Antihelmintička efikasnost odabranih etarskih ulja protiv gastrointestinalnih nematoda ovaca

Abstract

Gastrointestinalne nematode (GIN) u današnje vreme predstavljaju jednog od najvećih zdravstvenih i ekonomskih problema kod malih preživara. Kontrola ovih parazita se uglavnom zasniva na primeni komercijalnih preparata. Međutim, njihova neracionalna upotreba u smislu učestalih tretmana, niskih ili visokih doza ili kontinuirane upotrebe jednog leka je dovela do razvoja antihelmintičke rezistencije (AR). Savremene strategije borbe protiv AR podrazumevaju racionalnu upotrebu ovih lekova, uz korišćenje različitih alternativa poput botaničkih antihelmintika. Ciljevi ovih istraživanja su bili ispitati antihelmintičku aktivnost odabranih etarskih ulja (EOs) protiv GIN ovaca i sigurnost njihove primene, odrediti faktore koji utiču aktivnost, kao i oceniti mogućnost primene ispitivanih formulacija u veterinarskoj praksi kao alternativne metode u borbi protiv AR. Ispitivanje je vršeno u dve faze (istraživanja), tokom 2019. i 2021. godine, pri čemu je u prvom ispitano 12 uzoraka ulja a u drugom11 (od čega su tri ista kao i u prethodnoj fazi, odnosno dva od istog proizvođača, jedno od drugog). Hemijski sastav ulja je određen pomoću gasne hromatografije i masene spektrometrije (GC-MS analize). In vitro ovicidna aktivnost je ispitana uz pomoć testa izleganja larvi (eng. egg hatch test, EHT), u prvom istraživanju pri šest različitih koncentracija za svako ispitano ulje (50, 12,5, 3,125, 0,781, 0,195 i 0,049 mg/ml), a u drugom su pored navedenih dodate još dve niže koncentracije (0,025 i 0,0125 mg/ml). Za prvo in vivo istraživanje su odabrani ulje timijana (tip 1) i sinergistička kombinacija linalool:estragol, a za drugo ulje origana, pitome nane i rtanjskog čaja. Efikasnost ulja je ispitana uz pomoć testa redukcije broja jaja u fecesu (eng. faecal egg count reduction test, FECRT), pri čemu su EOs razblažena u suncokretovom ulju a formulacije aplikovane jednokratno, peroralno životinjama u različitim grupama, u prvom istraživanju pri dozi od 100 mg/kg, a u drugom 150 mg/kg. Toksični efekti in vivo primenjenih formulacija su ispitani putem kliničkog pregleda testiranih životinja, a u drugom istraživanju i analizom hematoloških i biohemijskih parametara krvi u cilju procene efekata na krvnu sliku, funkciju jetre i bubrega. Za oglede su korišćene dve farme u južnoj Italiji, gde je u oba istraživanja putem koprokulture određeno prisustvo GIN kod ovaca, a prisutni rodovi identifikovani uz pomoć odgovarajućih ključeva determinacije. Rezultati GC-MS analiza su pokazali složen hemijski sastav testiranih etarskih ulja sa jedinjenjima čiji se broj kretao od 10-35, i koja pripadaju različitim hemijskim grupama (terpeni, terpenoidi i fenilpropanoidna jedinjenja). Najvišu ovicidnu aktivnost na EHT su u prvom istraživanju pokazala ulja origana, rtanjskog čaja i morača sa maksimalnim inhibitornim efektom na izleganje larvi (100%) pri svim testiranim koncentracijama, a pored njih i čubar (99,3-100%), oba uzorka timijana (timijan 1, 95,3-100% i timijan 2, 98,5-100%), kleka (81-96,8%) i pitoma nana (72,5-99,8%). U drugom istraživanju je zbog nešto drugačije pripreme koncentracija (metodom razblaživanja) efekat bio znatno više dozno-zavistan, ali je ulje origana bilo ponovo najefikasnije (71,3-93,3%), a potom miloduh (42,7-91,3%), korijander (29-92,7%), bosiljak (23,3-93,3%), pitoma (20-90,3%) i divlja nana (13-92,7%). Kada je u pitanju FECRT, u prvom istraživanju postignut je određen antihelmintički efekat sa ukupnom redukcijom broja jaja nematoda od 25,23% i 24,42% (timijan) i 24,91% i 25,90% (linalool:estragol), ali je efekat bio bolji kod ulja iz drugog istraživanja s obzirom na aplikaciju formulacija direktno u rumen životinja, odnosno 43,21% i 60,13% (origano), 26,86 i 46,04% (pitoma nana) i 15,66% i 33,00% (rtanjski čaj), 7. i 14. dana nakon tretmana. Ni za jedno od primenjenih ulja, pri testiranim dozama, nisu zabeleženi nikakvi neželjeni ili toksični efekti prilikom kliničkog pregleda odnosno hematoloških i biohemijskih analiza krvi, što govori u prilog bezbednosti njihove upotrebe kod ovaca. Pokazana efikasnost ispitanih ulja potiče od jedinjenja koja ulaze u njihov sastav, odnosno pre svih karvakrola, anetola, timola, p-cimena, γ-terpinena, karvona, linaloola, mentola, α-tujona i cis-pinokamfona, imajući u vidu njihovu zastupljenost kod najefikasnijih ulja. Koprokulturom je pri tome na ispitivanim farmama u oba istraživanja identifikovano prisustvo četiri roda GIN ovaca, Haemonchus, Trichostrongylus, Teladorsagia i Chabertia, čija se procentualna zastupljenost u određenoj meri menjala nakon tretmana. Primena biljnih formulacija uključujući EOs u cilju kontrole parazita ima više prednosti. To uključuje složen hemijski sastav sa bioaktivnim supstancama iz različitih hemijskihgrupa, koji dovodi do njihove visoke antihelmintičke aktivnosti, kao i potencijalno nižeg stepena razvoja rezistencije. Tehnike inkapsulacije ili višestruka primena tokom nekoliko uzastopnih dana pri tome nude mogućnost dodatnog povećanja efikasnosti u terenskim uslovima. Sa druge strane, prirodno poreklo ovih formulacija može doprineti i njihovoj nižoj toksičnosti za životinje, manjim količinama rezidua u mesu i mleku i boljoj ekološkoj prihvatljivosti u poređenju sa komercijalnim preparatima. U svakom slučaju, dobijeni rezultati govore u prilog mogućnosti korišćenja ispitanih EOs u praksi, posebno origana, u integrisanom pristupu kontrole u cilju smanjenja upotrebe komercijalnih preparata i usporavanja razvoja rezistencije.

Gastrointestinal nematodes (GINs) nowadays represent one of the biggest health and economic issues in small ruminants. The control of these parasites is almost exclusively based on the application of commercial drugs. However, their irrational use in terms of frequent treatments, low or high doses or continuous use of one drug has led to the development of anthelmintic resistance (AR). Modern strategies for combating AR are based on the rational use of these drugs, along with the use of various alternatives such as botanical anthelmintics. The aims of these studies were to examine the anthelmintic activity of selected essential oils (EOs) against sheep GINs and the safety of their application, to determine the factors that influence the activity, as well as to evaluate the possibility of applying examined formulations in veterinary practice as an alternative method in combatting AR. The research was conducted in two phases (studies), in 2019. and 2021., whereby 12 oil samples were examined in the first and 11 in the second study (of which three are the same as in the previous study, two from the same manufacturer and one from the other). The chemical composition of the oils was determined by gaschromatography and mass spectrometry (GC-MS analysis). In vitro ovicidal activity was tested using the egg hatch test (EHT), in the first instance at six different concentrations for each tested oil (50, 12.5, 3.125, 0.781, 0.195 and 0.049 mg/ml), and in the second, in addition to the above, two lower concentrations (0.025 and 0.0125 mg/ml) were added. Thyme oil (type 1) and the synergistic combination of linalool:estragole in the first, and the oils of oregano, mint and winter savory in the second study were selected for further in vivo testing. The efficacy was tested using the faecal egg count reduction test (FECRT), whereby the EOs were diluted in sunflower oil and the formulations were applied once, perorally to animals in different groups, in the first case at a dose of 100 mg/kg and in the second at 150 mg/kg. The toxic effects of the in vivo applied formulations were examined through clinical observation of the tested animals, and in the second study also by analyzing the haematological and biochemical blood parameters in order to assess the side effects on the blood count, liver and kidney function. Two farms in southern Italy were used for the experiments, whereby the presence of GINs was determined by coproculture examination, and the present genera were identified with suggested determination keys. The results of GC-MS analyses showed the complex chemical composition of the tested EOs with the number of compounds varying from 10-35, which belong to different chemical groups (terpenes, terpenoids and phenylpropanoid compounds). The highest ovicidal effect in the first EHT was shown by the oils of oregano, winter savory and fennel with the maximum inhibitory effect on the egg hatchability (100%), along with summer savory (99.3-100%), both samples of thyme (sample 1, 95.3-100% and sample 2, 98.5-100%), juniper (81-96.8%) and mint (72.5-99.8%). In the second study, due to a different preparation of concentrations (dilution method), the effect was significantly more dose-dependent, but the most effective oil was again oregano (71.3-93.3%) along with hyssop (42.7-91.3%), coriander (29-92.7%), basil (23.3-93.3%), mint (20-90.3%) and wild mint (13-92.7%). In the first FECRT, a certain anthelmintic effect was achieved with a total reduction in the number of nematode eggs of 25.23% and 24.42% (thyme) and 24.91% and 25.90% (linalool:estragole), but due to the application of the formulations directly in the rumen of animals, the effect was better with oils from the second study, i.e. 43.21% and 60.13% (oregano), 26.86 and 46.04% (mint) and 15.66% and 33.00% (winter savory) on Days 7 and 14, respectively. No toxic or side effects to the sheep were observed for any of the applied oils at the tested doses, neither during the clinical examination of animals nor during haematological and biochemical blood tests, suggesting the safety of their use in sheep. The demonstrated efficacy of the tested oils is related to the compounds that are included in their composition, i.e. above all carvacrol, anethole, thymol, p-cymene, γ-terpinene, carvone, linalool, menthol, α-thujone and cis-pinocamphon, considering their presence in the most effective oils. Four genera of sheep GIN were identified by coproculture examination in both studies, Haemonchus, Trichostrongylus, Teladorsagia and Chabertia, whose percentage representation changed to some extent after the treatments. The use of herbal formulations including EOs in parasite control has numerous advantages. That includes a complex chemical composition with bioactive substances from different chemical groups, which leads to high anthelmintic activity, as well as potentially lower susceptibility to AR development. Furthermore, encapsulation techniques or multiple applications over several consecutive days offer the possibility of further increasing the efficacy in field conditions. On the other hand, the natural origin of these formulations may contribute to lower toxicity for animals and smaller amounts of residues in meat and milk, as well as better environmental acceptability in comparison with commercial drugs. The obtained results suggest the possibility of using the tested EOs in practice, especially oregano, in an integrated control approach designed to reduce the use of commercial drugs and slow down the development of resistance.