Histopathologic changes and larval recovery of <Emphasis Type="Italic">Toxocara cati</Emphasis> in experimentally infected chickens

This study was made to determine the distribution pattern of Toxocara cati larvae in chickens as a paratenic host and its potential zoonotic risk by consuming infected chickens. Two groups of chickens were fed with 1,000 and 3,000 embryonated eggs of T. cati. The chickens were necropsied 3, 7, 14, and 21 days postinfection. The liver, lungs, kidneys, spleen, small intestine, and half of all the striated muscles were digested for larval recovery. Squash method was used for brain. Larvae were recovered from the liver and brain of infected chickens with 1,000 embryonated eggs. Samples of these tissues were prepared for histopathologic studies. Experimental chickens exhibited hemorrhages in the liver, lungs, and kidneys on all days postinfections (dpi). White spots on the liver surfaces that showed necrotic foci, infiltration of eosinophils, and a few lymphocytes around necrotic areas were seen on 14 and 21 dpi. Remains of larvae were present in the liver on 14 dpi. Pathologic findings showed that larvae migrated in different organs of chickens. We suggest that chickens could be paratenic hosts, and human infection with T. cati might occur after consumption of raw or undercooked meat of infected chicken with T. cati.     

Toxocara canis in experimentally infected silver and arctic foxes

In two experiments, thirty-six farm foxes of two species were inoculated with various doses of infective Toxocara canis eggs or tissue larvae isolated from mice. In experiment I, six adult arctic foxes (Alopex lagopus; 11-month old) were each inoculated with 20,000 eggs and sacrificed 100, 220, or 300 days post infection (dpi), while ten silver fox cubs (Vulpes vulpes; 6–9-week old) were infected with varying doses of eggs (30–3000) and necropsied 120 dpi. In experiment II, two groups of five cubs and two groups of five adult silver foxes received both a primary inoculation and either one or two challenge inoculations: primary inoculation (day 0) with 400 embryonated eggs were administered to five cubs and five adults and another five cubs and five adults received 400 larvae. At 50 dpi, the first challenge inoculation (400 eggs) was inoculated in all animals. At 100 dpi, three animals from each group were necropsied. The remaining two animals in each group were received a second challenge inoculation of 400 tissue larvae on 100 dpi and were subsequently necropsied at 150 dpi. In both experiments, the highest numbers of larvae per gram (lpg) of tissue was found in the kidneys (100–300 dpi). In adult foxes receiving a high dose (20,000 eggs), increasing larval burdens were found in the kidneys over the course of the experiment (up to 300 dpi). The larval migration from the lungs to other tissues appeared to be dose-dependent with the highest larval burdens found in adult foxes. The faecal egg excretion, larval burden and intestinal worm burdens decreased from the first to the second challenge infection.     

Toxocara canis larvae viability after disinfectant—exposition

The effect of three routinely used disinfectants on the embryonary development of Toxocara canis eggs was evaluated both in vivo and in vitro. In the in vitro experiment, T. canis eggs were treated with the ethanol, sodium hypochlorite, and one commercial mix of benzalconium chloride and formaldehyde, and the embryonary development was assessed. After a period of 24 days incubation, ethanol was the best disinfectant because it prevented the development of the T. canis larvae 2 in the eggs, and sodium hypochlorite caused degeneration in 50% eggs. By using the commercial mix, 25% T. canis eggs developed to 2nd stage larvae. In the in vivo experiment, the embryonated eggs treated with the disinfectants were inoculated to mice, and their brain tissues were examined for larval presence on the 24th day postinfection. In addition, a control group was set up for comparison with the infected groups. No injury or T. canis larvae were observed in mice infected with sodium hypochlorite-treated eggs, opposite to that recorded in the animals infected with the commercial disinfectant-treated eggs. These results showed that both ethanol and sodium hypochlorite are very appropriate because of their full efficacy against infective T. canis eggs. Disinfection of kennels, animal shelters, cages, and veterinary clinics with one of these products to eliminate T. canis eggs and to avoid contamination is strongly recommended.     

Induction of matrix metalloproteinase-9 in mice during Toxocara canis larvae migration

The relationships between inflammation in organs with Toxocara canis larval migration and matrix metalloproteinase-9 (MMP-9) were investigated following the infection of mice with 1,000 infective eggs. Gelatinase activity was defined by gelatin zymography, optimum pH, inhibitor specificity and Western blot analysis. MMP-9 activity was present in the lungs, liver, muscles, and brain during T. canis larval migration. This enzyme had a molecular weight of about 94 kDa and showed maximum activity in the pH range of 6–8. The increased MMP-9 proteinases coincided with larval recovery and the degree of inflammation among the four organs. These results suggest that MMP-9 may be associated with the inflammatory reaction to larval toxocariasis during early migration, and may therefore be a useful marker during T. canis larvae migration.     

Fungi predatory activity on embryonated <Emphasis Type="Italic">Toxocara canis</Emphasis> eggs inoculated in domestic chickens (<Emphasis Type="Italic">Gallus gallus domesticus</Emphasis>) and destruction of second stage larvae

The objective of this study was to evaluate the infectivity of Toxocara canis eggs after interacting with isolated nematophagous fungi of the species Duddingtonia flagrans (AC001) and Pochonia chlamydosporia (VC4), and test the predatory activity of the isolated AC001 on T. canis second stage larvae after 7 days of interaction. In assay A, 5000 embryonated T. canis eggs previously in contact with the AC001 and VC4 isolated for 10 days were inoculated into domestic chickens (Gallus gallus domesticus), and then these animals were necropsied to collect material (digested liver, intestine, muscles and lungs) at 3-, 7-, 14-, and 21-day intervals after inoculation. In assay A, the results demonstrated that the prior interaction of the eggs with isolated AC001 and VC4 decreases the amount of larvae found in the collected organs. Difference (p?<?0.01) was observed in the medium larvae counts recovered from liver, lung, intestine, and muscle of animals in the treated groups when compared to the animals in the control group. At the end of assay A, a percentage reduction of 87.1 % (AC001) and 84.5 % (VC4) respectively was recorded. In the result of assay B, the isolated AC001 showed differences (p?<?0.01) compared to the control group, with a reduction of 53.4 % in the recovery of L₂. Through these results, it is justified to mention that prior interaction of embryonated T. canis eggs with the tested fungal isolates were efficient in reducing the development and migration of this parasite, in addition to the first report of proven predatory activity on L₂.     

Effect of various doses of infectiveToxocara canis andToxocara cati eggs on the humoral response and distribution of larvae in mice

The effect of 5–2,500 infectiveToxocara canis and 5–1,000T. cati eggs on the humoral immune response and on the distribution of larvae in the organism was studied in paratenic hosts — inbred C57BL6/J mice. With each dose ofT. canis eggs the maximal antibody level was recorded on day 56 post infection and was followed by a moderate decline that lasted until day 154 of the experiment. A correlation between the antibody level and the egg count was observed only with the infective dose of 5–50 eggs. A more rapid occurrence of antibodies was recorded in mice infected with a high dose of eggs. In those given 5 and 7T. cati eggs the antibody level exceeded the extinction threshold value only from day 21 to day 84. Low doses ofT. canis (n=5) andT. cati (n=7) eggs caused a comparable distribution of larvae in mice, and the larval recoveries on day 70 post infection ranged between 10.00% and 25.74%. Following a dose of 500T. cati eggs, 22.28% of the larvae were recovered, although only 1.08% were localized in the brain. A dose of 1,000T. canis eggs yielded, 36.37% of the larvae, with as much as 28.13% being found in the brain.     

Ascaridia galli in chickens: intestinal localization and comparison of methods to isolate the larvae within the first week of infection

This study was conducted to observe the localization and to compare methods for isolation of minute Ascaridia galli larvae in chicken intestine. Firstly, six 7-week-old layer pullets were orally infected with 2,000 embryonated A. galli eggs and necropsied either at 3, 5 or 7?days post infection (dpi). More than 95?% of the recovered larvae were obtained from the anterior half of the jejunoileum, suggesting this part as the initial predilection site for A. galli larvae. Secondly, the intestinal wall of one layer pullet infected with 20,000 A. galli eggs 3?days earlier was digested in pepsin?CHCl for 90?min. The initial 10?min of digestion released 51?% of the totally recovered larvae and the last 30?min of continuous digestion yielded only 5?%. This indicates that the majority of larvae were located superficially in the intestinal mucosa. Thirdly, 48 7-week-old layer pullets were infected with 500 A. galli eggs and necropsied at 3 dpi to compare three different larval isolation methods from the intestinal wall, viz., EDTA incubation, agar-gel incubation and pepsin?CHCl digestion, resulting in mean percentages of the recovered larvae: 14.4, 18.2 and 20.0?%, respectively (P?=?0.15). As conclusion, we recommended Pepsin?CHCl digestion as the method of choice for larval recovery from the intestinal wall in future population dynamics study due to high efficiency and quick and simple detection. The agar-gel method was considered to be a prerequisite for molecular and immunological investigations as the larvae were more active and fully intact.     

Studies on the development and role of some cell-mediated immune responses in experimental toxocarosis in mice

The studies were undertaken to investigate the development of some cell-mediated immune responses in experimental toxocarosis in mice and to assess the influence of these responses on the course of infection. Mice were infected orally with 350 eggs of Toxocara canis and reinfected with the same dose of parasites after 8 weeks. Groups of infected animals were killed each week of the experiments to obtain spleens, livers and brains for further studies. Lymphocytes from removed spleens were analysed by flow-cytometry for CD4 and CD8 expression and cultured in vitro to measure their responses to Concanavalin A and excretory-secretory (ES) antigen of T. canis in a lymphocyte transformation test. Pieces of livers were used to prepare paraffin sections to be stained later with haematoxylin and eosin, whereas whole brains of the infected animals were examined for the presence of parasite larvae. The results of the studies showed depression of T-cell responses to ConA in early stages of infection and significant increase in the blastogenic responses to the ES antigen from week 4 following infection. The depression of T-cell responses was accompanied by lowered CD4+/CD8+ ratio resulting from increased percentages of CD8+ T cells. Histopathological examination of liver sections revealed trapping of larvae in T. canis reinfected mice. The intensity of infection as measured by larval recoveries from the brains of mice increased gradually up to the 8th week of infection, but did not show significant changes after reinfection, testifying to the development of long-lasting protective immunity during primary infection.     

Matrix metalloproteinases activation in Toxocara canis induced pulmonary pathogenesis

BackgroundToxocara canis, a source of visceral larva migrans, causes toxocariasis and induces respiratory symptoms. The reasons by which the pulmonary pathological alteration in the lungs infected with T. canis remain unclear.MethodsThe involvement of the pulmonary pathological alteration by histology, enzyme activity, and Western blot analysis in the lungs of BALB/c mice after the infection of 2000 embryonated eggs.ResultsThe pathological effects gradually increased after the infection culminated in severe leukocyte infiltration and hemorrhage from days 4–14 post-inoculation. Gelatin zymography using substrate showed that the relative activity of matrix metalloproteinase (MMP) −9 and MMP-2 significantly increased in T. canis-infected mice. Western blot analysis indicated that the MMPs protein level of fibronectin monomer significantly increased in T. canis-infected mice compared with that in uninfected control. T. canis larvae mainly initiated leukocyte infiltration and hemorrhage in the lungs.ConclusionThese phenomena subsequently induced the activities of MMPs in parallel with the pathological changes in early stage pulmonary inflammation. In conclusion, T. canis larval migration activated the MMPs and caused pulmonary pathogenesis.     

Imaging of Toxocara canis larvae labelled by CFSE in BALB/c mice

Mice are used most often as a model for human toxocarosis caused by Toxocara canis larvae. Variety of symptoms developing during the infection reflects behaviour of the larvae, which are able to escape from the intestine and further invade and damage various host organs. In order to find an approach enabling observation on parasite behaviour in mouse in vivo, we used an epifluorescence method and a small animal imaging system (SAIS). Larvae of T. canis were labelled by carboxyfluorescein succinimidyl ester (CFSE) which incorporated on the parasite gastrointestinal tract. Following infection of BALB/c mice by CFSE-labelled larvae it has been observed that staining had no influence on viability and further migratory activity of the parasites through the host organs (the intestine, liver, lungs and brain) where they were detected by SAIS until day 17 p.i. In addition, the dye did not affect larval antigenic activity as well as the development of related immune response. Imaging of parasites labelled by CFSE, therefore, may represent a promising way to study behaviour of T. canis larvae in a paratenic host.     

Infection with the roundworm Toxocara canis leads to exacerbation of experimental allergic airway inflammation

Background Epidemiological studies performed in developing as well as in western countries suggest that infection with Toxocara canis contributes to the development of atopic diseases. Objectives To investigate the association between infection with this helminth and allergy, we examined the effect of T. canis infection on experimental allergic airway inflammation. Methods BALB/c mice were infected by oral administration with 500 embryonated T. canis eggs followed by ovalbumin (OVA) sensitization and challenge to induce allergic airway inflammation. Results Infection with T. canis in combination with OVA treatment leads to exacerbation of pulmonary inflammation, eosinophilia, airway hyperresponsiveness, OVA specific and total IgE. Relative quantification of cytokine expression in the lungs of these mice showed increased expression of IL‐4 compared with mice that were only T. canis infected or OVA treated. Increased expression of IL‐5 and IL‐10 was measured in the lungs of T. canis‐infected or OVA‐treated mice compared with controls; however, combining infection and OVA treatment did not significantly change the expression of these cytokines. Conclusion A previous infection with T. canis leads to exacerbation of experimental allergic airway inflammation. These results have important consequences for findings on the helminths–allergy association. Several factors, including parasite species, infection of definitive vs. accidental host, parasite load and timing of infection, may influence whether an infection with helminths protects one from or enhances allergic manifestations.     

The behavior and pathogenicity ofToxacara canis larvae in mice of different strains

In the present study the behavior and pathogenicity of second-stage larvae ofToxocara canis were examined in different mouse strains with special emphasis on the major histocompatibility complex (MHC). Mice of the inbred strains BALB, C3H, C57BL, and DBA and the outbred strain NMRI were infected orally with 1000 second-stage larvae ofT. canis. The clinical behavior of the animals: the numbers of larvae detected in the liver, lungs, brain and musculature; the hematological and serological parameters; and histological sections were examined. In mice of the BALB strain, no death occurred during the entire period of the investigation and the pattern of body-weight development of infected and uninfected animals was almost identical. The highest larval counts in the brain of all strains were found in BALB mice. The percentage of eosinophils in the blood of BALB mice increased after the 8th week postinfection, whereas it decreased in the other strains. Histological and pathophysiological changes developed to a lesser extent in this strain than in the other strains. In mice of the strains C3H, C57BL, DBA, and NMRI, deaths occurred from the 4th week postinfection onward. The infected animals lost weight in comparison with the uninfected controls; the numbers of larvae found in the brains of infected mice of the above-mentioned strains were lower than those detected in the BALB strain. There is no evidence that mechanical damage caused by migrating larvae in the brain tissue is mainly responsible for symptoms of central nervous toxocariasis. Likewise, the assumption that the MHC is involved in the allergicinflammatory response in the brain could not be proven: infected mice of the BALB and DBA strains reacted completely differently, although both are equipped with the same MHC haplotype.     

Enhanced expression of transforming growth factor-β1 in inflammatory cells and secretory granules in Paneth cells in the small intestine of mice infected with<Emphasis Type="Italic"> Toxocara canis</Emphasis>

The small intestine is the initial organ which Toxocara canis larvae invade. Information on intestinal pathological changes associated with transforming growth factor-1 (TGF-1) and secretory granules (SG) in Paneth cells (PCs) caused by T. canis is unclear. Mice orally inoculated with 250 T. canis infective eggs were evaluated by pathological and immunohistochemical assessments with a 294-day investigation. Pathologically, the inflammatory reactions with or without trapped larvae in the submucosa were observed only within the first 28 days post-infection (DPI), with inflammatory injury ranging from severe during 2 DPI to mild between 7 and 28 DPI. The crypts of Leiberkuhn were major larval penetration sites. Enhanced expression of SG in PCs appeared earlier than those of TGF-1 in infiltrating cells. The significance of both effectors might be related to the hosts defense against larval invasion in the intestinal phase of toxocaral infection.     

Anthelmintic efficacy of tinidazole against the progression of Toxocara canis larvae to the brain in mice

The anthelmintic effect of tinidazole (100 mg/kg per day for 3 successive days) was tested in male Swiss CF-1 mice infected with second-stage Toxocara canis larvae at challenge doses of 250, 500, 1000, and 1500 embryonated eggs per mouse. The drug was given orally on days 3–5 postinfection (p.i.) to one-half of the animals, and all mice were killed on day 40 p.i. The number of larvae recovered from each mouse's brain and skeletal muscle was then scored in both groups. Tinidazole yielded a highly significant reduction in the total recovery of larvae from the test animals' brains at the second and third inoculum levels but no statistically significant reduction at the highest larval dose as compared with the values obtained in the untreated control animals. Received: 8 March 1999 / Accepted: 22 March 1999     

Temperature and the development and survival of infective <Emphasis Type="Italic">Toxocara canis</Emphasis> larvae

The influence of temperature on the development and survival of Toxocara canis larvae was investigated under laboratory conditions, in water at 15, 20, 25, 30 and 35°C and at room temperature 22°C ± 1°C. T. canis eggs were able to develop to the larvated stage at all the tested temperatures. Development rate increased with temperature. Linear regression of development rate against temperature predicted a lower development threshold of 11.8°C. Eggs survived cooling to 1 and −2°C for 6 weeks, and could develop to the infective, larvated stage when transferred to higher temperatures, but their development rates were then retarded compared with non-chilled eggs. Larvated eggs remained viable after 7 weeks of incubation across the tested temperature range, with the highest percentage viability (47%) obtained at 25°C. Development of eggs to the infective larval stage required, on average, 121 degree days between 20°C and 30°C. Results provide a basis for predicting variation in the infectivity of eggs in the environment over time in different climates.     

<Emphasis Type="Italic">Trichinella spiralis</Emphasis> reinfection: macrophage activity in BALB/c mice

The role of macrophages and their products—nitric oxide (NO) and superoxide anion (O₂ ⁻)—were examined in BALB/c mice reinfected with Trichinella spiralis. Mice were infected twice with 400 T. spiralis larvae; the secondary infection was performed 60 days after the primary one. Adult T. spiralis in intestines were detected from 5 to 20 days postprimary infection (dpi), and postreinfection (dpri), but the intensity of worm expulsion was greater and quicker after the reinfection. The highest muscle larvae numbers were detected from 60 dpi till 90 dpi (30 dpri), and thereafter, a great reduction was noted. The high numbers of macrophages in the peritoneal cavity of infected mice were obtained at 20 dpi, but the highest numbers of these cells from 10 to 30 dpri were observed. The production of NO by macrophages in infected mice was suppressed at 5 dpi, and then, NO release increased until 60 dpi. On the contrary, the long-lasting (5–30 dpri) suppression of NO production after T. spiralis reinfection was observed. The levels of NO in plasma and urine were lower in infected mice till 20 dpi; there were no differences in plasma and urine NO level after the reinfection in comparison to control uninfected animals. The production of O₂ ⁻ in peritoneal macrophages was inhibited during the first 2 weeks after infection, but the reinfection caused great increase in O₂ ⁻ production lasting 30 days.     

Evaluation of the immunosuppressive effect of cyclophosphamide and dexamethasone in mice with visceral toxocariasis

Visceral toxocariasis is a serious public health problem with a cosmopolitan distribution. Children are susceptible due to their immature immune system and high risks of infection. Nevertheless, the few completed studies about immunosuppression have had controversial results. To evaluate the effect of two immunosuppressive drugs on the larval burden of Toxocara canis, four groups of ten Swiss strain mice each were inoculated on day 0 with 1,200 embryonated T. canis eggs. Fifteen days before the experimental infection, group 1 (control) was treated via intraperitoneal injection (IP) with sterile distilled water and groups 2 and 3 were treated with dexamethasone (DEX) at 1 and 5 mg/kg/day, respectively. Additionally, group 4 was treated IP with cyclophosphamide (CY) at 50 mg/kg at two times per week for 2 weeks. Sixty days following infection, the mice were euthanised to recover the larvae by means of the tissue digestion technique. The levels of antibodies detected by indirect ELISA were not associated with the larval burden. Administration of CY (50 mg/kg) and DEX (5 mg/kg) resulted in an increase of the larval burden of 162.1% and 50.8%, respectively, in relation to the control group. These two treatments, especially CY (50 mg/kg), promoted immunosuppression and the establishment of a significant larval burden, supporting its further utilisation in studies related to immunosuppression in visceral toxocariasis.     

Clinical, laboratory and pathological findings in dogs experimentally infected with Angiostrongylus vasorum

The aim of this comparative study was to investigate the development of clinical signs and accompanying haematological, coproscopic and pathological findings as a basis for the monitoring of health condition of Angiostrongylus vasorum infected dogs. Six beagles were orally inoculated with 50 (n = 3) or 500 (n = 3) A. vasorum third stage larvae (L3) obtained from experimentally infected Biomphalaria glabrata snails. Two dogs were treated with moxidectin/imidacloprid spot-on solution and two further dogs with an oral experimental compound 92 days post infection (dpi), and were necropsied 166 dpi. Two untreated control dogs were necropsied 97 dpi. Prepatency was 47–49 days. Dogs inoculated with 500 L3 exhibited earlier (from 42 dpi) and more severe respiratory signs. Clinical signs resolved 12 days after treatment and larval excretion stopped within 20 days in all four treated dogs. Upon necropsy, 10 and 170 adult worms were recovered from the untreated dogs inoculated with 50 and 500 L3, respectively. Adult worms were also found in two treated dogs, in the absence of L1 or eggs. Despite heavy A. vasorum infection load and severe pulmonary changes including vascular thrombosis, only mild haematological changes were observed. Eosinophilia was absent but the presence of plasma cells was observed. Neutrophilic leucocytes showed a transient increase but only after treatment. Signs for coagulopathies were slight; nevertheless coagulation parameters were inoculation dose dependent. Ten weeks after treatment pulmonary fibrosis was still present. Infections starting from 50 L3 of A. vasorum had a massive impact on lung tissues and therefore on the health of affected dogs, particularly after prepatency, although only mild haematological abnormalities were evident.     

Molecular and parasitological tools for the study of Ascaridia galli population dynamics in chickens

Experiments were first conducted to compare and evaluate different methods of Ascaridia galli larval recovery from the chicken intestine. The number of larvae recovered from the intestinal wall of chickens infected with 1000 embryonated A. galli eggs and killed 15 days post infection (p.i.) by three methods (ethylenediamine tetraacetic acid [EDTA], pepsin digestion and scraping) were compared. The EDTA and pepsin digestion were found to be the most efficient methods with no significant difference (P > 0.05) in the number of recovered larvae between the two. Subsequently, three different A. galli cohorts were established using the polymerase chain reaction-linked restriction fragment length polymorphism (PCR-RFLP) technique. A 533-bp long region of the cytochrome c oxidase subunit 1 gene of the mitochondrial DNA was targeted and 22 A. galli females were allocated to three different haplotypes. The four females with the highest embryonation rate from each haplotype group (total 12 females) were selected and used to inoculate each of 12 chickens with a dose of 1000 embryonated eggs. The chickens were killed 15 days p.i. and A. galli larvae were recovered from the small intestinal wall by the EDTA method and by sieving the lumen content on a 90 µm sieve. DNA of 40 larvae from each of the three different haplotypes was extracted using a worm lysis buffer, and PCR-RFLP analysis of these larvae revealed the same haplotype as that of their maternal parent. The identification of distinguishable cohorts may be a powerful tool in population studies of parasite turnover within the animal host.     

Immunoglobulin class of antibodies in monkeys infected with Toxocara canis

Sera of monkeys (Macaca sinica), experimentally infected with multiple doses of eggs of Toxocara canis, were subjected to Sephadex G-200 gel filtration and DEAE cellulose ion exchange chromatography. The distribution of antibodies in the various serum fractions was determined by the conglutinating complement absorption test (CCAT) and the agar-gel diffusion precipitin test using antigens prepared from the adult and the embryonated eggs of T. canis and by the formation of precipitates at the oral orifice of artificially hatched living 2nd stage larvae of T. canis.In serum samples taken 1 and 2 weeks after initial infection, CCAT antibodies were located mainly in the macroglobulin fractions of serum. By 1 to 4 weeks after a third dose of eggs such antibodies were located mainly in the 7S fraction of immunoglobulins.Antibodies detected by gel precipitation were located in the macroglobulin fraction of serum, while those that produced oral precipitates on 2nd stage larvae occurred in the 7S fractions.