Effects of Chronic Exposure to Molybdate and Tetrathiomolybdate Supplementation of Water on Immature Rats Acutely Infected with Nippostrongylus brasiliensis. 1. Effects on Outcome of Infection and Host Health.

Low molybdate (MoO4) exposure via drinking water in mature rats infected with Nippostrongylus brasiliensis raised liver and plasma copper (Cu) concentrations. The possibility that anthelmintic effects were attributable to conversion of MoO4 to tetrathiomolybdate (MoS4) in a non-ruminant species was investigated by giving three groups of 18 immature rats drinking water containing 70 mg Mo l-1 as MoO4 (group A), 5 mg Mo l-1 as MoS4 (group B) or no supplement (group C), while receiving a commercial cubed diet. After 41 days, 12 rats from each group were inoculated subcutaneously with 2,000 L3-stage N. brasiliensis larvae. Subgroups were killed 7, 8 or 9 days post infection (dpi), when adult worms are normally expelled, and enzyme markers for the inflammatory response to infection were measured in plasma or liver. Male rats given MoS4 prior to infection grew more slowly than those given MoO4. Eight dpi, females given MoS4 had lost more bodyweight than those in group C, while those given MoO4 had gained weight. Mean worm counts at 7 dpi were 160, 65 and 250 ± 30.6 (SE), respectively, in groups C, A and B, and differed significantly from each other (P <0.05) but only rats given MoO4 remained infected 9 dpi (mean worm count 52 ± 16.4): Faecal egg counts followed a broadly similar pattern. Both Mo sources pre-empted increases in liver and duodenal superoxide dismutase activity, induced by infection 7 and 9 dpi, respectively, in group C and enlarged the femur: neither source prevented hypertrophy of the small intestine and a rise in serum mast cell protease concentration caused by infection. Since data for plasma Cu concentration and caeruloplasmin oxidase activity, reported separately, indicated MoO4 was thiolated in vivo, differences between Mo sources may be attributable to differences in the degree of thiolation, extent of thiomolybdate exposure and rates of thiomolybdate degradation at critical times in host or parasite development.

Dietary protein supplementation results in molecular and cellular changes related to T helper type 2 immunity in the lung and small intestine in lactating rats re-infected with Nippostrongylus brasiliensis.

Acquired immunity to gastrointestinal nematodes reduces during late pregnancy and lactation which is known as periparturient relaxation of immunity (PPRI). Protein supplementation reduces the degree of PPRI in a rat model re-infected with Nippostrongylus brasiliensis, but the underlying molecular mechanisms have yet to be elucidated. Here, we hypothesized that protein supplementation will enhance T helper type 2 immunity (Th2) in the lung and small intestine. Nulliparous Sprague-Dawley rats were given a primary infection of N. brasiliensis prior to mating and restrictedly fed diets with either low protein (LP) or high protein (HP) during pregnancy and lactation. Dams were secondary infected with N. brasiliensis on day 2 post-parturition, and histology and gene expression were analysed for tissue samples collected at days 5, 8 and 11. Genes related to Th2 immunity in the lung, Retnla, Il13 and Mmp12, and in the intestine, Retnlb, were upregulated in HP dams compared to LP dams, which indicates the effect of dietary protein on Th2 immunity. HP dams also had increased splenic CD68+ macrophage populations compared to LP dams following secondary infection, suggesting enhanced immunity at a cellular level. Our data assist to define strategic utilization of nutrient supply in mammals undergoing reproductive and lactational efforts.

The phytonutrient cinnamaldehyde limits intestinal inflammation and enteric parasite infection.

Phytonutrients such as cinnamaldehyde (CA) have been studied for their effects on metabolic diseases, but their influence on mucosal inflammation and immunity to enteric infection are not well documented. Here, we show that consumption of CA in mice significantly down-regulates transcriptional pathways connected to inflammation in the small intestine, and alters T-cell populations in mesenteric lymph nodes. During infection with the enteric helminth Heligomosomoides polygyrus, CA treatment attenuated infection-induced changes in biological pathways connected to cell cycle and mitotic activity, and tended to reduce worm burdens. Mechanistically, CA did not appear to exert activity through a prebiotic effect, as CA treatment did not significantly change the composition of the gut microbiota. Instead, in vitro experiments showed that CA directly induced xenobiotic metabolizing pathways in intestinal epithelial cells and suppressed endotoxin-induced inflammatory responses in macrophages. Collectively, our results show that CA down-regulates inflammatory pathways in the intestinal mucosa and can limit the pathological response to enteric infection. These properties appear to be largely independent of the gut microbiota, and instead connected to the ability of CA to induce antioxidant pathways in intestinal cells. Our results encourage further investigation into the use of CA and related phytonutrients as functional food components to promote intestinal health in humans and animals.

Hydro-Ethanolic Extract of Mentha pulegium Exhibit Anthelmintic and Antioxidant Proprieties In Vitro and In Vivo.

INTRODUCTION: During recent decades, the emergence of chemoresistance among synthetic anthelmintic drugs has increased the interest in screening novel natural anthelmintic compounds derived from plants. The current study is aimed to determine the chemical profile, anthelmintic and antioxidant properties of Mentha pulegium hydro-ethanolic extract. MATERIALS AND METHODS: Two tests were used to assess the in vitro anthelmintic activity of the hydro-ethanolic extract of M. pulegium against Haemonchus contortus; egg hatch assay (EHA) and adult worm motility (AWM) assay. M. pulegium extracts at the doses of 500, 1000, 2000 and 4000 mg/kg were evaluated in vivo in mice infected with Heligmosomoides polygyrus. The anthelmintic efficacy was monitored using faecal egg count reduction (FECR) and total worm count reduction (TWCR). The antioxidant activity of M. pulegium extract was evaluated by testing the total antioxidant capacity and the DPPH free radical-scavenging ability. RESULTS: Chromatographic characterization of M. pulegium composition using RP-HPLC revealed the presence of phenolic acids such as syringic acid, ferulic acid and the presence of flavonoid compounds, such as isorhamnetin-3-O-glucoside and kaempferol-3-O-rutinoside. We observed 91.58% inhibition in the EHA at 8 mg/mL after 48 h of incubation (IC50=1.82 mg/mL). In the AWM assay, M. pulegium extract achieved 65.2% inhibition at 8 mg/mL after 8 h. The highest dose (4000 mg/kg) showed a significant nematicidal effect 7 days post-treatment by inducing 60.39% FECR and 71.6% TWCR. We also report strong in vivo antioxidant capacity of the extract, as revealed by a significant increase of the enzymatic activities of catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx) enzymes in mice infected with H. polygyrus. CONCLUSION: Together, the results in this paper suggest that M. pulegium possesses anthelmintic properties and could be a potential source of novel compounds for the control of helminth parasites as well as its associated oxidative damage.

Anthelmintic activity of crude powder and crude aqueous extract of Trachyspermum ammi on gastrointestinal nematodes in donkey (Equus asinus): An in vivo study.

ETHNOPHARMACOLOGICAL RELEVANCE: Trachyspermum ammi has been used traditionally as a popular ethnobotanical plant in human and animal parasitic infestations. Few scientific studies have been conducted on in vitro anthelmintic activity of T. ammi against various helminths and there is no study on its in vivo/in vitro anthelmintic properties against equine helminths. AIM OF THE STUDY: The present study aimed to evaluate in vivo anthelmintic activity of crude powder (CP) and crude aqueous extract (CAE) of T. ammi seeds against gastrointestinal nematodes in the donkey. MATERIALS AND METHODS: Thirty donkeys, naturally and severely infected with Strongyle-type nematodes, were randomly divided into six groups and administered orally with graded doses of CP and CAE at single dose as follow: Two groups were treated with 1 and 3 gkg-1 body weight of CP, two groups were treated with CAE at the equivalent dose rate 1 and 3 gkg-1 bw of CP and one group was treated with 200 µgkg-1 bw of Ivermectin 0.2% (Positive Control). One group received no medication (Negative Control). Efficacy was determined by faecal egg count reduction (FECR) test. The faecal samples were taken from the donkeys on days 7, 14 and 28 post-treatments (PT) and the egg per gram (EPG) of faeces was determined for each animal. FECR for donkeys treated with CP, CAE, and Ivermectin 0.2% was calculated on days 7, 14, and 28 PT. In the present study, CAE was analyzed using gas chromatography-mass spectrometry (GC-MS) to determine CAE chemical components. High-performance liquid chromatography (HPLC) using a marker (thymol) was applied to confirm the existence of thymol as an active principle. RESULTS: Based on the FECR findings, the maximum FECR (71.2%) was observed in the group treated with CAE3 on day 14 PT, while it was 66.2% for CP3 on the same day. The effects of different treatments (P < 0.001), time (P < 0.001) and their interactions (P < 0.05) on EPG showed a significant difference. A dose-dependent response in the anthelmintic activity of CP and CAE was observed. GC-MS analysis indicated that thymol was the major component of CAE (Area = 45.01%) with the respective peak at retention times (Rt) 10.41 min. On HPLC analysis of CAE, thymol was found to be the major constituent of CAE (Area = 58.76%) with the same Rt at 13.233 min. CONCLUSION: The present study indicated that both CP and CAE of T. ammi had the dose-dependent anthelmintic effects. Due to the development of drug resistance against synthetic anti-helminthics, T. ammi seems to be a promising alternative for the treatment and prevention of helminths in livestock.

Tanshinone IIA attenuates demyelination and promotes remyelination in A. cantonensis-infected BALB/c mice.

BACKGROUND: Angiostrongylus cantonensis infection can cause demyelination in the central nervous system, and there is no effective treatment. METHODS: We used dexamethasone, Tanshinone IIA (TSIIA) and Cryptotanshinone(Two traditional Chinese medicine monomers) in combination with albendazole (AB, a standard anti-helminthic compound) to observe their therapeutic effect on demyelination in A. cantonensis-infected mice. Luxol fast blue staining and electron microscope of myelin sheath, Oligodendrocyte (OL) number and myelin basic protein (MBP) expression in brain was detected in above groups. RESULTS: TSIIA+AB facilitated OL proliferation and significantly increased both myelin sheath thickness and the population of small-diameter axons. In addition, TSIIA treatment inhibited the expression of inflammation-related factors (interleukin [IL]-6, IL-1ß, tumor necrosis factor [TNF]-α, inducible nitric oxide synthase [iNOS]) rather than inhibiting eosinophil infiltration in brain. TSIIA also decreased microglial activation and shifted their phenotype from M1 to M2. CONCLUSIONS: Taken together, these results provide evidence that TSIIA combined with AB may be an effective treatment for demyelination caused by A. cantonensis infection and other demyelinating diseases.

Current anthelmintic treatment is not always effective at controlling strongylid infections in German alpaca herds.

BACKGROUND: Endoparasites are considered a major health problem of South American camelids as shown in a recent survey among German and Austrian camelid owners. Although prophylactic and therapeutic measures such as application of anthelmintics are commonly used, treatment efficacy is usually not assessed. Owners have expressed significant concerns regarding the effect of antiparasitic therapy, so this study aimed to evaluate the outcome of anthelmintic treatment in German alpaca herds with different drugs. RESULTS: Overall, 617 samples from 538 clinically healthy alpacas > 1 year-old from 27 farms (n = 11-157 animals/herd) were examined. The most common parasites detected by flotation were Eimeria spp. (75.1%) followed by strongylids (55.0%), Nematodirus spp. (19.3%), cestodes (3.1%) and Trichuris (2.7%). After initial coproscopical examination by flotation and strongylid egg quantification by the McMaster technique, positive animals excreting at least 150 eggs per gram of faeces were included in a faecal egg count reduction test (FECRT) using fenbendazole (n = 71 samples), moxidectin (n = 71) or monepantel (n = 66). Pre-treatment larval cultures (n = 23 positive pooled farm samples) revealed Haemonchus (87% of the farms), Cooperia (43.5%), Trichostrongylus (21.7%), Ostertagia (13.0%), Nematodirus and Oesophagostomum (4.3% each). Fenbendazole treatment reduced egg excretion by 45%, moxidectin by 91% and monepantel by 96%. On the farm level, 13/18 farms that used fenbendazole, 6/6 farms that used moxidectin and 2/5 farms that used monepantel had individual FECR values < 90% (fenbendazole) or < 95% (moxidectin, monepantel). Haemonchus and Cooperia were overrepresented on the farms with reduced treatment efficacy. CONCLUSIONS: Gastrointestinal strongylids are common in German alpacas and fenbendazole in particular was not sufficiently effective to reduce strongylid egg excretion. Although the FECRT could not unambiguously determine anthelmintic resistance in the present study, the finding that small ruminant strongylids, especially Haemonchus, are common in alpacas indicates that determination of effective anthelmintic doses, monitoring of efficacy and adapted (selective) treatment regimens must be implemented as part of sustainable deworming practices in this species in accordance with recommendations for ruminants.

Chamomile Methanolic Extract Mitigates Small Bowel Inflammation and ROS Overload Related to the Intestinal Nematodes Infection in Mice.

BACKGROUND: Chamomile (Matricaria recutita L.) is a plant which has been reported to be effective in treating several parasitic and digestive diseases. The present study was conducted to evaluate the anthelmintic activity of chamomile methanolic extract (CME). METHODS: In vitro, the anthelmintic activities of CME were investigated on the L3 larvae of Heligmosomoides polygyrus in comparison to albendazole. In vivo, Swiss albino mice were infected with infective third (L3) larval stage of H. polygyrus by intragastric administration. Moreover, the effect of CME and albendazole on worm eggs, adult worms, serum cytokine production, and oxidative stress was studied. RESULTS: All used doses of CME showed a potent anthelmintic activity both in vitro and in vivo and the effect being similar to treatment with albendazole. Moreover, H. polygyrus infestation was accompanied by an intestinal oxidative stress status characterized by an increased lipoperoxidation, a depletion of antioxidant enzyme activity, as well as an overload of hydrogen peroxide. We have also recorded an increase of pro-inflammatory mediator (TNF-α, IL-6, and IL-1ß) levels after treatment with CME (14 ± 0.8; 41 ± 2; 58 ± 4 pg/mg protein, respectively, with the concentration 800 mg/kg, body weight) when compared with infected control mice (20 ± 1; 59 ± 2, and 83 ± 4 pg/mg protein, respectively). However, extract treatment alleviated all the deleterious effects associated with H. polygyrus infection. CONCLUSION: These findings suggest that CME can be used in the control of gastrointestinal helminthiasis and associated oxidative stress.

Body Protein Reserves Sustain Maternal Performance in Early Lactation but Dietary Protein Is Necessary to Maintain Performance and Immune Responses to Nippostrongylus brasiliensis in Lactating Rats.

Background: It has been shown that dietary protein supplementation during lactation boosts immunity in Nippostrongylus brasiliensis-infected periparturient rats. It is not known whether body protein reserves accumulated during gestation have a similar effect during lactation. Objective: This study aimed to quantify the impact of body protein reserves and dietary protein supplementation on maternal performance and immune responses to N. brasiliensis during lactation. Methods: Multiparous female Sprague-Dawley rats were administered a primary infection of N. brasiliensis before mating and were restriction-fed either 60 g [low-protein diet gestation (Lge)] or 210 g [high-protein diet gestation (Hge)] crude protein (CP) per kilogram of dry matter (DM) until parturition. From parturition onward, dams were restriction-fed either 100 g [low-protein diet lactation (Lla)] or 300 g [high-protein diet lactation (Hla)] CP per kilogram of DM, generating 4 different dietary treatments. A subset of rats was sampled before parturition; postparturition, dams were secondarily infected with N. brasiliensis and samples were collected at days 5 and 11 postparturition. Results: Maternal performance until parturition, as measured by pup weight, was better in Hge rats than in Lge rats [Lge: 4.84 g; Hge: 6.15 g; standard error of the difference (SED): 0.19]. On day 11, pup weights of dams with reduced protein reserves fed protein during lactation (Lge-Hla; 20.28 g) were higher than their counterparts from Hge-Lla dams (17.88 g; SED: 0.92). Worm counts were significantly different between Lge-Lla-fed (253; 95% CI: 124, 382) and Hge-Hla-fed (87; 95% CI: 22, 104) dams on day 11 (P = 0.024). The expression of splenic interleukin 13 (Il13) and arachidonate 15-lipoxygenase (Alox15) was significantly higher (P < 0.05) in Hge-Hla dams compared with Lge-Lla dams on day 5. Conclusions: Although protein reserves were adequate to maintain maternal performance in the early stage of lactation in dams infected with N. brasiliensis, they were not adequate to maintain maternal performance and effective immune responses at later stages. Dietary protein supplementation was required to achieve this.

Hematopoietic cell-derived RELMα regulates hookworm immunity through effects on macrophages.

Resistin-like molecule α (RELMα) is a highly secreted protein in type 2 (Th2) cytokine-induced inflammation including helminth infection and allergy. In infection with Nippostrongylus brasiliensis (Nb), RELMα dampens Th2 inflammatory responses. RELMα is expressed by immune cells, and by epithelial cells (EC); however, the functional impact of immune versus EC-derived RELMα is unknown. We generated bone marrow (BM) chimeras that were RELMα deficient (RELMα-/- ) in BM or non BM cells and infected them with Nb. Non BM RELMα-/- chimeras had comparable inflammatory responses and parasite burdens to RELMα+/+ mice. In contrast, both RELMα-/- and BM RELMα-/- mice exhibited increased Nb-induced lung and intestinal inflammation, correlated with elevated Th2 cytokines and Nb killing. CD11c+ lung macrophages were the dominant BM-derived source of RELMα and can mediate Nb killing. Therefore, we employed a macrophage-worm co-culture system to investigate whether RELMα regulates macrophage-mediated Nb killing. Compared to RELMα+/+ macrophages, RELMα-/- macrophages exhibited increased binding to Nb and functionally impaired Nb development. Supplementation with recombinant RELMα partially reversed this phenotype. Gene expression analysis revealed that RELMα decreased cell adhesion and Fc receptor signaling pathways, which are associated with macrophage-mediated helminth killing. Collectively, these studies demonstrate that BM-derived RELMα is necessary and sufficient to dampen Nb immune responses, and identify that one mechanism of action of RELMα is through inhibiting macrophage recruitment and interaction with Nb. Our findings suggest that RELMα acts as an immune brake that provides mutually beneficial effects for the host and parasite by limiting tissue damage and delaying parasite expulsion.

Evaluation of the anthelminthic potential of Duranta erecta L. (Verbenaceae) fruits used in Nigerian ethnomedicine as a vermifuge.

ETHNOPHARMACOLOGICAL RELEVANCE: Duranta erecta L. fruits have been reported to have in vitro anthelminthic properties. People living in the tropical South-Eastern part of Nigeria use the plant in folk medicine for the treatment of malaria, abscesses and as a vermifuge. Although there are a few reports about its in vitro anthelminthic activity against different worm categories, scientific reports regarding its in vivo anthelminthic activity are limited. AIM OF THE STUDY: This study was designed with the aim of determining the potential of the plant as an anthelminthic and to verify the claims made by its local users. MATERIALS AND METHODS: Acute toxicity of the plant extract was determined using Lorke's method. Anthelminthic activity was investigated using adult male albino mice experimentally infected with Heligmosomoides bakeri infective L3. Graded ascending doses of the plant extract and Albendazole respectively were orally administered to the mice in the infected groups. Corprological and haematological parameters were recorded within the study period. Twenty-eight (28) days post-infection, all infected mice were humanely sacrificed and the Post-Mortem Adult Worm Burden (WB) was estimated and recorded. RESULTS: The results showed that the extract had an LD50 greater than 5000 mg/kg BW and therefore was not acutely toxic for oral use. It also showed that the plant extract was unable to eliminate the faecal egg output or adult worms in the gastrointestinal tract of infected animals even at the high doses used in the study. This was in contrast to Albendazole which significantly (p < .05) reduced faecal egg counts and worm burdens by 71% and 92% respectively in treated mice. Following infection, there was anaemia in all infected groups seen from results of erythrocytic parameters. Treatment with the plant extract, regardless of the dose, was unable to effectively reverse the effect of parasite infection on erythrocytic parameters. However, treatment with Albendazole positively reversed the anaemia, restoring the mice to pre-infection values by the end of the experiment. The results showed significant (p < .05) increase in WBC counts across all groups following infection with the parasite. Treatment with the plant extract and Albendazole respectively, significantly (p < .05) reduced the WBC counts to near pre-infection values in most treatment groups. CONCLUSION: As a result of the poor anthelminthic effects recorded in the study, it is therefore recommended that Duranta erecta L. fruits be explored for its other useful effects rather than as an anthelminthic.

Impact of heat treatment on antigen detection in sera of Angiostrongylus vasorum infected dogs.

BACKGROUND: In the last decade serological tests for detection of circulating Angiostrongylus vasorum antigen and specific antibodies have been developed and adopted for individual diagnosis and epidemiological studies in dogs. Although confirmed positive at necropsy, antigen detection was not possible in single experimentally, as well as naturally infected dogs, possibly due to immune complex formation. The aim of this study was to evaluate the effect of heat treatment on detection of A. vasorum antigen in sera of experimentally (n = 21, 119 follow-up sera) and naturally (n = 18) infected animals. In addition, sera of dogs showing clinical signs consistent with angiostrongylosis (n = 10), of randomly selected dogs (n = 58) and of dogs with other parasitic infections (n = 15) were evaluated. Sera were subjected to heat treatment at 100 °C after addition of 0.5 M EDTA (dilution 1:5) and tested with ELISAs for detection of circulating A. vasorum antigen before and after treatment. RESULTS: Between 5 and 11 weeks post-inoculation (wpi) the percentage of positive untreated samples (experimentally infected dogs) increased over time from 33.3 to 90%. Single samples were still negative between 12 and 15 wpi. Overall, between 5 and 15 wpi, 50.6% (45/89) of the available samples were seropositive. From 3 to 6 wpi EDTA/heat treatment caused a change in 8/34 (23.5%) of the samples, with most (n = 6, 17.6%) converting from positive to negative. In contrast, from 7 to 10 wpi, treatment induced a change in 19/52 (36.5%) samples, with all but one converting from negative to positive. Thirteen of 18 naturally infected dogs were antigen positive before and 15 after EDTA/heat treatment, respectively. Untreated samples of 3 dogs with suspected angiostrongylosis were antigen positive, of which only one remained positive after EDTA/heat treatment. One of 58 untreated random samples was antigen positive; this sample became negative after treatment, while another turned positive. One of 15 dogs infected with other parasites than A. vasorum was positive before but negative after treatment. CONCLUSION: Although heat treatment improves A. vasorum antigen detection between 7 and 10 wpi by immune complex disruption, we do not recommend systematic pretreating sera because of reduced antigen detection between 3 and 6 wpi and impairment of antibody detection, if performed contemporaneously.

An investigation into the effects of maternal supplementation with excess iodine on the mechanisms and impacts of reduced IgG absorption in the lamb postpartum.

An experiment was conducted to determine: (1) the effect of excess maternal I supplementation on the thyroid hormone status of the ewe and her progeny; (2) potential mechanisms underpinning the failure of passive transfer associated with excess I and (3) the growing lambs' response to natural gastrointestinal infection. Twin-bearing ewes received one of two treatments (n 32/treatment group): basal diet (C) or C plus 26·6 mg of iodine/ewe per d (I), supplied as calcium iodate. Ewes were individually fed from day 119 of gestation to parturition. Progeny of I ewes had lower (P<0·01) serum IgG concentrations from 24 h to 28 d postpartum but higher serum IgG concentrations at day 70 postpartum (P<0·05). I supplementation increased the relative expression of Fc receptor, IgA, IgM high affinity and polymeric Ig receptor in the ileum of the lamb at 24 h postpartum; however, thyroid hormone receptor-ß (THRB) and ß-2-microglobulin (B2M) expression declined (P<0·05). Progeny of I ewes had higher growth rates to weaning (P<0·05) and lower faecal egg count (FEC) for Nematodirus battus (P<0·05) between weeks 6 and 10 postpartum. In conclusion, excess maternal I supplementation negatively affected the thyroid hormone status, serum IgG concentration, ileal morphology and the gene expression of THRB and B2M in the ileum and ras-related protein (RAB) RAB25 and the mucin gene (MUC) MUC1 in the duodenum of the lamb postpartum. These effects were followed by an enhancement of average daily gain and lower N. battus FEC in the pre-weaning period of I-supplemented lambs.

Papaya latex supernatant has a potent effect on the free-living stages of equid cyathostomins in vitro.

The control of equid gastrointestinal nematodes in developed countries, in particular the cyathostomins, is threatened by high levels of anthelmintic resistance. In recent years, there has been increasing interest in the evaluation of traditional 'ethnoveterinary' medicines as alternatives to chemical anthelmintics. The cysteine proteinases (CPs), a group of enzymes derived from fruits such as papaya (Carica papaya), pineapple (Ananas comosus) and figs (Ficus spp.), have shown good efficacy against adult stages of a range of parasitic nematodes, in vitro and in vivo. The efficacy of CPs against cyathostomins remains to be explored. In this study, the efficacy of a crude preparation of CPs, papaya latex supernatant (PLS), against the free-living stages of cyathostomins was evaluated using two in vitro tests, the egg hatch test (EHT) and the larval migration inhibition test (LMIT). It was demonstrated that PLS had a potent effect in the EHT, with EC-50 values in the range of 0.12-0.22µM. At concentrations above 6.25µM the eggs did not develop, below this concentration the L1 developed but they lost integrity of the cuticle upon hatching. These effects were inhibited by pre-incubation of PLS with the CP inhibitor L-trans-epoxysuccinyl-l-leucylamido-(4-guanidino butane) (E64), indicating that CPs were responsible for the anti-parasitic activity. A dose-dependent inhibition of migration of third stage larvae (L3) in the LMIT was demonstrated at higher concentrations of PLS, with EC-50 values in the range of 67.35-106.31µM. Incubation of PLS with E64 prior to use in the LMIT did not reverse the anti-migratory effect, suggesting that CPs were not responsible for the reduced migration of cyathostomin L3 and that PLS also contains an additional active compound. This is the first report of PLS and/or CPs showing activity against the free-living stages of a parasitic helminth. In addition, it suggests that cyathostomins are highly sensitive to the effects of CPs and further evaluation of their efficacy against parasitic stages and in vivo are strongly indicated.

Selenoprotein Expression in Macrophages Is Critical for Optimal Clearance of Parasitic Helminth Nippostrongylus brasiliensis.

The plasticity of macrophages is evident in helminthic parasite infections, providing protection from inflammation. Previously we demonstrated that the micronutrient selenium induces a phenotypic switch in macrophage activation from a classically activated (pro-inflammatory; M1/CAM) toward an alternatively activated (anti-inflammatory; M2/AAM) phenotype, where cyclooxygenase (COX)-dependent cyclopentenone prostaglandin J2 (15d-PGJ2) plays a key role. Here, we hypothesize that dietary selenium modulates macrophage polarization toward an AAM phenotype to assist in the increasing clearance of adult Nippostrongylus brasiliensis, a gastrointestinal nematode parasite. Mice on a selenium-adequate (0.08 ppm) diet significantly augmented intestinal AAM presence while decreasing adult worms and fecal egg production when compared with infection of mice on selenium-deficient (<0.01 ppm) diet. Further increase in dietary selenium to supraphysiological levels (0.4 ppm) had very little or no impact on worm expulsion. Normal adult worm clearance and enhanced AAM marker expression were observed in the selenium-supplemented Trsp(fl/fl)Cre(WT) mice that express selenoproteins driven by tRNA(Sec) (Trsp), whereas N. brasiliensis-infected Trsp(fl/fl)Cre(LysM) selenium-supplemented mice showed a decreased clearance, with lowered intestinal expression of several AAM markers. Inhibition of the COX pathway with indomethacin resulted in delayed worm expulsion in selenium-adequate mice. This was rescued with 15d-PGJ2, which partially recapitulated the effect of selenium supplementation on fecal egg output in addition to increasing markers of AAMs in the small intestine. Antagonism of PPARγ blocked the effect of selenium. These results suggest that optimal expression of selenoproteins and selenium-dependent production of COX-derived endogenous prostanoids, such as Δ(12)-PGJ2 and 15d-PGJ2, may regulate AAM activation to enhance anti-helminthic parasite responses.

Effects of albendazole combined with TSII-A (a Chinese herb compound) on optic neuritis caused by Angiostrongylus cantonensis in BALB/c mice.

BACKGROUND: Angiostrongylus cantonensis (A. cantonensis) infection can lead to optic neuritis, retinal inflammation, damage to ganglion cells, demyelination of optic nerve and visual impairment. Combined therapy of albendazole and dexamethasone is a common treatment for the disease in the clinic, but it plays no role in vision recovery. Therefore, it has been necessary to explore alternative therapies to treat this disease. Previous studies reported the neuro-productive effects of two constituents of Danshen (a Chinese herb)-tanshinone II-A (TSII-A) and cryptotanshinone (CPT), and this study aims to evaluate the impacts of TSII-A or CPT combined with albendazole on optic neuritis caused by A. cantonensis infection in a murine model. METHODS: To assess the effects of TSII-A or CPT combined with albendazole on optic neuritis due to the infection, mice were divided into six groups, including the normal control group, infection group and four treatment groups (albendazole group, albendazole combined with dexamethasone group, albendazole combined with CPT group and albendazole combined with TSII-A group). The infection group and treatment groups were infected with A. cantonensis and the treatment groups received interventions from 14 dpi (days post infection), respectively. At 21 dpi, the visual acuity of mice in each group was examined by visual evoked potential (VEP). The pathologic alteration of the retina and optic nerve were observed by hematoxylin and eosin (H&E) staining and transmission electronic microscopy (TEM). RESULTS: Infection of A. cantonensis caused prolonged VEP latency, obvious inflammatory cell infiltration in the retina, damaged retinal ganglions and retinal swelling, followed by optic nerve fibre demyelination and a decreasing number of axons at 21 dpi. In treatment groups, albendazole could not alleviate the above symptoms; albendazole combined with dexamethasone lessened the inflammation of the retina, but was futile for the other changes; however, albendazole combined with CPT and albendazole combined with TSII-A showed obvious effects on the recovery of prolonged VEP latency, destruction and reduction of ganglion cells, optic nerve demyelination and axon loss. Compared with albendazole-CPT compound, albendazole combined with TSII-A was more effective. CONCLUSIONS: The current study demonstrates that albendazole combined with TSII-A plays a more effective role in treating optic neuritis caused by A. cantonensis in mice than with dexamethasone, as applied in conventional treatment, indicating that albendazole combined with TSII-A might be an alternate therapy for this parasitic disease in the clinic.

Comparison of coprological, immunological and molecular methods for the detection of dogs infected with Angiostrongylus vasorum before and after anthelmintic treatment.

Timely diagnosis of the nematode Angiostrongylus vasorum in dogs is important in view of severe and permanent lung and cardiovascular lesions that may occur. The performance of the classical Baermann coprological method was compared with ELISAs for the serological detection of circulating antigen and specific antibodies and with Polymerase chain reaction (PCR) performed on EDTA blood, feces and tracheal swabs of serial samples from experimentally inoculated dogs over 13 weeks post inoculation (wpi) (n = 16) and following anthelmintic treatment (n = 6). Patency was observed from 6.7 to 7.6 wpi in all dogs, Baermann results were then mostly positive (116/119, 97%) during the patent period, with wide variations in the numbers of first stage larvae numbers. Blood PCR was tested positive on 1-2 occasions in 11/16 dogs in the pre-patent period, while all tested positive by antibody-detection ELISA by 6 wpi. The proportion of dogs testing positive by fecal PCR and antigen-detection ELISA rose early in the patent period. Tracheal swabs were occasionally DNA-positive in 3/16 dogs starting from 10 wpi. Following treatment, larval excretion stopped within 3 weeks and blood PCR results became negative within 1 week (5/6 dogs), while 4/6 dogs were positive for parasite DNA in tracheal swabs. Parasite antigen and specific antibodies both persisted in the blood for 3-9 weeks after treatment, with average optical densities and the proportion of positive dogs falling gradually, while results using other tests were much more variable. Results indicate that the earliest and most consistent results are obtained by the ELISAs, which can also be used for monitoring dogs after anthelmintic treatment.

Factors affecting the anthelmintic efficacy of papaya latex in vivo: host sex and intensity of infection.

The development of plant-derived cysteine proteinases, such as those in papaya latex, as novel anthelmintics requires that the variables affecting efficacy be fully evaluated. Here, we conducted two experiments, the first to test for any effect of host sex and the second to determine whether the intensity of the worm burden carried by mice would influence efficacy. In both experiments, we used the standard C3H mouse reference strain in which papaya latex supernatant (PLS) consistently shows >80 % reduction in Heligmosomoides bakeri worm burdens, but to broaden the perspective, we also included for comparison mice of other strains that are known to respond more poorly to treatment with papaya latex. Our results confirmed that there is a strong genetic influence affecting efficacy of PLS in removing adult worm burdens. However, there was no effect of host sex on efficacy (C3H and NIH) and no effect of infection intensity (C3H and BALB/c). These results offer optimism that plant-derived cysteine proteinases (CPs), such as these from papaya latex, can function as effective anthelmintics, with neither host sex nor infection intensity presenting further hurdles to impede their development for future medicinal and veterinary usage.

How does the suppression of energy supplementation affect herbage intake, performance and parasitism in lactating saddle mares?

Agroecology opens up new perspectives for the design of sustainable farming systems by using the stimulation of natural processes to reduce the inputs needed for production. In horse farming systems, the challenge is to maximize the proportion of forages in the diet, and to develop alternatives to synthetic chemical drugs for controlling gastrointestinal nematodes. Lactating saddle mares, with high nutritional requirements, are commonly supplemented with concentrates at pasture, although the influence of energy supplementation on voluntary intake, performance and immune response against parasites has not yet been quantified. In a 4-month study, 16 lactating mares experimentally infected with cyathostome larvae either received a daily supplement of barley (60% of energy requirements for lactation) or were non-supplemented. The mares were rotationally grazed on permanent pastures over three vegetation cycles. All the mares met their energy requirements and maintained their body condition score higher than 3. In both treatments, they produced foals with a satisfying growth rate (cycle 1: 1293 g/day; cycle 2: 1029 g/day; cycle 3: 559 g/day) and conformation (according to measurements of height at withers and cannon bone width at 11 months). Parasite egg excretion by mares increased in both groups during the grazing season (from 150 to 2011 epg), independently of whether they were supplemented or not. This suggests that energy supplementation did not improve mare ability to regulate parasite burden. Under unlimited herbage conditions, grass dry matter intake by supplemented mares remained stable around 22.6 g DM/kg LW per day (i.e. 13.5 kg DM/al per day), whereas non-supplemented mares increased voluntary intake from 22.6 to 28.0 g DM/kg LW per day (13.5 to 17.2 kg DM/al per day) between mid-June and the end of August. Hence total digestible dry matter intake and net energy intake did not significantly differ between supplemented and non-supplemented mares during the second and third cycles. In conclusion, supplementing lactating mares at pasture should not be systematic because their adaptive capacities enable to increase herbage intake and ensure foal growth. Further research is needed to determine the herbage allowance threshold below which supplementation is required.

Evaluation of enhanced virgin coconut oil and senduduk (Melastoma malabathricum) as anthelmintics against caprine strongyle nematodes.

A study was conducted to evaluate the anthelmintic properties of enhanced virgin coconut oil (EVCO) and senduduk (Melastoma malabathricum) plant against strongyle nematodes in goats. Two preparations of 10% EVCO dissolved in 90% virgin coconut oil and 10% EVCO dissolved in 90% palm oil, were given orally to two groups of mixed breeds goats. The efficacy test indicated that EVCO was insufficiently active as an anthelmintic. Four concentrations of senduduk solution (1.25, 2.5, 5.0 and 10 mg ml(-1)) were compared with a control and albendazole in an in vitro test for larvicidal effect. There was no significant larval mortality using senduduk solution. An in vivo test of senduduk was conducted by comparing three groups of goats, namely control, levamisole and treatment groups that were given a daily oral dose of senduduk crude extract with 1g kg(-1) from Day 0 to Day 12 and 2 g kg(-1) from Day 13 to Day 30. This efficacy test with senduduk also gave negative results. The findings obtained indicated that EVCO and senduduk were ineffective as anthelmintics against caprinestrongyle nematodes at the concentrations used.