A new species, Toxocara lyncis, in the caracal (Lynx caracal)

Toxocara lyncis, sp. n. is described from Lynx caracal in Somalia. It most closely resembles T. cati, the only species of Toxocara reported from L. caracal. It differs from T. cati in the comparative length of the spicules and the esophagus, and in the shape of the cervical alae. Cervical alae have a nearly uniform width along their length in T. lyncis, while they are narrow anteriorly and broad posteriorly forming an arrow head shaped cephalic end in T. cati.     

Loop-mediated isothermal amplification assay for detection and discrimination of Toxocara canis and Toxocara cati eggs directly from sand samples

We developed a novel and simple method, using loop-mediated isothermal amplification (LAMP), for the detection and discrimination of Toxocara canis and Toxocara cati eggs. The new method employs 4 steps: (1) concentration of Toxocara eggs in a small amount of sand; (2) dissolution of the proteinaceous membrane of eggs and simultaneously separation of them from the sand using NaClO treatment; (3) extraction of DNA using NaOH treatment; and (4) detection of T. canis / T. cati DNA using a LAMP assay. All these steps are fast, easy to perform, and do not require expensive equipment or reagents. The novel method was tested both experimentally and in a field study. In the laboratory, we could reliably detect as few as 3 T. canis eggs in artificially contaminated sand, if the experiment was repeated twice. In the field trial, we were able to detect T. cati DNA from 4 natural sandpits having moderate to heavy contamination, although not in a single lightly contaminated sandpit. All of the examined sandpits were found to be contaminated with eggs of T. cati, but none appeared to contain T. canis. Our new method could extract DNA from T. canis and T. cati eggs directly from sand samples as well as detect and distinguish these 2 species in a few easy steps, with markedly reduced time and expense.     

Ocular larva migrans caused by Toxocara cati in Mongolian gerbils and a comparison of ophthalmologic findings with those produced by T. canis

To elucidate the pathogenic potential of Toxocara cari, we observed the ophthalmologic changes of the fundi in Mongolian gerbils, Meriones unguiculatus, after oral inoculation of 17 embryonated eggs/g body weight. Ophthalmic conditions in 8 T. cati-infected gerbils were monitored using an ophthalmoscope from day 0 to day 156 and were compared with those of 57 T. canis-infected gerbils. The results showed that T. cati larvae migrated into the eye of the gerbil and then elicited ophthalmic changes, including retinal (25%) and vitreous (50%) hemorrhaging, vasculitis (37.5%), and exudative lesions (25%). Lesions were less prevalent, however, in T. cati-infected than in T. canis-infected gerbils. Unlike in T. canis-infected gerbils, the hemorrhagic lesions did not reappear in T. cati-infected gerbils after they were absorbed. These findings suggested that T. cati larvae are a potentially hazardous pathogen for ocular toxocariasis and that Mongolian gerbils infected with T. cati may be a useful model for the study of human ocular toxocariasis caused by T. cati. This is the first study to report that T. cati larvae can induce ophthalmic lesions in the retina of gerbils.     

Toxocara infection in pigs. The use of indirect fluorescent antibody tests and an in vitro larval precipitate test for detecting specific antibodies.

An in vitro larval precipitate test using second-stage Toxocara canis larvae and an indirect fluorescent antibody (IFA) test employing cuticles of T. canis larvae as antigen were evaluated using antisera produced in pigs experimentally infected with T. canis, T. cati, Ascaris suum, Toxascaris leonina and Parascaris equorum. The former test was both specific and sensitive and is suggested as a reliable and simple method of detecting Toxocara antibodies in pigs. The latter test was considered unsuitable because of cross-reactions that occurred when sera from pigs infected with other ascarids were tested. An IFA test for Ascaris antibodies, employing cuticles of A. suum larvae as antigen, is described. The degree of specificity of this test suggests that it may be of value in the detection of antibodies to Ascaris in pigs under natural conditions.     

Toxocara canis: monoclonal antibodies to larval excretory-secretory antigens that bind with genus and species specificity to the cuticular surface of infective larvae

When maintained in culture, the infective-stage larvae of Toxocara canis produce a group of excretory-secretory antigens. Monoclonal antibodies to these antigens have been produced and partially characterized. Hybridomas were made using spleens from mice that had been given 250 embryonated eggs of T. canis followed by immunization with excretory-secretory antigens. Monoclonal antibodies were first screened against excretory-secretory antigens using an indirect enzyme-linked immunosorbent assay. Those antibodies positive in this assay were then screened against the surfaces of formalin-fixed, infective-stage larvae using an indirect fluorescent antibody assay. The two monoclonal antibodies showing fluorescence were also tested against the surfaces of infective-stage larvae of Toxocara cati, Baylisascaris procyonis, Toxascaris leonina, Ascaris suum, a Porrocaecum sp., and Dirofilaria immitis. One of these two antibodies bound to the surface of T. canis and T. cati while the other bound only to the surface of T. canis; neither were reactive with the other ascaridoid larvae or the larvae of D. immitis. Enzyme-linked immunoelectrotransfer blotting techniques were used to demonstrate that the cross-reactive antibody recognized antigens with molecular weights of about 200 kDa while the more specific monoclonal antibody recognized antigens with approximate molecular weights of 80 kDa. The specificity of these two antibodies for T. canis and T. cati should prove helpful in the development of more specific assays for the diagnosis of visceral and ocular larva migrans.     

Serologic evidence of toxocariasis in northeast Brazil

Sera from 54 Brazilian children with a history of tropical eosinophilia syndrome were screened for toxocariasis. The enzyme-linked immunosorbent assay detected 21 positive sera (40%). Many of these cases (15 of 21) were positive for Dilofilaria immitis as well. Of the six Toxocara-positive children with hypereosinophilia, two were asthmatic. Their sera were further examined by the Toxocara species-specific Ouchterlony's diffusion-in-gel test and one Toxocara canis and one T. cati infections were identified. This is the first report of serologically diagnosed toxocariasis in Brazil.     

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.     

Characterization of nematode glycoproteins: the major O-glycans of Toxocara excretory-secretory antigens are O-methylated trisaccharides.

Toxocara excretory-secretory antigens (TES) were isolated from the culture media of T.canis and T.cati larvae and their O-glycan content was investigated using fast atom bombardment-mass spectrometry (FAB-MS), gas chromatography and electron impact mass spectrometry. The major oligosaccharides released by reductive elimination of T.canis TES glycoproteins were shown to be two, approximately equi-abundant, trisaccharides: 2-O-Me-Fucp(alpha 1----2)-4-O-Me-Galp(beta 1----3)GalNAcitol and 2-O-Me-Fucp(alpha 1----2)-Galp(beta 1----3)GalNAcitol. In contrast T.cati TES O-glycans are predominantly one component, shown by FAB-MS to be a di-O-methylated trisaccharide, which is probably identical to the di-O-methylated trisaccharide from T.canis. The O-methylated trisaccharides are strong candidates for the carbohydrate epitopes recognized by a panel of monoclonal antibodies which exhibit multiple reactivity against TES antigens. This study constitutes the first rigorous characterization of glycans from a parasitic nematode.     

Modification of gDNA extraction from soil for PCR designed for the routine examination of soil samples contaminated with Toxocara spp. eggs

A modification of gDNA extraction was developed for the polymerase chain reaction (PCR) technique, intended for the detection and differentiation of Toxocara spp. eggs in soil or sediments. Sand samples from sandpits confirmed as being contaminated with Toxocara spp. eggs by the flotation technique were analysed by PCR. The use of proteinase K made it possible to obtain genomic DNA from the sample without needing to isolate eggs using flotation or to inactivate PCR inhibitors present in the sand. Specific primers in the PCR reaction allowed discrimination between T. canis and T. cati eggs. The modification simplified the procedure, thanks to eliminating the step of gDNA isolation from eggs, which is both laborious and difficult.     

Gastrointestinal parasites of cougars (Felis concolor) in Washington and the first report of Ollulanus tricuspis in a sylvatic felid from North America.

Gastrointestinal helminths including two species of cestodes (Taenia omissa and T. ovis krabbei) and three species of nematodes (Toxocara cati, Cylicospirura subequalis and Ollulanus tricuspis) are reported from two free-ranging cougars (Felis concolor) in Washington (USA). Ollulanus tricuspis is reported for the first time from cougars and represents the first occurrence of this parasite in a sylvatic felid from North America.     

The mitochondrial genome of Toxocara canis

Toxocara canis (Ascaridida: Nematoda), which parasitizes (at the adult stage) the small intestine of canids, can be transmitted to a range of other mammals, including humans, and can cause the disease toxocariasis. Despite its significance as a pathogen, the genetics, epidemiology and biology of this parasite remain poorly understood. In addition, the zoonotic potential of related species of Toxocara, such as T. cati and T. malaysiensis, is not well known. Mitochondrial DNA is known to provide genetic markers for investigations in these areas, but complete mitochondrial genomic data have been lacking for T. canis and its congeners. In the present study, the mitochondrial genome of T. canis was amplified by long-range polymerase chain reaction (long PCR) and sequenced using a primer-walking strategy. This circular mitochondrial genome was 14162 bp and contained 12 protein-coding, 22 transfer RNA, and 2 ribosomal RNA genes consistent for secementean nematodes, including Ascaris suum and Anisakis simplex (Ascaridida). The mitochondrial genome of T. canis provides genetic markers for studies into the systematics, population genetics and epidemiology of this zoonotic parasite and its congeners. Such markers can now be used in prospecting for cryptic species and for exploring host specificity and zoonotic potential, thus underpinning the prevention and control of toxocariasis in humans and other hosts.     

Utilizing a polymerase chain reaction method for the detection of Toxocara canis and T. cati eggs in soil

A polymerase chain reaction (PCR) technique has been used for the differentiation of T. canis and T. cati eggs isolated from soil and previously identified from microscopical observations. The method, using specific primers for the identification of the two Toxocara species, was assessed in both the field and laboratory. Successful results were obtained when only a single or large numbers of eggs were recovered from 40 g soil samples. The method is sensitive, allows analysis of material independent of the stage of egg development and can be adapted for the recovery of other species of parasites from soil.     

Prevalence of species of Toxocara in dogs, cats and red foxes from the Poznan region, Poland

The prevalence of toxocariasis was evaluated for 445 dogs, 105 cats and 92 foxes from the Poznan region during 1997-1998. Forty one cats were infected (39%), 140 dogs (32%) and 15 red foxes (16%). Toxocara canis was found most frequently in puppies up to 3 months old (58%) and T. cati in kittens 4-6 months old (64%). Toxocariasis was much more prevalent amongst adult foxes (14%) than adult dogs (3%). In contrast to cats, female dogs and foxes were less infected than males. The present study suggests that cats may constitute an underestimated risk of transmission of Toxocara spp. to humans and the progressive synatropization of red foxes may also increase the sources of environmental contamination with Toxocara eggs.     

Toxocara cati infections in stray cats in northern Iran

A cross-sectional survey was undertaken to study the prevalence and intensity of infection with Toxocara cati in 100 stray cats, from April to October 2004 in urban areas of Sari, northern Iran. A total of 44 cats (44%) were found to be infected with T. cati. There was a significant difference in the prevalence of infection relative to host age and weight (P = 0.000). There was also a significant difference in the intensity of infection relative to body weight and urban sites (P < 0.05). No significant difference was found between the prevalence of infection relative to host gender, urban sites and season (P>0.05), nor in the intensity of infection between host gender, age and season (P>0.05). The intensity of infection ranged from 1 to 32 worms per cat, with a mean of 7.30 +/- 6.82.     

Helminth fauna of Eurasian lynx (Lynx lynx) in Estonia

Thirty-seven carcasses of Eurasian lynx (Lynx lynx) collected and examined in Estonia during 1999-2001 had helminths. Parasites identified and their prevalence included Diphyllobothrium latum (5%), Taenia pisiformis (100%), Taenia laticollis (41%), Taenia hydatigena (3%), Taenia taeniaeformis (3%), Toxocara cati (68%), and Trichinella spp. (22%). The only significant relationships (P < or = 0.05) between occurrence of helminths and host age and sex were a greater number of T. pisiformis and T. laticollis in older than in youger male lynx, and older males had a greater number of species of helminth than did younger lynx. Sixty-one fecal samples collected during snow tracking of nine lynx were examined; eggs of T. cati were identified in 38 samples, and Capillaria spp were found in eight samples. This is the first systematic investigation of parasites of lynx in Estonia.     

Superoxide dismutase activity in nematodes

The activities of the enzymes superoxide dismutase (E.C.:1.15.1.1) and catalase (E.C.:1.11.1.6) were studied in purified extracts of four nematodes: Ascaris suum, Toxascaris leonina, Toxocara canis and T. cati adult males and females. No catalase activity was found in any of the extracts. The results reveal that the SOD activities of the four parasites presented species differences and also sexual differences within each species. Polyacrylamide gel electrophoresis pattern analysis confirmed that the mobilities, widths and band intensities varied according to the species and sex of the parasite from which the enzyme was obtained.     

The epidemiology of ocular toxocariasis

Ocular toxocariasis damages vision and may cause blindness. It is a relatively 'new' disease, the histological changes having been described in 1950 and the causative organism identified in 1956. Many aspects of the epidemiology of toxocariasis are unclear. It is generally accepted that the usual route of entry to the eye is via the blood stream. Once the eye is invaded characteristic changes are produced with comparative sparing of the anterior segment. Species differences in susceptibility can be very marked. In Mongolian gerbils 55% of infected animals exhibit ocular lesions. Humans are fortunate in that they are far less susceptible to ocular infection. While it has been established that ocular toxocariasis is caused by Toxocara canis, the role of T. cati in this condition is still unclear. It is perhaps surprising that the prevalence of such a disease in humans should remain in doubt. However, the wide variation in the few estimates of prevalence may well be a reflection of the wide variation in exposure as evidenced by the wide variation in seroprevalence. A further factor mitigating against an identification of prevalence rates is the fact that the condition is usually unilateral. Most surveys of blindness stipulate a vision of 6/60 or less in the better eye and so toxocariasis cases go unrecorded. There is a need for surveys which identify the level of vision in both eyes.     

Observations on the post-mortem migration of nematode larvae and its role in tissue digestion techniques

The importance of larval viability in relation to the success of recovery techniques and to the interpretation of histological and migration studies was tested using mice infected with Toxocara pteropodis, T. canis and T. cati. Little difference was found in total numbers of viable larvae recovered from tissues incubated for 18 hours in 0.85% saline, 2% trypsin or 1% pepsin, but the rates of larval egress did vary. The recovery of dead larvae by digestion techniques was much lower. Larvae migrated in tissues undergoing fixation, which influenced histological findings. Larvae of T. canis did not undergo significant peritoneal migration in acutely-infected mice, but did penetrate their intestinal walls in large numbers within 4 hours of death. The implications of these findings in experimental studies are discussed.     

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.     

Maternal transmission by Alaria marcianae (Trematoda) and the concept of amphiparatenesis

The term amphiparatenic host was coined originally for hosts of Alaria marcianae that as adults are paratenic hosts, but as juveniles serve as definitive hosts. In this study the concept of amphiparatenesis is placed in a theoretical context, maternal transmission shown to be the basic mechanism, and the concept extended to include Toxocara canis, T. cati, T. pteropodis, Neoascaris vitulorum, Ancylostoma caninum, Uncinaria lucasi, various species of Strongyloides, Pharyngostomoides procyonis, and P. adenocephala. Alaria marcianae mesocercariae were used in a feline model to show that male and nonlactating female cats are definitive hosts, but lactating cats are primarily paratenic hosts. Inoculation of 1 female cat resulted in the infection of 21 of her offspring via the milk over the course of 5 litters and after a 3-yr period she still had viable larvae in her tissues. The ability of parasites to remain immature in amphiparatenic hosts is believed to be an adaptation on the part of the parasite to promote dissemination through maternal transmission and not the result of resistance, immunological or otherwise, on the part of the host. The amphiparatenic concept has important implications that include: the use of pregnant and lactating females as reservoirs of infection for the offspring; infection transmitted through a contagious transplacental or transmammary pathway; a parasite population structure in which adult worms are in greater abundance in neonate than adult hosts; and the effective control of parasites utilizing this strategy proving to be very difficult.