Can an entomopathogenic nematode serve, as proxy for strongyles, in assessing the anthelmintic effects of phenolic compounds?

As gastro-intestinal nematodes (GINs) become increasingly resistant to chemical anthelmintics, and because consumers scrutinize chemical residues in animal products, the use of herbal anthelmintics and in particular, phenolic compounds, has become attractive. Most life stages of GINs cannot be grown in the lab as they are obligatory parasites, which limits our understanding of the effects of phenolic compounds on their parasitic stages of life. We hypothesized that a species phylogenetically close to GINs and grown in vitro, the insect-parasitic nematode Heterorhabditis bacteriophora (Rhabditida; Heterorhabditiade), when fed with Photorhabdus luminescens exposed to plant phenolics, can serve, as proxy for strongyles, in assessing the anthelmintic effects of phenolic compounds. We compared the development of H. bacteriophora infective juveniles (IJ) and the exsheathment rate of L3 larvae of the strongyle Teladorsagia circumcincta and Trichostrongylus colubriformis when exposed to catechin, rutin, chlorogenic and gallic acids, and myricetin. Gallic acid had the highest impact in terms of IJ mortality but the highest impairment of IJ development to adulthood was imposed by myricetin. The studied compounds were not lethal to GINs stricto sensu but we consider that the practical implications of total exsheathment inhibition and mortality on GIN populations are similar. Catechin and rutin had similar effects on rhabditid and strongyles: they imposed ca. 90% lethality of IJs at concentrations higher than 1200 ppm and the remaining live IJs did not develop further, and they also totally inhibited strongyle L3 exsheathment in a dose-response fashion. Gallic acid was 100% lethal to IJs exposed above 300 ppm and chlorogenic acid caused 87% mortality above 1200 ppm, with no development for the surviving IJs but for all lower concentrations, all the IJs developed to adult stages. Likewise, gallic and chlorogenic acids did not affect the exsheatment of GIN L3 larvae. Therefore, a discrepancy between the effects of gallic and chlorogenic acids on the development of rhabditid IJs and exsheathment of GIN L3 larvae was found only when they were exposed to high concentrations. A dose-response of IJ lethality to myricetin was found, with no IJ development between 150 and 2400 ppm; but contrary to the other compounds, myricetin also impaired IJ development of IJs above 10 ppm in a dose-response manner and showed dose-responses in the L3 exsheathment. Apart for the high rates of lethality imposed on IJs by gallic and chlorogenic acids at high concentration, these results suggest that H. bacteriophora fed P. luminescens exposed to phenolics shows potential to serve as model in studies of the anthelmintic effects of phenolics in GIN.

Phylogenetic analysis of Spirocerca lupi and Spirocerca vulpis reveal high genetic diversity and intra-individual variation.

BACKGROUND: Spirocerca lupi is a parasitic nematode of canids that can lead to a severe and potentially fatal disease. Recently, a new species, Spirocerca vulpis, was described from red foxes in Europe, suggesting a high genetic diversity of the Spirocerca spp. infecting canids. The genetic variation and phylogenetic relationships of S. lupi collected from naturally-infected domestic dogs from Australia, Hungary, Israel, Italy, India and South Africa, and S. vulpis from red foxes from Bosnia and Herzegovina, Italy and Spain, was studied using mitochondrial and rDNA markers. RESULTS: A high intra-individual variation was found in the first internal transcribed spacer (ITS1) locus in all Spirocerca spp., ranging between 0.37-2.84%, with up to six haplotypes per specimen. In addition, a combination of phylogenetic and haplotype analyses revealed a large variability between S. lupi specimens collected from different geographical locations using the ITS1 (0.37-9.33%) and the cytochrome c oxidase subunit 1 (cox1) gene (1.42-6.74%). This genetic diversity led to the identification of two S. lupi genotypes circulating among dogs (PTP support > 0.829), including genotype 1 found in S. lupi from Australia, India, Israel and South Africa, and genotype 2 represented by specimens from Hungary and Italy. These genotypes presented pairwise nucleotide distances of 0.14%, 8.06% and 6.48 ± 0.28% in the small rDNA subunit (18S), ITS1 and cox1 loci, respectively. Additionally, Nei's genetic distance in the ITS1 showed a further subdivision of genotype 1 worms into 1A (Israel and South Africa) and 1B (Australia and India). A morphological analysis of the anterior and posterior extremities of genotype 1 and genotype 2 worms using scanning electron microscopy did not show any differences between the specimens, contrary to the morphological differences between S. lupi and S. vulpis. CONCLUSIONS: These findings demonstrate the high genetic variability among Spirocerca spp. from different geographical locations, thereby expanding our understanding of the epidemiology, evolution and phylogenetic variability within the genus.

Effects of selected Palestinian plants on the in vitro exsheathment of the third stage larvae of gastrointestinal nematodes.

BACKGROUND: Gastrointestinal parasites are one of the main restrictions to small ruminant production. Their pathological importance is primarily related to the major production losses, in quantity or quality, induced by the direct action of worms. Control of these parasites is based exclusively on the frequent use of anthelmintic drugs. However, the resistance to anthelmintics in worm populations after commercialisation of chemical drugs is now widespread. Therefore, there is a need to find new natural resources to ensure sustainable and effective treatment and control of these parasites. The aim of this study was to evaluate the anthelmintic activity, as minimum inhibitory concentration (IC 50 mg/mL), of different plant extracts using larval exsheathment inhibition assay using a two-species but steady population of parasitic nematodes (ca. 20% Teladorsagia circumcinta and 80% Trichostrongylus colubriformis). RESULTS: The study showed that the ethanolic extracts of 22 out of the 48 plant extracts, obtained from 46 plant species, have an inhibitory effect >50% (at concentrations of 100 mg/mL) on the third stage larvae (L3) of the nematodes exhibited the strongest inhibition activity (94%) with IC 50 of 0.02 mg/mL, where other members of the Rhamnaceae family have shown to possess strong anthelmintic activity (70-89%). CONCLUSIONS: Plant extracts are potential rich resources of anthelmintics to combat helminthic diseases. Our results suggest that extracts from Rhamnus elaternus, Epilobium hirsutum, Leucaena leucocephala and Rhamnus palaestinus have promising anthelmintic activity, with potential applications in animal therapeutics and feed.

Detection and quantification of Spirocerca lupi by HRM qPCR in fecal samples from dogs with spirocercosis.

BACKGROUND: Spirocerca lupi, the dog oesophageal nematode, causes a potentially fatal disease in domestic dogs, and is currently clinically diagnosed by coproscopy and oesophagoscopy. To date, a single molecular method, a semi-nested PCR, targeting the cox1 gene, has been developed to aid in the diagnosis of spirocercosis. The present study describes three novel high-resolution melt (HRM) quantitative PCR (qPCR) assays targeting fragments of the ITS1, 18S and cytb loci of S. lupi. The performance of these molecular assays in feces was compared to fecal flotation and to the previously described cox1 gene semi-nested PCR in 18 fecal samples from dogs with clinical oesophageal spirocercosis diagnosed by oesophagoscopy. RESULTS: The HRM qPCR for ITS1 and 18S were both able to detect 0.2 S. lupi eggs per gram (epg), while the HRM qPCR for the cytb and the semi-nested PCR for the cox1 detected 6 epg and 526 epg, respectively. Spirocerca lupi was detected in 61.1%, 44.4%, 27.8%, 11.1% and 5.6% of the fecal samples of dogs diagnosed with spirocercosis by using the ITS1 and 18S HRM qPCR assays, fecal flotation, cytb HRM qPCR and cox1 semi-nested PCR, respectively. All dogs positive by fecal flotation were also positive by ITS1 and 18S HRM qPCRs. Quantification of S. lupi eggs was successfully achieved in the HRM qPCRs and compared to the fecal flotation with no significant difference in the calculated concentrations between the HRM qPCRs that detected the 18S and ITS1 loci and the fecal flotation. The HRM qPCR for the 18S cross-amplified DNA from Toxocara canis and Toxascaris leonina. In contrast, the HRM qPCR for ITS1 did not cross-amplify DNA from other canine gastrointestinal parasites. CONCLUSIONS: This study presents two new molecular assays with significantly increased sensitivity for confirming and quantifying fecal S. lupi eggs. Of these, the HRM qPCR for ITS1 showed the best performance in terms of the limit of detection and absence of cross-amplification with other parasites. These assays will be useful in detecting infection and for follow-up during therapy.

Influence of physical and chemical factors on the embryonation, hatching and infectivity of Spirocerca lupi.

Spirocerca lupi is a parasitic nematode which causes spirocercosis, a severe disease of dogs. Its life cycle involves dung beetles as intermediate hosts and canids as definitive hosts. The effect of different physical conditions and chemical factors on the embryonation and hatching of S. lupi eggs were investigated in this study in order to understand the triggers for progression in the early development of this parasite. Exposure to potassium dichromate significantly enhanced the embryonation of eggs compared to formaldehyde and controls (p<0.0001), reaching the maximum embryonation level of 83% within 2days of incubation. Hatching of eggs was significantly (p<0.05) enhanced in the presence of 2.5% trypsin, pH 6.0 and 8.0, a temperature of 26°C, 20% CO2 and mechanical force by stirring with 3-mm beads. Dissection of Onthophagus sellatus beetles 8h post-feeding with eggs showed that 13% of the ingested eggs hatched in the buccal cavity and the midgut. Finally, the pH range of the beetle's gut was 6.0-6.2 compared to 7.2±0.4 in dog feces suggesting that this pH change may induce hatching in the beetle. These findings contribute to the understanding of the early steps in the life cycle of S. lupi and may be used in the future to block the development of S. lupi and prevent dog infection and disease.

Ethanolic extracts of Inula viscosa, Salix alba and Quercus calliprinos, negatively affect the development of the entomopathogenic nematode, Heterorhabditis bacteriophora - A model to compare gastro-intestinal nematodes developmental effect.

Heterorhabditis bacteriophora can represent a model system for herbal medication against gastro-intestinal strongylid parasites in determining the recovery and development due to their unique parasitic infectious cycle. The fact that plant extracts impair nematode development is known but their differential impact on stages of the life cycle of H. bacteriophora has never been investigated. We examined the developmental stages resumed from eggs, young juveniles (J1-3), infective juveniles (IJs), young and adult hermaphrodites of H. bacteriophora upon exposure to crude ethanolic extracts of Inula viscosa, Salix alba, and Quercus calliprinos at concentrations of 600, 1200, and 2400ppm. Our results showed that plant extracts were highly toxic to the survival of the eggs and young juveniles J1 to J3 at all concentrations. The plant extracts inhibited their development and were associated with low reproduction parameters (i.e. fecundity and viability of eggs). The IJs, J4, young and developed hermaphrodites displayed concentration-dependent negative effect on development with less egg count, poor vulval muscle development, loss of egg laying capacity and progeny development by matricidal hatching. Plant extract of I. viscosa at low (600ppm) concentration did not impair vulval development. These results suggest that these plant extracts show potential for the control of parasitic rhabditids.

Molecular characterization of Trichinella species from wild animals in Israel.

Trichinellosis is a worldwide disease caused by nematode worms of the genus Trichinella, frequently diagnosed in Israel. However, the identity of the Israeli isolates have not been studied. Here we describe the molecular characterization of 58 isolates collected from jackals (Canis aureus), wild boar (Sus scrofa), foxes (Vulpes vulpes) and a wolf (Canis lupus) in central and northern Israel. Isolates were analyzed using the multiplex PCR analysis encompassing expansion segment V (ESV) and internal sequence 1 (ITS-1) markers, which identified 52 of the 58 samples. Out of the six unidentified samples, four were successfully identified using extended PCR assays for ESV and ITS-1, developed in this study. Our analysis identified 44 isolates as T. britovi, 8 as T. spiralis, four mixed infections, and two isolates were not identified. Clonal analysis of the ITS-1 sequences from six isolates confirmed the initial identification of four mixed infections. These results show that the prevalent species in Israel are T. britovi and T. spiralis, with nearly 7% (4 of 58) incidence of mixed infection.

Effects of tannin-rich host plants on the infection and establishment of the entomopathogenic nematode Heterorhabditis bacteriophora.

Parasitized animals can self-medicate. As ingested plant phenolics, mainly tannins, reduce strongyle nematode infections in mammalian herbivores. We investigated the effect of plant extracts known to be anthelmintic in vertebrate herbivores on the recovery of the parasitic entomopathogenic nematode Heterorhabditis bacteriophora infecting African cotton leafworm (Spodoptera littoralis). Nematode infective juveniles (IJs) were exposed to 0, 300, 900, 1200, 2400 ppm of Pistacia lentiscus L. (lentisk), Inula viscosa L. (strong-smelling inula), Quercus calliprinos Decne. (common oak) and Ceratonia siliqua L. (carob) extracts on growth medium (in vitro assay). In control treatments, 50-80% of IJs resumed development to J4, young and developed adult hermaphrodites, whereas all extracts, except for C. siliqua at 300 ppm, impaired IJ exsheathment and development. The highest concentration of I. viscosa extract (2400 ppm) had the strongest effect, killing 95% of exposed nematodes. Surviving nematodes did not recover, remaining at the IJ stage. Over the whole cycle, I. viscosa extract inhibited recovery to 25% or less, and did not allow full development to adulthood, whereas 65% of IJs in the control treatment recovered and resumed development, 12% reaching complete maturation within 72 h of incubation. When herbivorous S. littoralis larvae were fed with different plant extracts in vivo, I. viscosa had the strongest effect at concentrations above 300 ppm, with 90% of insect-invading IJs not developing to parasitic stages, whereas in the control treatment, 85% of IJs resumed development. Exposure to C. siliqua extract also inhibited exsheathment and development of 75% of the IJs. Half of those that resumed development reached full maturation. P. lentiscus and Q. calliprinos extracts also inhibited development of 50% IJs. Our results suggest that H. bacteriophora can be used to study herbal medication against parasites in animals.

A novel bacterial symbiont in the nematode Spirocerca lupi.

BACKGROUND: The parasitic nematode Spirocerca lupi (Spirurida: Thelaziidae), the canine esophageal worm, is the causative agent of spirocercosis, a disease causing morbidity and mortality in dogs. Spirocerca lupi has a complex life cycle, involving an obligatory coleopteran intermediate host (vector), an optional paratenic host, and a definitive canid host. The diagnosis of spirocercosis is challenging, especially in the early disease stages, when adult worms and clinical signs are absent. Thus, alternative approaches are needed to promote early diagnosis. The interaction between nematodes and their bacterial symbionts has recently become a focus of novel treatment regimens for other helminthic diseases. RESULTS: Using 16S rDNA-based molecular methods, here we found a novel bacterial symbiont in S. lupi that is closely related to Comamonas species (Brukholderiales: Comamonadaceae) of the beta-proteobacteria. Its DNA was detected in eggs, larvae and adult stages of S. lupi. Using fluorescent in situ hybridization technique, we localized Comamonas sp. to the gut epithelial cells of the nematode larvae. Specific PCR enabled the detection of this symbiont's DNA in blood obtained from dogs diagnosed with spirocercosis. CONCLUSIONS: The discovery of a new Comamonas sp. in S. lupi increase the complexity of the interactions among the organisms involved in this system, and may open innovative approaches for diagnosis and control of spirocercosis in dogs.

Characterization of Onthophagus sellatus as the major intermediate host of the dog esophageal worm Spirocerca lupi in Israel.

Spirocerca lupi is a parasitic nematode of dogs, that causes significant morbidity and mortality. Its intermediate hosts in Israel have never been described. The aim of this study was therefore to identify the intermediate hosts of S. lupi in Israel and to describe their abundance and annual infection rate with the nematode, in different microenvironments (i.e., the effects of irrigation and shade) in an endemic area. Dog dung pads were collected every 2 months from two different public parks for 1 year. Dung beetles were identified to the species level in infested dog feces and were examined for the presence of S. lupi larvae through dissection. The Scarabid beetle Onthophagus sellatus was the most abundant dung beetle species in dog dung pads and the only one infected with S. lupi larvae. The minimal period for development of the S. lupi L3 infective stage was 7 days. Significant differences were observed between the two different microenvironments and along the year. The highest risk for infection of dogs with the nematode was during the summer, in an irrigated, shady microenvironment.

The development of a molecular approach for coprodiagnosis of Toxoplasma gondii.

Copro-diagnostic methods for Toxoplasma gondii infected cats have been traditionally based on the identification of oocysts by light microscopy or by bioassays. The first method is not sensitive and also unable to differentiate between Toxoplasma oocysts from other coccidian parasites in cats, and the second is cumbersome, time consuming and expensive. We have adapted a polymerase chain reaction (PCR) method to detect T. gondii oocyst DNA in fecal samples. Oocysts were successfully disrupted by freeze thawing coupled with mechanical means, and DNA extraction was subsequently accomplished. The test, based on amplifying a 529 bp repeated sequence, proved sensitive for detecting 1-2 oocysts in 200 microg of stool sample. The test specificity was established by showing that DNA from other cat coccidia tested negative. Specificity was reconfirmed by Southern hybridization of the PCR products with a specific probe. Of 122 stool samples from Jerusalem cats surveyed for the presence of Toxoplasma oocysts, 11 were found positive by PCR while none was detected by microscopy.

Emergence of distinct genotypes of Cryptosporidium parvum in structured host populations.

Cryptosporidium parvum is an apicomplexan parasite that infects humans and ruminants. C. parvum isolated from cattle in northeastern Turkey and in Israel was genotyped using multiple polymorphic genetic markers, and the two populations were compared to assess the effect of cattle husbandry on the parasite's population structure. Dairy herds in Israel are permanently confined with essentially no opportunity for direct herd-to-herd transmission, whereas in Turkey there are more opportunities for transmission as animals range over wider areas and are frequently traded. A total of 76 C. parvum isolates from 16 locations in Israel and seven farms in the Kars region in northeastern Turkey were genotyped using 16 mini- and microsatellite markers. Significantly, in both countries distinct multilocus genotypes confined to individual farms were detected. The number of genotypes per farm was higher and mixed isolates were more frequent in Turkey than in Israel. As expected from the presence of distinct multilocus genotypes in individual herds, linkage disequilibrium among loci was detected in Israel. Together, these observations show that genetically distinct populations of C. parvum can emerge within a group of hosts in a relatively short time. This may explain the frequent detection of host-specific genotypes with unknown taxonomic status in surface water and the existence of geographically restricted C. hominis genotypes in humans.