BIOLOGICAL CONTROL OF HAEMONCHUS CONTORTUS BY FUNGAL ANTAGONISTS IN SMALL RUMINANTS

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1 Khttk et l.: Biologicl control of Hemonchus contortus by fungl ntgonists in smll ruminnts BIOLOGICAL CONTROL OF HAEMONCHUS CONTORTUS BY FUNGAL ANTAGONISTS IN SMALL RUMINANTS KHATTAK, B. 1* SAFI, A. U. R. 1 SINDHU, Z. U. D. 2 ATTAULLAH, M. 3 JAMAL, Q. 1 KHAN, T. A. 1 HUSSAIN, M. 1 ANJUM, S. I. 4 ISRAR, M. 5 KHAN, I. A. 6 1 Deprtment of Microbiology, Koht University of Science nd Technology, Pkistn 2 Deprtment of Prsitology, University of Agriculture Fislbd, Pkistn 3 Deprtment of Zoology, University of Mlknd 4 Deprtment of Zoology, Koht University of Science nd Technology, Pkistn 5 Pkistn Science Foundtion Islmbd, Pkistn 6 Deprtment of Weed Science, The University of Agriculture Peshwr, Pkistn *Corresponding uthor e-mil: bhrkk75@gmil.com; phone: (Received 10 th My 2018; ccepted 5 th Jul 2018) Abstrct. Hemonchus contortus is highly pthogenic nemtode tht cuses hemonchosis in ruminnts. As H. contortus is reported to be resistnt to nthelmintic drugs, biologicl control might be the lterntive pproch. The study ws conducted in Pkistn on different prmeters to control H. contortus biologiclly by fungi. Alternri lternt, Fusrium oxysporum, Aspergillus niger, Trichoderm hrzinum nd Ochroconis gllopv were isolted from the feces nd boms of infected domestic sheep (Ovis ries) nd gots (Cpr eggrus hircus). Different concentrtions of the extrcted spore-free cultures of these fungi were pplied on eggs, lrve nd dults of H. contortus for eggs htching inhibition ssy, lrvl mortlity ssy nd dult mortlity ssy. In eggs htching inhibition ssy, the higher concentrtions i.e. 100% nd 50% of A. lternt, F. oxysporum, A. niger, T. hrzinum nd O. gllopv cused 100% inhibition of eggs. In dult mortlity ssy, ll the culture filtrtes cused no mortlity in the first nd second hour while in the third hour of the post tretment mortlity vried with concentrtions. In lrvl mortlity ssy, higher concentrtions i.e. 100% nd 50% of T. hrzinum nd A, niger cused 100% mortlity of L 3 lrve of H. contortus. All the extrcted spore-free cultures significntly cused eggs htching inhibition. Higher concentrtions of Trichoderm nd Aspergillus significntly cused lrvl mortlity (100%) while dult mortlity ssys were dose nd time dependent nd insignificntly cused dult mortlity of H. contortus. Keywords: nemtode prsite, hemonchosis, biocontrol, fungl extrcts, egg htch ssy, dult mortlity ssy Introduction Livestock plys n importnt role in the economy of Pkistn. Agriculture contributes 21% in ntionl gross domestic product (GDP) in which shre of livestock is 11.9%. The estimted popultion of domestic sheep (Ovis ries) ws 29.1 million while domestic gots (Cpr eggrus hircus) were 66.6 million during According to survey in Pkistn, bout tons of met ws obtined from sheep during the yer (Economic Survey of Pkistn ). Smll ruminnt livestock re highly susceptible to gstrointestinl nemtodes (GINs). Among GINs, Hemonchus contortus, member of the fmily Trichostrongylide (Rdostits et l., 2006), is highly pthogenic nemtode prsite tht cuses hemonchosis in sheep nd got, n infection chrcterized ISSN (Print) ISSN (Online) DOI: , ALÖKI Kft., Budpest, Hungry

2 Khttk et l.: Biologicl control of Hemonchus contortus by fungl ntgonists in smll ruminnts by nemi, protein deficiency, bottle jw nd digestive disturbnces throughout the world (Getchew et l., 2007; Bowmn et l., 2009). It is estimted tht n infected sheep with 5000 H. contortus worms my lose 250 ml blood/dy. Life cycle of H. contortus tkes bout 21 dys to complete. H. contortus life cycle is direct i.e. without the involvement of secondry host. Every dult femle prsite lys between ,000 eggs/ dy which re pssed out in the feces (Hepworth, 2006) s shown in Figure 1. Figure 1. Life cycle of Hemonchus contortus. (Courtesy: pinterest.com) As H. contortus is reported to be resistnt to nthelmintic drugs (Miller et l., 1987; Sngster, 1999; Jckson nd Coop, 2000) biologicl control might be the lterntive pproch. Mny fungl species hve been isolted from nemtodes in which Duddingtoni flgrns hs proven to be the most effective nemtode-destroying fungus (Lrsen et l., 1997) whose spores re found to survive while pssing through the gstro-intestinl trct of grzing livestock (Hertzberg et l., 2001). Trichoderm, Fusrium nd Acremonium hve ovicidl effect on Toxcr cnis ov (Mi Filho et l., 2011). The present study ws crried out in the light of previous work done to isolte nd identify fungi from the feces of infected sheep nd gots to evlute the fungl filtrtes bilities of suppression the popultion of H. contortus on eggs htching level, lrvl developmentl level nd dult level. These fungl extrcts cn be used s biologicl control ginst this prsite s spry on grzing pstures or cn be fed to nimls in the form suspensions or pellets. Moreover, ll fungi investigted in this study re of less toxigenic nture to nimls helth so mking them more suitble cndidtes for the purpose of the study. Mterils nd methods This reserch work crried out in lbortory of the Deprtment of Microbiology, Koht University of Science nd Technology, Koht, Pkistn. Smpling 100 fecl smples of rectum from live gots nd sheep nd 100 boms smples from freshly slughtered gots nd sheep were collected from different res of Koht- ISSN (Print) ISSN (Online) DOI: , ALÖKI Kft., Budpest, Hungry

3 Khttk et l.: Biologicl control of Hemonchus contortus by fungl ntgonists in smll ruminnts Pkistn ( N E). The smples were nlyzed for the detection of eggs (fecl smples) nd worms (boms smples) of H. contortus s shown in Tble 1. All the smples were processed in the deprtment of Microbiology, Koht University of Science nd Technology Koht, Pkistn. Tble 1. Smples collected nd nlyzed for the detection of eggs nd worms of H. contortus Ruminnts Fecl smples Positive (eggs seen) Negtive (no eggs) Aboms Positive (worms seen) Negtive (no worms) Gots Sheep Totl Hemonchus contortus (eggs, dults nd lrve) Eggs of H. contortus were identified nd concentrted from fecl smples by simple test tube flottion method (Dryden et l., 2005), Mc Mster counting chmber (Soulsby, 1968) nd isoltion of eggs from feces s described by (Hubert nd Kerboeuf, 1992). Adult worms were collected from boms bsed on their typicl brber pole ppernce of femle worms in clen continer s shown in Figure 2. Collected worms were wshed with distilled wter nd concentrted in continer hving freshly prepred phosphte buffer sline (PBS). Third stge lrve (L 3 ) were cultivted from the eggs recovered from dult femle worms following the protocol of (Amrnte, 2011) with slight modifiction. The L 3 were morphologiclly differentited nd identified s described by (Vn Wyk et l., 2004). Figure 2. Hrvesting worms from bomusm Fungi (isoltion, identifiction nd extrction) All positive fecl nd boms smples were processed for the isoltion of fungi. One-grm smples contents were serilly diluted nd poured on Sbourud dextrose gr (SDA) medi for fungl growth. After 72 h, the grown colonies were sub cultured to isolte pure cultures. For identifiction, both colony morphology nd microscopic chrcteristics were deeply compred with the identifiction keys provided by (Nvi et l., 1999) nd (Lrone nd Lrone, 1987). Among mny other isoltes, Alternri ISSN (Print) ISSN (Online) DOI: , ALÖKI Kft., Budpest, Hungry

4 Khttk et l.: Biologicl control of Hemonchus contortus by fungl ntgonists in smll ruminnts lternt, Fusrium oxysporum, Aspergillus niger, Trichoderm hrzinum nd Ochroconis gllopv were the min isoltes present in ll positive smples. So these fungi were selected for fungl extrcts. For extrction, fungi in broth medi were incubted in shker incubtor for 7 dys t 25 C in drk. After 7 dys, they were filtered by common Whtmn filter ppers nd the filtrtes were gin filtered through Millex HA syringe driven filters (0.45 µm). Different concentrtion i.e. 100%, 50%, 25%, 12.5% nd 6.25% were mde from ech in phosphte buffer sline (PBS) nd distilled wter seprtely. Finl filtrtes were tested on potto dextrose gr (PDA) nd SDA medi to confirm the bsence of fungl spores. Egg htch ssy (EHA) EHA ws performed in triplicte following the protocol of (Coles et l., 1992) with slight modifiction. Isolted eggs were dissolved in 15 ml distilled wter with concentrtion djusted to eggs/75 µl. Then 75 µl egg suspension ws dded to ech well of 24-welled titrtion plte hving 2.5 ml of ech concentrtion. For positive control, mg/ml of Oxfendzole (Glxo-welcome) ws prepred in 0.3% dimethyl sulfoxide (DMSO). Wells hving egg suspension nd 2.5 ml tp wter with no extrcts were used s negtive control. The plte ws incubted t 25 C for 48 h. After 48 h, one drop of Lugol s oil ws dded to stop the rection. Then the plte ws observed under 10X of inverted microscope s shown in Figure 3. The percent htching inhibition ws observed s: PHITest Htching Inhibition % 100 P Totl P HI Test = number of unhtched eggs, P HI Totl = number of unhtched eggs + number of htched eggs. Adult mortlity ssy (AMA) In AMA, ten worms were treted in triplicte to ech filtrte concentrtion t room temperture. 25 µg/ml of Closntel ws used s positive control. Phosphte buffer sline with worms nd no fungl extrcts were used s negtive control. After ech one hour of post tretment, ll the pltes were observed to check the mortlity till ll worms were ded in negtive control. Becuse dult worms cnnot survive without host longer thn 9 h. The percent mortlity ws clculted s: PAMTest Adult Mortlity % 100 P Totl P AM Test = number of ded worms, P AM Totl = number of ded worms + number of live worms. Lrvl mortlity ssy (LMA) One ml of L 3 lrvl suspension contining 10 lrve were dded in triplicte to ech well of titrtion plte hving 2.5 ml of fungl filtrtes. Negtive control well received AM HI ISSN (Print) ISSN (Online) DOI: , ALÖKI Kft., Budpest, Hungry

5 Khttk et l.: Biologicl control of Hemonchus contortus by fungl ntgonists in smll ruminnts only one ml L 3 suspension nd 2.5 ml phosphte buffer sline while positive control well received one ml L 3 suspension nd 2.5 ml closntel (25 µg/ml). The plte ws incubted t room temperture for 3 h. After 3 h, the plte ws observed under 10X of inverted microscope. Percent mortlity ws clculted s: PLMTest Lrvl Mortlity % 100 P Totl P LM Test = number of ded lrve, P LM Totl = number of ded lrve + number of live lrve. LM Figure 3. H. contortus eggs under inverted microscope Sttisticl nlysis Dt obtined from bio-ssys i.e. EHA, AMA nd LMA were nlyzed by ONE Wy ANOVA nd TWO-Wy ANOVA through Sttistix version 9 nd GrphPd Prism version 5 softwre. The p vlues <0.05 ws considered significnt. Results Egg htch ssy After three dys incubtion, the effect of different concentrtion of ll fungl filtrtes on eggs, were observed s shown in Figure 4. Higher concentrtions i.e. 100% nd 50% of ll filtrtes cused 100% inhibition while inhibition by lower concentrtions vried from specie to specie. There were 100% nd no inhibition (0%) in positive nd negtive control respectively. Adult mortlity ssy All concentrtions of fungl filtrtes treted with dult worms cused no mortlity in the first three hours of post tretment. After three hours, the results were dose nd time dependent nd cused insignificnt percent mortlity s shown in Figure 5. ISSN (Print) ISSN (Online) DOI: , ALÖKI Kft., Budpest, Hungry

6 Percent (%) Inhibition fter 3 dys Khttk et l.: Biologicl control of Hemonchus contortus by fungl ntgonists in smll ruminnts P vlue = <0.05 = significnt 100 Conc 50 Conc 25 Conc 12.5 Conc 6.25 Conc T. hrzinum O. gllopv F. oxysporum A.lternt A.niger -ve control +ve control Figure 4. Percent inhibition of eggs htching vi fungl filtrtes ISSN (Print) ISSN (Online) DOI: , ALÖKI Kft., Budpest, Hungry

7 Percent (%) Mortlity fter 3 hours Khttk et l.: Biologicl control of Hemonchus contortus by fungl ntgonists in smll ruminnts Figure 5. Adult worms post-exposure ginst different concentrtion of fungl extrcts. A. F. Oxysporum. B. A. lternte, C. T. hrzinum, D. A. niger, E. O. gllopv Lrvl mortlity ssy After 3 h post tretment of L 3 lrve with fungl filtrtes, higher concentrtions i.e. 100% nd 50% of the Trichoderm hrzinum nd Aspergillus niger cused 100% mortlity while 100% concentrtions of F. oxysporum nd A. lternt cused 94% nd 91% mortlity respectively. Mortlity cused by lower concentrtions vried from species to species. 100% nd no mortlity in positive nd negtive control respectively s shown in Figure 6. = significnt 100 Conc Conc 25 Conc 12.5 Conc 6.25 Conc T. hrzinum O. gllopv F. oxysporum A. lternt Fungi A. niger - control Figure 6. Percent mortlity of L 3 lrve fter 3 h post tretment + control Overll effect on Hemonchus contortus When the verges of ll concentrtions of ech fungl tretment were nlyzed by Lest Significnt Difference (LSD) nd All-Pirwise Comprison (APC) tests, the ISSN (Print) ISSN (Online) DOI: , ALÖKI Kft., Budpest, Hungry

8 Averge effect of fungl tretments Khttk et l.: Biologicl control of Hemonchus contortus by fungl ntgonists in smll ruminnts results showed tht ll the tested fungi significntly ffected ll stges of H. contortus. Pirwise comprison of fungi showed tht ll fungi hve equl significnt effect on eggs of H. contortus while the significnce of efficcy on dults nd lrve vries from fungl to fungl species s shown in Figure F. oxysporum A. lternt T. hrzinum A. niger O. gllopv ADULTS LARVAE EGGS H. contortus stges Figure 7. Averge effect of fungl tretments on dults, lrve nd eggs of H. contortus Discussion To control Hemonchus contortus, common nthelmintics re pplied but not enough to erse this prsite completely s it is reported to be resistnt to ll three brod spectrum fmilies of nthelmintics viz. benzimidzole, lmidzothizole nd ivermectin nd ginst drugs with nrrow spectrum of ctivity such s slicylnilides nthelmintics (Kpln nd Vidyshnkr, 2012). Keeping in view the importnce of this blood sucker, there is need of lterntive methods other thn common nthelmintics to prevent sheep nd gots from this highly dngerous nemtode prsite. Biologicl control is n environment friendly lterntive pproch towrds these nemtodes. Among the microorgnisms tht prsitize on nemtodes, fungi hold n importnt position in continuously eliminting prsitic nemtodes (McSorley et l., 2008). About 70 gener nd 200 species of fungi hve been investigted tht they re ssocited with nemtode prsites. Till now, only few of them re successful biocontrol gents (Nordbring- Hertz et l., 2011). In the present study, it ws tried to investigte the fungl bilities biologiclly to suppress the H. contortus popultion on eggs htching, lrvl nd dult levels. Fungi ISSN (Print) ISSN (Online) DOI: , ALÖKI Kft., Budpest, Hungry

9 Khttk et l.: Biologicl control of Hemonchus contortus by fungl ntgonists in smll ruminnts were isolted from the feces of smll ruminnts nd different concentrtions of culture extrcts were mde from the isolted fungi. The spore-free cultures were pplied on eggs, L 3 lrve nd dult worms of H. contortus to find out the percent inhibition nd percent mortlity. The results demonstrted tht the higher concentrtions i.e.100% nd 50% of ll the extrcted spore-free cultures of A. lternt, F. oxysporum, A. niger, T. hrzinum nd O. gllopv showed 100% eggs htching inhibition of H. contortus. For the first time these fungi were pplied on eggs of H. contortus lthough in previous studies mny other nemtophgous nd filmentous fungi such s Arthrobotrys oligospor, A. musiformis, Cldosporium spp, A, flvus nd Duddingtoni flgrns were treted with H. contortus eggs nd showed similr results (Mi Filho et l., 2011). Also, different plnt extrcts hve been reported previously to ffect the biology of the prsitic eggs in the sme rtio (Hounzngbe-Adote et l., 2005). Adult worms of H. contortus in this study were treted with different concentrtions i.e. 100%, 50%, 25%, 12.5% nd 6.25% of the extrcted spore-free cultures. The results were dose nd time dependent s compred to similr study conducted on nimls grzing on pstures where D. flgrns ws present which showed 97% efficcy of the fungus ginst this nemtode prsite (Mri et l., 2011). Another study conducted by Chndrwthni nd collegues (Chndrwthni et l., 2003) in which they fed sheep during 24 dys with either minerl blocks contining dose of 500,000 chlmydospores/kg bw/dy or pellets with the sme fungl dose, nd obtined reduction in dults of H. contortus of >95% in the first cse nd 98% in the second cse. The third stge lrve (L 3 ) of H. contortus in the present study were treted with different concentrtions of ech extrcted spore-free cultures for three hours. After three hours, it ws observed tht higher concentrtions i.e. 100% nd 50% of the Trichoderm hrzinum nd Aspergillus niger cused 100% mortlity in the L 3 lrve while the mortlity cused by lower concentrtions of extrcts vried from species to species. These results resemble recent study conducted in Irn by Zrrin nd collegues in which they demonstrted tht some sprophytic fungi spred in the environment cn be effective in reducing the number of third stge lrve of Trichostrongylide fmily. However, the rte of mortlity ws different becuse of the fungl species nd isoltes. They concluded tht there ws considerble decrese in lrve popultion by Cldosporium spp. (98.8%), Trichoderm spp. (98.5%), nd Aspergillus spp. (96.7%) culture. Their results indicted gret potentil of using pproprite sprophyte fungi for biologicl control of H. contortus lrve in sheep (Zrrin et l., 2015). A similr study conducted on stge 3 lrve of H. contortus by Ivonneet l., 2011 investigted tht mortlity of infective lrve (L 3 ) by A. oligospor ws 90.3%, A. musiformis ws 97%, nd M. thumsium ws 62.3% (Ivonne et l., 2011). Also, Menduz De Gives nd Vzquez-prts investigted tht A. oligospor, Moncrosporium cermtoum nd A. robust cused mortlity of the popultion of H. contortus infective L 3 lrve with 93.30%, 95.70% nd 10.10%, respectively (Mendoz- De Gives nd Vzquez-Prts, 1994). Conclusion nd recommendtions From the study, it is concluded tht higher concentrtions of ll the extrcted sporefree culture filtrtes from the isolted fungi significntly cused eggs htching inhibition. Higher concentrtions of Trichoderm hrzinum nd Aspergillus niger significntly cused lrvl mortlity (100%) while dult mortlity ssys were dose nd ISSN (Print) ISSN (Online) DOI: , ALÖKI Kft., Budpest, Hungry

10 Khttk et l.: Biologicl control of Hemonchus contortus by fungl ntgonists in smll ruminnts time dependent nd insignificntly cused dult mortlity of Hemonchus contortus. Keeping in view the importnce of this problem nd encourging results obtined from the study, there is need to explore the chemistry of the fungl filtrtes to find out their ctive compounds nd their mechnism of ctions (both in vitro nd in vivo). The effects of these fungi t moleculr level like DNA dmge nd genetic ltertions in H. contortus need further explortions. Acknowledgements. We sincerely cknowledge Pkistn Science Foundtion Islmbd for providing us finncil support with grnt number (PSF/NSLP/KP-KUST-298). Also, thnks go to fculty nd stff of Deprtment of Microbiology, Koht University of Science nd Technology Koht nd Deprtment of Prsitology, University of Agriculture Fislbd for their Technicl support. REFERENCES [1] Amrnte, A. F. (2011): Why is it importnt to correctly identify Hemonchus species? Revist Brsileir de Prsitologi Veterinári 20(4): [2] Bowmn, D. D. (2009): Georgis Prsitology for Veterinrins. 9 th ed. Sunders Elsevier, St; Louis, pp [3] Chndrwthni, P., Jmnh, O., Wller, P. J., Lrsen, M., Gillespie, A. T., Zhri, W. M. (2003): Biologicl control of nemtode prsites of smll ruminnts in Mlysi using the nemtophgous fungus Duddingtoni flgrns. Veterinry Prsitology 117(3): [4] Coles, G. C., Buer, C., Borgsteede, F. H. M., Geerts, S., Klei, T. R., Tylor, M. A., Wller, P. J. (1992): World Assocition for the Advncement of Veterinry Prsitology (WAAVP) methods for the detection of nthelmintic resistnce in nemtodes of veterinry importnce. Veterinry Prsitology 44(1): [5] De Souz, M. F., Vieir, N., Berne, J. A., Stoll, F. E., Nscente, P., Potter, L. (2013): Fungl ovicidl ctivity on Toxocr cnis eggs. Revist Iberomricn de Micologi 30: [6] Dryden, M. W., Pyne, P. A., Ridley, R., Smith, V. (2005): Comprison of common fecl flottion techniques for the recovery of prsite eggs nd oocysts. Veterinry Therpeutics 6(1): [7] Economic Survey of Pkistn ( ): [8] Getchew, T., Dorchies, P., Jcquiet, P. (2007): Trends nd chllenges in the effective nd sustinble control of Hemonchus contortus infection in sheep. Prsite 14(1): [9] Hepworth, K., Nery, M., Hutchens, T. (2006): Mnging Internl Prsitism in Sheep nd Gots. Purdue University Coopertive Extension Service, West Lfyette, IN, pp [10] Hertzberg, H., Lrsen, M., Murer, V. (2002): Biologicl control of helminths in grzing nimls using nemtophgous fungi. Berliner und Munchener Tierrztliche Wochenschrift 115(7): [11] Hounzngbe-Adote, M. S., Polini, V., Moutirou, I. K., Hoste, H. (2005): In vitro effects of four tropicl plnts on three life-cycle stges of the prsitic nemtode, Hemonchus contortus. Reserch in Veterinry Science 78(2): [12] Hubert, J., Kerbouef, D. (1992): A micro lrvl development ssy for the detection of nthelmintic resistnce in sheep nemtodes. Veterinry Record 130(20): [13] Ivonne, C. A. G., de Gives, P. M., Hernández, E. L., Arellno, E. L., Vlero Coss, R. O., Velázque, M. H. (2011): Nemtophgous fungi (Orbililes) cpturing, destroying nd ISSN (Print) ISSN (Online) DOI: , ALÖKI Kft., Budpest, Hungry

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