Quantitative comparison of various methods for detecting eggs of Toxocara canis in samples of sand

Six techniques for recovering unembryonated Toxocara canis eggs from sand samples were tested for efficiency and suitability for routine use. The tests were done under standardised conditions on 50g of sand samples contaminated experimentally with 10, 100 and 500 eggs of T. canis. Best result was achieved by the method of Dunsmore et al. [Dunsmore, J.D., Thompson, R.C.A., Bates, I.A., 1984. Vet. Parasitol. 16, 303-311]. The results were expressed as the number of T. canis eggs recorded and percentage rates of recovery in sand samples.     

Contamination of dog hair with eggs of Toxocara canis

Toxocara canis, the common intestinal nematode of dogs and foxes, is the parasite responsible for human toxocarosis. It has recently been shown that dogs may harbour eggs of the parasite in their fur. To further investigate this claim a population of 100 stray dogs was examined to establish the prevalence and intensity of adult toxocaral worm infection in the intestines and eggs harboured in the hair. A novel method of washing the eggs from the hair was used. Sixty-seven percent of dogs were found to have T. canis eggs on their hair with a mean egg retrieval of nearly 584 eggs per gram from positive dogs. The age of the dog was found to be the only significant factor to influence the prevalence and intensity of eggs, with 95% of all the eggs recovered found on puppies. Thirty-nine percent of dogs were found to have adult T. canis worms in their intestine, although a significantly higher percentage of puppies (80%) were infected with worms than adults (22.5%). Puppies also had more worms per infection than adults and have a strong positive correlation between egg and worms numbers whereas adults did not. These studies show that stray dogs, particularly puppies, potentially harbour considerable numbers of eggs on their hair, at densities far higher than those reported in the soil or the general environment.     

Toxocara canis: proteinases in perivitelline fluid from hatching eggs

Developing larvae of Toxocara canis may secrete several kinds of enzymes within the egg perivitelline fluid (EPF) prior to and during hatching. In particular, proteinases in EPF could play a role in larval emergence within the host gastrointestinal lumen but its presence and nature is unknown. In this work, proteolytic activities in hatching fluid of T. canis were identified and analysed by substrate gel electrophoresis at different pH values and by using type specific protease inhibitors. Three bands of 91, 68 and 38 kDa showed gelatinolytic activity and all proteinase activity from EPF was of the aspartic-type since it was inhibited by pepstatin A. Interestingly, a significantly higher proteolytic activity was observed at acidic pH (< or =5.5). These data suggest that T. canis developmentally secretes and accumulates in EPF aspartic proteinases with a pH-dependent activity that might help the parasite to take advantage of conditions in the host gastrointestinal microenvironment where egg hatching is induced and executed.     

Quantitative comparison of different purification and detection methods for Cryptosporidium parvum oocysts

Cryptosporidium parvum (C. parvum) is the causal agent of cryptosporidiosis in many animals, mainly cattle, and possesses a high zoonotic potential. It occurs worldwide and ubiquitously. Detection of C. parvum is mainly performed directly but purification of the oocysts is useful to increase sensitivity and to obtain oocyst material for further use. The study was designed to compare (a) three different direct diagnostic methods, namely modified Ziehl-Neelsen staining, carbol fuchsin staining and conventional PCR, and (b) three routine oocyst purification methods, in particular flotation with saturated sodium chloride solution, Sheather's sucrose solution and a Percoll(?) gradient. During comparison of purification methods, special regard was paid to the ability to separate morphologically intact oocysts from the morphologically degenerated fraction or viable from non-viable oocysts, respectively. Results: (a) Diagnostic methods: Most effective in C. parvum oocysts detection in calf faeces was PCR; carbol fuchsin and modified Ziehl-Neelsen stainings achieved comparable results. (b) Purification methods: Oocyst flotation using sodium chloride solution showed to be superior to Percoll(?) gradient centrifugation and sugar flotation in terms of purification quality, recovery efficacy (yield) and reduction of the proportion of degenerated or non-viable oocysts.     

Renal involvement in mice experimentally infected with Toxocara canis embryonated eggs.

Histological examination of kidneys from mice experimentally infected with Toxocara canis embryonated eggs demonstrated the presence of a segmental or diffuse mesangioproliferative glomerulonephritis. Immunohistochemical studies established that renal alterations were associated with glomerular deposits of IgG, IgM and third component of complement (C3). These findings suggest that an immunomediated mechanism might possibly be involved in the genesis of kidney damage observed in mice infected with T. canis embryonated eggs.     

Risk of Toxocara canis eggs in stray and domestic dog hair in Egypt

The aim of the study was to assess whether the hair of stray and domestic dogs in Egypt was contaminated with the eggs of the zoonotic parasite Toxocara canis, and also to identify risk factors for T. canis for contamination. Paired samples of hair and feces were collected from 53 stray and 47 domestic dogs, and hair samples were obtained from a further 11 stray and 9 domestic dogs. All samples were examined to identify T. canis eggs and, if eggs were found, their maturation stage. Eggs were identified in 26.6% of stray and 10.7% of domestic dog's hair samples. A significantly increased risk of embryonated T. canis eggs in hair samples was found in stray dogs (p=0.04), stray dogs had 3.18 (95% CI: 1.04-9.74) times the odds of having T. canis eggs present compared with domestic dogs. There was also a significant difference (p=0.02) between the mean quantity of eggs per gram in stray (77.6±6.54) and domestic (48.7±6.65) dog's hair. Fecal examination found a T. canis egg prevalence of 35.8% and 21.3% in stray and domestic dogs, respectively. As no domestic dogs which were positive from hair samples had negative fecal samples, this indicates that the presence of T. canis eggs in hair is probably due to self contamination. Two stray dogs had positive hair samples but negative fecal samples indicating that contamination may also be environmental. As both non-embryonated and embryonated T. canis eggs were found in the hair of domestic dogs, direct contact with dogs may be a potential risk factor for transmission of T. canis eggs to humans.     

Infectivity and pathogenicity of 14-month-cultured embryonated eggs of Toxocara canis in mice

Infectivity and pathogenicity to mice of embryonated eggs of Toxocara canis, that had been maintained in 2% formalin for 14 months at 4 degrees C, were evaluated by immunological and pathological assessment at 1, 4, 8, 12, 16, 20, 24, 28, 42 and 67 weeks post-infection (WPI). On each date, three infected mice and two age-matched uninfected mice were sacrificed for serum collection and histological processing of the liver, lungs, musculature, and brain. Infectivity assessment by enzyme-linked immunosorbent assay (ELISA) revealed that the overall immunological pattern of infected mice tended to be towards the Th2 type response. Serum IgG1 antibody titers in infected mice were significantly higher than that of the uninfected control mice throughout the trial (P<0.05). On the other hand, no significant difference in titers of IgG3 antibody, an indicator for the Th1 type response, was observed between the infected and control mice, except at eight WPI (P<0.05). Pathogenicity was assessed semiquantitatively by comparing the mean number or diameter of inflammatory foci as well as histopathological changes in the liver, musculature, brain, or lungs of the infected mice and the control mice. Each hematoxylin and eosin (H&E) stained tissue section slide was examined under 100x magnification and 15 random fields were counted. Degree of inflammatory injury among the four organs was scored and categorized into four levels: normal (0), mild (1+), moderate (2+), and severe (3+). An index of inflammatory injury (mean score of experimental group/mean score of 10 control groups of 20 uninfected mice) of 2-3 is considered as moderate to severe, 1-2 as mild to moderate, and 0-1 as normal to mild. Histopathological changes were moderate to severe in the liver and lungs, mild to moderate in the musculature, and only normal to mild in the brain throughout the trial. It is noteworthy that apocrine-like change in epithelial cells of the bile duct was observed in most of the infected mice from eight WPI onward. Furthermore, larvae trapped by organized granulomas were found in soft tissue near the musculature at 12, 20, and 28 WPI. Altogether, not only were the infectivity and pathogenicity of the 14-month-cultured T. canis embryonated eggs retained, the hatched larvae were also capable of eliciting some special pathological changes in the murine host.     

Parasitism of Toxocara canis larvae in Japanese quails by inoculation of the ascarid eggs.

The distribution of T. canis larvae and pathological changes caused by them were studied in Japanese quails orally inoculated with 1,500, 4,000 or 15,000 embryonated eggs. Larvae were distributed mainly in the liver and, to lesser extent, in the muscles, brain, eyes and other organs. The number of larvae varied from 7 to 3,346, and from 1 to 288 in the liver and muscles (breast and legs), respectively. A small number of larvae were also recovered from the heart, gizzard, brain and eyes. In the groups of quails inoculated with 4,000 or 15,000 eggs, small white foci were observed on the surface of the liver 6 or 12 hr after inoculation. Histopathological examinations revealed necrotic lesions, leukocytic infiltration, granuloma and nodular lesions. The pathological changes became more serious with the large size of inoculum and days after inoculation.     

The investigation of Toxocara canis eggs in coats of different dog breeds as a potential transmission route in human toxocariasis

This study was undertaken to determine the prevalence of Toxocara canis eggs on the coats of dogs (a potential etiological factor for human toxocariasis) and to see if there were mainly a dog breed and coat type effects for the presence of eggs on the coat. Hair samples were collected from the different breeds of 51 domestic pet dogs and examined for the presence of T. canis eggs. A total of 62 T. canis eggs (all viable) were found in 21.56% of the dogs. Forty-nine (79.03%) of the eggs recovered were unembryonated, 8 (12.90%) were embryonating, and 5 (8.06%) were embryonated. The maximum densities of the embryonating and embryonated eggs were 93 and 8.45 eggs per gram (epg) of hair, respectively. The number of eggs recovered was much higher than those previously reported for soil samples. Although the statistical analysis for all dogs in this study showed that there were no breed (P>0.4), coat type (P>0.8), sex (P>0.1), age group (P>0.1) and hair length (P>0.3) effects for the presence of T. canis eggs per gram of hair, the majority of dogs (82%) with T. canis eggs in their coats were breeds that had double coats with thick undercoats suggesting that the coat characteristic may play a role for providing a suitable environment for the development of T. canis eggs. Also 82% of infected dogs were under 1 year of age indicating that the age of dog is a very important risk factor. The present study indicates that direct contact with T. canis infected dogs may be a potential etiological factor for human toxocariasis.     

Differentiation of Toxocara canis and T. cati eggs by light and scanning electron microscopy

In this study, we investigated the morphological identification of Toxocara canis and T. cati eggs on the basis of light and scanning electron microscopic (SEM) observations. T. canis and T. cati eggs used in this study were recovered from the uteri of respective gravid female worms. Measurement of egg size was not helpful in the differentiation of these species, because approximately 90% of eggs measured were of similar size. Using SEM, we were able to differentiate T. canis eggs from T. cati eggs based on their respective characteristic surface structures. Both species have subspherical eggs with markedly pitted surfaces like those of a golf ball, but the surface pitting in T. canis is more coarse than that in T. cati. In this study, however, these differences were not absolute, as 16% of T. canis and 29% of T. cati eggs showed surface pitting that was uncharacteristic of their species. Of the 16% of T. canis eggs that could not be differentiated by surface structure, 3% had pitting resembling T. cati, and the remaining 13% showed intermediate type surface pitting. Similarly, 5% of T. cati eggs resembled T. canis, and 25% of these were of intermediate type. Light microscopic observation yielded results similar to those of SEM, indicating that light microscopy is also a useful tool for the identification of Toxocara eggs.     

Larval development of Toxocara canis in dogs

The parasitic roundworm Toxocara canis is present in dog populations all over the world. Due to its zoonotic potential, this roundworm is of special interest not only for veterinarians, but also for medical practitioners. In the present review, current knowledge of infection routes and the subsequent development of larvae within the canine host is summarised. Furthermore, information about the clinical, pathological, enzymatic, haematological and histopathological changes was collected, giving a broad overview of current knowledge of the infection. Although the data collected over the years give an idea of what happens during the larval development of T. canis, many questions remain open. Nevertheless, it is important that we continue our efforts to further understand the biology of this versatile and compelling parasite and try to improve and optimise strategies to prevent the infection in dogs and thereby to protect humans from this infection.     

Seroprevalence of Toxocara canis in sheep in Wales

Antibody levels to Toxocara canis L2 excretory/secretory antigens were examined by ELISA in 400 serum samples from sheep in Powys and Gwent, Wales. A positive OD value was set at the mean +/-3S.D. of 45 control samples. Seroprevalence increased with age. Seven percent and 13% of 6-month-old sheep showed positive OD values as did 16% of 10-month-old, 27% and 31% of 15-month-old and 47% of cull ewes. Analysis of variance showed a significant increase in ELISA OD values among the seropositive sheep with increasing age of sheep.     

Patent Toxocara canis infections in previously exposed and in helminth-free dogs after infection with low numbers of embryonated eggs

The outcome of Toxocara canis infections in the canine host depends on the migratory pathway of parasite larvae (somatic or tracheal) which is considered to be related to the host's age and its immune status. However, field studies attest high prevalences of patent T. canis infections in adult animals. The controlled induction of patent infections with low doses of embryonated eggs was investigated in 18 beagles in a 7-month study until their 16th life month. The animals were assigned to three groups, each consisting of three vertically infected dogs (with a short patent infection as pups before anthelmintic treatment) and three helminth-free dogs. At study days 10 and 40, the animals of groups 1 and 3 were given each 100 embryonated T. canis eggs. In each case, group 1 was treated 10 days post-infection with Milbemax, while dogs of group 3 remained untreated. Control group 2 was not experimentally infected but treated as group 1. Two weeks after first egg administration, a sharp increase of specific antibody reactions in ELISA and increased eosinophilic counts indicated larval invasion in all infected dogs. 42-56 days following first infection, patent infections were detected coproscopically in all animals of group 3, but in none of the uninfected dogs (group 2) or the infected and treated dogs (group 1). Following a 3-month observation period, all animals of the three groups were treated with piperazine citrate to eliminate intestinal infections and all were administered 100 embryonated eggs. Subsequently, patent infections developed in animals of all groups: in one of the infected and treated animals of group 1, in five of the so far not infected control group 2 and in four of the dogs with previous patent infections (group 3). Susceptibility to patent infections was not significantly altered in T. canis-free dogs compared to dogs with previous patent infection (vertically acquired or experimentally induced). However, dogs of group 1 treated with Milbemax after repeated egg administration developed a significantly increased resistance to patent infections as compared to control dogs (group 2). Observed prepatency periods were between 40 and 56 days and did not differ in the three groups. Even in urban areas, facing high infection pressure with Toxocara eggs maintained by a high dog and fox population, dogs of all ages are at risk to develop patent T. canis infections.     

A comparison of the protein constituents of the major body compartments of the dog roundworm, Toxocara canis

An analysis of the protein profiles of intact worms and isolated tissues of adult male and female Toxocara canis worms was conducted. Soluble proteins recovered from homogenized whole specimens and dissected tissues (body wall, reproductive tract, esophagus and intestine) of T. canis adults from several different canine hosts were separated by size using gradient sodium dodecyl sulfate electrophoresis (SDS-PAGE) and visualized with silver staining. SDS-PAGE profiles of worms from different hosts were found to be virtually identical irrespective of sex or tissue type. Recovered proteins ranged in size from 3.4 to 325 kDa. As expected, variations existed between the protein profiles of different body tissues, with only slight variations between the sexes. The largest number of recovered proteins was present in the female reproductive tract extracts.     

A field evaluation of treatment with febantel for the control of Toxocara canis in pups

The faecal egg count depression (FECD) of febantel (Rintal vet. 100 mg tablets, Bayer AG, Veterin?r-Bereich, Leverkusen), against Toxocara canis was tested in suckling pups treated at 2 weeks of age. The dose rate was 30 mg kg-1 body weight given orally, once every 12 h, three times. The effect of a further treatment of 6- and 12-week-old pups on excreted eggs was also evaluated. The FECD of 6-week-old pups was 100%. However, some of the 12- and 17-week-old pups had low eggs per gram (epg) values indicating that shorter intervals between the treatments should have been used in order to minimize the risk of spreading T. canis eggs. The control pups of the first treatment group were untreated litter mates. They were treated when 4 weeks old and then followed a similar regimen to the experimental animals. At 6 weeks of age, their FECD was 100%, but low epg values were observed among 12- and 17-week-old pups, similar to the test group.     

Transplanted infections with Toxocara canis as a model for the effectiveness testing of anthelmintics

Patent infections of adult dogs with Toxocara canis induced by transplantation of immature, intestinal stages were examined for their suitability for testing of anthelmintics. Each of 5 dogs were infected four times by transplantation of 80 immature, intestinal stages of Toxocara canis. The dogs were treated with various anthelmintics of well established efficacy (pyrantel, nitroscanate, mebendazole, piperazine) 20 dpi. All anthelmintics tested showed the same efficacy as had been assessed earlier by treatment of dogs infected prenatally with Toxocara canis.