized periodate-resistant epitopes present on 55-kDa ES components of B. procyonis and B. melis. Bapr3 primarily recognized periodate-resistant epitopes on 33-45-kDa components of B. procyonis and B. melis ES. Heterologous rabbit antisera cross-reacted with many B. procyonis ES antigens on western blots, but recognition of the 33-45-kDa components was genus-specific. Normal human sera and T. canis-positive human sera also cross-reacted with many B. procyonis ES antigens, including those of 33-45 kDa. However, periodate oxidation markedly decreased cross-reactions and allowed for differential immunodiagnosis of B. procyonis versus T. canis. These studies demonstrated that antibody recognition of carbohydrate epitopes on ES components is an important cause of cross-reactions in antibody detection assays. Recognition of periodate-resistant (protein) epitopes on the 33-45-kDa B. procyonis ES components appears to be useful for genus-specific immunodiagnosis of larva migrans caused by Baylisascaris spp.     

Ecto- and endoparasites of the black bear in northern Wisconsin

Parasites collected from free-ranging black bears, Ursus americanus, in northern Wisconsin included Dermacentor variabilis, D. albipictus, Ixodes scapularis, Demodex sp., Trichodectes pinguis euarctidos, Baylisascaris transfuga and Dirofilaria ursi. Mange, possibly caused by the Demodex sp., also was observed. Dental caries were common and periodontal disease was observed in one animal.     

Helminths recovered from black bears in the Peace River region of northwestern Alberta.

A total of 91 free-ranging black bears (Ursus americanus) from the Peace River region of northwestern Alberta was examined for helminths. Four species, Baylisascaris transfuga, Taenia krabbei, Taenia hydatigena and Dirofilaria ursi, were found. None of the bears was heavily infected. Results are compared with other similar North American surveys.     

Fatal Baylisascaris larva migrans in a colony of Japanese macaques kept by a safari-style zoo in Japan

A colony of Japanese macaques (Macaca fuscata fuscata) kept by a safari-style zoo in Japan experienced 9 sporadic cases of fatal neurological diseases, such as epilepsy and posterior paralysis, during the 12 yr from 1989 to 2001. This macaque colony consisted of approximately 30 animals, on average, during this period, and the macaques shared their living space with II American black bears (Ursus americanus) harboring zoonotic roundworms (Baylisascaris transfuga). Close to this enclosure, a cote for 2-3 raccoons (Procyon lotor) was placed, and raw sewage from this cote ran into a shallow drain in the area for macaques and bears. However, fecal examinations in recent years did not detect the infection of raccoons with zoonotic roundworms (Baylisascaris procyonis). Postmortem histological examination of the latest 2 ill macaques detected multifocal malacia in the brain; 2 ascarid larvae of 60 microm maximum width were encapsulated in the cerebrum and lungs of 1 of the animals. To determine the causative ascarid species of the fatal larva migrans, we analyzed 2 additional encapsulated Baylisascaris larvae collected from formalin-fixed lungs by morphological and molecular approaches. This sporadic outbreak is the second record of Baylisascaris larva migrans in animals in Japan.     

Comparative efficacy of ivermectin and levamisole for reduction of migrating and encapsulated larvae of Baylisascaris transfuga in mice

The comparative efficacy of 2 anthelmintics (ivermectin and levamisole) against Baylisascaris transfuga migrating and encapsulated larvae was studied in mice. A total of 60 BALB/c mice inoculated each with about 1,000 embryonated B. transfuga eggs were equally divided into 6 groups (A-F) randomly. Mice of groups A and B were treated with ivermectin and levamisole, respectively, on day 3 post-infection (PI). Mice of groups A-C were killed on day 13 PI. Similarly, groups D and E were treated with ivermectin and levamisole, respectively, on day 14 PI, and all mice of groups D-F were treated on day 24 PI. The groups C and F were controls. Microexamination was conducted to count the larvae recovering from each mouse. The percentages of reduction in the number of migrating larvae recovered from group A (ivermectin) and B (levamisole) were 88.3% and 81.1%, respectively. In addition, the reduction in encapsulated larvae counts achieved by ivermectin (group D) and levamisole (group E) was 75.0% and 49.2%, respectively. The results suggested that, to a certain extent, both anthelmintics appeared to be more effective against migrating larvae than encapsulated larvae. However, in the incipient stage of infection, ivermectin may be more competent than levamisole as a larvicidal drug for B. transfuga.     

Studies on endoparasites of the black bear (Ursus americanus) in the southeastern United States.

Examination of 53 black bears (Ursus americanus) from six states in the southeastern United States revealed at least 17 species of endoparasites, including Sarcocystis sp., Spirometra mansonoides (spargana), Macracanthorhynchus ingens, Ancylostoma caninum, Arthrocephalus lotoris, Baylisascaris transfuga, Capillaria aerophila, Capillaria putorii, Crenosoma sp., Cyathospirura sp., Dirofilaria immitis, Gnathostoma sp., Gongylonema pulchrum, microfilariae, Molineus barbatus, Physaloptera sp. and Strongyloides sp. Twelve of these represent new host records for black bear, and two are considered to be new species. Data are presented on prevalence, intensity and geographic distribution of each species. Pathologic effects were associated with infections of spargana of S. mansonoides and adults of C. aerophilia.     

Migration behaviour and pathogenesis of five ascarid nematode species in the Mongolian gerbil Meriones unguiculatus

To understand the characteristic features of the Mongolian gerbil, Meriones unguiculatus, as an animal model of ascarid infections, the migration behaviour and pathogenesis of larvae were investigated in experimentally infected gerbils. Embryonated eggs from each of Toxocara canis, Baylisascaris procyonis, B. transfuga, Ascaris suum, and A. lumbricoides were orally inoculated into gerbils and larvae were recovered from various organs at designated periods. In T. canis-infected gerbils, larvae were present in the liver 3 days after infection and in the skeletal muscle and brain via the heart and lungs at a similar rate. In B. procyonis- and B. transfuga-infected gerbils, larvae were present in the lungs within 24 h after infection, with some having reached the brain by that time. After 24 h, larvae of B. procyonis tended to accumulate in the brain, while those of B. transfuga accumulated in skeletal muscles. In A. suum- and A. lumbricoides-infected gerbils, larvae remained in the liver on day 5 post-infection and elicited pulmonary haemorrhagic lesions, which disappeared 7 days after initial infection. Thereafter, no larvae of any type were recovered. Ocular manifestations were frequently observed in T. canis- and B. procyonis infected gerbils, but were rare in B. transfuga-infected gerbils. In the cases of A. suum and A. lumbricoides, migration to the central nervous system and eyes was extremely rare, and larvae had disappeared by 2 weeks post-infection. Fatal neurological disturbances were observed in B. procyonis-infected gerbils, whereas irreversible non-fatal neurological symptoms were observed in the case of B. transfuga.     

Parasites of black bears of the Lake Superior region.

Parasites collected from free-ranging black bears, Ursus americanus Pallas, 1780, from northeastern Minnesota or northern Michigan include the dog tick, Dermacentor variabilis (Say, 1821), the winter tick, D. ALBIPICTUS (Packard, 1869), a louse, Trichodectes pinguis euarctidoes Hopkins, 1954, an ascarid worm, Baylisascaris transfuga (rudolphi, 1819), a filarial worm, Dirofilaria ursi Yamaguti, 1941, taeniid tapeworms, and unidentified fleas. The broad fish tapeworm, Diphyllobothrium latum (linnaeus, 1785), was reported to be common in man and black bears in northeastern Minnesota earlier in this century, but now it apparently is uncommon in black bears in norther Wisconsin. Cannibalism of carcasses is common in the black bear and may play a major role in the transmission of Trichinella.     

Molecular characterization and phylogenetic analysis of ascarid nematodes from twenty-one species of captive wild mammals based on mitochondrial and nuclear sequences

SUMMARY Although ascarid nematodes are important parasites of wild animals of public health concern, few species of ascarids from wild animals have been studied at the molecular level so far. Here, the classification and phylogenetic relationships of roundworms from 21 species of captive wild animals have been studied by sequencing and analysis of parts of the ribosomal 18S and 28S genes and the mitochondrial (mt) 12S gene. Phylogenetic relationships were inferred by 3 methods (NJ/MP/ML) based on the data of single gene sequences and concatenated sequences. Homology analysis indicated that the 18S sequences were conserved among roundworms from all 21 species and that 28S showed interspecies variability. Divergence levels displayed in 12S suggested that 12S appears to be either intra- or interspecifically variable. Evolutionary trees indicated that the ascarids split into 2 families, 4 genera and 7 species, with high bootstrap support for each clade. Combined trees suggested that Baylisascaris ailuri is more closely related to B. transfuga than to B. schroederi. This study provides useful molecular markers for the classification, phylogenetic analysis and epidemiological investigation of roundworms from wild animals.     

Phylogenetic study of Baylisascaris schroederi isolated from Qinling subspecies of giant panda in China based on combined nuclear 5.8S and the second internal transcribed spacer (ITS-2) ribosomal DNA sequences

The nuclear ribosomal DNA (rDNA) region spanning 5.8S rDNA and the second internal transcribed spacer (ITS-2) of Baylisascaris schroederi isolated from the Qinling subspecies of giant panda in Shaanxi Province, China were amplified and sequenced. Sequence variations in the two rDNA regions within B. schroederi and among species in the family Ascarididae were examined. The lengths of B. schroederi 5.8S and ITS-2 rDNA sequences were 156 bp and 327 bp, respectively, and no nucleotide variation was found in these two rDNA regions among the 20 B. schroederi samples examined, and these ITS-2 sequences were identical to that of B. schroederi isolated from giant panda in Sichuan province, China. The inter-species differences in 5.8S and ITS-2 rDNA sequences among members of the family Ascarididae were 0-1.3% and 0-17.7%, respectively. Phylogenetic relationships among species in the Ascarididae were re-constructed by Bayesian inference (Bayes), maximum parsimony (MP), and maximum likelihood (ML) analyses, based on combined sequences of 5.8S and ITS-2 rDNA. All B. schroederi samples clustered together and sistered to B. transfuga with high posterior probabilities/bootstrap values, which further confirmed that nematodes isolated from the Qinling subspecies of giant panda in Shaanxi Province, China represent B. schroederi. Because of the large number of ambiguously aligned sequence positions (difficulty of inferring homology by positions), ITS-2 sequence alone is likely unsuitable for phylogenetic analyses at the family level, but the combined 5.8S and ITS-2 rDNA sequences provide alternative genetic markers for the identification of B. schroederi and for phylogenetic analysis of parasites in the family Ascarididae.     

Baylisascaris procyonis (Nematoda: Ascarididae) larva migrans in free-ranging wildlife in Orange County,California

Baylisascaris procyonis is well recognized as a cause of visceral (VLM), ocular, and neural (NLM) larva migrans in birds and mammals, including man. A study of the prevalence of larva migrans in free-ranging wildlife associated with raccoon latrines as well as a retrospective study of wildlife mortalities with neurological disease was conducted in 2000 in Orange County, California. Eighty-seven birds of 18 species and 64 mammals of 8 species were found to have NLM or VLM or both. NLM clinical signs included convulsions, torticollis, opisthotonus, head-tilts, circling, ataxia, paralysis, and visual defects. NLM lesions were characterized by focally disseminated, frequently linear, "tracklike" areas of parenchymal degeneration with varying degrees of astrocytosis in the white matter of the cerebrum, cerebellum, brain stem, and spinal cord. Larvae were rarely found in these lesions but were rather isolated in the brain-spinal cord parenchyma. At least 1 larva was isolated by digestion from each case of NLM and identified as Baylisascaris sp., most likely B. procyonis. VLM lesions consisted of granulomatous reactions surrounding intact or degenerative larvae in the parenchyma of the liver, kidney, diaphragm, and, occasionally, the lymph nodes. This report broadens the range of species of wild birds and mammals that have been found to be susceptible to larva migrans caused by B. procyonis and reaffirms the importance of raccoon latrine sites as contaminative foci for wildlife.     

Identity of Cysticercoides menidiae Chandler, 1935 (Cestoda: Dilepididae)

Cysticercoides menidiae Chandler, 1935, described as a metacestode of an unknown dilepidid cestode from the intestinal wall and mesenteries of the silversides Menidia menidia (Pisces: Atherinidae), from Galveston Bay, Texas, was found to be conspecific with Ascodilepis transfuga (Krabbe, 1869), a tapeworm described from the spoonbill. Platalea ajaja Linnaeus (syn. Ajaja ajaja), from Brazil. Consequently. C. menidiae becomes a junior synonym of A. transfuga.     

The inducibility of TNF-alpha production is different in the granulocytic and monocytic differentiated forms of wild type and CGD-mutant PLB-985 cells

Chronic granulomatous disease is an inherited disorder associated with a defect in phagocytic cell oxidative metabolism resulting in ineffective microbicidal activity. Consequently, patients with chronic granulomatous disease suffer from recurrent infections. Published data show that besides the failure to produce superoxide and its derivatives, other functional problems can also be found in chronic granulomatous disease-mutant cells. Since in innate immune responses other mediators, such as cytokines, also play an important role, we hypothesized that there may be a disturbance in cytokine production by chronic granulomatous disease-mutant cells as well. To prove this hypothesis, the production of tumour necrosis factor-alpha, an important proinflammatory cytokine, was determined by enzyme-linked immunosorbent assay in wild-type and chronic granulomatous disease-mutant myelomonoblastic PLB-985 cells in their immature, granulocytic and monocytic/macrophage differentiated forms. Tumour necrosis factor-alpha production was induced with N-formyl-L-methionyl-L-leucyl-L-phenylalanine (100 nmol/L), lipopolysaccharide (10 micro g/mL), opsonized zymosan (100 micro g/mL) or phorbol 12-myristate 13-acetate (100 nmol/L) for 24 h. We could demonstrate that: (i) there were marked differences in tumour necrosis factor-alpha production only in the differentiated forms of both wild-type and chronic granulomatous disease-mutant cells, while there were no differences in the case of their immature counterparts; (ii) only chronic granulomatous disease-mutant cells retained sensitivity to phorbol 12-myristate 13-acetate both in their granulocytic and monocytic forms, although phorbol 12-myristate 13-acetate responsiveness was a characteristic of both types of immature cells; (iii) the granulocytic form of wild-type cells produced tumour necrosis factor-alpha after opsonized zymosan stimulation, but such a response was not observed in cells originating from the chronic granulomatous disease-mutant cell line; (iv) with the monocytic forms, significantly higher tumour necrosis factor-alpha production could be induced by lipopolysaccharide in the wild-type cells than in the chronic granulomatous disease-mutant cells, although there was no difference in their lipopolysaccharide receptor CD14 expression. In summary, these data show an altered inducibility of tumour necrosis factor-alpha production by chronic granulomatous disease-mutant cells. Our observations suggest a further defect in differentiated chronic granulomatous disease-mutant cells in addition to the known defect in reduced nicotinamide adenine dinucleotide phosphate oxidase, which may contribute to the development of susceptibility to infections in people with chronic granulomatous disease.     

Suppression of experimental allergic encephalomyelitis in rats by mercuric chloride

Brown-Norway (BN) rats are uniquely susceptible to development of autoimmune phenomena and enlargement of lymph nodes and spleen after repeated injections of mercuric chloride. Despite its ability to produce autoimmunity, HgCl2 inhibited the development in BN rats of experimental allergic encephalomyelitis (EAE), another autoimmune process. The inhibition by mercury was probably due to lack of the normal absorption and granulomatous reaction to the EAE inoculum in the enlarged lymph nodes draining the inoculation site. Lewis rats did not develop enlarged nodes from HgCl2 treatment. Lewis lymph nodes absorbed the EAE inoculum abundantly and developed an extensive granulomatous reaction despite the mercury treatment, and there was only a slight inhibition of EAE. Therefore, the ability of HgCl2 to produce lymphadenopathy in BN rats may be responsible for the inability of these rats to absorb the inoculated antigen. The mercury-induced failure of absorption was manifested as an inhibition of EAE in BN rats.