Migratory pattern of zoonotic Toxocara cati and T. canis in experimentally infected pigs.

BACKGROUND: Over a billion people are infected with Toxocara canis or T. cati, the roundworms of dogs and cats. Historically, T. canis has been considered the main species responsible for human toxocarosis, but as serodiagnosis cannot discriminate between the two species, this remains unresolved. We used pigs as a relevant large animal model for human infection to assess the migratory pattern of T. cati and T. canis. METHODS: Pigs were inoculated with T. cati or T. canis eggs or PBS (negative controls) and necropsied 14 or 31 days later. Different organs and tissues were examined for parasites and pathological changes. RESULTS: Overall, the two parasite species had a similar migration pattern reaching multiple organs and tissues, including the mesenteric lymph nodes, liver, lungs, and diaphragm. We recovered larvae of both species in the brain, suggesting that T. cati also can cause neurological toxocarosis in humans. Both species induced systemic eosinophilia and histopathological changes in the lungs, livers, and mesenteric lymph nodes. CONCLUSION: This study emphasises the importance of T. cati as a zoonotic agent and the need to develop diagnostic methods that can differentiate between sources of infection in humans.

Evaluation of a novel multiplex qPCR method for rapid detection and quantification of pathogens associated with calf diarrhoea.

AIMS: Diarrhoea is a common health problem in calves and a main reason for use of antimicrobials. It is associated with several bacterial, viral and parasitic pathogens, most of which are commonly present in healthy animals. Methods, which quantify the causative agents, may therefore improve confidence in associating a pathogen to the disease. This study evaluated a novel commercially available, multiplex quantitative polymerase chain reaction (qPCR) assay (Enterit4Calves) for detection and quantification of pathogens associated with calf-diarrhoea. METHODS AND RESULTS: Performance of the method was first evaluated under laboratory conditions. Then it was compared with current routine methods for detection of pathogens in faecal samples from 65 calves with diarrhoea and in 30 spiked faecal samples. The qPCR efficiencies were between 84%-103% and detection limits of 100-1000 copies of nucleic acids per sample were observed. Correct identification was obtained on 42 strains of cultured target bacteria, with only one false positive reaction from 135 nontarget bacteria. Kappa values for agreement between the novel assay and current routine methods varied between 0.38 and 0.83. CONCLUSION: The novel qPCR method showed good performance under laboratory conditions and a fair to good agreement with current routine methods when used for testing of field samples. SIGNIFICANCE AND IMPACT OF STUDY: In addition to having fair to good detection abilities, the novel qPCR method allowed quantification of pathogens. In the future, use of quantification may improve diagnosis and hence treatment of calf diarrhoea.

Extracts of pine bark (Pinus sylvestris) inhibit Cryptosporidium parvum growth in cell culture.

The widespread apicomplexan parasite Cryptosporidium parvum is responsible for severe gastrointestinal disease in humans and animals. The treatment options are limited, and the efficacy of available drugs is low. Bark contains condensed tannins (CT), which are bioactive compounds previously shown to inhibit parasite development. Here, we examined the anti-cryptosporidial properties of bark extract of Scots pine (Pinus sylvestris) against C. parvum by means of an in vitro growth inhibition test. We hypothesised that bark extracts would have dose-dependent inhibitory effects on the development of C. parvum in cell culture.Bark extracts from Scots pine extracted with acetone, methanol, and water as solvents were investigated using human colorectal adenocarcinoma cells infected with C. parvum. Oocysts were inoculated onto the cell monolayer and bark extract was added at seven different concentrations. Parasite growth inhibition was quantified by qPCR.The acetone and methanol extracts demonstrated a sigmoid dose-dependent inhibition of C. parvum. The IC50 values were 244.6 and 279.1 µg dry matter extract/mL, and 25.4 and 24.1 µg CT/mL, for acetone and methanol extracts, respectively. The IC50 for both extracts were similar, both with regard to the dry matter concentration of each extract and to CT concentrations.Given the limited treatment options available for Cryptosporidium spp., the evidence generated in our study encourages further investigation into the in vitro and in vivo effects of pine bark extracts against C. parvum.

Assessment of the societal cost of Taenia solium in Angónia district, Mozambique.

BACKGROUND: The zoonotic parasite Taenia solium is endemic in Angónia district, Tete province, Mozambique, though the burden of the disease complex is unknown. METHODS: As part of two cross-sectional studies on human and porcine cysticercosis in the area, unique epidemiological and cost data were collected in Angónia district, Mozambique in 2007. These data provided the basis for the assessment of the societal cost of T. solium in the district, which estimates the impact of the disease on human and pig populations and includes both health and economic approaches in the analysis. RESULTS: Approximately 0.7% (95% Uncertainty Interval (UI), 0.4-0.9) and 0.4% (95% UI, 0.2-0.6) of the total population in the district was estimated to suffer from neurocysticercosis (NCC)-associated epilepsy and headache. The estimated average number of disability-adjusted life years (DALYs) due to NCC-associated epilepsy and headache was 6 (95% UI, 4-8) per thousand persons per year. The total annual costs due to T. solium cysticercosis were estimated at 90,000 USD (95% UI, 39,483-201,463) of which 72% (95% UI, 45-91) were costs linked to human cysticercosis and 28% (95% UI, 9.5-55) to pig production losses. The annual economic burden per NCC-associated epilepsy case in the district amounted to 33 USD (95% UI, 10-76). CONCLUSIONS: In this highly endemic area of Mozambique a large number of individuals suffer from symptoms associated with NCC. Healthy years of life are lost and people are left living with disabilities. Infected pork poses a serious risk to the community and affects the economy of smallholder farmers. Cost for treatment and hospitalization of patients with NCC-associated epilepsy, and lack of productivity and inability of suffering patients to work, further hinder socioeconomic development. Feasible solutions framed within a country specific algorithm and stepwise approaches are needed to control the parasite in the country.

In vitro hatching of Trichuris suis eggs.

Eggs of the pig whipworm, Trichuris suis ova (TSO), are currently tested in human clinical trials for their potential immunomodulatory capacity. The biological potency of TSO (egg viability and infectivity) is traditionally assessed in Göttingen minipigs as the establishment of intestinal larvae after inoculation with a known number of eggs. To minimize testing in animal models, development of an in vitro egg hatching assay is proposed as a reliable, cost-effective, and a faster alternative to test the egg viability. The present study aimed to investigate the influence of different chemical, physical, and biological factors on egg hatching. Thus, in a series of experiments and in different combinations, the eggs were stimulated with glass beads, artificial gastric juice, bile salt and trypsin solution, fermentation gut medium, or stimulated with mucosal scrapings from the ileum and the large intestine of the infected and uninfected Göttingen minipig. Mechanical stimulation with glass beads presented a simple and reproducible method for egg hatching. However, incubation of eggs with mucosal scrapings from the ileum, caecum, and colon for 24 h at 38 °C significantly increased hatching.

The jejunal cellular responses in chickens infected with a single dose of Ascaridia galli eggs.

This histopathological study was carried out in order to investigate the cellular response in the jejunum to Ascaridia galli during the first 7 weeks of infection. Fourty-two ISA Brown chickens (7 weeks old) were infected orally with 500 embryonated A. galli eggs each while 28 chickens were left as uninfected controls. Six infected and four control chickens were necropsied at each time point 3, 7, 10, 14, 21, 28 and 42 days post-infection (dpi). Samples for histopathology were taken from three sites of the jejunoileum. Significantly higher eosinophil counts were seen in infected chickens compared to uninfected at 3, 7, 10, 14 and 28 dpi (P < 0.01). In both groups, the initial number of mast cells was high, but this high level of mast cells remained for a longer period in the infected group compared to the control group. Significantly higher counts were thus found in the infected group at 21 (P < 0.001), 28 (P < 0.01) and 42 dpi (P < 0.05). A. galli infection induced changes in the mucosal thickness as reduced villi length at 7, 10, 14, 21 and 28 dpi and in the degree of general cellular infiltration in the lamina propria of the mucosal layer. No adult worms were seen during the experiment; therefore, A. galli larvae have elicited a moderate cellular response in the lamina propria, mainly consisting of eosinophils in the early phase and later of mast cells.

Productivity and parasitic infections of pigs kept under different management systems by smallholder farmers in Mbeya and Mbozi districts, Tanzania.

An on farm experiment was carried out to assess the effects of production systems on the performance of local pigs kept by smallholder farmers. Six villages from Mbeya and Mbozi districts, Tanzania were purposely selected based on the prominent pig production systems: free range, semi-confinement and total confinement. Fifteen pig keeping households were randomly selected from each village to participate in the study. A participatory rural appraisal and structured questionnaire were used for collecting information from the households on pig production and reproduction performance. In addition, a total of 180 weaner pigs, 2-3 months old, were purchased and randomly allocated to the 90 participating households. The pigs were subjected to three production systems: free range (M1), confinement with local diet (M2) and confinement with a compounded diet and anthelmintic treatment (M3). The anthelmintic treatment (piperazine citrate) was administered at 1 g per kg body weight. Faecal and blood samples were collected at month three of the experiment to assess the burden of intestinal helminths and sero-prevalence of Taenia solium cysticercosis, respectively. Sows kept under free range system were reported to have smaller litter size both at farrowing and at weaning compared to those kept under confinement. The experiment showed pigs under M3 had higher (P < 0.05) liveweight gains (136 g/day) compared to pigs in M2 (73 g/day) and M1 (68 g/day). In addition, pigs in M3 had higher body length and heart girth size with the feed to gain ratio of 8.5. Free range pigs tended to have lower faecal egg counts for most worm species compared to permanently confined pigs. Sero-prevalence of Taenia solium cysticercosis was 26%, with village prevalence ranging from 8 to 52%. Although pigs kept in M3 performed better than the rest, the compounded feed was too expensive for the farmers to afford. Locally available feed types combined with vitamin and mineral supplements may be a more sustainable option.

Development and survival of Ascaris suum eggs in deep litter of pigs.

SUMMARY Indoor transmission of Ascaris suum partly depends on the physico-chemical conditions in bedding material. Temperature, pH, aqueous ammonia, moisture, occurrence and development of A. suum eggs were therefore compared in different areas (resting, intermediate and latrine) of two deep litter pens on an organic farm in four seasons. There was some variation, but mean ammonia levels were generally very low (1·0-2·6 mm) and pH levels were moderate (8·04-8·88) in all three areas. Relatively, resting areas were characterized by overall moderate moisture (36%) and moderately high temperature (35·7 °C) levels. The area contained few eggs (50 eggs g-1 DM) of which 17% were viable, and though only 4% were larvated and 0·7% appeared infective, it was more than in the other areas. Intermediate areas had moderate moisture (43%) and high temperature (43·6 °C) levels. There were many eggs (523 eggs g-1 DM), but overall viability was very low (5%) and few eggs were larvated (0·004%) or even infective (0·002%). Latrines typically had high moisture (79%) and moderate temperature (30 °C) levels. The concentration of eggs was very high (1444 egg g-1 DM) and though 32% were viable, none had developed larval stages. The large majority of A. suum eggs appear to die and only few become infective while in the deep litter. However, a large fraction of eggs may remain viable for some time and could thus contaminate agricultural land and develop to infectivity, if the manure is not composted appropriately.

Viability of Ascaris suum eggs in stored raw and separated liquid slurry.

Separation of pig slurry into solid and liquid fractions is gaining importance as a way to manage increasing volumes of slurry. In contrast to solid manure and slurry, little is known about pathogen survival in separated liquid slurry. The viability of Ascaris suum eggs, a conservative indicator of fecal pollution, and its association with ammonia was investigated in separated liquid slurry in comparison with raw slurry. For this purpose nylon bags with 6000 eggs each were placed in 1 litre bottles containing one of the two fractions for 308 days at 5 °C or 25 °C. Initial analysis of helminth eggs in the separated liquid slurry revealed 47 Ascaris eggs per gramme. At 25 °C, egg viability declined to zero with a similar trend in both raw slurry and the separated liquid slurry by day 308, a time when at 5 °C 88% and 42% of the eggs were still viable in separated liquid slurry and raw slurry, respectively. The poorer survival at 25 °C was correlated with high ammonia contents in the range of 7.9-22.4 mM in raw slurry and 7.3-23.2 mM in liquid slurry compared to 3.2-9.5 mM in raw slurry and 2.6-9.5 mM in liquid slurry stored at 5 °C. The study demonstrates that at 5 °C, A. suum eggs have a higher viability in separated liquid slurry as compared to raw slurry. The hygiene aspect of this needs to be further investigated when separated liquid slurry is used to fertilize pastures or crops.

Population dynamics of Ascaridia galli following single infection in young chickens.

The population dynamics of Ascaridia galli was studied in 70 ISA Brown layer pullets, 42 of them were each experimentally infected with 500 embryonated A. galli eggs and 28 chickens were kept as uninfected controls. Six chickens from the infected group and 4 from the control group were necropsied at 3, 7, 10, 14, 21, 28 and 42 days post-infection (d.p.i.). The mean worm recovery varied from 11-20% of the infection dose with the highest recovery at 3 d.p.i. and the lowest at 21 and 42 d.p.i. (P < 0·05). More larvae were recovered from the intestinal wall than from the content (P < 0·0001) and intestinal content larvae were longer than those from the wall (mean length 1·6 and 1 mm, respectively, P < 0·0001). Although larvae were growing over time, a population of small-sized larvae (length < 1 mm) was recovered at all d.p.i. During the first week of infection most of the larvae were located in the anterior half of the jejunoileum but they moved posteriorly with the age of infection. Thus, a subpopulation of larvae mainly in the lumen grew with time while another subpopulation remained small and associated with the mucosa. During the infection both subpopulations moved to a more posterior localization in the gastrointestinal (GI) tract.

Assessing welfare risks in unowned unsocialised domestic cats in Denmark based on associations with low body condition score.

BACKGROUND: Populations of unowned unsocialised cats are present worldwide. Generally, there is concern about their welfare. Low body condition score (BCS) is a potentially relevant indicator that is relatively easy to assess: emaciated cats are likely to have welfare problems while thin cats may be at risk of becoming emaciated. The objective of this study was to assess the association of low BSC with a selection of factors relating to the host, disease, and infection in unowned unsocialised domestic cats. We necropsied 598 euthanised unowned unsocialised cats from Denmark. We recorded each cat's age-group, sex, and neuter status, together with its pregnancy status, the season and location of trapping, as well as gross lesions at necropsy. We also tested for feline immunodeficiency virus and feline leukaemia virus, recorded presence of ectoparasites, and a subsample of the cats were also tested for endoparasites. Cats with no or sparse adipose deposits were categorised as having low BCS, and logistic regression was used to determine the factors associated with low BCS. RESULTS: Of the cats, 11.4% had low BCS. Season, age-group and sex were associated with low BCS and confounded potential associations with other variables. Intact adult males and females in spring and early summer were at highest risk of low BCS. When these factors were taken into account, cats with biting lice had 2.8 (95% confidence interval (CI) 1.4-5.4) times higher odds of low BCS, and cats with gastro-intestinal findings (i.e., enlarged mesenteric lymph nodes, abdominal hernia, diarrhoea, obstructive foreign bodies, or diaphragmatic hernia) had 50 (95% CI 10-417) times higher odds of low BCS, than cats with no such findings. Cats with low BCS were primarily adult intact cats with tooth lesions and skin lesions, ear mite infection, and positive test result for feline immunodeficiency virus. CONCLUSIONS: The results highlight associations that can be used to define a risk profile: low BCS, notably in summer-autumn, in an unowned unsocialised cat was associated with underlying, less visible problems. Thus, low BCS can be more than a step towards being emaciated; it can also be an indicator of other underlying welfare problems.

Condensed tannins, novel compounds and sources of variation determine the antiparasitic activity of Nordic conifer bark against gastrointestinal nematodes.

The antiparasitic potential of plants could offer a vital solution to alleviating the costs of gastrointestinal nematode (GIN) infections in ruminant production globally. Leveraging known bioactive molecules, however, is complex, where plant species, extraction processes and seasonality impact bioavailability and efficacy. This study assessed the impact of a comprehensive set of factors on the antiparasitic activity of Norwegian conifers to identify bark compounds specific against GIN. Antiparasitic activity was determined using in vitro assays targeting morphologically distinct life stages of ovine GIN: the egg hatch assay and larval motility assay. In depth characterisation of the chemical composition of the bark extracts was carried out using chromatographic separation, UV-absorbance, and molecular mass profiles to identify compounds implicated in the activity. Three key findings emerged: (1) the activity of bark extracts varied markedly from 0 to 100% antiparasitic efficacy, owing to tree species, extraction solvent and seasonality; (2) the GIN exhibited species-and stage-specific susceptibility to the bark extracts; (3) the presence of condensed tannins, amongst other compounds, was associated with anthelmintic activity. These findings add new insights into urgently needed alternative parasite control strategies in livestock.

Dietary seaweed (Saccharina latissima) supplementation in pigs induces localized immunomodulatory effects and minor gut microbiota changes during intestinal helminth infection.

Brown seaweeds have a rich bioactive content known to modulate biological processes, including the mucosal immune response and microbiota function, and may therefore have the potential to control enteric pathogens. Here, we tested if dietary seaweed (Saccharina latissima) supplementation could modulate pig gut health with a specific focus on parasitic helminth burdens, gut microbiota composition, and host immune response during a five week feeding period in pigs co-infected with the helminths Ascaris suum and Oesophagostomum dentatum. We found that inclusion of fermented S. latissima (Fer-SL) at 8% of the diet increased gut microbiota α-diversity with higher relative abundances of Firmicutes, Tenericutes, Verrucomicrobia, Spirochaetes and Elusimicrobia, and lower abundance of Prevotella copri. In the absence of helminth infection, transcription of immune-related genes in the intestine was only moderately influenced by dietary seaweed. However, Fer-SL modulated the transcriptional response to infection in a site-specific manner in the gut, with an attenuation of infection-induced gene expression in the jejunum and an amplification of gene expression in the colon. Effects on systemic immune parameters (e.g. blood lymphocyte populations) were limited, indicating the effects of Fer-SL were mainly localized to the intestinal tissues. Despite previously documented in vitro anti-parasitic activity against pig helminths, Fer-SL inclusion did not significantly affect parasite egg excretion or worm establishment. Collectively, our results show that although Fer-SL inclusion did not reduce parasite burdens, it may modify the gut environment during enteric parasite infection, which encourages continued investigations into the use of seaweeds or related products as novel tools to improve gut health.

The role of cooking practices in the transmission of the foodborne parasite Taenia solium: A qualitative study in an endemic area of Southern Tanzania.

The pork tapeworm Taenia solium is a zoonotic food-borne parasite endemic in many developing countries causing human cysticercosis and taeniosis as well as porcine cysticercosis. It mainly affects the health of rural smallholder pig farmers and their communities, resulting in lower health status, reduced pork quality, and economic loss due to condemnation of pigs or low pricing of pork. This qualitative study aimed to identify key food related practices linked to consumption of pork at village level, of importance for transmission of taeniosis. We used an interpretivist-constructivist paradigm in a multiple case study of exploratory qualitative research design. Data was acquired through guided and probing interviews with 64 pork cooks, and 14 direct observations in four villages in a T. solium endemic area of Mbeya Region in the Southern Highlands of Tanzania. The study showed that the informants were members of communities of practice through their pork cooking practices, one community of practice for the restaurant cooks and one for the home cooks, learning, sharing, and distributing their cooking skills. Furthermore, the analysis showed that the pork cooks generally had some awareness of there being something undesirable in raw pork, but they had very diverse understandings of what it was, or of its potential harm. Major potential transmission points were identified in restaurants and in home kitchens. It appears that the pork cooks act according to socio-cultural and economic factors guiding them in their actions, including pressure from customers in restaurants, the family values of tradition in the home kitchens, and the culturally guided risk perception and appraisal. These practices might generate potential transmission points. Future research on interventions aimed at preventing the spread of T. solium taeniosis should recognise the importance of tradition and culture in risky food practices.

Administration of spruce bark (Picea abies) extracts in young lambs exhibits anticoccidial effects but reduces milk intake and body weight gain.

BACKGROUND: Eimeria spp. are widespread apicomplexan parasites known to cause coccidiosis in livestock, resulting in reduced animal welfare and productivity, particularly in sheep. The treatment options are limited, and there is an emerging development of resistance against registered pharmaceuticals. Spruce bark is rich in plant secondary metabolites (PSM), such as condensed tannins, which are bioactive compounds previously shown to have antiparasitic activity. Here, we examined the anticoccidial properties of bark extract of Norway spruce (Picea abies) against a field isolate of ovine Eimeria spp. by treating Eimeria-infected pre-ruminant lambs with water-extracted bark daily for 12 days. We hypothesised that the bark extract would reduce the faecal oocyst excretion and, consequently, the severity of diarrhoea. RESULTS: Oral administration of spruce bark extract significantly reduced the excretion of Eimeria oocysts in milk-fed lambs post treatment till the end of the trial 22 days post infection. This difference in oocyst excretion between the treated and the untreated infected animals increased with time. Compared to the untreated and the sham-infected control group, the group treated with bark extract had softer faeces and reduced milk intake during the treatment period. After discontinuing the treatment, the treated animals got a more solid and formed faeces compared to that of the untreated control group, and the milk intake increased to the level of the sham-infected, untreated control group. The bark extract treated animals had a lower body weight and a lower mean daily body weight gain throughout the whole duration of the experiment. CONCLUSIONS: Bark extract from Norway spruce showed marked anticoccidial properties by reducing the faecal oocyst count and associated diarrhoea in young lambs. Simultaneously we experienced detrimental effects of the treatment, displayed as reduced feed intake and daily body weight gain. Therefore, we suggest conducting similar studies with lower bark extract dosage to explore the possibilities of a better trade-off to reduce the negative impact while maintaining the antiparasitic effect.

Anthelmintic resistance in small ruminants in the Nordic-Baltic region.

Gastrointestinal nematodes (GIN) in small ruminants result in production losses, and consequently economic losses, and are an animal welfare problem in most countries in the Nordic-Baltic region. Intensive use of anthelmintics to control helminth infections has led to anthelmintic resistance (AR), which has become a major issue in many European countries. Several studies have been performed in countries in the Nordic-Baltic region (e.g. Denmark, Sweden, Norway and Lithuania) showing increasing/emerging levels of AR. The aim of this paper is to provide an overview of the problem of AR on sheep and goat farms in the Nordic-Baltic region. This region has a limited number of registered anthelmintics. However, researchers in this area have discovered some surprising findings, such as ivermectin (IVM) resistance on a farm that had never used IVM. In Sweden there is evidence of macrocyclic lactone (ML)-resistant Haemonchus contortus being introduced with sheep imported from the Netherlands. As elsewhere in the world, the livestock trade appears to be contributing to the spread of AR in the region and isolated cases of multidrug-resistant cases have also been reported. This is surprising given that the frequency of treatments here is much lower than in other countries where sheep production is economically more important. The prevailing nematodes are Haemonchus, Teledorsagia and Trichostrongylus, while on some farms Haemonchus is dominant and clinical haemonchosis has increasingly been observed in recent decades. The reasons for this are unclear, but are probably related to this parasite's propensity to rapidly develop drug resistance and a general lack of awareness of the problem, possibly in combination with global warming and the increased livestock trade within the EU. In addition, domestic interactions through contacts with wildlife ruminants, alpacas may also be a contributing factor for transmission of AR.

Bio-Guided Fractionation and Molecular Networking Reveal Fatty Acids to Be Principal Anti-Parasitic Compounds in Nordic Seaweeds.

Widespread use of antimicrobial drugs has led to high levels of drug-resistance in pathogen populations and a need for novel sources of anti-bacterial and anti-parasitic compounds. Macroalgae (seaweed) are potentially a rich source of bioactive compounds, and several species have traditionally been used as vermifuges. Here, we investigated the anti-parasitic properties of four common cold-water Nordic seaweeds; Palmaria palmata (Rhodophyta), Laminaria digitata, Saccharina latissima and Ascophyllum nodosum (Ochrophyta, Phaeophyceae). Screening of organic extracts against helminths of swine (Ascaris suum) and sheep (Teladorsagia circumcincta) revealed that S. latissima and L. digitata had particularly high biological activity. A combination of molecular networking and bio-guided fractionation led to the isolation of six compounds from extracts of these two species identified in both fermented and non-fermented samples. The six isolated compounds were tentatively identified by using MS-FINDER as five fatty acids and one monoglyceride: Stearidonic acid (1), Eicosapentaenoic acid (2), Alpha-Linolenic acid (3), Docosahexaenoic acid (4), Arachidonic acid (5), and Monoacylglycerol (MG 20:5) (6). Individual compounds showed only modest activity against A. suum, but a clear synergistic effect was apparent when selected compounds were tested in combination. Collectively, our data reveal that fatty acids may have a previously unappreciated role as natural anti-parasitic compounds, which suggests that seaweed products may represent a viable option for control of intestinal helminth infections.

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 microm 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.

Mebendazole treatment persistently alters the size profile and morphology of Trichuris trichiura eggs.

The soil transmitted whipworm, Trichuris trichiura affects about half a billion people globally. Diagnosis is based on identification of characteristic lemon-shaped eggs in stool samples. Occasionally large Trichuris eggs have been reported and have been ascribed to variation within T. trichiura or zoonotic infection with T. vulpis from dogs. We observed that the egg size profile changed markedly after anthelmintic treatment, and remained so, in a human individual self-infected with T. trichiura. The large eggs were detected with two standard diagnostic methods, Kato-Katz thick smear and salt-flotation (McMaster) method. It is therefore important to bear in mind that the morphology of parasite eggs may vary, e.g. in response to previous drug treatment. Large Trichuris eggs in human stool samples should not be diagnosed as T. vulpis infection without confirmation by other means.

Evidence for mitochondrial pseudogenes (numts) as a source of contamination in the phylogeny of human whipworms.

Trichuris trichiura and T. suis are whipworms of humans and pigs, respectively, but it has recently been suggested that humans may be infected with multiple genotypes or species of Trichuris and cross-infection with Trichuris of pig origin has also been reported. In addition, the species status of Trichuris in non-human primates is unsettled and it is unknown how many whipworm species we share with other primates. Herein, we inferred the phylogeny of Trichuris collected from human, baboon and pig based on nuclear (18S and beta-tubulin) and mitochondrial (cox1) genes and evaluated the use of three PCR linked restriction fragment length polymorphism (PCR-RFLP) to identify worms. We found that all baboon worms clustered with human worms and that all these primate worms are different from T. suis. In general, there was an agreement between the phylogeny established based on the nuclear and mtDNA genes. However, we found evidence for non-targeted cox1 gene amplification for a subset of the human worms and suggest the presence of mitochondrial pseudogenes (numts) of pig cox1 gene in the human Trichuris genome. In conclusion, phylogenetic characterization of human whipworm based on the cox1 gene alone may be problematic without suitable preceded measures to avoid the numts amplification.