Infection of Japanese quail with Toxocara canis larvae and establishment of patent infection in pups

Migration and distribution of Toxocara canis larvae in the tissues of Japanese quail, infected orally with 5 X 10(3) infective eggs, and the establishment of patent infection from the quails to the definitive host, were studied. Larval yield at necropsy from various tissues and organs of quail varied from 4.72 to 7.54% of the infective eggs inoculated within the period 1-60 days post-infection (PI). The total number of larvae recovered on different days showed a gradual increase. The percent inoculum recovered at necropsy was highest on Day 60. Most of the larvae were found in the liver throughout the period and only a few migrated to other tissues, such as lung, heart, muscle and brain. The establishment of patent infections in the definitive host was studied by feeding the 15-day infected livers of Japanese quail (400 larvae) to Toxocara-free pups. Eggs first appeared in the faeces 38 days post-infection, the mean worm burden at necropsy was 87 and the percentage of infection established was 21.75%. Thus the role of Japanese quail as a paratenic host of T. canis is established.     

Experimental infection of Japanese quail with Toxocara canis larvae through earthworms

The migration and distribution of Toxocara canis larvae in the tissues of earthworms exposed to 5 x 10(5) infective eggs in soil, as well as the reinfectivity of the larvae in Japanese quail fed with three earthworms, were studied. The average number of larvae recovered from an earthworm gradually declined from 9 +/- 3.21 to 4.5 +/- 2.00 at 4-16 days. No larvae were recovered at Day 20. When compared between three portions of the earthworms' body, the middle part showed the greatest recovery in comparison with the anterior and posterior parts. At necropsy, the average larval burden of quail at 15 days post-infection was 6 +/- 3.60 in the liver and no larvae were recovered from other tissues. The role of the earthworm in relation to paratenism is discussed.     

Toxocara cati larvae persist and retain high infectivity in muscles of experimentally infected chickens

The distribution of Toxocara cati larvae in the organs of chickens (n=31), experimentally inoculated with 3000 embryonated eggs, was examined 1, 2, 3, 7, 29, 86 and 175-176 days post-infection (dpi), and the infectivity of recovered larvae was evaluated by bioassay in mice. The duodenum, liver, lungs, heart, brain, pectoral muscles (white meat), and hindlimb muscles (red meat) of the chickens were HCl-pepsin digested for larval recovery. Larvae were recovered from all chickens [mean=220.4 ± 114.9 (SD)], and although no decrease of total larval recovery was observed over time, predilection sites changed: liver (92.6% of larval recovery) at 1 dpi; lungs (77.3%) and liver (20.9%) at 2 dpi; lungs (80.8%), muscles (9.4%), and liver (8.5%) at 3 dpi; muscles (52.0%) and lungs (45.6%) at 7 dpi, whereafter most larvae were recovered from muscles: 29 dpi (99.5%), 86 dpi (99.3%) and 175-176 dpi (99.6%). In the bioassay, 52.9% of larvae from 175 to 176 days old infections in chickens established in mice, which demonstrates that T. cati larvae retain infective in the muscles of chickens for half a year. These results highlight the zoonotic potential of poultry meat as a causative agent of human toxocarosis.     

Development of Centrocestus armatus in different final hosts

In this study, Centrocestus armatus metacercariae were fed orally to hamsters, albino rats, mice, and chicks. Animals were sacrificed and dissected at 1, 3, 5, 7, and 14 days post-infection to determine the development and recovery rate of worms. Results indicated that the average worm recovery rate in hamsters was 25% on the first day post-infection and recovery continued until the 14th day with a gradual decrease in the percentage. Worms were also recovered from mice and albino rats from the first until the third day post-infection, but no worms were recovered thereafter. In chicks, worms were not observed on first day but recovery was positive at 12 h post-infection. Among the four animal hosts, feces from hamsters were the only ones positive for eggs; these were initially observed from the third day and recovery continued until 14 days post-infection. In our study, hamsters are the animal model most suitable for the study of C. armatus when compared to rats, mice, and chicks.     

Autoradiographic quantification of the efficacy of niridazole in mice infected with 75Se-labelled cercariae of Schistosoma mansoni.

The effects of the anti-schistosomal drug, niridazole, on the migration of Schistosoma mansoni larvae, biosynthetically radioisotope-labelled with 75[Se]-selenomethionine, was evaluated by autoradiography of compressed tissues of mice treated daily from Days 6 to 10 post-infection with 200 mg kg-1 niridazole. The results were compared with the migration of schistosomula in untreated controls. The distribution of schistosomula was altered in niridazole-treated mice, where there was a delayed migration from the lungs relative to the controls and significantly fewer schistosomula in total appeared to reach the liver. The total percentage of schistosomula detected as autoradiographic foci was significantly lower in treated mice than in the untreated controls. Niridazole-treated mice were free of any foci 10 days after the last treatment and no adult worms were recovered on perfusion of the hepatic portal system relative to control mice from which 5.8% of the infective cercariae were recovered as adult worms at Day 42 post-infection.     

Systemic responses to challenge infection withHaemonchus contortus in immune Merino sheep

Some systemic responses to single-dose infection with 10 000Haemonchus contortus infective larvae were examined in sheep already shown to have protective immunity against the parasite. The major haematological finding was a neutrophil leukocytosis that occurred after the infections became patent but not during the pre-patent period. There was no definitive eosinophilia and no discernible change in the erythrocyte parameters. Systemic hyperthermia was not conclusively evident during the pre-patent period. Enzyme-linked-immunosorbent assays (ELISA) were used to measure the secondary anti-helminth antibody response in serum during the pre-patent period when the establishment of patent infection is resisted. These ELISAs employed preparations from adult worms to represent the parasitic stages of the worm, preparations from infective larvae to represent the pre-parasitic stages of the worm, and exsheathing fluid, which is the soluble material obtained whenH. contortus larvae undergo ecdysis and transform from the pre-parasitic to the parasitic phase. Antibody responses to the three preparations differed qualitatively, indicating the presence of three different but perhaps overlapping sets of antigens. The three peaks in antibody against exsheathing fluid may reflect the pulses of antigen delivered to sheep as the parasite undergoes its three moults within the host.Abbreviations ELISA enzyme-linked immunosorbent assay - EDTA ethylene diamine tetraacetate     

Sarcocystis neurona infections in raccoons (Procyon lotor): evidence for natural infection with sarcocysts, transmission of infection to opossums (Didelphis virginiana), and experimental induction of neurologic disease in raccoons

Equine protozoal myeloencephalitis (EPM) is a serious neurologic disease of horses in the Americas and Sarcocystis neurona is the most common etiologic agent. The distribution of S. neurona infections follows the geographical distributions of its definitive hosts, opossums (Didelphis virginiana, Didelphis albiventris). Recently, cats and skunks were reported as experimental and armadillos as natural intermediate hosts of S. neurona. In the present report, raccoons (Procyon lotor) were identified as a natural intermediate host of S. neurona. Two laboratory-raised opossums were found to shed S. neurona-like sporocysts after ingesting tongues of naturally-infected raccoons. Interferon-gamma gene knockout (KO) mice fed raccoon-opossum-derived sporocysts developed neurologic signs. S. neurona was identified immunohistochemically in tissues of KO mice fed sporocysts and the parasite was isolated in cell cultures inoculated with infected KO mouse tissues. The DNA obtained from the tongue of a naturally-infected raccoon, brains of KO mice that had neurological signs, and from the organisms recovered in cell cultures inoculated with brains of neurologic KO mice, corresponded to that of S. neurona. Two raccoons fed mature S. neurona sarcocysts did not shed sporocysts in their feces, indicating raccoons are not likely to be its definitive host. Two raccoons fed sporocysts from opossum feces developed clinical illness and S. neurona-associated encephalomyelitis was found in raccoons killed 14 and 22 days after feeding sporocysts; schizonts and merozoites were seen in encephalitic lesions.     

Experimental trichinellosis in horses: biological and parasitological evaluation

Three groups of three horses each were, respectively, infected with 5000, 20,000 and 50,000 larvae of Trichinella spiralis. The strain used was isolated from a human biopsy during horsemeat-related outbreaks of trichinellosis in France. Transient muscular disorders were only observed in two of the horses infected with 50,000 larvae but none of the horses had fever. A significant increase in blood eosinophils was noticed in 5 horses. Serum LDH, aldolase and CPK peaked at the fifth week post-infection. Specific IgG assayed by indirect immunofluorescence and ELISA, appeared 2-5 weeks post-infection and disappeared between 16 and 40 weeks. The distribution of T. spiralis larvae was maximal in the tongue, masseters and diaphragm, but a large decrease in the number of larvae recovered from the muscles was noticed among the horses slaughtered at the beginning and end of the experiment. In muscular histological sections, larvae were observed in an intramyofibrillar position and were surrounded by a mild to severe inflammatory reaction.     

Experimental infection of laboratory animals and sheep with Gongylonema pulchrum in Japan

Japanese White rabbits, Wistar rats, ddY mice, Suffolk sheep, and a domestic cat were each orally inoculated with 20-140 third-stage larvae (L3) of Gongylonema pulchrum, isolated from naturally infected dung beetles captured in Aomori Prefecture. Worm recovery rates were 40.0-72.0% in rabbits at 7, 14, and 19 weeks post-infection (PI) and 3.3-25.0% in rats at 19 weeks PI. Those in 2 sheep at 7 weeks PI showed 53.6% and 29.3%. No worms were recovered from the mice and the cat. In the susceptible animals, many worms were found in the esophagus, and a few were present in the pharyngeal mucosa, tongue, buccal mucosa, and cardiac portion of the stomach wall. No distinct morphological differences were observed in the worms from rabbits and sheep. These results indicate that rabbits are very suitable experimental definitive hosts for G. pulchrum.     

Host resistance and faecal sporocyst excretion in dogs exposed to repeated infection with Sarcocystis levinei

A marked reduction in the faecal excretion of sporocysts was observed in experimental pups, following the repeated oral administration to them of buffalo cardiac muscle infected with Sarcocystis levinei. Sporocysts excreted from days 9 to 25 post-infection (pi) exhibited a gradual reduction in the quantum. Maximum intensity of excretion of sporocysts was recorded between days 9 and 16 pi, becoming moderate after day 16, light after day 21 and completely absent after day 36. After the subsequent feeding to pups of S. levinei infected buffalo cardiac tissues at 40 day intervals the quantity of sporocysts shed was less, the prepatent period was prolonged and the patent period was considerably shortened. The peak period of excretion varied depending upon the number of exposures of the pups to the infected S. levinei tissues from buffaloes (Bubalus bubalis).     

Gallus gallus domesticus: Paratenic host of Angiostrongylus vasorum

Angiostrongylus vasorum, a parasite of the cardiorespiratory system in canids, has a heteroxenous biological cycle in which the intermediate hosts are terrestrial and aquatic mollusks. Generally, canids become infected by ingesting the intermediate host or paratenic hosts, such as amphibians, that contain infective larvae (L3). However, there are no reports of birds as paratenic hosts of A. vasorum. To evaluate the susceptibility and viability of Gallus gallus domesticus as a paratenic host of A. vasorum, 17 Cobb chickens were randomly divided into two groups. The animals in group A were inoculated with third stage larvae of A. vasorum, and those in group B ate snails inoculated with A. vasorum L3. At 30 days post-infection, the chickens were killed, and the muscles and organs were placed in a pepsin–HCl solution (1% HCl (37%), 1% pepsin) for 3 h in an oven at 40 °C to recover the L3. In group A, 1863 L3 were recovered per chicken. In group B, 2585 L3 were recovered. A dog that ingested organs and tissues from a chicken from group A released first-stage larvae of A. vasorum in its feces 51 days after infection; the dynamics of this process were monitored for 107 days, when treatment with 25 mg fenbendazole/kg body weight was performed for 21 days. Chickens nourished with infected snails or with infective L3 may be a source of infection for dogs indicate that G. gallus is a potential paratenic host for this parasite.     

IgG response in guinea pigs to Trichinella spiralis infection

An enzyme-linked immunosorbent assay (ELISA) using excretory-secretory antigens was developed to study the dynamics of the IgG antibody response to varying levels of Trichinella spiralis infection in the guinea pig. Four groups of four Hartley guinea pigs each were infected with 1250, 250, 50 or 10 T. spiralis infective muscle larvae. They were bled every 15 days for 6 months and the IgG antibody response determined by ELISA. The time of seroconversion was dose dependent as the larger the dose, the earlier the response occurred. Significant differences in antibody response between the dose groups were evident at 30 days post-infection (P less than 0.05). Beyond 60 days post-infection, the response was similar in the four groups. The antibody response in the groups infected with 250 and 50 infective larvae was similar, but was significantly different from that of the high (1250) and low (10) dose groups from 30 days post-infection (P less than 0.01). Once seroconversion occurred, the antibody titer rose to the same level, irrespective of the initial dose. To compare the antibody response according to muscle larvae recovered, the guinea pigs were grouped into four categories: less than 10 larvae; 10-25 larvae, 50-80 larvae, greater than 100 larvae. A significant positive correlation (P less than 0.05) was observed at 60 days post-infection when these groups were compared.     

Exploring the life cycle of Besnoitia besnoiti - experimental infection of putative definitive and intermediate host species

The biology of Besnoitia besnoiti, the cause of bovine besnoitiosis, is poorly understood. Its definitive host is unknown, and information on potential intermediate hosts is scarce. In order to investigate potential definitive and intermediate hosts for European isolates of B. besnoiti, domestic dogs, cats, rabbits, guinea pigs (Cavia porcellus), gerbils (Meriones unguiculatus), common voles (Microtus arvalis) and NMRI-mice were inoculated with B. besnoiti isolated from naturally infected German cattle. Dogs and cats were fed 5×10(6)B. besnoiti tachyzoites (isolate Bb-GER1), or tissue cysts containing at least 2×10(7)B. besnoiti bradyzoites obtained from the skin of a naturally infected Limousin cow from the same herd where strain Bb-GER1 was isolated. Rodents and rabbits were subcutaneously inoculated with either 5×10(5) Bb-GER1 tachyzoites or 5×10(5) bradyzoites. Groups of 2-4 non-inoculated animals of each species were monitored as negative controls. Feces from all dogs and cats were daily examined by a sedimentation-flotation technique for at least 11 weeks after inoculation but no B. besnoiti oocysts were identified. Cats fed tachyzoites and dogs did not seroconvert, but specific antibodies to B. besnoiti tachyzoites were detected by IFAT (titer≥100) in 2 out of 3 cats fed tissue cysts since 5-7 weeks post infection. By immunoblot, these two cats exhibited a reaction pattern against tachyzoite antigens similar to that observed in naturally infected cattle. Antibodies against B. besnoiti tachyzoites were detected in all inoculated rodent species and rabbits by both, IFAT and immunoblot since 3 weeks post-inoculation. Rabbits and rodents, subcutaneously inoculated with same doses of inactivated bradyzoites remained serologically negative (IFAT titer<50). Clinical signs observed in the inoculated rabbits included fever, serous conjunctivitis and transient swelling of the testes. No clinical abnormalities were noticed in the other tested animal species. Voles developed pneumonia as observed by histological examination. B. besnoiti-DNA was detected by PCR in blood from rabbits, gerbils and voles at 9 days post-infection, and in skin, heart, lung, striated muscle and kidney tissues from voles at 19-21 weeks post-infection. Domestic dogs and cats could not be shown to be definitive hosts of B. besnoiti, but cats seroconverted after feeding on B. besnoiti tissue cysts indicating that B. besnoiti stages had invaded the cats' tissues. The molecular and serological results from this study indicate that European B. besnoiti isolates may infect cats, rabbits, guinea pigs, gerbils, mice and voles; however a persistence of the parasite could be demonstrated only in voles.     

Intestinal establishment and reproduction of adult Trichinella spp. in single and mixed species infections in foxes (Vulpes vulpes)

Intestinal establishment and reproduction of adult Trichinella spiralis, Trichinella nativa, Trichinella britovi and Trichinella pseudospiralis were examined as single species or mixed species infections in foxes. This is the first study of intestinal dynamics of Trichinella spp. in a carnivore model and the results suggest that the intestinal phase is relatively short as only very few worms were recovered 10 days post-inoculation (dpi). In mixed species infection with equal doses of T. nativa and T. spiralis, molecular typing demonstrated that 64% of the intestinal worms and 78% of the muscle larvae were T. nativa. Conversely, T. spiralis dominated in the mixed species infections with T. pseudospiralis, constituting 66% of the intestinal worms and 94% of the muscle larvae. Although, the individual recoveries of intestinal worms were only up to 5.6% on day 1, and up to 1.5% on day 4 post-infection, the muscle larvae establishment was comparable to other fox studies. Infectivity, measured as muscle larvae burden did not differ among the four species of Trichinella, which is in contrast to other models with mice, rats, pigs or herbivores. Although statistically significant differences in intestinal worm burdens were found for some days, no distinct species were recovered in consistently higher numbers than the others.     

The efficacy of milbemycin oxime against pre-adult Spirocerca lupi in experimentally infected dogs

The aim of this investigation was to determine the efficacy of milbemycin oxime in preventing the oesophageal encapsulation of Spirocerca lupi, following the experimental infection of dogs. Two studies were conducted which involved a total of 21 purpose-bred Beagles. Each dog was infected with approximately 40, third stage infective S. lupi larvae. The larvae were dissected from scarabaeid beetles that had been collected from areas endemic for spirocercosis. In the first study, milbemycin oxime (minimum dose 0.5mg/kg body weight) was administered to seven dogs on day 30 post-infection. Seven other dogs served as untreated controls. In the second study, milbemycin oxime (also at a minimum dose of 0.5mg/kg body weight) was administered to four of seven infected dogs on day 28 post-infection. Treatment was repeated at 14- or 28-day intervals. All of the dogs, from both studies, were euthanized 168 or 169 days after infection. All S. lupi were recovered, and lesions in the thoracic aorta and oesophagus were described and quantified. A single treatment with milbemycin oxime was 79.8% effective in preventing the establishment of S. lupi in the oesophagus. This treatment significantly (p<0.05) reduced both the number of S. lupi within the oesophagus and the size of the oesophageal nodules. The efficacy of anthelmintic treatment was increased to 100% when repeat doses of milbemycin oxime were administered at 14- or 28-day intervals. These repeat treatments completely prevented the establishment of S. lupi within the oesophagus and thereby averted the development of oesophageal nodules. As expected, none of the treatment protocols reduced S. lupi related damage within the aorta because the administration of milbemycin oxime only began after the larvae had completed their first stage of migration.     

Acquired immunity to Toxocara canis infection in mice

Acquired immunity develops against Toxocara canis infection in mice, and NIH mice are more immunoresponsive than CD1 mice. Twice infected NIH female mice showed 27% reduction in the total larval recoveries compared with non-sensitized controls. Twice-infected NIH male, and CD1 (both sexes) mice showed a negligible reduction in the total recoveries, though a significant (P less than 0.05) number of larvae were retained in the liver compared with the non-sensitized controls. All twice-infected mice showed a significant reduction in the number of larvae recovered from the brain compared with once-infected mice. Vaccination using ultraviolet irradiated embryonated eggs gave the best protection against reinfection. Excretory/secretory antigen afforded less protection, whilst whole adult worm vaccine and whole L2 culture vaccine gave no protection. Vaccinated mice had a higher 'free:penetrating ratio' of larvae in their intestine than similarly challenged but non-vaccinated mice. When the ileum was examined histologically 9 h post-infection, an inflammatory reaction was seen around the penetrating larvae in the sensitized and vaccinated mice but not in untreated controls, suggesting a role played by the intestine in the resistance against T. canis infection in mice.     

Zoonotic risk of Toxocara canis infection through consumption of pig or poultry viscera

The potential zoonotic risk of Toxocara canis infections from consumption of swine or poultry viscera containing larvae was assessed using a pig model. Two groups of six pigs were fed either fresh swine viscera (group FS) or poultry viscera (FP) containing around 3500 Toxocara larvae. Another two groups of six pigs were fed swine viscera (PS) or poultry viscera (PP) preserved at 4 degrees C for 1 week. All pigs were necropsied 14 days after the exposure. Liver white spots were counted and T. canis specific IgG antibodies were measured by ELISA. Larval burdens were assessed in the mesenteric lymph nodes, liver, lungs, brain, tongue, and eyes. All recipient pigs exhibited several white spots on the liver surface and detectable antibody levels. Larvae were recovered predominantly from the lungs, but also from the mesenteric lymph nodes and the liver, a few larvae were found in the brain and tongue of the pigs. Two larvae were found in the eyes of two pigs in group FS. Mean percentages of total larval recoveries in groups FS, FP, PS, and PP were 75.3, 63.6, 42.6, and 18.8%, respectively. Significantly higher numbers of larvae were recovered from pigs given swine viscera than pigs given poultry viscera. The preservation at 4 degrees C for 1 week caused a significant reduction in the larval infectivity overall, nevertheless, the recoveries remained substantial. The fact that larvae migrating in swine or poultry organs and tissues have high infectivity in pigs even after preservation at 4 degrees C for 1 week, suggests that human infection with T. canis might easily occur following consumption of raw or undercooked dishes, either fresh or refrigerated, prepared from swine or poultry organs and tissues harbouring T. canis larvae.     

Impeded establishment of the infective stage of Trichinella in the intestinal mucosa of mice by passive transfer of an IgA monoclonal antibody

Our previous study showed that the IgA monoclonal antibody (mAb) HUSM-Tb1 forms immunoprecipitates on the cuticular surface of infective larvae of Trichinella britovi, and that intraperitoneal injection of this mAb to mice 5 hr before challenge infection confers a high level of protection against intestinal T. britovi. The same treatment produced a similar effect in BALB/c mice inoculated orally with Trichinella pseudospiralis larvae, indicating that the effects may be seen upon most members of the genus Trichinella. Worms recovered from the intestinal mucosa at 1 hr after challenge infection with T. pseudospiralis was few in mice passively immunized with the mAb, whereas a substantial number of worms were recovered from the mucosa of control groups. These results suggest that the IgA mAb impedes establishment of infective Trichinella worms in the intestinal mucosa. Trichinella worms inoculated orally into BALB/c mice vaccinated with ultraviolet-irradiated muscle larvae 3 weeks earlier were expelled between days 4 and 7 after challenge infection. Although the mAb HUSM-Tb1 originated from the mesenteric lymph node cells of mice vaccinated repeatedly with such irradiated larvae, IgA-mediated expulsion does not seem to play an important role in this vaccination model.     

A Trichinella murrelli infection in a domestic dog in the United States

Trichinella murrelli infection was diagnosed in a naturally infected Beagle bitch from VA, USA, where encapsulated larvae were found in histological sections of several skeletal muscles. A laboratory reared dog fed infected muscles resulted in viable muscle larvae that were subsequently infective to Swiss-Webster mice. Multiplex PCR using larvae from the experimentally infected dog demonstrated two distinct bands migrating at 127 bp and 316 bp which together are diagnostic for T. murrelli; the isolate was assigned the ISS code: ISS1608 by the International Trichinella Reference Centre. This is the first report of T. murrelli infection in a companion animal.