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.

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.

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.

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.

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.

In vitro anthelminthic efficacy of Dichrocephala integrifolia (Asteraceae) extracts on the gastro-intestinal nematode parasite of mice: Heligmosomoides bakeri (Nematoda, Heligmosomatidae).

OBJECTIVE: To evaluate the ovicidal and larvicidal activities of aqueous and ethanolic extracts of leaves of Dichrocephala integrifolia (D. integrifolia) against the eggs (fresh and embryonnated), the first and second larval stages of Heligmosomoides bakeri. In order to verify if this medicinal plant possesses active compounds capable of inhibiting the embryonation and hatching of eggs or to induce the mortality of larvae (L1 and L2). METHODS: dried extracts were diluted in distilled FIV water to obtain five different concentrations: 625, 1,250, 2,500, 3,750 and 5,000 µg/mL. Fresh eggs obtained from artificially infected mice feces were exposed to these different concentrations for 48 h. Time of contact for embryonated eggs was 6 h while L1 and L2 larvae were exposed for 24 h. Distilled water (placebo) and 1.5% DMSO were used as negative controls. RESULTS: Distilled water, and 1.5% DMSO had no effect on embryonation, hatching and larval survival. Aqueous extracts of D. integrifolia showed a weak activity against all stages of the parasite at all concentrations tested. On the contrary, the ethanolic extract of D. integrifolia inhibited the embryonation of 87.5% of fresh eggs, the hatching of 81.1% of embryonated eggs and induced the mortality of 98.1% and 98% of L1 and L2 larvae respectively at 5,000 µg/mL. CONCLUSIONS: The results of the present study indicate that the ethanolic extracts of D. integrifolia contained compounds with ovicidal and larvicidal properties. In spite of these results, in vivo tests, studies on toxicity and mechanism of action of active compounds are also needed to validate the utilisation of this medicinal plant by population of Dschang-Cameroon to treat gastro-intestinal parasites.

New epidemiological and molecular insights into feline lungworm infection.

Aelurostrongylus abstrusus (Nematoda, Strongylida) lives in the terminal respiratory bronchioles and alveolar ducts of cats. The infection may present with respiratory signs of varying severity and can be life threatening. Despite the importance of A. abstrusus in feline medicine and recent reports of clinical cases of aelurostrongylosis in Italy, epidemiological data are sparse and molecular information available on the parasite are lacking. The present work reports recent insights into the epidemiology of feline lungworm infection in Italy and contributes to the molecular knowledge of A. abstrusus. Individual stool samples were collected from 100 animals and examined with flotation and Baermann methods. The risk factors were analyzed by binary logistic multiple-regression models. All positive fecal samples (n= 16) were subjected to the PCR-coupled sequencing of the rDNA internal trancribed spacer 2 (ITS2). The detected infection rate differs significantly from data reported in the literature, and stray and free-ranging cats and the presence of respiratory symptoms were risk factors for the infection. The molecular characterization showed that the ITS2 sequence is a promising genetic target for a holistic approach to the study of cat lungworm infection. Epidemiological and molecular insights and avenues are discussed.

Acquired cervical scoliosis attributed to Parelaphostrongylus tenuis infection in an alpaca.

CASE DESCRIPTION: A 2-year-old alpaca was evaluated because of acute onset of cervical scoliosis. CLINICAL FINDINGS: Physical examination revealed severe scoliosis of the caudal portion of the cervical vertebral column with a C-shaped curvature to the right side. No gait deficits were observed. Cervical radiography confirmed severe curvature of C4 to C6 but did not reveal any bony changes. Cerebrospinal fluid had high total protein concentration and extremely high nucleated cell count with a high proportion of eosinophils, suggesting parasitic infection. TREATMENT AND OUTCOME: The alpaca was treated for suspected parelaphostrongylosis with ivermectin, fenbendazole, flunixin, vitamin E, thiamine, physical therapy, and a custom-made neck brace. The alpaca's condition continued to deteriorate, and it developed tetraparesis and ataxia and was euthanized after approximately 1 month. Microscopic evaluation of the cervical spinal cord revealed marked vacuolar changes in the left medial portion of the ventral funiculus, mild lymphoplasmacytic infiltration, and multifocal granulomas. The lesions were continuous from C1 to C7 and were compatible with parasite migration. CLINICAL RELEVANCE: To the authors' knowledge, this is the first report of acquired scoliosis in an alpaca, which appears to represent an unusual manifestation of parelaphostrongylosis that was reported in horses.

Improving resilience against natural gastrointestinal nematode infections in browsing kids during the dry season in tropical Mexico.

The objective was to determine the effect of supplementary feeding on the resilience and resistance of Criollo kids against natural gastrointestinal nematode (GIN) infections, when browsing native vegetation during the dry season in tropical Mexico. Thirty-three two-month-old Criollo kids, raised nematode free, were included at weaning in a 20-week trial. The kids were placed into four groups. Two groups of eight kids were offered 100g/day soybean and sorghum meal (26%:74% respectively fresh basis) (treated/supplemented (T-S) and infected/supplemented (I-S)). Two groups remained with no supplement for the duration of the trial (infected/non-supplemented (I-NS) (n=9) and treated/non-supplemented (T-NS) (n=8)). Kids in groups T-S and T-NS were drenched with 0.2mg of moxidectin/kg body weight orally (Cydectin, Fort Dodge) every 28 days. Groups I-S and I-NS were naturally infected with GIN. The animals browsed native vegetation for an average of 7h/day together with a herd of 120 naturally infected adult goats. Cumulative live weight gain (CLWG), packed cell volume (PCV), haemoglobin (Hb), total plasma protein and plasma albumin were recorded every 14 days as measurements of resilience. Resistance parameters (faecal egg counts (FEC) and peripheral eosinophil counts (PEC)) were also measured. Bulk faecal cultures were made for each group every 28 days. Every month a new pair of initially worm-free tracer kids assessed the infectivity of the vegetation browsed by the animals. Tracer kids and faecal cultures showed that kids faced low mixed infections (Haemonchus contortus, Trichostrongylus colubriformis and Oesophagostomum columbianum). Under conditions of scarce vegetation, such as those in the present study, supplemented groups (I-S and T-S) had higher growth rates compared to the non-supplemented groups independently of the control of GIN infection with anthelmintic (AH) treatment (P<0.001). Supplementary feeding did not affect FEC or PEC. In the absence of supplementation, lack of AH treatment may lead to outbreaks of clinical nematodosis. The supplementary feeding was economically feasible.