Effect of pomegranate (Punica granatum) anthelmintic treatment on milk production in dairy sheep naturally infected with gastrointestinal nematodes.

Anthelmintic drug resistance has proliferated across Europe in sheep gastrointestinal nematodes (GINs). Sheep welfare and health are adversely impacted by these phenomena, which also have an impact on productivity. Finding alternatives for controlling GINs in sheep is thus of utmost importance. In this study, the anthelmintic effectiveness (AE) of a Calabrian ethnoveterinary aqueous macerate based on Punica granatum (whole fruits) was assessed in Comisana pregnant sheep. Furthermore, an examination, both qualitative and quantitative, was conducted on milk. Forty-five sheep were selected for the investigation. The sheep were divided by age, weight, physiological state (pluripara at 20 days before parturition), and eggs per gram of feces (EPG) into three homogeneous groups of 15 animals each: PG received a single oral dosage of P. granatum macerate at a rate of 50 mL per sheep; AG, treated with albendazole, was administered orally at 3.75 mg/kg/bw; and CG received no treatment. Timelines were as follows: D0, treatments, group assignment, fecal sampling, and AE assessment; D7, D14, D21, fecal sampling, and AE evaluation. The FLOTAC technique was used to evaluate the individual GIN fecal egg count (FEC) using a sodium chloride flotation solution (specific gravity = 1.20) and 100 × (1-[T2/C2]) as the formula for evaluating FEC reduction. Following the lambs' weaning, milk was collected on the following days (DL) in order to quantify production: DL35, DL42, DL49, DL56, DL63, DL70, DL77, and DL84. The amount of milk produced by every animal was measured and reported in milliliters (ml) for quantitative evaluations. Using MilkoScan TM fT + foss electric, Denmark, the quality of the milk (casein, lactose, protein concentration, and fat, expressed as a percentage) was assessed. The macerate demonstrated a considerable AE (51.8%). Moreover, its use has resulted in higher milk production rates quantitatively (15.5%) and qualitatively (5.12% protein, 4.12% casein, 4.21% lactose, and 8.18% fat). The study showed that green veterinary pharmacology could be the easiest future approach to counteracting anthelmintic resistance in sheep husbandry.

A general framework to support cost-efficient fecal egg count methods and study design choices for large-scale STH deworming programs-monitoring of therapeutic drug efficacy as a case study.

BACKGROUND: Soil-transmitted helminth (STH) control programs currently lack evidence-based recommendations for cost-efficient survey designs for monitoring and evaluation. Here, we present a framework to provide evidence-based recommendations, using a case study of therapeutic drug efficacy monitoring based on the examination of helminth eggs in stool. METHODS: We performed an in-depth analysis of the operational costs to process one stool sample for three diagnostic methods (Kato-Katz, Mini-FLOTAC and FECPAKG2). Next, we performed simulations to determine the probability of detecting a truly reduced therapeutic efficacy for different scenarios of STH species (Ascaris lumbricoides, Trichuris trichiura and hookworms), pre-treatment infection levels, survey design (screen and select (SS); screen, select and retest (SSR) and no selection (NS)) and number of subjects enrolled (100-5,000). Finally, we integrated the outcome of the cost assessment into the simulation study to estimate the total survey costs and determined the most cost-efficient survey design. PRINCIPAL FINDINGS: Kato-Katz allowed for both the highest sample throughput and the lowest cost per test, while FECPAKG2 required both the most laboratory time and was the most expensive. Counting of eggs accounted for 23% (FECPAKG2) or ≥80% (Kato-Katz and Mini-FLOTAC) of the total time-to-result. NS survey designs in combination with Kato-Katz were the most cost-efficient to assess therapeutic drug efficacy in all scenarios of STH species and endemicity. CONCLUSIONS/SIGNIFICANCE: We confirm that Kato-Katz is the fecal egg counting method of choice for monitoring therapeutic drug efficacy, but that the survey design currently recommended by WHO (SS) should be updated. Our generic framework, which captures laboratory time and material costs, can be used to further support cost-efficient choices for other important surveys informing STH control programs. In addition, it can be used to explore the value of alternative diagnostic techniques, like automated egg counting, which may further reduce operational costs. TRIAL REGISTRATION: ClinicalTrials.gov NCT03465488.

Potential New Therapeutic Approaches Based on Punica granatum Fruits Compared to Synthetic Anthelmintics for the Sustainable Control of Gastrointestinal Nematodes in Sheep.

Parasites, in particular, gastrointestinal nematodes (GINs) represent one of the main burdens affecting small ruminant farming and pose a serious threat to their health, welfare, productivity, and reproduction. The correct management of animals and the correct use of anthelmintic drugs are the pillars of the GIN control programs for small ruminants. However, globally due to the indiscriminate use of synthetic anthelmintics, there is a significant increase in anthelmintic resistance phenomena to one or more classes of drugs. Even if such a problem never represented a serious threat in southern Italy because of the favourable environmental conditions and because of the good farm management, the phenomenon is actually showing a steep increasing trend and requires alternative treatment measures and constant monitoring. The use of phytotherapies is considered a valuable alternative approach for GIN control in small ruminants and could help with reducing the amount of synthetic drugs used and the forthcoming anthelmintic resistance. From this perspective, the Calabria territory offers a wide number of plants with anthelmintic efficacy that could be helpful for this purpose. The aim of this study was to evaluate the anthelmintic efficacy of aqueous pomegranate (Punica granatum L.) macerate compared to the treatment with Ivermectin and Albendazole in sheep naturally infected with GINs. The pomegranate macerate derives from the ethnoveterinary knowledge of the Calabria region, Southern Italy. The anthelmintic efficacy was evaluated according to the faecal egg count reduction test (FECRt) using the FLOTAC techniques in two sheep farms in Southern Italy. The FECR was calculated from individual samples using the formula FECR = 100 × (1 - [T2/C2]). The treatment with Albendazole in the first farm showed an efficacy of 99.8% after 14 days and 94.8% after 21 days, while the treatment with Ivermectin in the second farm showed an efficacy of 99.9% after 14 days and 96.5% after 21 days of treatment. The pomegranate macerate, in both farms, showed a value of efficacy of around 50% from day 7 to day 21 after the treatment. Previous studies highlighted the presence of gallic acid as the main component in the pomegranate macerate, and its efficacy in nematode control has been as well previously demonstrated in other plant extracts. This in vivo study demonstrated the unequivocal efficacy of plant macerate in easily reducing 50% of the number of GIN eggs in sheep faeces. These results, obtained without the use of synthetic anthelmintics, indicate the use of green veterinary pharmacology as a sustainable alternative to the use of synthetic drugs to reduce the increase in drug resistance phenomena and the environmental impact.

Anthelmintic Properties of Essential Oils to Control Gastrointestinal Nematodes in Sheep-In Vitro and In Vivo Studies.

Herbal products such as essential oils may play a promising role in the treatment of infections caused by gastrointestinal nematodes (GINs). The aim of this study was to evaluate the in vitro potential of 11 essential oils (EOs) and one binary combination of isolated EO compounds, as well as the in vivo anthelmintic efficacy of two EO formulations. Four GIN genera were identified in the coproculture examination: Haemonchus, Trichostrongylus, Teladorsagia and Chabertia. The in vitro egg hatch test (EHT) was performed at six different concentrations (50, 12.5, 3.125, 0.781, 0.195 and 0.049 mg/mL) for each EO, whereas in the in vivo faecal egg count reduction test (FECRT), each EO sample was diluted in sunflower oil and orally administrated at a dose of 100 mg/kg to the different group of animals. In the EHT, the EOs of Origanum vulgare, Foeniculum vulgare, Satureja montana, Satureja hortensis and two types of Thymus vulgaris were the most effective. The dominant compounds of these EOs were carvacrol, thymol, anethol, p-cymene and γ-terpinene, indicating their importance for the anthelmintic activity. In the FECRT, both T. vulgaris EO type 1 and linalool:estragole combination show an anthelmintic potential with a mean effect on FECR of approximately 25%. The results suggest the possible role of tested EOs as anthelmintic agents in sheep farms, although further in vivo tests are needed.

Green Veterinary Pharmacology Applied to Parasite Control: Evaluation of Punica granatum, Artemisia campestris, Salix caprea Aqueous Macerates against Gastrointestinal Nematodes of Sheep.

Resistance to anthelmintic drugs in gastrointestinal nematodes (GIN) of sheep is of high concern for livestock production worldwide. In Calabria (southern Italy), many plants have been used in ethnoveterinary medicine for parasite control in small ruminants. Here, we present an in vivo evaluation of anthelmintic efficacy of three plant extracts. The first was based on bark and leaves of Salix caprea, the second and the third were based on the whole plant Artemisia campestris and whole fruit (seeds and peel) of Punica granatum, respectively. Anthelmintic efficacy was evaluated according to the fecal egg count reduction test (FECRT) performed with the FLOTAC technique. The results showed a significant anthelmintic effect of Punica granatum macerate (50%), a low effectiveness of the Artemisia campestris macerate (20%), and a complete ineffectiveness of Salix caprea macerate (0.1%). With these outcomes, we report a P. granatum-based remedy reducing 50% GIN egg output. This result was obtained without using any synthetic drug, paving the way for the employment of green veterinary pharmacology (GVP) as a complementary and sustainable method to reduce the use of chemicals and to counteract anthelmintic resistance.

The global progress of soil-transmitted helminthiases control in 2020 and World Health Organization targets for 2030.

Soil-transmitted helminth (STH) infections are the most widespread of the neglected tropical diseases, primarily affecting marginalized populations in low- and middle-income countries. More than one billion people are currently infected with STHs. For the control of these infections, the World Health Organization (WHO) recommends an integrated approach, which includes access to appropriate sanitation, hygiene education, and preventive chemotherapy (i.e., large-scale, periodic distribution of anthelmintic drugs). Since 2010, WHO has coordinated two large donations of benzimidazoles to endemic countries. Thus far, more than 3.3 billion benzimidazole tablets have been distributed in schools for the control of STH infections, resulting in an important reduction in STH-attributable morbidity in children, while additional tablets have been distributed for the control of lymphatic filariasis. This paper (i) summarizes the progress of global STH control between 2008 to 2018 (based on over 690 reports submitted by endemic countries to WHO); (ii) provides regional and country details on preventive chemotherapy coverage; and (iii) indicates the targets identified by WHO for the next decade and the tools that should be developed to attain these targets. The main message is that STH-attributable morbidity can be averted with evidence-informed program planning, implementation, and monitoring. Caution will still need to be exercised in stopping control programs to avoid any rebound of prevalence and loss of accrued morbidity gains. Over the next decade, with increased country leadership and multi-sector engagement, the goal of eliminating STH infections as a public health problem can be achieved.

Use of Complementary Natural Feed for Gastrointestinal Nematodes Control in Sheep: Effectiveness and Benefits for Animals.

The treatments of gastrointestinal nematodes (GIN) infection in sheep is almost exclusively based on the use of synthetic drugs. In some European regions the intensive use of antiparasitic drugs is leading to widespread development of anthelmintic resistance (AR). Currently in southern Italy AR is rare, but a constant monitoring of anthelmintic efficacy and the use of effective alternative therapies is strongly recommended. The aim of our study was to evaluate the effectiveness of a complementary natural feed (natural vegetable mixture), based on natural extracts, registered for GIN treatment in sheep, and its comparison with the drug ivermectin. The study was conducted in two sheep breeding farms in southern Italy and 75 sheep were divided in groups of 15 animals each (treated and untreated groups), homogeneous by GIN eggs per gram (EPG) of faeces, using the natural anthelmintic administered at full dose (10 g/sheep/orally) in the first breeding and at double dose (20 g/sheep/orally) in the second. In the latter we compared the effectiveness of mixture with ivermectin administered at full dose (200 µg/kg/BW). To determine the effectiveness, individual faecal samples were collected to evaluate the faecal eggs count (FEC) using FLOTAC technique and FEC reduction (FECR) on different days. The formula used FECR = 100 × (1 - (T2/C2)), based on the comparison of post-treatment EPG mean of the treated and untreated group (T2 and C2, respectively), is the one recommended by World Association for the Advancement of Veterinary Parasitology (W.A.A.V.P.) guidelines to monitor drug efficacy against GIN in livestock. The results reported that complementary natural feed, at two different dosages, was ineffective against GIN, while the drug, at conventional dosage, showed good anthelmintic efficacy, also confirming the importance of in vivo effectiveness studies.

Therapeutic efficacy of albendazole against soil-transmitted helminthiasis in children measured by five diagnostic methods.

BACKGROUND: Preventive chemotherapy (PC) with benzimidazole drugs is the backbone of soil-transmitted helminth (STH) control programs. Over the past decade, drug coverage has increased and with it, the possibility of developing anthelmintic resistance. It is therefore of utmost importance to monitor drug efficacy. Currently, a variety of novel diagnostic methods are available, but it remains unclear whether they can be used to monitor drug efficacy. In this study, we compared the efficacy of albendazole (ALB) measured by different diagnostic methods in a head-to-head comparison to the recommended single Kato-Katz. METHODS: An ALB efficacy trial was performed in 3 different STH-endemic countries (Ethiopia, Lao PDR and Tanzania), each with a different PC-history. During these trials, stool samples were evaluated with Kato-Katz (single and duplicate), Mini-FLOTAC, FECPAKG2, and qPCR. The reduction rate in mean eggs per gram of stool (ERR) and mean genome equivalents / ml of DNA extract (GERR) were calculated to estimate drug efficacy. PRINCIPAL FINDINGS AND CONCLUSIONS: The results of the efficacy trials showed that none of the evaluated diagnostic methods could provide reduction rates that were equivalent to a single Kato-Katz for all STH. However, despite differences in clinical sensitivity and egg counts, they agreed in classifying efficacy according to World Health Organization (WHO) guidelines. This demonstrates that diagnostic methods for assessing drug efficacy should be validated with their intended-use in mind and that other factors like user-friendliness and costs will likely be important factors in driving the choice of diagnostics. In addition, ALB efficacy against STH infections was lower in sites with a longer history of PC. Yet, further research is needed to identify factors that contribute to this finding and to verify whether reduced efficacy can be associated with mutations in the ß-tubulin gene that have previously been linked to anthelmintic resistance. TRIAL REGISTRATION: ClinicalTrials.gov NCT03465488.

In Vitro Anthelminthic Efficacy of Hypoestes forskaolii (Vahl) R.Br (Acanthaceae) Extracts on Gastrointestinal Nematodes of Sheep.

The anthelminthic efficacy of the crude extracts of Hypoestes forskaolii (Vahl) R.Br (Acanthaceae) against gastrointestinal nematodes (GIN) in sheep was investigated using the in vitro egg hatch inhibition assay. Faecal samples were collected from sheep with naturally occurring infection of GIN (Trichostrongylus spp., Chabertia ovina, Cooperia spp., Haemonchus contortus and Teladorsagia spp.). Crude leaf extracts of H. forskaolii was obtained using increasing polarity solvents: n-hexane, chloroform, chloroform:methanol 9:1, methanol. Thiabendazole (0.2 µg/mL and 0.5 µg/mL) was used as a positive control and untreated GIN eggs in deionised water served as the negative control. All the extracts exhibited a weak ovicidal activity against GIN (less than 50% of egg hatch). Noteworthy, the n-hexane extract showed a percentage of inhibition of egg hatching greater than other extracts inhibiting the 30.8% at the concentration of 1 mg/mL showing a dose-dependent effect on nematode eggs hatching. Further studies are needed to investigate the effects of extracts used and to evaluate the ovicidal effects of other extracts of H. forskaolii.

Comparison of three copromicroscopic methods to assess albendazole efficacy against soil-transmitted helminth infections in school-aged children on Pemba Island.

BACKGROUND: The diagnostic accuracy of three faecal egg count techniques (Kato-Katz, McMaster and FLOTAC) to assess albendazole efficacy against soil-transmitted helminth (STH) infections was compared. METHODS: The study is registered with Current Controlled Trials [identifier: ISRCTN90088840]. During September-November 2009, 304 school-aged children on Pemba Island, Tanzania, were screened and those infected with Ascaris lumbricoides, hookworm or Trichuris trichiura were treated with a single dose of albendazole (400 mg). Twenty-one days post-treatment, children provided a single stool sample which was examined using the same diagnostic methods. All stool samples were divided into two aliquots and one was fixed in 5% formalin and examined using FLOTAC and McMaster approximately 6 months after collection. RESULTS: Using fresh stool samples, comparable prevalences were demonstrated for the three methods at baseline (90-92.2% for T. trichiura, 41.1-52.8% for hookworm, 32.9-37.2% for A. lumbricoides); FLOTAC was the most sensitive method at baseline and follow-up. Albendazole showed high cure rate (CR) against A. lumbricoides (90-97%), moderate CR against hookworm (63-72%) and very low CR against T. trichiura (6-9%), regardless of the technique used. Egg counts (eggs per gram) at baseline were similar for A. lumbricoides and for hookworm among the three methods, and higher using McMaster and Kato-Katz compared with FLOTAC for T. trichiura. All methods were similar for hookworm and A. lumbricoides egg reduction rate (ERR) estimation, but Kato-Katz indicated a significantly higher ERR than McMaster and FLOTAC for T. trichiura. Preserved stool samples revealed consistently lower FECs at baseline and follow-up for all STHs. CONCLUSION: Further development and validation of standard protocols for anthelminthic drug efficacy evaluation must be pursued.