Developmental capacity of Ascaridia galli eggs is preserved after anaerobic storage in faeces.

The reliability of the results of in vitro studies such as detection of anthelmintic resistance often depends on the ability of the parasite eggs to develop under laboratory conditions. The aim of this study was to assess the embryonation capability of the chicken roundworm Ascaridia galli eggs after storage under different conditions. Two storage media for parasite eggs were used; faeces or water. Eggs in petri dishes (90 dishes in total) containing faces or water media were first exposed either to aerobic or anaerobic conditions at different temperatures (4 °C /+O2, 4 °C /-O2, 25 °C/-O2) for a maximum of 72 days. Every second week, materials from petri dishes in triplicates were recovered and incubated aerobically for two weeks at 25 °C. After the incubation, 200-300 eggs from each petri shish (sampling unit) were counted and the number of embryonated eggs was determined. Data was analyzed in R (version 3.4.3) A logistic regression model with the probability of an egg to embryonate as dependent variable and conditions, storage medium and time points as fixed effects with quasibinomial distribution was run. Least-square means were calculated and pairwise comparisons were made with the fixed effect factors (condition, storage medium and time point). Eggs in faeces had a significantly (p ˂ 0.05) higher embryonation than those in water, irrespective of storage conditions. At 4 °C embryonation tended to decline over time under aerobic conditions irrespective of the storage medium, whereas it remained constant following storage at anaerobic conditions. In contrast, anaerobic storage at the 25 °C negatively affected egg development in both media, except for day 14 in faeces. Our major finding was that eggs in faeces under anaerobic conditions and at 4 °C retained the highest rate of development, with a minimum decline in their developmental capacity over time compared to cleaned eggs stored in water.

Survival and development of chicken ascarid eggs in temperate pastures.

Eggs of chicken ascarids (Ascaridia galli and Heterakis spp.) are believed to be hardy and survive for long periods. However, this has not been evaluated quantitatively and our study therefore aimed to determine development and recovery of chicken ascarid eggs after burying in pasture soil. Unembryonated eggs were mixed with soil, placed in sealed nylon bags and buried at 7 cm depth in pasture plots April (spring, n = 72) and December 2014 (winter, n = 72). Eight randomly selected bags per season were used to estimate pre-burial egg recovery [0 week post-burial (wpb)]. Eight random bags were removed at 5, 12, 23, 38, 52, 71 wpb per season and additionally at 104 wpb for spring burial. The content of each bag was analysed for numbers and development stages of eggs. Eggs buried in spring were fully embryonated within 12 wpb. In contrast, eggs buried in winter were developing between 23 and 38 wpb, so that all viable eggs seemed to be fully developed by 38 wpb. About 90% eggs disappeared within 23 wpb (spring) and 38 wpb (winter). Small proportions (2-3%) of seemingly viable and infective eggs were still recovered up to 2 years after deposition. In conclusion, most eggs buried in temperate pasture soil seem to experience a heavy mortality within a few months after the deposition, especially during warm periods. However, a small proportion of eggs may survive and remain infective for at least 2 years.

Evaluation of benzimidazole resistance status in Ascaridia galli.

Susceptability of Ascaridia galli to benzimidazole (BZ) was investigated using faecal egg count reduction test (FECRT), in ovo larval development test (LDT) and genetic markers (mutations at codons 167, 198 and 200 of ß-tubulin gene). Six flocks (F1-F6) of a commercial laying hen farm with different number of exposure to BZ were recruited. The FECR was calculated by analyzing individual faeces (F1, F2, F4 and F5) before and 10 days after treatment. The LDT was performed on parasite eggs from pooled samples from F1 to F6 and LC50 and LC99 were calculated. DNA was extracted from 120 worms and sequenced for ß-tubulin gene. In all flocks, the FECRs were above 95% (lower CI above 90%). No significant difference was observed (p > 0·05) among obtained LC50 (F1/F4 and F2/F5 vs F3/F6) in the LDT. However, LC50 and LC99 were higher than suggested values for declaration of resistance in other nematode species. No variation was observed in codon positions involved in BZ resistance. Overall, our results indicated lack of evidence of resistance to BZ in A. galli. More research is needed to confirm these results and to further optimize the existing tools for detection and monitoring of anthelmintic resistance in A. galli.

Pochonia chlamydosporia fungal activity in a solid medium and its crude extract against eggs of Ascaridia galli.

The present study aimed to evaluate the ovicidal activity (type 3 effect) of VC1 and VC4 isolates of Pochonia chlamydosporia in a solid medium and the action of a crude extract of P. chlamydosporia against eggs of Ascaridia galli. To evaluate ovicidal activity in culture medium, 1000 A. galli eggs were plated on Petri dishes containing 2% water-agar with grown fungal isolates (VC1 or VC4) and without fungus (control group) and were examined at 1, 3 and 5 days post-inoculation (assay A). Then, to test the action of crude extracts of P. chlamydosporia (VC1 or VC4), 500 eggs of A. galli were plated on Petri dishes of 4.5 cm diameter with 5 ml of fungal filtrate from each tested isolate. The control group consisted of 500 eggs of A. galli with 10 ml of distilled water on each Petri dish (assay B). Fungal isolates were effective (P < 0.01) at destroying these eggs, showing a type 3 effect at the studied intervals. On the other hand, the crude extract of isolates (VC1 or VC4) reduced the number of A. galli eggs in the treated group compared with the control group by 64.1% and 56.5%, respectively. The results of the present study show that P. chlamydosporia is effective at destroying eggs of A. galli and could therefore be used in the biological control of nematodes.

Age-related differences of Ascaridia galli egg output and worm burden in chickens following a single dose infection.

Ninety white chickens (Lohmann LSL) were reared under helminth-free conditions and divided into five groups. Four groups were artificially infected with 250 embryonated Ascaridia galli eggs at the age of 6, 12, 18 or 24 weeks. Ten birds were kept as uninfected controls. Six and 10 weeks after infection (p.i.), individual faecal egg counts (FEC) were performed. The birds were slaughtered after the second sampling and their gastrointestinal tracts were examined for the presence of adult A. galli. The FEC increased from the first to the second sampling significantly in all the infected groups. The highest increase was shown in the group infected at 12 weeks of age, whereas the increase in the other groups was relatively moderate. However, the total worm burden and mean FEC at the second sampling were highest (p<0.01) in those birds infected at an age of 12 or 18 weeks. The serum protein and triiodothyronine (T3) levels did not differ significantly (p>0.05) between any of the groups. Thyroxine (T4) was significantly different between the groups infected at 6 and 18 weeks of age (p<0.05), and those at 6 and 24 weeks of age (p<0.01). The thyroid hormone levels correlated significantly with the FEC. Age does not seem to play a major role in resistance to A. galli infections in layers, whereas a bird's hormonal and immune status, related to laying activity, seems to have a significant negative impact on resistance.

Host age only partially affects resistance to primary and secondary infections with Ascaridia galli (Schrank, 1788) in chickens.

Two experiments were conducted to compare the effect of chickens' age on resistance to primary and secondary infections with Ascaridia galli. In Experiment I, three groups, each of 80 female Lohman Brown chickens, aged one day, one month, or four months were compared. Within each group, 54 chickens were infected orally with 500 embryonated eggs and 26 were kept as non-infected controls. Weights were recorded weekly and five chickens in each group were slaughtered every 2 weeks for worm counts. At week 10 post-infection, 17 of the infected chickens and 18 of the controls were challenged with 500 eggs. In a replicate experiment (Experiment II), 35 one-day-old and 53 one-month-old female Lohman Brown chickens were infected orally with 500 A. galli eggs. Weights and fecal egg counts were recorded every week and infected chickens were necropsied every two weeks for determination of the worm burden. Chickens infected at one month of age excreted significantly fewer A. galli eggs when measured at 14 weeks of inoculation. The worms recovered from the one-month-old age group were significantly shorter than those from the chickens infected at one day of age in the first experiment. Worm burden and female fecundity values, however, were not significantly different between age groups in both Experiments I and II. Weight gains of infected chickens were not significantly different from the controls' and only a few chickens exhibited occasional slight diarrhea in both experiments. The results from these experiments demonstrate that the chickens' age only partially influences resistance to A. galli infection.

Comparative genetic resistance to Ascaridia galli infections of 4 different commercial layer-lines.

1. The objective of the study was to compare the establishment and effect of Asaridia galli infections in 4 different layer-lines. 2. A total of 160 birds comprising 4 different commercial layer-lines, ISA Brown, New Hampshire, Skalborg and a cross of New Hampshire(NH) and Skalborg (Sk), were infected with A. galli eggs. The birds were examined for the presence of parasite eggs and parasites at weeks 3, 6 and 9 post infection (pi). 3. At week 6 pi the chickens of the NH line harboured more larvae compared with the three otherlines. The Sk line chickens excreted more A. galli eggs throughout the study compared with the other lines. Female worms in the Sk line were more fecund than the worms in the other lines. Male and female worms recovered from the Sk line at week 9 pi were longer. Male worms recovered from the NH line 6 weeks pi were shorter than male worms from the other lines. Female worms recovered from the NH line were shorter than the female worms from the ISA line and the Sk line. No differences were seen in weight gain among the 4 lines. 4. The results suggest that genetic factors are involved in the establishment and survival of A. galli in the intestine of layers. Further studies are needed to elucidate the genetic mechanisms behind the observed parasitological findings.

Genetic differences of Ascaridia galli egg output in laying hens following a single dose infection.

Groups of 20-week-old white (Lohmann LSL) and brown (Lohmann Brown) hens were reared under helminth-free conditions and both divided into two subgroups. One subgroup was artificially infected with 250 embryonated Ascaridia galli eggs, the other subgroup were kept as uninfected controls. During the following 12-month laying period individual faecal Ascaridia egg counts (FEC) were performed and bodyweight measured at monthly intervals. Laying performance and egg weights were determined daily. The mean FEC were significantly (P < 0.01) higher in infected white hens than in infected brown hens. The growth rate of the white hens was significantly (P < 0.05) higher in the control group, whereas in brown hens no significant group difference was observed. The laying performances and egg weights did not differ significantly (P > 0.05) between control and infected animals for either line. The estimated repeatabilities for mean log(10) FEC of different samples were reasonably high (0.16-0.54). Heritabilities for mean log(10) FEC were between 0.13 and 0.19 for white hens and between 0.0 and 0.10 for brown hens. Thus, it should be possible to select for A. galli resistance in chickens, which will be of importance for birds kept in alternative and organic farming systems.

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 10(5)-10(6) 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.

Effect and repeatability of Ascaridia galli egg output in cockerels following a single low dose infection.

Three groups of caged 20 Tetra-SL cockerels aged 1 day were orally infected with 30, 60 or 125 embryonated Ascaridia galli eggs. After 11, 12 and 13 weeks, faecal egg counts (FECs) were determined. All birds were slaughtered after the last sampling. A group of 25 control birds was sampled and slaughtered in parallel. The gastrointestinal tracts were examined for the presence of adult stages of A. galli. A random sample of 10% was also examined for the presence of immature stages of A. galli. The group with an infection dose of 125 eggs showed the highest average worm burden (p<0.05) and number of females (p<0.05), but the mean establishment rate was the lowest in this group. There was no significant difference in the mean logFEC between the groups. The logFEC per female worm was the highest in the low infection group (p<0.05). The average worm length and weight and the birds body weight were not significantly different among the groups. The estimated repeatabilities for mean logFEC of the different samples were reasonably high (0.55-0.87). This may open a way of genetic selection for A. galli resistance in chickens, which will be of importance for birds kept in alternative and organic farming systems.

Kinetic model of parasite development and of the host microelement content under combined drug treatment.

The content of nine microelements has been investigated in a parasite-host system by nondestructive neutron activation analysis. A mathematical model has been proposed for the development of Ascaridia galli in the "Leghorn" chicks intestines and for the microelement content dynamics under combined treatment with antiparasite agent and microelements. An analytical solution of the system of nonlinear differential equations has been obtained for the case of simple invasion. The model explains well the experimentally observed process of the change in the microelement content. The recovery constant has been introduced for the damaged by the helminthosis tissue and its value has been determined.

[The survival ability of salmonella, coccidia oocysts and ascarid eggs in laying hen feces from different housing systems]. / Zur Uberlebensfähigkeit von Salmonellen, Kokzidienoozysten und Spulwurmeiern im Legehennenkot unterschiedlicher Haltungssysteme.

The time of survival of Salmonella typhimurium, coccidia oocysts and ascaris eggs in manure of layer was determined in 5 different housing systems and 2 storing places for litter. The experiments were carried out in a stable of experimental station Frankenforst of the university of Bonn with a flock of 2200 hens. The effects of the environment conditions temperature, dry matter content, pH-value and intestinal microflora of the manure have also been studied. The time of survival was different depending on the housing system. A recovery of viable coccidia oocysts was possible after 13-370 days, ascaris eggs 53-347 days and Salmonella typhimurium 2-175 days. The tenacity of the investigated test organism mainly depend on the dry matter content of the manure. The longest period of survival of salmonellas was found in dry environment conditions, were as coccidia oocysts and ascaris eggs have been observed with the shortest period of survival. The possibility of the examined resistant parasite stages to develop was disturbed. Only few of them were able to develop and with a longer development time than those examined in the control suspension. The results of this study indicate that chicken manure, before using it in plant production, should be stored long enough to prevent men or animals from possible infections.

Effect of ascariasis and respiratory diseases on growth rates in swine.

Growth rates, measures of ascarid burden, and the levels of anteroventral pneumonia and atrophic rhinitis at slaughter were determined for 352 hogs born between March 8 and March 28, 1987 on 15 farms located in Prince Edward Island. Regression analyses were used to determine associations between average daily gain (ADG) and independent variables controlling for sex, farm, and litters nested within farm. The regression model accounted for 75.4% of the variation in ADG. The number of intestinal ascarids at slaughter did not affect ADG. However, the "life-time burden" (a composite measure based on fecal egg counts and duration of infection) was associated with ADG (p less than 0.05) in a quadratic manner. Although heavy ascarid burdens decreased the growth rate of swine, the magnitude of the effect was minimal. The maximum improvement one could expect from reducing the ascarid burden on heavily infected farms would be less than 1%. Severe atrophic rhinitis and the presence of anteroventral pneumonia each had a detrimental effect on ADG (p less than 0.001). The corresponding reductions in mean ADG were 7.7% and 2.8% respectively. There was significant interaction between the effects of atrophic rhinitis and anteroventral pneumonia on ADG (p less than 0.05). Hogs with both anteroventral pneumonia and severe atrophic rhinitis had a 17.6% lower ADG than hogs with neither disease. There is much greater potential for improvement in ADG through control of respiratory diseases than through control of ascariasis.

Establishment of Ascaridia galli in betamethasone-treated chickens.

Twenty-five day-old White Leghorn chickens were each infected orally with 500 (Group I), 1000 (Group II) and 2000 (Group III) infective eggs of Ascaridia galli and were killed 30 days after the infection. A high percentage of the infecting dose (6.5%) established as adult worms in the intestine of chickens receiving the lowest level of primary infection, but as the amount of primary infection given to birds increased, there was a significant fall in the percentage establishment of adult worms in the intestine. A similar pattern of worm establishment was shown by chickens of the same age and receiving similar levels of primary infections, but which were treated with betamethasone at a dose of 2 mg/kg body weight commencing 5 days before and continuing up to 15 days after the infection. Betamethasone-treated birds, however, showed more establishment of worms in the intestine, but lower weight gains in comparison to the birds which were not treated. Different levels of primary infections given, with or without treatment with betamethasone, had no effect on the sex ratio of the resultant male/female worm populations, which became established in almost equal numbers in the intestine of chickens.

In vitro hatching of the infective eggs of Ascaridia galli in tissue extracts.

Extracts of proventriculus (PE) and small intestine (IE) of fowl were used, for the first time, as media for the in vitro hatching of infective eggs of Ascaridia galli. Hatching was successful in a mixture of equal parts of PE and IE (MIP) under normal air and under different concentrations of CO2, but not under 100% CO2. A very highly significant retardation (P less than 0.001) in the rate of hatching appeared as the concentration of CO2 was increased, indicating that high levels of CO2 inhibit the process of hatching. 100% of the eggs hatched in PE in 40.5 min whereas they did not hatch at all in IE, suggesting that IE alone has no influence on egg hatching. The shortest time taken for 100% of the eggs to hatch in this study suggests that the present method of hatching is faster than all previous methods used by different workers.

Ascaris lumbricoides and Ascaridia galli: biogenic amines in adults and developmental stages.

Ascaris lumbricoides var. hominis and Ascaridia galli contain 5-hydroxytryptamine, histamine, dopamine, and norepinephrine. The chick parasite showed lower levels of monoamines compared to human ascaris. Amine concentrations in females were higher than in males. In all specimens, 5-hydroxytryptamine was the highest while norepinephrine was found to be uniformly low. The female reproductive organ contained the maximum amount of dopamine while intestine was rich in histamine. A progressive increase in the concentrations of biogenic amines was noticed during development.

Nippostrongylus brasiliensis and Ascaridia galli: mitochondrial respiration in free-living and parasitic stages.

Aerobic respiratory pathways have been delineated and respiratory efficiency has been assessed in mitochondria isolated from embryonated eggs, infective larvae, and adult Nippostrongylus brasiliensis and Ascaridia galli. Mitochondrial respiration in free-living stages of N. brasiliensis is mediated mainly by a mammalian-like antimycin A- and cyanide-sensitive pathway; specific respiratory activity is high and oxidative phosphorylation efficient. In mitochondria of adult N. brasiliensis, antimycin A- and cyanide-sensitive respiration is decreased relative to respiration though an alternative pathway, and specific respiratory activity and mitochondrial efficiency are lower. Respiration in mitochondria from embryonated eggs and tissues of adult A. galli is comparable, and apparently mediated by an antimycin A- and cyanide-insensitive alternative respiratory pathway; no evidence for the presence of a mammalian-like respiratory pathway in embryonated eggs of A. galli was found. The results of this study are compared to mitochondrial respiration in eggs, larvae, and adult body wall muscle of Ascaris suum.

[Data on the biological cycle of the ascaridoid Ophidascaris trichuriformis, parasite of snakes]. / Données sur le cycle biologique de l'ascaridide Ophidascaris trichuriformis, parasite de serpents.

Ophidascaris trichuriformis is an ascaridoid commonly found in some species of Colubridae snakes. Similarly to what has been observed with several ascaridoid species, in O. trichuriformis the larvae, when still inside the egg-shells, reach the 3th snake stage around the 8th day of incubation in cultures kept at around 25 degrees C. Tentative assays to infect mice, snakes, toads, fish, and tadpoles of Hyla fuscovaria and Bufo sp. was made through the administration of embryonated eggs. Eggs didn't even eclode in mice, snakes or toads in such experiments; while in fish, although the eggs ecloded, its larvae were eliminated after the 4th day. In H. fuscovaria and Bufo sp. tadpoles, larvae were liberated from egg-shells and their development was observed up to the 36th day. They grew from the 6th hour to the above mentioned day, after which larvae were longer in Bufo sp. than in H. fuscovaria. By the 36th day distinction of sexes was possible through examination of genital primordia whose morphology and location differed in male and female larvae. From the 30th day on, following the recognizable beginning of metamorphosis in anuran amphibians and going up to the 65th day after experimental infection, an intense growth of the larvae infecting Bufo sp. was observed; the growth of such larvae was negligible between the 65th and the 85th days. Five snakes (Waglerophis merremii) were given per os larvae obtained from Bufo sp. after the 65th day of infection; these snakes died on the 53th, 54th, 60th, 110th and 116th days after their own infection, respectively, and in each of then 4th stage O. trichuriformis larvae were found in the stomach. The gastric location of such larvae in each snake was identical to the one in which adult worms of this same species are regularly found in natural infections: all of them had the middle part of the body embedded into the stomach wall while both anterior and posterior extremities kept free towards the lumen of the organ.

Experimental life cycle of Ascaridia columbae in intravenously infected pigeons, Columba livia.

The fate and routes of migration of infective larvae of Ascaridia columbae were studied in intravenously (i.v.) infected pigeons. Larvae were able to complete a trachael migration, and arrived at the small intestine where they established a patent infection. Granulomas were observed and histopathologically described in the lungs of i.v. infected birds.