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.     

Effect of host age in the distribution of adult Trichinella zimbabwensis in the small intestines of golden hamsters (Mesocricetus auratus) and Balb C mice

Golden hamsters (Mesocricetus auratus) and Balb C mice were experimentally infected with Trichinella zimbabwensis to determine the effect of host age in the distribution of adult stages in the small intestines. The hamsters and mice were divided into two groups of young and old animals. Hamsters aged 90 days were designated as young and those aged 360 days were designated as old while mice of 30 days of age were designated as young and those aged 90 days as old. To recover the adult parasites of T. zimbabwensis, the small intestines of each animal were separated and divided into four equal parts and each part was slit open longitudinally. The contents were incubated in 0.85% saline for 4 h at 37 degrees C before the adult worms were recovered from the saline. They were fixed in 70% alcohol and counted under a dissecting microscope. In both young and old hamsters and mice, T. zimbabwensis adult worm counts were significantly higher (P < 0.05) in the second segment of the intestines thus invariably reflecting a significantly high count (P < 0.05) in the first (anterior) half of the small intestines. From this study it was demonstrated that host-age had no effect on the distribution of T. zimbabwensis adult worms in the different segments of the small intestines of golden hamsters and Balb C mice.     

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.     

Experimental infection of pups with Ancylostoma caninum-infected mouse tissues

Migration and distribution of Ancylostoma caninum larvae in the tissues of mice orally infected with 1000 larvae, and establishment of patent infection from the mice to the definitive host, were studied. Larval yield from different organs of mice, after digestion with artificial gastric juice, indicated that the highest recovery was at 4 h post-infection (62.8%), and thereafter a slight decline occurred up until 30 days post-infection (51.5%). Migration of larvae to the lungs occurred within 4 h, to the liver within 12 h and into the heart within 24 h. No larvae were recovered from spleen and kidney tissues. From the 9th day onwards larvae were also recovered from the brain. Migration in the muscles of head and neck occurred within 4 h, in the thoracic and abdominal muscles at 24 h and in lumbosacral and leg muscles at 48 h. The establishment of patent infections in the definitive host was studied by feeding the orally- and percutaneously-infected mice to hookworm-free pups at 10 and 30 days post-infection. The mean necropsy worm burden in the pups fed with the orally-infected mice was comparatively higher than in the pups fed cutaneously-infected mice.     

Susceptibility of laboratory animals to experimental infection with Ife virus

The susceptibility of rabbits, domestic chickens and albino rats to experimental infection with Ife virus was investigated. Neither pyrexia nor clinical signs of disease were observed in infected rabbits or chickens. Low-grade viraemia (10(1.0) mouse lethal doses per 0.02 ml) occurred in intracerebrally (i.c.) inoculated chicks on the second day post-infection. Complement-fixing antibody was detected on the 14th day post-inoculation in rabbits and on the 7th day in chickens. Infant rats less than 3 and 5 days of age died after subcutaneous (s.c.) and i.c. inoculation, respectively; older rats survived infection. Ife virus titres were highest in the brain following both i.c. and s.c. inoculation.     

Enhanced survival of rats concurrently infected with Trypanosoma brucei and Strongyloides ratti

The interaction between the blood protozoan parasite, Trypanosoma brucei and the gastrointestinal nematode parasite, Strongyloides ratti was studied in outbred white albino rats. Rats were grouped and given either single infection with T. brucei or S. ratti or concurrently infected with both parasites. Blood parasitaemia and packed cell volume, faecal egg/larva output, adult worm burden and survivability were monitored in order to assess the interactive effects of the infections. All trypanosome-infected rats became parasitaemic within 1 week of infection but surprisingly parasitaemia was higher in the single than concurrently infected group of rats. In addition all animals with single T. brucei infection had died by 14 days after the infection, whereas animals with concurrent infection were still alive by day 28 after the infection when the experiment was terminated. Concurrent infection resulted in significant increase in daily S. ratti egg/larval output in faeces (P < 0.01), but lesser number of adult worms were recovered from the intestine of sacrificed rats on day 8 post-infection. Taken together these results suggest that T. brucei and S. ratti interact in a manner that ameliorates their pathogenic effects resulting in a decrease in the level of parasitaemia and intestinal worm burden and in increased life span of the infected rats. These results differ from the classical immunosuppressive attributes of T. brucei and the results are discussed in the context of the possible immune responses that might have contributed to this outcome and the potential significance of the findings in alternative control method of trypanosomosis.     

Experimental Final Hosts of Metagonimus Hakubaensis (Trematoda: Heterophyidae) and Their Suitability to the Fluke

Seven laboratory mammal and bird species were orally inoculated with 200–1,000 encysted Metagonimus hakubaensis metacercariae that had been isolated from naturally infected lampreys (Lethenteron reissneri) captured in Aomori Prefecture. At 8 and 15 days post-infection, adult flukes were recovered from all of the laboratory animals tested, and therefore, hamster, rat, mouse, dog, cat, chicken and quail were considered as final hosts of M. hakubaensis. Recovery rates of the fluke were higher in dogs and hamsters than in cats, rats, mice, chickens and quails. The flukes recovered from dogs and hamsters showed increased body length and higher fecundity than those recovered from the other hosts. These results indicate that the suitability of dogs and hamsters for M. hakubaensis infection is higher than that of the other laboratory animals.     

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.     

Efficacy of thiabendazole, mebendazole, levamisole and ivermectin against gullet worm, Gongylonema pulchrum: in vitro and in vivo studies

In vitro and in vivo studies were conducted to evaluate the effects of thiabendazole, mebendazole, levamisole and ivermectin against Gongylonema pulchrum. For in vitro assays, third-stage larvae (L3) incubated with the drugs were administered orally to mice and the ability of larvae to invade the gastric mucosa of the animals was examined. After incubation, only those larvae treated with high concentrations of levamisole (1 and 10 microg/ml) were tightly coiled with intestines exhibiting morphological abnormalities. Good dose-response data for the drugs tested was observed at the time of worm recovery from mice, with no worms recovered at the two highest concentrations of levamisole. In vivo efficacy of the drugs against adult worms was evaluated in six groups of three rabbits, each of which was infected with 30 L3 of G. pulchrum and treated with thiabendazole at 100 mg/kg for 3 days, mebendazole at 70 mg/kg for 3 days, levamisole as a single dose of 8 mg/kg, and subcutaneously injected ivermectin as a single dose of 0.2 mg/kg or vehicles of the drugs (control) at 4 months post-infection. Necropsy 14 days after treatment revealed that levamisole, mebendazole and ivermectin reduced worm burdens by 63.2%, 22.8% and 25.8%, respectively, with no reductions in worms observed with thiabendazole. The surviving worms were principally found in the esophagus with the remainder distributed among the buccal mucosa, the tongue, and/or pharyngeal mucosa in all groups. A number of morphologically abnormal eggs were observed within the uterus and ovijector in female worms recovered from the thiabendazole-treated group. These findings suggest that levamisole exhibits in vivo efficacy against G. pulchrum infection and that the larval invasion tests using mice could be used to screen for anthelmintic susceptibility of nematodes.     

Effect of feeding selected carbohydrates on the in vivo attachment of Salmonella typhimurium in chick ceca.

Two-day-old chicks were orally inoculated with 1 ml of Salmonella typhimurium (10(5) colony-forming units/ml) and divided into four groups. Three groups were fed 2.5% carbohydrates starting on day 1 (arabinose, galactose, and lactose), while the fourth group served as the control. Ceca were obtained from each group at 7, 14, and 21 days. At the end of 14 days, all three carbohydrates statistically reduced Salmonella recovery. However, lactose failed to reduce recovery between day 14 and day 21. Arabinose and galactose continued to show significant reductions of recovery through 21 days. No statistical difference was found between Salmonella recovery from whole ceca (with cecal material) and inverted ceca (washed free of cecal material).     

Humoral immune responses following experimental infection of goats with Mycoplasma capricolum subsp. capripneumoniae.

Goats housed in microbiologically secure facilities were experimentally endobronchially infected with Mycoplasma capricolum subsp. capripneumoniae (Mccp), causal agent of contagious caprine pleuropneumonia (CCPP). The animals were monitored over an 8-week period post-infection (p.i.). Elevated temperatures were observed 2-7 days p.i., reaching a maximum of 41.5 degrees C in one animal (1884). By 8 weeks p.i. the infection was successfully cleared, with no Mccp being recovered from the lungs, serum or nasal passages. Mccp was not isolated from serum throughout the experiment, either directly by culture or indirectly via polymerase chain reaction (PCR). Humoral immune responses against Mccp capsular polysaccharide (CPS) were generally poor when measured by ELISA. CPS antigen was present in the serum of all infected animals early in the infection (day 14 p.i.), although in one animal (1855) CPS antigen persisted throughout. This was the only animal to exhibit a serious cough (day 5-19 p.i.). Successful diagnosis of CCPP was achieved using two different types of latex agglutination test (CPS antibody and CPS antigen detection test), immunoblotting and a blocking ELISA, although the latter lacked sensitivity until later in the infection (35-40 days p.i.). Only a single animal (1855) was detected positive using the current complement fixation test (CFT). Strong immune responses to protein antigens were detected by IgG and IgM immunoblotting from the first time point at day 14 p.i. IgM immunodominant bands of 220, 85, 62 and 40kDa were observed in the 3 infected animals and from CFT-positive CCPP field sera. Band intensity gradually diminished throughout the experiment. IgG immunodominant bands of 108, 70, 62, 44, 40 and 23kDa were shared between experimentally-infected and field sera, with band intensity either remaining unchanged or increasing from day 14 p.i. These bands were not present using pre-infection sera. Of the diagnostic tests used, only the CPS antibody detection latex agglutination test and IgG immunoblotting gave positive diagnoses throughout the entire period post-infection (days 14-53 p.i.).     

Relationship between egg output and parasitic burden in lambs experimentally infected with different doses of Dicrocoelium dendriticum (Digenea)

The relationship between egg elimination and parasitic burden was studied in two groups of 12 lambs experimentally infected with 1000 and 3000 Dicrocoelium dendriticum metacercariae, respectively. Half the animals in each group were slaughtered 2 months post-infection (p.i.) and the other half 6 months p.i. In order to detect and follow elimination of D. dendriticum eggs by the lambs, faeces samples collection started one and a half months p.i. and continued fortnightly until the end of the experiment. Egg elimination was first detected between days 49 and 79 p.i. (mean = 59 +/- 1.6 SE). Mean eggs per gram (epg) was higher in the lambs infected with 3000 metacercariae (347.2 +/- 42.4 epg) than in those infected with 1000 (194.8 +/- 14.4), although no significant differences were detected between both groups using the Student 't' test. Egg elimination was higher in the faeces samples taken in the afternoon (mean = 357.8 +/- 47.6 epg) than in those from the morning (mean = 215.7 +/- 21.3). The percentage of metacercariae which became established as worms was higher in the animals dosed with 1000 metacercariae (21.6%) than in those infected with 3000 (16.3%). The number of worms recovered on necropsy of each animal varied between 30 and 2063 (mean = 346.6 +/- 80.5) and their length between 2.6 and 7.1 mm (mean = 5.2 +/- 0.1). The mean number of parasites for lambs infected with 3000 metacercariae (489.3 +/- 163.1) was higher than that obtained from those dosed with 1000 (215.7 +/- 41.4), although more worms were collected in some cases from the lambs infected with the latter dose than the former. In general there was an increase in the number of epg eliminated as days p.i. and parasitic burden increased. A positive relationship was observed via the correlation coefficient between the number of epg eliminated by each of the lambs throughout the experiment and that of worms recovered. This relationship was more intense on considering only the number of epg eliminated between days 120 and 180 p.i.     

Efficacy of ivermectin against the swine kidney worm, Stephanurus dentatus

Ivermectin in solution was given subcutaneously (in the neck) at the rate of 0.5 mg/kg of body weight to 5 sows naturally infected with kidney worm (Stephanurus dentatus). Six similar sows were used as nontreated controls. A total of 114 kidney worms were recovered from daily urine samples of 4 of the 5 treated sows (the 5th sow's samples were negative) during the first 4 days after treatment. Ten kidney worms were recovered from daily urine samples of 1 control sow on the 2nd and 3rd days of the post-treatment period. Examination of urine samples from treated sows for kidney worm eggs showed a precipitous numerical decrease. Treated animals became negative between the 5th and 12th days after treatment, and remained negative until necropsy on the 61st day. Compared with control sows, hatchability of eggs and survival of larvae from treated sows was reduced by 9% and 7%, respectively. Kidney worms were not recovered at necropsy from the treated sows, and a total of 392 kidney worms were recovered from the control sows (av 65).     

Effects of milbemycin on adult Toxocara canis in dogs with experimentally induced infection

To determine the efficacy of a formulation of milbemycins in treating patent infection with Toxocara canis, 8 male and 7 female, 10-week-old, ascarid-free Beagles each were given 125 embryonated eggs of T canis. All dogs developed patent infection within 56 days. On post-infection day 70, the dogs were assigned to 1 of 3 groups of 5 dogs each; members of 1 group were given a placebo, while dogs of the other 2 groups were given either 5.68 or 34.08 mg of the milbemycin formulation, respectively. In both groups of dogs given the drug, the number of eggs passed per gram of feces decreased precipitously. However, a few eggs still were found in the feces of several dogs of each group on the day of necropsy (postinfection day 75). Worms or fragments of worms were passed by the treated dogs from the day of treatment until the day on which necropsy was performed; however, most worms were passed during the first 2 days after treatment. At necropsy, only dogs of the control group were found to harbor adult T canis.     

Changes in the levels of eicosanoids in cats naturally and experimentally infected with Dirofilaria immitis

Feline heartworm (Dirofilaria immitis) infection is a severe, life-threatening disease. The eicosanoids are lipid mediators derived from the metabolism of the arachidonic acid, involved in the regulation of the immune response and of inflammatory reactions. In this study, naturally infected cats showed significant higher levels of prostaglandin E(2) (PGE2), thromboxane B(2) (TXB(2)) and leukotriene B(4) (LTB4) than uninfected cats. Changes in the levels of eicosanoids during the infection were observed in experimentally infected cats. PGE2 increased significantly during the first 60 days post-infection, then progressively decreased until day 180 post-infection. At this time, PGE2 values are still significantly higher than those observed before the infection. TxB2 and LTB4 increased progressively from the beginning of infection and reached their maximum levels 180 days post-infection. In experimentally infected, ivermectin-treated cats, 15 days after treatment (45 days after infection) both PGE2 and LTB4 levels were similar to those observed in experimentally infected, untreated cats. No significant differences of PGE2 levels were found before the infection and at the end of the experiment (165 days post-treatment, 195 days post-infection). Increased levels of LTB4 were found 15 days post-treatment, afterward they progressively decreased. These data show that D. immitis infection influences the production of intravascular eicosanoids in cats. The high levels of PGE2 observed in the early phase of infection could be related to the survival of the worms, while those of TxB2 and LTB4 detected at the end of the study could mediate the inflammatory reactions and thrombi formation during the feline dirofilariosis.     

Adaptability of Japanese quail chicks to conditions of simulated weightlessness

The objective of this study was to evaluate the adaptability of young Japanese quail chicks to the simulated weightlessness, represented by hypodynamy. Unsexed hatchlings were subjected to hypodynamy on either the first, second or third day of age and reared under these conditions to 21 days of age. During this period, the control quail chicks were housed in a floor box. The effect of hypodynamy on adaptability of chicks was significant (P < 0.001). Approximately 75% of all chicks exposed to hypodynamy were not able to adapt in three experimental groups, although significant differences in adaptability were not found between these groups. Those birds were considered as non-adapted (eliminated from experiment) that manifested hyperactivity, escape attempts, turning 180 degrees in the sling, soaking in the water from the drinker, as well as the total apathy, at least three times per day. This experiment confirmed that some quail chicks are capable of adapting to conditions simulating weightlessness to 21 days of age and that the first 2-weeks after hatching may be a critical period of quail sensitivity to hypodynamy. This finding raises a key issue relevant to rearing quails in simulated weightlessness until the age of sexual maturity.     

Immunolocalization and pathological alterations following Strongyloides venezuelensis infection in the lungs and the intestine of MHC class I or II deficient mice

The present study, investigated the mechanisms involved in the immune responses of Major Histocompatibility Complex class I or class II knockout mice, following Strongyloides venezuelensis infection. Wild-type C57BL/6 (WT), MHC II(-/-) and MHC I(-/-) mice were individually inoculated with 3000 larvae (L3) of S. venezuelensis and sacrificed on days 1, 3, 5, 8, 13 and 21 post-infection (p.i.). Samples of blood, lungs and small intestines were collected. The tissue samples were stained with hematoxylin-eosin for the pathological analysis. The presence of the parasite was demonstrated by immunoperoxidase analysis. MHC II(-/-) mice presented a significantly higher number of adult worms recovered from the small intestine on day 5p.i. and presented elevated numbers of eggs in the feces. The infection by S. venezuelensis was completely eliminated 13 days after infection in WT as well as in MHC I(-/-) mice. In MHC II(-/-) mice, eggs and adult worms were still found on day 21 p.i., however, there was a significant reduction in their numbers. In the lung, the parasite was observed in MHC I(-/-) on day 1 p.i. and in MHC II(-/-) mice on days 1 and 5 p.i. In the small intestine of WT mice, a larger number of parasites were observed on day 8 p.i. and their absence was observed after day 13 p.i. Through immunohistochemistry analysis, the parasite was detected in the duodenum of WT on days 5 and 8 p.i., and in knockout mice on days 5, 8 and 13 p.i.; as well as in posterior portions of the small intestine in MHC I(-/-) and MHC II(-/-) on day 13 p.i., a finding which was not observed in WT mice. We concluded that immunohistochemistry analysis contributed to a more adequate understanding of the parasite localization in immunodeficient hosts and that the findings aid in the interpretation of immunopathogenesis in Strongyloides infection.     

Enhanced protection against the migratory phase,but defective protection against the intestinal phase of Strongyloides venezuelensis infection in cotton rats,Sigmodon hispidus

The protective capacity of the cotton rat, Sigmodon hispidus, against the migratory and intestinal phases of Strongyloides venezuelensis infection was examined. After subcutaneous infection with infective larvae (L(3)), adult worm recovery rates from male and female animals on Day 71 were only 0.10% and 0.06% of initial dose, respectively. To determine whether this enhanced protection was expressed during the migratory phase or the intestinal phase, larval recovery from the lungs of cotton rat was evaluated 3 days after subcutaneous L(3) infection. The larval recovery rate was only 0.5% of initial dose and about 40-fold lower than that from control mice. Protection in the intestine was also evaluated after intraduodenal implantation of adult worms. About 30% of implanted worms became established and worm burden remained constant until Day 28. Despite a high worm burden on Day 28, EPG was about 25-fold lower than the peak count. To evaluate expulsive capacity and monitor the cellular responses in the intestine of cotton rats, adult Nippostrongylus brasiliensis worms were implanted in addition to S. venezuelensis. Cotton rats were unable to expel adult S. venezuelensis worms, even after 21 days of observation. Although the number of mucosal mast cells increased significantly, the intraepithelial migration of mast cells was not observed. In contrast, N. brasiliensis was expelled by Day 6 in association with goblet cell hyperplasia. These results suggest that in cotton rats, the defective intestinal protection against adult S. venezuelensis worms results from dysfunction of mucosal mast cells.     

Pathogenicity of a recently isolated strain of Tritrichomonas foetus in mice

The pathogenicity of a recently isolated strain of T. foetus in mice was studied. The parasite produced local abscesses 3 days post subcutaneous inoculation (p.i.) in albino mice. The abscesses in mice, inoculated with large numbers (4 × 10⁶) of organisms, continued increasing in size until the 14th day. Some absceses ruptured between day 11 to 14 p.i., while others formed a point. Secondary abscesses were also found in some mice. the abscesses in mice, inoculated with small numbers (1 × 10⁶) of organisms reached their maximum on the 5th day and decreased thereafter. All the abscesses had motile trichomonads with no bacterial contamination. The trichomonads, inoculated intraperitoneally in mice, persisted in that cavity till day 5 or 6 p.i., but did not multiply. Subcutaneous inoculations appear to be more reliable than the intraperitoneal ones for studies on pathogenicity. The comparison of present observations with those using a similar model and T. foetus or other trichomonads, suggests that the strain of T. foetus employed was of low pathogenicity.     

Dose dependency of prednisolone on the establishment of Echinococcus multilocularis infection in an alternative definitive host, Mongolian gerbil

This study revealed the dose dependency of prednisolone tertiary-butylacetate (PTBA) treatment on the establishment of Echinococcus multilocularis in the small intestine of Mongolian gerbil (Meriones unguiculatus) and that some of the physiological parameters of host were correlated with the doses of PTBA and establishment of the worm. Twenty Mongolian gerbils were divided into 5 groups, according to the doses of PTBA; 0 mg, 0.5 mg, 2 mg, 5 mg and 10 mg per head. All animals were injected intraperitoneally with PTBA every other day from 6 days before to 6 days after infection. Doses of PTBA and the number of worms recovered at 7 days post-infection showed a positive correlation (r = 0.929, P < 0.0001). The increase of total protein (TP) and the decrease of the percentage of lymphocytes in the peripheral leukocytes were dependent on doses of PTBA (TP: r = 0.811, P < 0.0001, percentage of lymphocyte: r = -0.92, P < 0.0001). The TP and the percentage of lymphocyte also correlated with the number of worms recovered (TP: r = 0.617, P = 0.0049; percentage of lymphocyte: r = -0.800, P < 0.0001).