Ovicidal Potential of Five Different Essential Oils to Control Gastrointestinal Nematodes of Sheep

The development of resistance to commercial anthelmintics, particularly in different species of gastrointestinal nematodes (GINs), requires the search for alternatives. Within that context, the aim of this study was to evaluate the in vitro ovicidal activity of five different essential oils (EOs): Origanum vulgare, Satureja hortensis, Thymus vulgaris, Mentha x piperita and Helichrysum arenarium against sheep GINs. For this purpose, the nematode eggs were collected from naturally infected sheep by GINs in two farms located in southern Italy. The 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. Gas chromatography-mass spectrometry chemical analyses of tested EOs, as well as coproculture examination of tested faecal samples, were also conducted. The results of EHT showed the greatest ovicidal activity of O. vulgare EO with a maximum effect on egg hatching (100%) for all tested concentrations. A similar effect was also shown by S. hortensis and T. vulgaris EOs with an activity of 99.3-100% and 98.5-100%, respectively. M. piperita EO showed medium, dose-dependent ovicidal activity with an inhibitory effect of 72.5-99.8% on the egg hatchability, while the least effective was H. arenarium EO with an activity of 59.8-69.3%. For the anthelmintic activities of the tested EOs are responsible their ingredients, above all carvacrol, thymol, p-cymene and gamma-terpinene. The present study demonstrated the significant anthelmintic potential of the tested EOs and emphasizes the possible importance of medicinal plant products for the control of gastrointestinal parasites in sheep

Epidemiology and spatial distribution of Echinococcus granulosus in sheep and goats slaughtered in a hyperendemic European Mediterranean area

Background: Cystic echinococcosis (CE) is a worldwide parasitic zoonosis caused by the larval stage of Echinococcus granulosus sensu lato affecting livestock, particularly sheep and goats. However, often this parasitosis is underestimated. For this reason, this study aimed to evaluate the epidemiological features and spatial distribution of CE in sheep and goats slaughtered in a hyperendemic Mediterranean area. Methods: A survey was conducted in the Basilicata region (southern Italy) from 2014 to 2019. A total of 1454 animals (1265 sheep and 189 goats) from 824 farms were examined for hydatid cyst detection by visual inspection, palpation and incision of target organs. All the CE cysts were counted and classified into five morphostructural types (unilocular, multiseptate, calcified, caseous and hyperlaminated). Molecular analysis was performed on 353 cysts. For spatial analysis, a kriging interpolation method was used to create risk maps, while clustering was assessed by Moran’s I test. Results: CE prevalence of 72.2% (595/824) and 58.4% (849/1454) was observed at the farm and animal levels, respectively, with higher values in sheep (62.9%) than goats (28.0%). The liver and lungs were the most frequently infected organs in both sheep and goats. Most of recovered cysts were of the calcified and multiseptate morphotypes. All the isolates were identified as E. granulosus sensu stricto (genotypes G1–G3). Spatial distribution showed a moderate clustering of positive animals. Conclusion: The findings of this study can be used to better understand the eco-epidemiology of echinococcosis and to improve CE surveillance and prevention programs in regions highly endemic for CE.Fil: Bosco, Antonio. Università degli Studi di Napoli Federico II; ItaliaFil: Alves, Leucio Camara. Universidad Federal Rural Pernambuco; BrasilFil: Cociancic, Paola. Università degli Studi di Napoli Federico II; Italia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Estudios Parasitológicos y de Vectores. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Centro de Estudios Parasitológicos y de Vectores; ArgentinaFil: Amadesi, Alessandra. Università degli Studi di Napoli Federico II; ItaliaFil: Pepe, Paola. Università degli Studi di Napoli Federico II; ItaliaFil: Morgoglione, Maria Elena. Università degli Studi di Napoli Federico II; ItaliaFil: Maurelli, Maria Paola. Università degli Studi di Napoli Federico II; ItaliaFil: Ferrer Miranda, Edyniesky. Universidad Federal Rural Pernambuco; BrasilFil: Santoro, Kleber Régis. Universidad Federal Rural Pernambuco; BrasilFil: Nascimento Ramos, Rafael Antonio. Universidad Federal Rural Pernambuco; BrasilFil: Rinaldi, Laura. Università degli Studi di Napoli Federico II; ItaliaFil: Cringoli, Giuseppe. Università degli Studi di Napoli Federico II; Itali

In vitro ovicidan efekat etarskog ulja kleke (Juniperus communis L.) na gastrointestinalne nematode ovaca

The negative influence of gastrointestinal parasitism in sheep is growing today due to the development of anthelmintic resistance to commercial drugs. For this reason, researchers around the world are looking for new, alternative strategies for controlling these parasites. In this context, medicinal plants and their products are increasingly mentioned. The aim of this study was to evaluate in vitro ovicidal activity of common juniper (Juniperus communis L.) essential oil against sheep gastrointestinal nematodes. For that purpose, the egg hatch test was used, and the obtained results were analyzed by analysis of variance (ANOVA) followed by comparison with Tukey’s test (p<0.05). Faecal samples were collected from two independent, natural-infected farms located in Eboli (SA), Italy, whereby the coproculture examination identified the presence of four genera of sheep gastrointestinal nematodes: Haemonchus (53%), Trichostrngylus (29.5%), Teladorsagia (14.5%) and Chabertia (3%). Main components of common juniper essential oil identified by GC-MS analysis were α-pinene (40.46%), sabinene (14.04%), myrcene (8.87%) and limonene (4.95%). In vitro ovicidal activity was evaluated at six different concentrations (50, 12.5, 3.125, 0.781, 0.195 and 0.049 mg/mL), whereby J. communis essential oil showed high activity with the inhibitory effect on hatchability of 81-96.75%, depending on the concentration. For all concentration tested, the effect was statistically significantly higher compared to the negative control, while for the three highest concentrations the effect was similar to the positive control. The obtained results suggested that J. communis essential oil have high anthelmintic potential. However, these results need confirmation in further field examination.Negativni uticaj gastrointestinalnih parazita kod ovaca u današnje vreme raste usled razvoja antihelmintičke rezistencije na komercijalne preparate. Iz tog razloga, istraživači širom sveta tragaju za novim, alternativnim strategijama u cilju kontrole ovih parazita. U tom kontekstu, lekovite biljke i njihovi proizvodi se sve češće spominju. Cilj ovog istraživanja je bio ispitati in vitro ovicidnu aktivnost esencijalnog ulja kleke (Juniperus communis L.) protiv gastrointestinalnih nematoda ovaca. Za ovu svrhu je upotrebljen egg hatch test, nakon čega su dobijeni rezultati obrađeni analizom varijanse i potom upoređeni Tukijevim testom (p<0.05). Uzorci fecesa su uzeti sa dve odvojene farme koje se nalaze u oblasti Eboli (SA), Italija, pri čemu je tokom ispitivanja koprokulture identifikovano prisustvo četiri roda gastrointestinalnih nematoda ovaca: Haemonchus (53%), Trichostrongylus (29.5%), Teladorsagia (14.5%) i Chaberita (3%). Najzastupljenije komponente etarskog ulja kleke identifikovane biohemijskim analizama su bile α-pinen (40.46%), sabinen (14.04%), mircen (8.87%) i limonen (4.95%*). In vitro ovicidna aktivnost je testirana za šest različitih koncentracija (50, 12.5, 3.125, 0.781, 0.195 i 0.049 mg/mL), pri čemu je etarsko ulje kleke pokazalo visoku aktivnost sa inhibitornim efektom na izleganje larvi od 81-96.75%, u zavisnosti od koncentracije. Kod svih ispitivanih koncentracija efekat je bio statistički značajno veći u poređenju sa negativnom kontrolom, dok je kod tri najveće koncentracije efekat bio sličan pozitivnoj kontroli. Dobijeni rezultati ukazuju na to da etarsko ulje kleke ima visok antihelmintički potencijal. Međutim, potrebno je ove rezultate potvrditi u budućim ispitivanjima u terenskim uslovima

In vitro ovicidna aktivnost dva tipa etarskog ulja hajdučke trave (Achillea millefolium L.) protiv jaja gastrointestinalnih nematoda ovaca

Economic losses caused by the gastrointestinal nematodes of small ruminants have been on increase mainly due to the development of anthelmintic resistance. Therefore, researchers from all around the world are searching for new, alternative strategies to control these parasites. Being a valuable natural resource, medicinal plants and their products have emerged as a viable option. This study aimed to evaluate the in vitro ovicidal activity of two chemotypes of yarrow (Achillea millefolium L.) essential oil against ovine gastrointestinal nematode eggs. Nematode eggs obtained from the faeces of naturally infected sheep on two farms located in Southern Italy were subjected to the egg hatch test. On both farms, the coproculture examination identified the presence of species belonging to four genera of sheep gastrointestinal nematodes: Haemonchus, Trichostrongylus, Teladorsagia and Chabertia. The main components of the yarrow essential oil identified by GC-MS analysis were 1,8-cineole (41.69%), camphor (8.37%) and trans-chrysanthenyl acetate (4.90%) in the oil type 1 and β-pinene (28.53%), β-caryophyllene (18.71%) and 1,8-cineole (11.69%) in type 2. The in vitro ovicidal activity was evaluated at six oil concentrations (50, 12.5, 3.125, 0.781, 0.195 and 0.049 mg/mL), whereby the inhibitory effect of the essential oil on egg hatchability varied from 46.5-99.5% (type 1) and from 69.6-97.25% (type 2). All concentrations tested showed a significantly higher efficacy compared to the negative control (p0.05) to the positive control (98.0%) at concentrations of 50 mg/mL (99.5%), 12.5 mg/mL (98.0%) and 3.125 (95.25%) of the oil type 1, and at concentrations of 50 mg/mL (97.25%) and 12.5 mg/mL (90.0%) of the type 2. The obtained results suggested that the A. millefolium essential oil has high anthelmintic potential, especially the chemotype rich in 1,8-cineole and camphor, which requires confirmation in further in vivo studies.Ekonomski gubici koje prouzrokuju gastrointestinalne nematode malih preživara u poslednje vreme rastu usled razvoja rezistencije na antihelmintike. Zbog toga istraživači širom sveta tragaju za novim, alternativnim strategijama za kontrolu ovih parazita. Kao dragocen prirodan resurs, lekovite biljke i njihovi proizvodi nametnuli su se kao moguća opcija. Cilj ovog istraživanja je bio utvrditi in vitro ovicidnu aktivnost dva hemotipa etarskog ulja hajdučke trave (Achillea millefolium L.) protiv jaja gastrointestinalnih nematoda ovaca. Jaja nematoda su izolovana iz fecesa prirodno inficiranih ovaca sa dve farme locirane u južnoj Italiji kako bi se sproveo test izleganja larvi (eng. egg hatch test). Na obe farme, koprokulturološkim ispitivanjima je utvrđeno prisustvo vrsta četiri roda gastrointestinalnih nematoda ovaca: Haemonchus, Trichostrongylus, Teladorsagia i Chabertia. Najzastupljenije komponente etarskog ulja hajdučke trave identifikovane GC-MS analizama bile su 1,8-cineol (41,69%), kamfor (8,37%) i trans-hrizantenil acetat (4,90%) u ulju tipa 1, odnosno β-pinen (28,53%), β-kariofilen (18,71%) i 1,8-cineol (11,69%) u tipu 2. In vitro ovicidna aktivnost je testirana u šest različitih koncentracija (50; 12,5; 3,125; 0,781; 0,195 i 0,049 mg/mL). Inhibitorni efekat etarskog ulja na izleganje jaja je varirao od 46,5-99,5% (ulje tipa 1), odnosno 69,6-97,25% (tip 2), dok je kod svih ispitivanih koncentracija efekat bio značajno veći u poređenju sa negativnom kontrolom (p0.05) pozitivnoj kontroli (98,0%) pri koncentracijama 50 mg/mL (99,5%), 12,5 mg/mL (98,0%) i 3,125 mg/mL (95,25%) ulja tipa 1, odnosno pri koncentracijama od 50 mg/mL (97,25%) i 12,5 mg/mL (90,0%) kod tipa 2. Dobijeni rezultati ukazuju na to da etarsko ulje hajdučke trave, naročito tipa 1 koje je bogato 1,8-cineolom i kamforom, poseduje visok antihelmintički potencijal, što zahteva potvrdu u daljim in vivo ispitivanjima

In Vivo Potential of Thyme (Thymus vulgaris L.) Essential Oil and Synergistic Combination of Linalool:Estragole to Control Sheep Gastrointestinal Nematodes

Economic losses caused by sheep gastrointestinal nematodes (GIN) are increasing nowadays due to the development of anthelmintic resistance, therefore requiring a search for alternatives. In doing so, field testing of new potential substances provides credible and realistic insight of current possibility of their use. The aim of this study was to evaluate the in vivo anthelmintic potential of thyme, Thymus vulgaris L. essential oil (EO) and synergistic combination of two compounds, linalool:estragole on days 7 and 14 post-treatment using the faecal egg count reduction test (FECRT). The experiment was conducted on two separate farms located in southern Italy, whereby the total number of sheep (n=96, n=48 per farm) was divided into four groups on each farm (n=12). Groups received the following treatments perorally: G1 – 100 mg kg-1 T. vulgaris; G2 – 100 mg kg-1 linalool:estragole; G3 – standard dose of fenbendazole and G4 – 50 ml per animal of sunflower oil. Main compounds of thyme EO identified by gas chromatographymass spectrometry (GC-MS) analyses were p-cymene (41.72%), thymol (31.59%) and α-terpineol (11.71%), and the coproculture examination showed the presence of four genera of sheep GIN on tested farms: Haemonchus, Trichostrongylus, Teladorsagia and Chabertia. In total, results of the FECRT showed the following reductions of GIN eggs on day 7: 24.95% (T. vulgaris); 24.64% (linalool:estragole) and 82.49% (fenbendazole). On day 14, reductions of GIN eggs were: 23.72% (T. vulgaris), 25.91% (linalool:estragole) and 88.82% (fenbendazole). Furthermore, no toxic effects were observed in animals at these doses. The present study reveals the anthelmintic potential of tested substances. However, a new trial with modified conditions is needed to improve their effectiveness: increased dosage, a different way of administration or possible use of encapsulated or nanoencapsulated forms of these substances, especially if bearing in the mind anatomical-physiological specifics of the ruminant gastrointestinal tract

Chemical Composition of Thyme Essential Oil and its Anthelmintic Properties

Thyme (Thymus vulgaris L.) is a well-known member of the genus Thymus with healing properties known since ancient times. Thyme has application in traditional medicine and pharmaceutical preparations, but also in the food and cosmetic industry. The aim of this study was to examine the chemical composition and in vitro anthelmintic potential of thyme essential oils from Serbia and compare it with sample from France. Chemical characterization of the samples was done by GC-MS and antihelmintic potential of tested samples was evaluated using in vitro egg hatch test on sheep gastrointestinal nematodes (concentration range 0.049-50 mg/mL) for each sample. The most represented compounds in sample from Serbia were: p-cymene (41.72%), thymol (31.59%), α-terpineol (11.71%), linalool (4.37%) and α-pinene (2.47%) and it inhibited egg hatchability for 95.25-100% and in sample from France thymol (54.48%), p-cymene (21.01%), γ-terpinene (8.11%), carvacrol (3.95%) and linalool (2.77%) were the dominant compounds and inhibited egg hatchability for 98.5-100%. The results indicate that chemical composition of thyme essential oil is dependant on geographic origin but regardless it poseses antihelmintic potential and may be used in veterinary medicine as anthelmintic agent

In vitro ovicidna aktivnost smeše linaloola i estragola protiv gastrointestinalnih nematoda ovaca

Based on the results in different studies conducted so far, plant essential oils (EOs) are recently been marked as a possible solution for the problem of anthelmintic resistance (AR). However, their efficacy against a wide range of livestock parasites is due to active ingredients. In that context, the aim of this study was to evaluate the in vitro ovicidal effect of a mixture of linalool and estragole (14% : 86%, respectively) against sheep gastrointestinal nematodes (GINs) using the egg hatch test (EHT). The study was conducted using faecal samples of naturally infected sheep by GINs from two different farms located in Southern Italy. Coproculture of tested faecal samples was performed, as well as chemical analyses of the tested mixture. The egg hatch test (EHT) was performed at six different concentrations (50, 12.5, 3.125, 0.781, 0.195 and 0.049 mg/mL) and the obtained values were compared to the positive control (thiabendazole, 0.025 mg/mL) and the negative control (3% Tween 80, v/v). The tested binary combination showed high and dose-dependent ovicidal activity varied from 29.5% to 100%. The inhibitory effect on egg hatchability was similar (p>0.05) to the positive control (98.0%) at concentrations of 50 mg/mL (100%), 12.5 mg/mL (100%) and 3.125 (99.8%). Moreover, all tested concentrations showed significantly higher (p<0.001) activity compared to the negative control. The obtained results suggest that the combination of linalool:estragole has the potential to be used against sheep GINs. These also highlight the possible role of different combinations of certain constituents of essential oils in combating AR. However, these findings need confirmation in further in vivo studies.Na osnovu rezultata do sada sprovedenih istraživanja, etarska ulja biljaka su označena kao jedna od mogućih opcija za rešenje problemarezistencije na antihelmintike. Pri tome, za njihovu efikasnost protiv različitih parazita domaćih životinja odgovorni su njihovi aktivni sastojci. U tom kontekstu, cilj ovog istraživanja je bio ispitati ovicidnu efikasnost smeše linaloola i estragola (14% i 86%, tim redom) protiv gastrointestinalnih nematoda (GIN) ovaca koristeći test izleganja jaja (egg hatch test (EHT)). U studiji su korišćeni uzorci fecesa ovaca prirodno inficiranih GIN sa dve različite farme u južnoj Italiji. Koprokultura testiranih uzoraka je sprovedena, kao i hemijske analize testirane smeše. EHT je sproveden za šest različitih koncentracija (50; 12,5; 3,125; 0,781; 0,195 i 0,049 mg/mL), pri čemu su dobijeni rezultati upoređeni sa pozitivnom (tiabendazol, 0.025 mg/mL) i negativnom (3% Tween 80) kontrolom. Testirana binarna kombinacija je pokazala snažnu, dozno-zavisnu ovicidnu aktivnost koja je varirala između 29,5% i 100%. Inhibitorni efekat na izleganje larvi je bio sličan (p<0,05) pozitivnoj kontroli (98,0%) pri koncentracijama od 50 mg/mL (100%), 12,5 mg/mL (100%) i 3,125 mg/mL (99,8%). Pri tome, kod svih testiranih koncentracija efekat je bio značajno veći (p<0,001) u poređenju sa negativnom kontrolom. Dobijeni rezultati ukazuju na to da ispitana smeša linaloola i estragola ima potencijal da se koristi protiv GIN ovaca. Takođe, ova studija ukazuje na moguću ulogu različitih sastojaka biljnih etarskih ulja u borbi protiv rezistencije na antihelmintike. Međutim, ovi nalazi zahtevaju dodatnu potvrdu u budućim in vivo ispitivanjima

Ovicidal Potential of Essential Oils of Juniperus communis and Achillea millefolium to Control Gastrointestinal Nematodes in Sheep

The worldwide increased difficulty to contrast gastrointestinal nematode (GIN) infections in sheep, due to growing anthelmintic resistance, has led to find alternative helminth control strategies. This study aimed to evaluate the in vitro ovicidal activity of essential oils (EOs) of Juniperus communis and Achillea millefolium (two chemotypes) against GINs of sheep. For this purpose, the nematode eggs were collected from naturally infected sheep by GINs in two farms located in Southern Italy. The 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 and compared to the positive control (thiabendazole, 0.025 mg/mL) and the negative control (Tween 80 3%, v/v). In both farms, coprocultures revealed the presence of four genera of GINs: Haemonchus, Trichostrongylus, Teladorsagia and Chabertia. The inhibitory effect on GIN eggs’ hatchability varied from 81% to 96.75% for J. communis EO. When using A. millefolium EO, the ovicidal activity against GIN was 46.5-99.5% (type 1) and 69.6-97.25% (type 2). Furthermore, the effect of tested EOs was very high at concentrations of 50 mg/mL (96.75%), 12.5 mg/mL (95.5%) and 3.125 mg/mL (94.75%) of J. communis EO, whilst for A. millefolium EO at concentrations of 50 mg/mL (99.5%), 12.5 mg/mL (98.0%) and 3.125 (95.25%) (type 1), and 50 mg/mL (97.25%) and 12.5 mg/mL (90.0%) (type 2), showing a similar effect (p>0.05) to the positive control (98%). Therefore, the findings of this study showed the anthelmintic potential of the tested EOs and highlight the importance of ethnopharmacology to control GIN infection in sheep

A FLOTAC and Mini-FLOTAC automated system for parasitological diagnosis in animals. New tools for Faecal Egg Count of gastrointestinal nematodes in ruminants

This PhD project has allowed to design, project and validate the Kubic FLOTAC Microscope, a new compact, low-cost, versatile digital microscope able to automatically scan the fecal specimens prepared with Mini-FLOTAC or FLOTAC techniques, used for Faecal Egg Count (FEC). This microscope will improve significantly the performances of diagnostic techniques. In this thesis, technical, mechanical, electronical and informatic aspects of Artificial Intelligence and Machine Learning were described. CHAPTER I provides an examination of the most common techniques for copromicroscopic diagnosis of gastrointestinal nematodes (GIN) in ruminants. Moreover, the limitations for each technique and the factors that led to the development of the FLOTAC and Mini-FLOTAC techniques are discussed. This chapter highlights also the factors that influence the variability of FEC techniques based on the flotation principle (e.g. method of preservation of faecal sample, choosing of flotation solution) and that are important for the standardization of methods. CHAPTER II shows the results of three studies aimed to evaluate and implement the performances of the Mini-FLOTAC techniques. These studies permitted to integrate and strengthen the data on Mini-FLOTAC techniques, in order to develop an automated system for the recognition and counting of GIN eggs in ruminants. In CHAPTER III is presented the Kubic FLOTAC Microscope (KFM), a digital microscope for the use in field and in laboratory for the reading of FLOTAC and Mini-FLOTAC through the use of Smartphone, Tablet and other newdevices, which allow to send via Internet the captured images for a real-time consultation, in vision of the Tele-Parasitology. The fully automated recognition and counting of the KFM system are in development. In addition, the first validations of the KFM were performed, using faecal samples collected from cattle experimentally infected with Cooperia oncophora and Ostertagia ostertagi at the Department of Virology, Parasitology and Immunology of the University of Ghent (Belgium). As described in the general discussion (CHAPTER IV), this doctoral thesis has allowed the development of a digital microscope which will be integrated, in the future, in a system that will permit the automation of the FLOTAC and Mini-FLOTAC techniques from the sampling to the reading phase