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.     

Establishment and migration pattern of<Emphasis Type="Italic"> Toxocara canis</Emphasis> larvae in chickens

Migrations of Toxocara canis larvae were observed in experimentally infected chickens. Three groups of three chickens were inoculated orally with T. canis eggs. Within each group, individual chickens received either 5,000, 10,000, or 20,000 eggs. A group of infected chickens was then necropsied at either 1, 3 or 6 days post infection (dpi). The entire duodenum, spleen, liver, heart, lungs, right inner pectoral muscle, and brain were subjected to pepsin digestion for larval recovery. Larvae were predominately (>87%) recovered from the liver and lungs, and only a few larvae were seen in other organs or tissues in all chickens, with the exception of the duodenum at 1 dpi of chickens inoculated with 20,000 eggs. The percentage of total larval recovery varied widely among chickens (range: 0.4–16.7%). Similar numbers of larvae were distributed in the liver and lungs at 1 dpi. Subsequently, more larvae were found in the lungs than the liver at 3 dpi, whereas the larval distributions in the liver and lungs were reversed at 6 dpi. These observations suggest that T. canis larvae can migrate by a hepatopulmonary route in the chicken, and reinforces the possibility that chickens harboring migrating T. canis larvae may pose a zoonotic risk, especially if the liver is consumed.     

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.     

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.     

Mechanical recovery of inhibited cyathostomin larvae from equine intestinal tissue

The Stomacher? is very widely used in food and medical research for extracting tissues. To determine whether nematode larvae were disrupted by the Stomacher?, L₃ larvae of Haemonchus contortus were homogenised for up to 40 min at full power but no larval disruption occurred. Therefore, tissue from the mucosa and submucosa of the caecum of horses collected from a licenced abattoir was treated to determine whether inhibited cyathostomin larvae could be extracted. The optimum time on full power for a 10-g sample was 20 min, and in three out of five caecal samples from different horses, significantly more larvae were recovered than with 6 h pepsin HCl digestion. It is concluded that the Stomacher? provides a simple fast method of extracting inhibited nematode larvae from gastrointestinal tissues in the horse that could replace digestion with pepsin HCl.     

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.     

Evidence for bacteria-independent hatching of Trichuris muris eggs

Hatching of infective larvae from embryonated eggs in the intestine is an essential first step in Trichuris infections. There are three isolates of the murine parasitic nematode Trichuris muris: E, E-J (the E isolate maintained in Japan), and S. Incubation of T. muris embryonated eggs with the intestinal bacteria Escherichia coli and Staphylococcus aureus induced in vitro hatching of the eggs, but Enterococcus faecalis failed to induce hatching. Bacteria-induced in vitro hatching of embryonated eggs occurred in the E and E-J isolates, whereas the S isolate was unresponsive to bacteria. T. muris worms recovered from infected mice showed no significant difference between the E-J and S isolates in their infectivity to susceptible B10.BR mice (P?>?0.05). In vivo hatching of embryonated eggs occurred at 30 min post-infection in the upper and lower halves of the small intestine of ddY mice infected with the E-J or S isolates, and on average, 65 % of embryonated eggs recovered from the upper half of the small intestine were hatched in both the E-J and S isolates. In comparison with Enterococcus, the bacteria E. coli and S. aureus represent relatively minor components of the flora of the upper half of the small intestine of mice. These observations point to the possibility that bacteria-independent hatching might also occur in vivo, at least for the S isolate, and imply the existence of a very different system of induction of hatching in vivo.     

On the life cycle and morphology of development stages of Paraspiralatus sakeri Gibbons et al., 2004 (Nematoda: Spiroidea,Spirocercidae), a heteroxenic stomach parasite of falcons

Pitted darkling beetles (Adesmia cancellata) were infected with nematode eggs found in the alimentary tract of a gyrfalcon (Falco rusticolus) naturally infected with Paraspiralatus sakeri. Third-stage larvae in numbers between 1 and 84 were removed from the beetles 5 weeks postinfection and were used for morphological studies as well as to infect domestic chicken, yellow-bellied geckos (Hemidactylus flaviviridis) and fringe-toed lizards (Acanthodactylus schmidti). All experimental animals, necropsied 4–38 weeks later, were positive for spirally coiled nematode larvae located under the skin and in the interstitium of skeletal muscles. Despite similarities in general morphology, larvae from beetles and reptiles and chicken differed strikingly in the total body length and body width. Differences in length of the muscular oesophagus and distances of cervical papillae, nerve ring and excretory pore from the anterior end were less distinct. Morphology of these larvae matched with larvae found in subcutaneous cysts in naturally infected houbara bustards (Chlamydotis macqueeni) from Pakistan and UAE as well as with those detected in the muscles of an ocellated skink (Chalcides ocellatus).     

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.     

Investigation of the parasitic nematode Ascaridia galli (Shrank 1788) as a potential vector for Salmonella enterica dissemination in poultry

During recent years, the level of organically farmed poultry in Denmark has increased. Subsequent investigations have demonstrated an incidence of 64% of Ascaridia galli infections in layers established in organic farming systems. Studies to determine the interaction of Salmonella enterica with the parasitic nematode A. galli associated with poultry were undertaken to establish the significance of A. galli in the dissemination of S. enterica. A. galli was isolated from 40-week-old Lohmann Brown Salmonella-free layers. Worms were subsequently maintained in vitro and exposed to S. e. serovar Typhimurium at concentrations of 10⁵–10⁶ colony forming units/ml for varying times (24–144 h). Eggs were harvested aseptically from the worms and the associations of S. e. Typhimurium in relation both to the eggs and to structures on the surface of the worm were studied, using immunofluorescence, viable counts and in situ hybridisation. Results show attachment of S. e. Typhimurium to the outer coating of the eggs and possible internalisation. Evidence of association of the bacteria with the nematode eggs was further substantiated by establishing Salmonella infection in day-old chicks after dosing them with eggs harvested from parasitic worms infected in vitro with Salmonella. Received: 11 September 2000 / Accepted: 14 September 2000     

On the life-cycle of subulura suctoria,a caecal nematode of poultry in egypt

Summary By random examination of various insects for cysticercoides of tapeworms, larvae of round worms were encountered attached to the intestinal wall of beetles, Ocnera hispida and Blaps polycresta. The morphological features of the larvae were discussed and materials from infected beetles were fed to chicken and we obtained embryonated eggs from their droppings and on making post-mortum examination of chicken, adult Subulura suctoria worms were found.With 3 Figures in the text     

Immunopathogenesis of Ascaridia galli infection in layer chicken

Gastro-intestinal nematode infections in mammals are associated with local T lymphocyte infiltrations, Th2 cytokine induction, and alterations in epithelial cell secretion and absorption. This study demonstrates that Ascaridia (A.) galli infection in chicken also elicits local gut-associated immune reactions and changes in the intestinal electrogenic nutrient transport. In A. galli-infected birds we observed infiltrations of different T cell populations in the intestinal lamina propria and accumulation of CD4+ lymphocytes in the epithelium. The Th2 cytokines IL-4 and IL-13 dominated the intestinal immune reactions following A. galli infection. A. galli-specific systemic IgY antibodies were detected after two weeks post infection, and did only poorly correlate with detected worm numbers. Electrogenic transport of alanin and glucose was impaired in A. galli-infected chicken. Our data provide circumstantial evidence that local immune responses and electro-physiological intestinal functions may be connected and contribute to the elimination of worm infection.     

Gasterophilosis in horses in Sardinia (Italy): effect of meteorological variables on adult egg-laying activity and presence of larvae in the digestive tract,and update of species

Gasterophilus larvae are common obligate parasites of the digestive tract of the equids. Horses become infected with this parasite by ingesting the larvae hatched from eggs laid by the female flies. In this study carried out monthly, we (i) counted the Gasterophilus eggs deposited by female flies on the coat of 30 grazing horses, (ii) counted and identified the Gasterophilus larvae retrieved from the digestive tract of 128 slaughtered horses, and (iii) compared these results to meteorological data. Eggs were deposited on all monitored horses, and were present from October to January and from May to September, whereas they were absent from February to April. The number of laid eggs was significantly different between the months, body regions, genders, and age classes (p?<?0.05). Larvae were recovered in 112 (87.5 %) horses, and 6 species of Gasterophilus were identified. The prevailing species were Gasterophilus intestinalis (recovered in 110 horses; 85.9 %) and Gasterophilus nasalis (69 horses; 53.9 %), recovered in all months. Gasterophilus inermis (5 horses; 3.9 %), Gasterophilus pecorum (3 horses; 2.3 %), Gasterophilus haemorrhoidalis (3 horses; 2.3 %)¸ and Gasterophilus meridionalis (2 horses; 1.6 %) larvae were also found. Significant differences were found among monthly larval burdens for both Gasterophilus spp. and G. intestinalis (p?<?0.05), but not for G. nasalis (p?>?0.05). Larval burdens and prevalences did not differed significantly between both genders and age classes (p?>?0.05). Monthly eggs and larvae trends were not significantly correlated (p?>?0.05). With regard to the meteorological variables, minimum air temperature was significantly correlated with the eggs trend (rho?=?1.000; p?<?0.001) and maximum air temperature with the Gasterophilus spp. (rho?=?0.972; p?<?0.001) and G. intestinalis (rho?=?0.972; p?<?0.001) larvae trends. In addition, the number of hours with a temperature below +10 °C was significantly correlated with G. intestinalis larvae trend (rho?=?0.602; p?<?0.05). Our findings confirmed that in Sardinia, Gasterophilosis is an important parasitosis in the horses, and it needs more attention and extensive and/or correct treatment to reduce its prevalence.     

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.     

Clinical,laboratory and pathological findings in cats experimentally infected with Aelurostrongylus abstrusus

Aelurostrongylus abstrusus parasitizes the respiratory tract and can heavily affect the breathing and general condition of cats. Experimental infections of six cats were initiated by intragastric administration with 100 or 800 third-stage larvae (L3) obtained from the terrestrial snail Helix aspersa. First-stage larvae were isolated from faecal samples after 35–41 days post infection (dpi) in five animals and until end of study (84 dpi) in two cats. Cough and respiratory sounds were observed starting from 28 to 41 dpi and dyspnoea and panting starting from 52 dpi. All cats had enlarged lymph nodes and, starting from 56 dpi, reduced body weight, and four cats showed intermittent reduced general condition with apathia and anorexia. Eosinophilia and leucocytosis partially with massive lymphocytosis, and occasional basophilia and monocytosis were observed. Mild anaemia was present in five cats, while alterations in coagulation parameters suggested stimulation of the coagulation cascade with increased consumption of coagulation factors (delayed PT, hypofibrinogenemia). Adult A. abstrusus specimens were isolated from the five patent cats at necropsy and all six cats showed pathological changes in the lungs, including disseminated inflammatory cell infiltrates, often associated with incorporated larvae and eggs. There was some degree of overlap between the severity and the inoculation doses. Infections starting from 100 L3 of A. abstrusus had an impact on the lung tissues and on the health of the cats, despite the presence of only mild haematological abnormalities. Due to the worldwide occurrence of feline lung worms, parasitic infections should be considered in the differential diagnosis of lung diseases regardless of the presence of clinical signs and larval excretion.     

Intestinal IgA response and immunity to rotavirus infection in normal and antibody-deficient chickens

Rotavirus inoculation by oesophageal cannulation resulted in subclinical infection without decreasing intestinal D-xylose absorption in both intact and embryonally bursectomised, antibody-deficient (EBx) 8-week-old specific-pathogen-free chickens. In intact chickens, rotavirus-specific IgM, IgG and IgA responses were detected in serum, while the intestinal antibody response consisted almost entirely of IgA Serum IgG and intestinal IgA levels were increased for at least 70 days following a single inoculation with the virus. Intact chickens recovered from a primary rotavirus infection between 4 and 14 days post inoculation (dpi) and developed resistance to homotypic challenge between 14 and 28 dpi. These responses were only slightly delayed in EBx birds, which recovered from primary infection between 8 and 28 dpi and developed resistance between 14 and 42 dpi. This suggested that the intestinal IgA response in chickens participated in both recovery from and resistance to rotavirus infection, but that it was not the only mediator of recovery and resistance.     

Response to Ascaridia galli infection in growing chickens in relation to their body weight

It was hypothesized that chickens with extremely varying body weights (BW) from an otherwise homogeneous host sample cope differently with Ascaridia galli (Schrank 1788) infection. Small and large birds, falling into either the lower or the upper 5 % quantiles of BW distribution of a parent stock flock, were selected at an age of 4 weeks, housed separately and fed restrictively with the same amount of feed. At week 5, all the small and large birds (635 and 1,297 g/bird, respectively; P?<?0.001) were inoculated with 1,000 A. galli eggs and euthanized 52 days post-infection. Small birds had higher daily weight gains (P?=?0.004) but final BWs of larger birds were still higher (P?<?0.001) at slaughter. Prevalence, intensity of infection as well as worm abundance were higher in small birds when compared with the large birds (P?<?0.05), whereas plasma concentrations of A. galli-specific antibodies and worm length remained unaffected (P?>?0.05). In conclusion, large birds resist A. galli infection more effectively than do small ones, possibly through different mechanisms acting on allocation of available nutrient and body reserves under the exposure of the infection.     

Selenium Status Alters the Immune Response and Expulsion of Adult Heligmosomoides bakeri Worms in Mice

Heligmosomoides bakeri is a nematode with parasitic development exclusively in the small intestine of infected mice that induces a potent STAT6-dependent Th2 immune response. We previously demonstrated that host protective expulsion of adult H. bakeri worms from a challenge infection was delayed in selenium (Se)-deficient mice. In order to explore mechanisms associated with the delayed expulsion, 3-week-old female BALB/c mice were placed on a torula yeast-based diet with or without 0.2 ppm Se, and after 5 weeks, they were inoculated with H. bakeri infective third-stage larvae (L3s). Two weeks after inoculation, the mice were treated with an anthelmintic and then rested, reinoculated with L3s, and evaluated at various times after reinoculation. Analysis of gene expression in parasite-induced cysts and surrounding tissue isolated from the intestine of infected mice showed that the local-tissue Th2 response was decreased in Se-deficient mice compared to that in Se-adequate mice. In addition, adult worms recovered from Se-deficient mice had higher ATP levels than worms from Se-adequate mice, indicating greater metabolic activity in the face of a suboptimal Se-dependent local immune response. Notably, the process of worm expulsion was restored within 2 to 4 days after feeding a Se-adequate diet to Se-deficient mice. Expulsion was associated with an increased local expression of Th2-associated genes in the small intestine, intestinal glutathione peroxidase activity, secreted Relm-β protein, anti-H. bakeri IgG1 production, and reduced worm fecundity and ATP-dependent metabolic activity.     

Fowl aviadenovirus serotype 1 confirmed as the aetiological agent of gizzard erosions in replacement pullets and layer flocks in Great Britain by laboratory and in vivo studies

An investigation into the aetiology and pathogenesis of adenoviral gizzard erosion has been conducted following three natural outbreaks affecting one flock of 6-week-old replacement pullets and two consecutive placements of free range layers at the age of 21 and 23 weeks. Affected flocks showed increased mortality (0.12–0.30% per week), and gizzard lesions were consistent with fowl aviadenovirus (FAdV) involvement. To substantiate the initial findings, a selection of archived formalin-fixed paraffin-embedded gizzard samples from another 12 pullet and layer flocks, for which macroscopic and histopathological diagnosis of the disease were recorded in Great Britain during the period 2009–2016, were also investigated. In situ hybridization (ISH), virology and/or PCR confirmed the presence of FAdV species-A, serotype-1 (FAdV-A, FAdV-1) DNA in gizzard samples of all 15 cases investigated. Co-infections with additional FAdV serotypes including FAdV-8a were detected by serology and/or virology in two of the pullet flocks. However, species-specific in situ hybridization revealed that pathological changes of affected gizzards were only associated with the detection of FAdV-A. A subsequent in vivo study infecting 21-day-old SPF pullets with FAdV-1 or FAdV-8a strains isolated from the 6-week-old replacement pullets revealed characteristic pathomorphological changes only in the gizzards from birds infected with FAdV-1. While infection with FAdV-8a was confirmed by virology and serology, infected SPF birds did not develop pathomorphological changes. Therefore, the aetiological involvement of the isolated FAdV-8a in the development of adenoviral gizzard erosion in commercial pullets has been ruled out.