Research ArticleCoadministration of Nematophagous Fungi for BiologicalControl over Nematodes in Bovine in the South-Eastern Brazil

Faacutebio Dias Luns1 Rafaela Carolina Lopes Assis 1 Laryssa Pinheiro Costa Silva2

Carolina Magri Ferraz2 Faacutebio Ribeiro Braga2 and Jackson Victor de Arauacutejo1

1Department of Veterinary Medicine Laboratory of Parasitology Federal University of Vicosa Av PH RolphsVicosa Campus 36570-000 Vicosa MG Brazil2Department of Veterinary Medicine University Vila Velha Avenue Comissario Jose Dantas de Melo 21Boa Vista 29102-920 Vila Velha ES Brazil

Correspondence should be addressed to Rafaela Carolina Lopes Assis rafaelalopesassisyahoocombr

Received 13 October 2017 Revised 19 January 2018 Accepted 20 February 2018 Published 26 March 2018

Academic Editor Juan F J Torres-Acosta

Copyright copy 2018 Fabio Dias Luns et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

This study compared the coadministration among the three nematode predatory fungi Duddingtonia flagrans Monacrosporiumthaumasium and Arthrobotrys robusta in the biological control of cattle gastrointestinal nematodiasis in comparison with the useof the fungusD flagrans alone Five groups consisting of eight Girolando heifers were kept in paddocks of Brachiaria decumbens forsix months Each heifer received 1 g10 kg of pellets containing the fungi (02 g of fungus10 kg bw) Group 1 (G1) received pelletswith D flagrans and M thaumasium in coadministration G2 received D flagrans and A robusta G3 received M thaumasiumA robusta and D flagrans and G4 received the fungus D flagrans alone Group 5 (control) received pellets without fungi Themonthly mean of fecal egg count (FEC) of Groups 1 2 3 and 4 were 938 853 827 and 964 smaller than the mean of controlgroupThe treatments with pellets containingD flagrans orD flagrans +M thaumasium produced significantly better results thanthe D flagrans + A robusta or the combination of the three fungi The associations which include A robusta were less efficient inthis study than D flagrans alone or associated withM thaumasium

1 Introduction

Nematode-trapping fungi are the most studied group offungal nematode antagonistsThey have the greatest potentialfor destroying infectious forms of gastrointestinal nema-tode parasites among both animals and humans [1 2]The fungal species Duddingtonia flagrans Monacrosporiumthaumasium and Arthrobotrys robusta were identified aspredators of nematodes and they have been studied asbiological control agents for these parasites [3ndash5] Researchon the application of the nematode-trapping fungiD flagrans[6 7] A robusta [5 8] and M thaumasium [9 10] in thetreatment of gastrointestinal nematodiasis in bovines hasdemonstrated the potential of these fungi in reducing thefree-living stages of parasitic nematodes (L3) in the fieldThe use of more than one biocontrol agent is considered aprimary suppressive measure that contributes to controllingthe presence of infectious agents in soil [11] The use of a

combination of several nematophagous fungi can minimizeany potential flaws in their individual administration orit may even enhance their actions as biocontrol agents[12] Furthermore numerous biological control mechanisms(including the production and use of substances that exertfungicidal effects) may vary among species and even amongisolates of the same species resulting in interactions betweenfungi that may interfere with their antagonistic performance[13] However most of the studies examining biologicalcontrol in cattle have been conducted with fungal isolatesapplied alone There are no reports of previous in vivostudies that have evaluated the biological control achievedfollowing coadministration of D flagrans A robusta andMthaumasium in extensive systems of dairy cattle It is unclearwhether the coadministration of a few of these species couldyield additive effects from a biological standpoint [14] Manystudies have already proven the effectiveness of the fungusD flagrans in the biological control of nematodes of several

HindawiBioMed Research InternationalVolume 2018 Article ID 2934674 6 pageshttpsdoiorg10115520182934674

2 BioMed Research International

species [1ndash3 6 7] We then chose this fungus to comparethe treatment groups with fungal association In this contextthe objective of this study was to evaluate the effects of thecoadministration of nematode-trapping fungi A robusta Dflagrans andM thaumasium on gastrointestinal helminths incombination would be synergistic or antagonistic in naturecompared to Duddingtonia flagrans used alone

2 Materials and Methods

21 Area of Study Theexperimentwas carried out at a privatefarm located in the municipality of Ouro Branco state ofMinas Gerais in the south-eastern region of Brazil 20∘3110158401510158401015840south latitude and 43∘4110158403110158401015840 west longitude from April toSeptember 2012 The paddockrsquos topography is undulating tohilly (5 flat 60 undulating and 35 hilly) with an meanaltitude of 1052m (maximum 1568m minimum 1099m)and featuring native vegetation indicative of a transitionzone between the Atlantic forest and savanna The climate istropical (Koppen-Geiger climate classification Aw) with anannualmeanmaximum temperature of 22∘C and aminimumtemperature of 7∘C and featuring an mean annual rainfall of1200mm

22 Fungi and the Production of a Mycelial Mass Isolates ofthree fungal species that are known predators of nematodeswere used A robusta (I31) D flagrans (AC001) and Mthaumasium (NF34) These isolates were obtained from soilsin the Zona da Mata region of the state of Minas GeraisBrazil Mycelium was obtained by transferring disks (sim4mmin diameter) which were cultured with the fungal isolatesin 2 water agar (2 WA) to Erlenmeyer flasks (250mL incapacity) containing 150mL of liquid glucose-yeast-peptone(GYP) medium [3] These flasks were then incubated inthe dark under agitation at 120 rpm at 26∘C for 10 daysFollowing this period the fungal mycelium was removedand weighed on an analytical balance All of the proceduresfollowed the methodology of Araujo et al [10]

23 Experimental Animals At the beginning of the exper-iment a total of 40 6-month-old Girolando heifers withan mean body weight (bw) of 120 kg were pretreated with10 albendazole (Mogivet Lab Brazil) which was orallyadministered at a dose of 1mL20 kg of bw Fifteen days afterthe antihelminthic treatment the heifers were separated intoone of five groups (Groups 1 2 3 4 and 5) consisting of eightheifers each based on the animalsrsquo mean weight

The heifers were allocated in five paddocks of Brachiariadecumbens that had been previously grazed by young andadult animals and which were naturally infected with gas-trointestinal helminth parasites Each paddock had an areaof 15 ha Each group was allocated in only one paddockwithout rotational grazing during the experimental periodEach animal from all groups treated (G1 G2 G3 and G4)received 1 g of pellets (02 g of fungal mycelium) per 10 kg ofbw The animals from Group 5 received 1 g of fungus-freepellets per 10 kg of bw All of the animals received the pelletsorally twice a weekThe pellets were mixed in a concentrated

and balanced ration provided for dairy cattle (accounting for18 of the cattlersquos total protein Federal University of Vicosa)The cattle were given water ad libitum for 6 months startingfrom April 2012

The differences between the groups were in the compo-sition of the pellets Pellets of group 1 (G1) contained thefungi D flagrans and M thaumasium while the pellets ofGroup 2 (G2) contained the fungi D flagrans and A robustaGroup 3 (G3) received pellets containing the three fungiD flagrans A robusta and M thaumasium Group 4 (G4)pellets contained only theD flagrans fungusThe pellet dosesof the different groups were all comparable with respectto the proportions of fungi species included in each pelletGroups 1 and 2 contained 50 of each of two fungi whilethe pellets combining the three fungal isolates (Group 3) werecomprised of one-third of each fungus

After allocating the heifers to the paddocks the animalsrsquofecal samples were collected directly from the rectum once aweek to determine the number of nematode fecal egg count(FEC) as described by Gordon and Whitlock [15]

Meteorological data were recorded daily at a specializedstation in the region the mean of the maximum mean andminimummonthly temperatures as well as themean rainfallwere noted

Fecal samples were collected to observe fungal growthonce a week 2 days after the animals were treated with thefungi The feces were incubated in plates containing 2WA100 L3 were recovered from the coproculture and they wereplaced into a drying oven at 25∘C for 10 days to confirm thepassage and predatory ability of the fungi through the cattlersquosgastrointestinal tract as well as to assess fungal growth in thefeces [3]

Coproculture was evaluated together with FEC counts20 g of feces was mixed with autoclaved wood shavings andkept moist at a controlled temperature (25∘C) for 7 days toobtain trichostrongylid larvae Identification of the infectivelarvae in the coproculture was performed according to Keith[16]

The FEC and larvae recovered from the coproculture ofanimals in both the treated and control groupswere recordedand the percentage of larval reduction was determinedaccording to De Gives et al [17] reduction () = mean L3recovered from control group minus mean L3 recovered fromtreated group times 100mean L3 recovered from the controlgroup

The FEC number of infective larvae recovered fromthe feces were statistically analyzed on a weekly basis andcompared over the experimental period

The data were transformed into log(119909 + 1) prior to theanalysis and subjected to analysis of variance (ANOVA)with repeated measures Tukeyrsquos test and regressions usinga randomized design at probability levels of 1 and 5

The animalsrsquo weights were also compared throughoutthe experiment starting from April 2012 The correlationanalyses were performed using Pearsonrsquos correlation (119875 lt0001) The analyses were performed using the BioEstat 30Software

The Ethics Committee of the Federal University of Vicosaprotocol number 662012 approved this study

BioMed Research International 3

0

200

400

600

800

1000

1200

Rain

fall

(mm

3)

SepAugJulJunMayApr

Group 1Group 2Group 3Group 4Group 5Rainfall

1830 a2070 a1750 a1930 a2220 a1113

163 a157 a177 a188 a160 a611

587 b600 b516 b600 b1200 a170

381 c400 b525 b369 c740 a0

160 c250 b280 b128 c750 a0

84 c120 b150 b71 c570 a228

0

500

1000

1500

2000

2500

3000

Feca

l egg

coun

t (FE

C)

Figure 1 Monthly mean of the fecal eggs count (FEC) among heifers in the groups treated with various combinations of coadministerednematophagous fungi D flagrans +M thaumasium (G1) D flagrans + A robusta (G2) D flagrans +M thaumasium + A robusta (G3) Dflagrans alone (G4) and the control group All samples were collected fromApril to September 2012 inOuro BrancoMG Brazil (abcNumbersfollowed by different letters present statistical difference)

3 Results

The monthly mean values of FEC counts are shown inFigure 1 In the first month of the experiment no statisticallysignificant differences were found (119875 gt 005) between thegroups treated with fungi (G1 G2 G3 and G4) and thecontrol group (group 5) In the first month of treatment (May2012) the low FEC number was likely due to the previouslyadministered anthelmintic treatment The FEC of animalstreated with D flagrans and M thaumasium (Group 1) Dflagrans andA robusta (Group 2)D flagransA robusta andM thaumasium (Group 3) and D flagrans (Group 4) thatis all treated groups were significantly lower than those ofthe control group from June to September 2012 (119875 lt 005)However the FEC of the animals treated withD flagrans andM thaumasium (Group 1) and with D flagrans alone (Group4) were significantly lower than those treated withD flagransandA robusta (Group 2) andwithD flagransA robusta andM thaumasium (Group 3) (119875 lt 005) (Figure 1)

The monthly mean FEC of the animals in the grouptreated with pellets containing the fungus D flagrans aloneas well as those treated with pellets containing both Dflagrans and M thaumasium were 964 and 938 lowerrespectively than the FEC of the animals in the controlgroup at the end of the experiment The animals from Group2 which were treated with pellets containing the fungi Dflagrans and A robusta as well as the animals from Group3 (treated with pellets containing the three fungi) exhibitedFEC reductions of 853 and 827 respectively whencompared with the animals in the control group Moreoverthe FEC counts were significantly lower in Groups 1 and4 when compared with Groups 2 and 3 at the end of theexperiment (119875 lt 005)

Figure 2 shows the maximum mean and minimumtemperatures as well as the mean monthly rainfall Overallit was found that the meteorological data correlated with

0

200

400

600

800

1000

1200

05

101520253035

April

May

June July

Augu

st

Sept

embe

r

T MaxT Min

T Mean

Tem

pera

ture

(∘C)

Rain

fall

(mm

3)

Rainfall (mm3)

Figure 2Themean of themaximummean andminimummonthlytemperatures (∘C) andmonthly rainfalls (mm3) recorded fromAprilto September 2012 Ouro Branco MG Brazil

the parasitological findings as temperatures and rainfallinfluenced the environmental parasite load

The coproculture showed that the Cooperia sp was themost prevalent gastrointestinal parasitic nematode in allgroups throughout the experiment which was observed atpercentages of 68 676 606 462 and 451 forGroups 1 2 3 4 and 5 respectively this was followed byHaemonchus which was found at rates of 228 248304 455 and 458 and Oesophagostomum whichwas observed at rates of 92 84 84 92 and 87respectively No significant differences (119875 gt 001) were foundwith respect to the proportion of the different genera amongthe five groups The percent reduction of L3 recovered fromeach coproculture of the treated groups was significantlylower when compared with that of the control group Thereductions were 923 907 815 and 783 for Groups1 2 3 and 4 respectively

4 BioMed Research International

May Jun Jul Aug Sep Mean

Group 1Group 2Group 3

Group 4Group 5

0100200300400500600700800900

Mea

n w

eigh

t gai

n (g

day

)

lowast

Figure 3 Mean weight gains (gday) from each group Measure-ments were taken from April to September 2012 in Ouro BrancoMG Brazil Significant differences between each treated group andthe control group are indicated by an asterisk (Tukeyrsquos test)

Analysis of the culture plates confirmed the fungalgrowth the specific conidia of each isolate and the abilityof D flagrans A robusta and M thaumasium to predate L3in all treated groups confirming the passage of the isolatesthrough the animalsrsquo gastrointestinal tracts The presence ofnematophagous fungi was not detected in the feces of thecontrol group animals during the experiment

Figure 3 shows the mean weight gains for the animalsin the five groups The weight gains of the animals in thetreated groups (G1 G2 G3 and G4) differed from those ofthe animals in the control group (G5) (119875 lt 005) in the lastmonth of the study

4 Discussion

Studies evaluating the coadministration of nematophagousfungi are scarce and this work was the first to evaluatethe combined use of the fungi A robusta D flagrans andM thaumasium in bovines In this study the heifers fromthe group treated with D flagrans alone exhibited an FECreduction of 964 when compared with the heifers in thecontrol group Several studies using the fungus D flagrans inruminants also reported smaller monthly mean FEC countsamong the treated animals in relation to the control group[3 18ndash20] In studies using the same D flagrans isolate inBrazil others researchers also obtained significant reductionsin FEC in treated crossbred Holstein-Zebu and Nellore bulls31 and 57

There was a 938 reduction in the FEC among thegroup treated with D flagrans and M thaumasium whencompared with the FEC of the control group Studies usingM thaumasium in bovines reported that the monthly meanFECwere lower in treated animals In studieswhere crossbredHolstein-Zebu heifers and Nellore bulls were treated withthis same Monacrosporium thaumasium isolate the authorsobtained FEC reductions of 888 and 478 respectively[3 9]

Furthermore the coadministration of pellets containingD flagrans and M thaumasium employing the same formu-lation as that used in this experiment was tested in sheepand the author demonstrated that this treatment was effectivein controlling gastrointestinal helminths in young and adultsheep in the semiarid region of northeastern Brazil [21] Inaddition the researchers found that the FEC rates remainedstatistically significantly lower throughout the study withoutthe administration of salvage deworming reaching a 76reduction in the FEC of treated animals when compared withthe FEC of controls which still required to be dewormedseven times [21]

In this study the group treated with D flagrans and Arobusta showed an FEC reduction of 853 in relation to thatof the control group Other study reported a 519 reductionin the FEC of crossbred Holstein-Zebu calves treated with anisolate of A robusta

Moreover the compatibility between isolates A robustaand D flagrans was evaluated under laboratory conditionsWith the aid of direct confrontation and antibiosis andvolatile metabolite tests the authors verified that the Arobusta isolate colonized approximately two-thirds of theplate suggesting that there was competition (and subsequentantagonism) between these two fungi Specifically A robustareduced the growth of D flagrans suggesting the actionof volatile antibiotics in inhibiting mycelial growth Theseresults corroborate the findings of the in vivo experimentdescribed herein since the groups of animals that receivedthe coadministration treatments containing the A robustaisolate demonstrated lower nematode reduction results whencompared to the other treatments with statistical significance(119875 lt 005)

The heifers in the group treated with the combinationof the three fungi had an FEC reduction of 827 whencompared with the animals in the control group There areno previous records in the literature describing the coadmin-istration of the three nematophagous fungi in the biologicalcontrol of nematode parasites in vivo This is the first reportof its kind the fungal combinations that were tested wereeffective in reducing the FEC in cattle Furthermore thecoadministration of these three isolates was less effective thanthe coadministration of D flagrans and M thaumasium orthe administration of theD flagrans isolate alone at reducingthe FEC

Trap formation and L3 predation by fungal isolates wereconfirmed by in vitro assays Braga et al [1] reported that Dflagrans showed greater predatory activity in vitro (803)on L1 Angiostrongylus vasorum when compared with Mthaumasium (745) and A robusta (718) In another invitro study Braga et al [22] compared the predatory abilityof the same isolates used in this study on L3 Strongyloidesstercoralis The L3 reductions were 837 (D flagrans) 755(M thaumasium) and 732 (A robusta) A study conductedto examine the interaction between L3 H contortus in goatand the fungi M thaumasium and A conoides showed thatboth strains were able to reduce the larval population butMthaumasium proved to be more efficient [23] These resultsare in agreement with the findings of this work the greatestreduction in L3 observed at the end of the experiment was

BioMed Research International 5

due to D flagrans while combinations featuring A robustawere less efficient at reducing L3 and the FEC counts

In the present study the percent reduction of L3 achievedby the M thaumasium isolate alone was 923 SimilarlyAraujo et al [10] recorded larval reduction in the coprocul-ture of animals treated with M thaumasium in the Braziliansemiarid region In their study the combination ofD flagransand M thaumasium resulted in a 907 decrease in thenumber of larvae in the coproculture

Furthermore the in vitro action of fungal isolates Arobusta and M thaumasium on L3 cyathostome in horseswas compared and the percent reduction obtained with Mthaumasium 313 (934) was higher than that obtained withthe A robusta isolate (863) at 25∘C The combination ofthe same isolates (A robusta +M thaumasium) in this studyreduced the L3 recovered in the coproculture by 815 whilea combination of isolates A robusta + M thaumasium + thefungus D flagrans decreased L3 by 783

It is important to note that climate plays an important rolein the ability of fungi to trap nematodes particularly sincethe optimum growth temperature varies with each fungalspeciesMorgan et al observed that temperatures in the rangeof 20∘Cndash33∘C influenced the larvae trapping percentage indifferent species of fungi [24] Thus optimal rainfalls andpredation temperatures that is specific environmental cli-matic conditions may be directly related to the results of thisstudy To support this Castro et al reported that A robustaexhibited the best larval trapping results at temperaturesranging from 25∘C to 28∘C while M thaumasium was notaffected by temperatures of 25∘Cndash30∘C which confirms thattemperature influences the degree of trapping depending onthe cyathostome species or genus [25]

In addition Castro et al obtained a 9336 reductionin the number of cyathostome larvae after administering Mthaumasium at 25∘CThe authors found that temperatures of25∘C 28∘C and 30∘C did not affect the performance of Mthaumasium which demonstrated an mean efficiency of 94[25] The results obtained in our study corroborate the find-ings of these previous works and suggest that the efficiencywith which larvae are controlled by nematophagous fungiessentially depends on the choice of the fungal species as wellas on their suitability for specific temperature conditions

The fungus Monacrosporium demonstrated unvaryingperformance in vitro at a temperature range of 15∘Cndash30∘Cas reported by Mendoza-de Gives and Vazquez-Prats [26]and Castro et al [25] even at 30∘C a common temperaturein the tropics thus this fungi would be better adapted to theconditions of the Brazilian climateThese results may supportand explain the higher degree of efficiency exhibited by thecombinationM thaumasium and D flagrans in reducing theFEC and L3 in this study particularly when compared withthe administration of A robusta and D flagrans Moreoverthe results also corroborate the reduced percentage of L3observed in the coproculture after administering combina-tions featuring A robusta In this study the regionrsquos tempera-tures ranged from 243∘C to 305∘C whichmay be consideredunsuitable for optimal predation by A robusta

Of note all of the treated groups showed a similar patternof weight gain during the study Even though notable weight

gain differences were found between heifers in the treatedgroups there was a significant difference (119875 lt 001) in weightgain between the treated groups which varied in an inverselyproportional manner to both the number of larvae recoveredin pasture and the FEC Specifically the heifers treated withD flagrans and D flagrans + M thaumasium showed greatermean weights This reinforces the fact that administeringpellets containing fungi was favored when the animals werepretreated Moreover greater weight gains in the treatedanimals (as compared to those in the control group) werealso observed by Araujo et al when testing the fungusM thaumasium in goats in the Brazilian semiarid regionFurthermore Braga et al [1] studied horses in the fieldthe researchers administered the nematophagous fungus Dflagrans and they observed significantweight gain differencesbetween the groups treated with the fungus and the controlanimals The animals that were treated with the fungusdemonstrated greater weight gains than those in the controlgroup

5 Conclusion

Treating dairy cattle with alginate pellets containing andcoadministering the nematophagous fungi D flagrans Arobusta andM thaumasium resulted in the biological controlof gastrointestinal nematodes in bovines although the levelof control was not increased when compared with the useof the fungus D flagrans alone Coadministration with theA robusta isolate was not considered a good alternative Theadministration of D flagrans alone was found to be morepromising than coadministration for continuous use in dairycattle in this tropical region in Brazil

Ethical Approval

The authors assert that all procedures contributing to thiswork comply with the ethical standards of the relevantnational and institutional committees on human experimen-tation and with the Helsinki Declaration of 1975 as revised in2008

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

The authors would like to thank the author of the thesisldquoViability of Predatory Fungi M 421 thaumasium and Dflagrans and Moxidectin on Gastrointestinal Nematodes ofBeef 422 Cattlerdquo a member of this research group whosemethodology forms the basis of this paper 423 and theyacknowledge Fundacao de Amparo a Pesquisa de MinasGerais (FAPEMIG) for the support in 424 project financingand in scientific dissemination for the publication of thispaperThis work was financially supported by FAPEMIG andCAPES

6 BioMed Research International

References

[1] F R Braga J V Araujo A R Silva et al ldquoBiological controlof horse cyathostomin (Nematoda Cyathostominae) usingthe nematophagous fungus Duddingtonia flagrans in tropicalsoutheastern Brazilrdquo Veterinary Parasitology vol 163 no 4 pp335ndash340 2009

[2] F R Braga J V Araujo A K Campos et al ldquoIn vitro evaluationof the action of the nematophagous fungi Duddingtonia fla-grans Monacrosporium sinense and Pochonia chlamydosporiaon Fasciola hepatica eggsrdquo World Journal of Microbiology andBiotechnology vol 24 no 8 pp 1559ndash1564 2008

[3] R C L Assis A Viabilidade dos fungos predadoresMonacrosporium thaumasiume Duddingtonia flagrans eMoxidectina sobre nematoides gastrintestinais de bovinos decorte [Tese de Doutorado] UFV Vicosa Brazil 2013

[4] R O Carvalho J V Araujo F R Braga et al ldquoBiologicalcontrol ofAncylostomosis in dogs using the nematode-trappingfungus Monacrosporium thaumasium in southeastern BrazilrdquoVeterinary Parasitology vol 165 no 1-2 pp 179ndash183 2009

[5] J V Araujo A P S Gomes and M P Guimaraes ldquoBiolog-ical control of bovine gastrointestinal nematode parasites insouthern Brazil by the nematode-trapping fungus Arthrobotrysrobustardquo Revista Brasileira de Parasitologia Veterinaria vol 7pp 117ndash122 1998

[6] M Larsen P Nansen J Wolstrup J Groslashnvold S A Henriksenand A Zorn ldquoBiological control of trichostrongyles in calves bythe fungus Duddingtonia flagrans fed to animals under naturalgrazing conditionsrdquoVeterinary Parasitology vol 60 no 3-4 pp321ndash330 1995

[7] A S Dias J V Araujo A K Campos F R Braga and T AFonseca ldquoApplication of a formulation of the nematophagousfungus Duddingtonia flagrans in the control of cattle gas-trointestinal nematodiosisrdquo World Journal of Microbiology andBiotechnology vol 23 no 9 pp 1245ndash1252 2007

[8] J VAraujo andWM Sampaio ldquoEffects of temperaturemineralsalt and passage through gastrointestinal tract of calves on algi-nate formulation of Arthrobotrys robustardquo Revista Brasileira deParasitologia Veterinaria vol 9 pp 55ndash59 2000

[9] P Alves J Araujo M Guimaraes R L Assis P Sarti andA Campos ldquoAplicacao de formulacao do fungo predador denematoides Monacrosporium thaumasium (Drechsler 1937)no controle de nematoides de bovinosrdquo Arquivo Brasileiro deMedicina Veterinaria e Zootecnia vol 55 no 5 pp 568ndash5732003

[10] J V Araujo M P Guimaraes A K Campos N C Sa PSarti and R C Assis ldquoControl of bovine gastrointestinalnematode parasites using pellets of the nematode-trappingfungus Monacrosporium thaumasiumrdquo Ciencia Rural vol 34no 2 pp 457ndash463 2004

[11] P A Ferreira S Ferraz E A Lopes and L G Freitas ldquoPara-sitismo de ovos deMeloidogyne exigua por fungos nematofagose estudo da compatibilidade entre os isolados fungicosrdquo RevistaTropical de Ciencias Agrarias e Biologicas vol 2 no 21 2008

[12] M Dalla-Pria and S Ferraz ldquoControle biologico de Meloidog-yne incognita por seis especies deMonacrosporium isoladas oucombinadas comVerticillium chlamydosporiumrdquo FitopatologiaBrasileira vol 21 pp 30ndash34 1996

[13] T de Hollanda Ayupe T S A Monteiro F R Braga etal ldquoAssessment of compatibility between the nematophagousfungi Arthrobotrys robusta and Duddingtonia flagrans underlaboratory conditionsrdquo Revista Iberoamericana de Micologıavol 33 no 2 pp 129-130 2016

[14] A D O Tavela J V de Araujo F R Braga et al ldquoCoad-ministration of sodium alginate pellets containing the fungiDuddingtonia flagrans and Monacrosporium thaumasium oncyathostomin infective larvae after passing through the gas-trointestinal tract of horsesrdquo Research in Veterinary Science vol94 no 3 pp 568ndash572 2013

[15] HMGordon andHVWhitlock ldquoNew technique for countingnematode eggs in sheep faecesrdquo Journal of the CSIR vol 12 pp50ndash52 1939

[16] R K Keith ldquoThe differentiation of the infective larvae of somecommon nematode parasites of cattlerdquo Australian Journal ofZoology vol 1 no 2 pp 223ndash235 1953

[17] P M De Gives K G Davies S J Clark and J M BehnkeldquoPredatory behaviour of trapping fungi against srf mutants ofCaenorhabditis elegans and different plant and animal parasiticnematodesrdquo Parasitology vol 119 no 1 pp 95ndash104 1999

[18] S-O Dimander J Hoglund and P J Waller ldquoSeasonal trans-lation of infective larvae of gastrointestinal nematodes of cattleand the effect of Duddingtonia flagrans A 3-year plot studyrdquoVeterinary Parasitology vol 117 no 1-2 pp 99ndash116 2003

[19] M E Fontenot J E Miller M T Pena M Larsen and AGillespie ldquoEfficiency of feeding Duddingtonia flagrans chlamy-dospores to grazing ewes on reducing availability of parasiticnematode larvae on pasturerdquo Veterinary Parasitology vol 118no 3-4 pp 203ndash213 2003

[20] A R Silva J V Araujo F R Braga et al ldquoBiological controlof sheep gastrointestinal nematodiasis in a tropical region ofthe southeast of Brazil with the nematode predatory fungiDuddingtonia flagrans and Monacrosporium thaumasiumrdquoParasitology Research vol 105 no 6 pp 1707ndash1713 2009

[21] V Longo Ribeiro Vilela T F Feitosa F R Braga et al ldquoCoad-ministration of nematophagous fungi for biological control overgastrointestinal helminths in sheep in the semiarid region ofnortheastern Brazilrdquo Veterinary Parasitology vol 221 pp 139ndash143 2016

[22] F R Braga A R e Silva J M Araujo R O Carvalho JV de Araujo and L N Frassy ldquoPredatory activity of thenematophagous fungi duddingtonia flagrans monacrosporiumthaumasium and arthrobotrys robusta on strongyloides sterco-ralis infective larvaerdquo Journal of the Brazilian Society of TropicalMedicine vol 43 no 5 pp 588ndash590 2010

[23] M Mota C Bevilacqua and J V Araujo ldquoAtividade predatoriados fungos Arthrobotrys conoides e Monacrosporium thau-masium sobre larvas infectantes de Haemonchus contortus decaprinosrdquo Ciencia Animal vol 10 pp 37ndash41 2000

[24] MMorgan J M Behnke J A Lucas and J F Peberdy ldquoIn vitroassessment of the influence of nutrition temperature and larvaldensity on trapping of the infective larvae of Heligmosomoidespolygyrus by Arthrobotrys oligospora Duddingtonia flagransand Monacrosporium megalosporumrdquo Parasitology vol 115no 3 pp 303ndash310 1997

[25] A A Castro C R C Oliveira D H S Anjos et al ldquoPotencialdos fungos nematofagos Arthrobotrys sp e Monacrosporiumthaumasium para o controle de larvas de ciatostomıneos deequinos (Nematoda Cyathostominae)rdquo Revista Brasileira deParasitologia Veterinaria vol 12 no 2 pp 53ndash57 2003

[26] P Mendoza-de Gives and V M Vazquez-Prats ldquoReduction ofHaemonchus contortus infective larvae by three nematophagousfungi in sheep faecal culturesrdquo Veterinary Parasitology vol 55no 3 pp 197ndash203 1994

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

BioMed Research International 3

0

200

400

600

800

1000

1200

Rain

fall

(mm

3)

SepAugJulJunMayApr

Group 1Group 2Group 3Group 4Group 5Rainfall

1830 a2070 a1750 a1930 a2220 a1113

163 a157 a177 a188 a160 a611

587 b600 b516 b600 b1200 a170

381 c400 b525 b369 c740 a0

160 c250 b280 b128 c750 a0

84 c120 b150 b71 c570 a228

0

500

1000

1500

2000

2500

3000

Feca

l egg

coun

t (FE

C)

Figure 1 Monthly mean of the fecal eggs count (FEC) among heifers in the groups treated with various combinations of coadministerednematophagous fungi D flagrans +M thaumasium (G1) D flagrans + A robusta (G2) D flagrans +M thaumasium + A robusta (G3) Dflagrans alone (G4) and the control group All samples were collected fromApril to September 2012 inOuro BrancoMG Brazil (abcNumbersfollowed by different letters present statistical difference)

3 Results

The monthly mean values of FEC counts are shown inFigure 1 In the first month of the experiment no statisticallysignificant differences were found (119875 gt 005) between thegroups treated with fungi (G1 G2 G3 and G4) and thecontrol group (group 5) In the first month of treatment (May2012) the low FEC number was likely due to the previouslyadministered anthelmintic treatment The FEC of animalstreated with D flagrans and M thaumasium (Group 1) Dflagrans andA robusta (Group 2)D flagransA robusta andM thaumasium (Group 3) and D flagrans (Group 4) thatis all treated groups were significantly lower than those ofthe control group from June to September 2012 (119875 lt 005)However the FEC of the animals treated withD flagrans andM thaumasium (Group 1) and with D flagrans alone (Group4) were significantly lower than those treated withD flagransandA robusta (Group 2) andwithD flagransA robusta andM thaumasium (Group 3) (119875 lt 005) (Figure 1)

The monthly mean FEC of the animals in the grouptreated with pellets containing the fungus D flagrans aloneas well as those treated with pellets containing both Dflagrans and M thaumasium were 964 and 938 lowerrespectively than the FEC of the animals in the controlgroup at the end of the experiment The animals from Group2 which were treated with pellets containing the fungi Dflagrans and A robusta as well as the animals from Group3 (treated with pellets containing the three fungi) exhibitedFEC reductions of 853 and 827 respectively whencompared with the animals in the control group Moreoverthe FEC counts were significantly lower in Groups 1 and4 when compared with Groups 2 and 3 at the end of theexperiment (119875 lt 005)

Figure 2 shows the maximum mean and minimumtemperatures as well as the mean monthly rainfall Overallit was found that the meteorological data correlated with

0

200

400

600

800

1000

1200

05

101520253035

April

May

June July

Augu

st

Sept

embe

r

T MaxT Min

T Mean

Tem

pera

ture

(∘C)

Rain

fall

(mm

3)

Rainfall (mm3)

Figure 2Themean of themaximummean andminimummonthlytemperatures (∘C) andmonthly rainfalls (mm3) recorded fromAprilto September 2012 Ouro Branco MG Brazil

the parasitological findings as temperatures and rainfallinfluenced the environmental parasite load

The coproculture showed that the Cooperia sp was themost prevalent gastrointestinal parasitic nematode in allgroups throughout the experiment which was observed atpercentages of 68 676 606 462 and 451 forGroups 1 2 3 4 and 5 respectively this was followed byHaemonchus which was found at rates of 228 248304 455 and 458 and Oesophagostomum whichwas observed at rates of 92 84 84 92 and 87respectively No significant differences (119875 gt 001) were foundwith respect to the proportion of the different genera amongthe five groups The percent reduction of L3 recovered fromeach coproculture of the treated groups was significantlylower when compared with that of the control group Thereductions were 923 907 815 and 783 for Groups1 2 3 and 4 respectively

4 BioMed Research International

May Jun Jul Aug Sep Mean

Group 1Group 2Group 3

Group 4Group 5

0100200300400500600700800900

Mea

n w

eigh

t gai

n (g

day

)

lowast

Figure 3 Mean weight gains (gday) from each group Measure-ments were taken from April to September 2012 in Ouro BrancoMG Brazil Significant differences between each treated group andthe control group are indicated by an asterisk (Tukeyrsquos test)

Analysis of the culture plates confirmed the fungalgrowth the specific conidia of each isolate and the abilityof D flagrans A robusta and M thaumasium to predate L3in all treated groups confirming the passage of the isolatesthrough the animalsrsquo gastrointestinal tracts The presence ofnematophagous fungi was not detected in the feces of thecontrol group animals during the experiment

Figure 3 shows the mean weight gains for the animalsin the five groups The weight gains of the animals in thetreated groups (G1 G2 G3 and G4) differed from those ofthe animals in the control group (G5) (119875 lt 005) in the lastmonth of the study

4 Discussion

Studies evaluating the coadministration of nematophagousfungi are scarce and this work was the first to evaluatethe combined use of the fungi A robusta D flagrans andM thaumasium in bovines In this study the heifers fromthe group treated with D flagrans alone exhibited an FECreduction of 964 when compared with the heifers in thecontrol group Several studies using the fungus D flagrans inruminants also reported smaller monthly mean FEC countsamong the treated animals in relation to the control group[3 18ndash20] In studies using the same D flagrans isolate inBrazil others researchers also obtained significant reductionsin FEC in treated crossbred Holstein-Zebu and Nellore bulls31 and 57

There was a 938 reduction in the FEC among thegroup treated with D flagrans and M thaumasium whencompared with the FEC of the control group Studies usingM thaumasium in bovines reported that the monthly meanFECwere lower in treated animals In studieswhere crossbredHolstein-Zebu heifers and Nellore bulls were treated withthis same Monacrosporium thaumasium isolate the authorsobtained FEC reductions of 888 and 478 respectively[3 9]

Furthermore the coadministration of pellets containingD flagrans and M thaumasium employing the same formu-lation as that used in this experiment was tested in sheepand the author demonstrated that this treatment was effectivein controlling gastrointestinal helminths in young and adultsheep in the semiarid region of northeastern Brazil [21] Inaddition the researchers found that the FEC rates remainedstatistically significantly lower throughout the study withoutthe administration of salvage deworming reaching a 76reduction in the FEC of treated animals when compared withthe FEC of controls which still required to be dewormedseven times [21]

In this study the group treated with D flagrans and Arobusta showed an FEC reduction of 853 in relation to thatof the control group Other study reported a 519 reductionin the FEC of crossbred Holstein-Zebu calves treated with anisolate of A robusta

Moreover the compatibility between isolates A robustaand D flagrans was evaluated under laboratory conditionsWith the aid of direct confrontation and antibiosis andvolatile metabolite tests the authors verified that the Arobusta isolate colonized approximately two-thirds of theplate suggesting that there was competition (and subsequentantagonism) between these two fungi Specifically A robustareduced the growth of D flagrans suggesting the actionof volatile antibiotics in inhibiting mycelial growth Theseresults corroborate the findings of the in vivo experimentdescribed herein since the groups of animals that receivedthe coadministration treatments containing the A robustaisolate demonstrated lower nematode reduction results whencompared to the other treatments with statistical significance(119875 lt 005)

The heifers in the group treated with the combinationof the three fungi had an FEC reduction of 827 whencompared with the animals in the control group There areno previous records in the literature describing the coadmin-istration of the three nematophagous fungi in the biologicalcontrol of nematode parasites in vivo This is the first reportof its kind the fungal combinations that were tested wereeffective in reducing the FEC in cattle Furthermore thecoadministration of these three isolates was less effective thanthe coadministration of D flagrans and M thaumasium orthe administration of theD flagrans isolate alone at reducingthe FEC

Trap formation and L3 predation by fungal isolates wereconfirmed by in vitro assays Braga et al [1] reported that Dflagrans showed greater predatory activity in vitro (803)on L1 Angiostrongylus vasorum when compared with Mthaumasium (745) and A robusta (718) In another invitro study Braga et al [22] compared the predatory abilityof the same isolates used in this study on L3 Strongyloidesstercoralis The L3 reductions were 837 (D flagrans) 755(M thaumasium) and 732 (A robusta) A study conductedto examine the interaction between L3 H contortus in goatand the fungi M thaumasium and A conoides showed thatboth strains were able to reduce the larval population butMthaumasium proved to be more efficient [23] These resultsare in agreement with the findings of this work the greatestreduction in L3 observed at the end of the experiment was

BioMed Research International 5

due to D flagrans while combinations featuring A robustawere less efficient at reducing L3 and the FEC counts

In the present study the percent reduction of L3 achievedby the M thaumasium isolate alone was 923 SimilarlyAraujo et al [10] recorded larval reduction in the coprocul-ture of animals treated with M thaumasium in the Braziliansemiarid region In their study the combination ofD flagransand M thaumasium resulted in a 907 decrease in thenumber of larvae in the coproculture

Furthermore the in vitro action of fungal isolates Arobusta and M thaumasium on L3 cyathostome in horseswas compared and the percent reduction obtained with Mthaumasium 313 (934) was higher than that obtained withthe A robusta isolate (863) at 25∘C The combination ofthe same isolates (A robusta +M thaumasium) in this studyreduced the L3 recovered in the coproculture by 815 whilea combination of isolates A robusta + M thaumasium + thefungus D flagrans decreased L3 by 783

It is important to note that climate plays an important rolein the ability of fungi to trap nematodes particularly sincethe optimum growth temperature varies with each fungalspeciesMorgan et al observed that temperatures in the rangeof 20∘Cndash33∘C influenced the larvae trapping percentage indifferent species of fungi [24] Thus optimal rainfalls andpredation temperatures that is specific environmental cli-matic conditions may be directly related to the results of thisstudy To support this Castro et al reported that A robustaexhibited the best larval trapping results at temperaturesranging from 25∘C to 28∘C while M thaumasium was notaffected by temperatures of 25∘Cndash30∘C which confirms thattemperature influences the degree of trapping depending onthe cyathostome species or genus [25]

In addition Castro et al obtained a 9336 reductionin the number of cyathostome larvae after administering Mthaumasium at 25∘CThe authors found that temperatures of25∘C 28∘C and 30∘C did not affect the performance of Mthaumasium which demonstrated an mean efficiency of 94[25] The results obtained in our study corroborate the find-ings of these previous works and suggest that the efficiencywith which larvae are controlled by nematophagous fungiessentially depends on the choice of the fungal species as wellas on their suitability for specific temperature conditions

The fungus Monacrosporium demonstrated unvaryingperformance in vitro at a temperature range of 15∘Cndash30∘Cas reported by Mendoza-de Gives and Vazquez-Prats [26]and Castro et al [25] even at 30∘C a common temperaturein the tropics thus this fungi would be better adapted to theconditions of the Brazilian climateThese results may supportand explain the higher degree of efficiency exhibited by thecombinationM thaumasium and D flagrans in reducing theFEC and L3 in this study particularly when compared withthe administration of A robusta and D flagrans Moreoverthe results also corroborate the reduced percentage of L3observed in the coproculture after administering combina-tions featuring A robusta In this study the regionrsquos tempera-tures ranged from 243∘C to 305∘C whichmay be consideredunsuitable for optimal predation by A robusta

Of note all of the treated groups showed a similar patternof weight gain during the study Even though notable weight

gain differences were found between heifers in the treatedgroups there was a significant difference (119875 lt 001) in weightgain between the treated groups which varied in an inverselyproportional manner to both the number of larvae recoveredin pasture and the FEC Specifically the heifers treated withD flagrans and D flagrans + M thaumasium showed greatermean weights This reinforces the fact that administeringpellets containing fungi was favored when the animals werepretreated Moreover greater weight gains in the treatedanimals (as compared to those in the control group) werealso observed by Araujo et al when testing the fungusM thaumasium in goats in the Brazilian semiarid regionFurthermore Braga et al [1] studied horses in the fieldthe researchers administered the nematophagous fungus Dflagrans and they observed significantweight gain differencesbetween the groups treated with the fungus and the controlanimals The animals that were treated with the fungusdemonstrated greater weight gains than those in the controlgroup

5 Conclusion

Treating dairy cattle with alginate pellets containing andcoadministering the nematophagous fungi D flagrans Arobusta andM thaumasium resulted in the biological controlof gastrointestinal nematodes in bovines although the levelof control was not increased when compared with the useof the fungus D flagrans alone Coadministration with theA robusta isolate was not considered a good alternative Theadministration of D flagrans alone was found to be morepromising than coadministration for continuous use in dairycattle in this tropical region in Brazil

Ethical Approval

The authors assert that all procedures contributing to thiswork comply with the ethical standards of the relevantnational and institutional committees on human experimen-tation and with the Helsinki Declaration of 1975 as revised in2008

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

The authors would like to thank the author of the thesisldquoViability of Predatory Fungi M 421 thaumasium and Dflagrans and Moxidectin on Gastrointestinal Nematodes ofBeef 422 Cattlerdquo a member of this research group whosemethodology forms the basis of this paper 423 and theyacknowledge Fundacao de Amparo a Pesquisa de MinasGerais (FAPEMIG) for the support in 424 project financingand in scientific dissemination for the publication of thispaperThis work was financially supported by FAPEMIG andCAPES

6 BioMed Research International

References

[1] F R Braga J V Araujo A R Silva et al ldquoBiological controlof horse cyathostomin (Nematoda Cyathostominae) usingthe nematophagous fungus Duddingtonia flagrans in tropicalsoutheastern Brazilrdquo Veterinary Parasitology vol 163 no 4 pp335ndash340 2009

[2] F R Braga J V Araujo A K Campos et al ldquoIn vitro evaluationof the action of the nematophagous fungi Duddingtonia fla-grans Monacrosporium sinense and Pochonia chlamydosporiaon Fasciola hepatica eggsrdquo World Journal of Microbiology andBiotechnology vol 24 no 8 pp 1559ndash1564 2008

[3] R C L Assis A Viabilidade dos fungos predadoresMonacrosporium thaumasiume Duddingtonia flagrans eMoxidectina sobre nematoides gastrintestinais de bovinos decorte [Tese de Doutorado] UFV Vicosa Brazil 2013

[4] R O Carvalho J V Araujo F R Braga et al ldquoBiologicalcontrol ofAncylostomosis in dogs using the nematode-trappingfungus Monacrosporium thaumasium in southeastern BrazilrdquoVeterinary Parasitology vol 165 no 1-2 pp 179ndash183 2009

[5] J V Araujo A P S Gomes and M P Guimaraes ldquoBiolog-ical control of bovine gastrointestinal nematode parasites insouthern Brazil by the nematode-trapping fungus Arthrobotrysrobustardquo Revista Brasileira de Parasitologia Veterinaria vol 7pp 117ndash122 1998

[6] M Larsen P Nansen J Wolstrup J Groslashnvold S A Henriksenand A Zorn ldquoBiological control of trichostrongyles in calves bythe fungus Duddingtonia flagrans fed to animals under naturalgrazing conditionsrdquoVeterinary Parasitology vol 60 no 3-4 pp321ndash330 1995

[7] A S Dias J V Araujo A K Campos F R Braga and T AFonseca ldquoApplication of a formulation of the nematophagousfungus Duddingtonia flagrans in the control of cattle gas-trointestinal nematodiosisrdquo World Journal of Microbiology andBiotechnology vol 23 no 9 pp 1245ndash1252 2007

[8] J VAraujo andWM Sampaio ldquoEffects of temperaturemineralsalt and passage through gastrointestinal tract of calves on algi-nate formulation of Arthrobotrys robustardquo Revista Brasileira deParasitologia Veterinaria vol 9 pp 55ndash59 2000

[9] P Alves J Araujo M Guimaraes R L Assis P Sarti andA Campos ldquoAplicacao de formulacao do fungo predador denematoides Monacrosporium thaumasium (Drechsler 1937)no controle de nematoides de bovinosrdquo Arquivo Brasileiro deMedicina Veterinaria e Zootecnia vol 55 no 5 pp 568ndash5732003

[10] J V Araujo M P Guimaraes A K Campos N C Sa PSarti and R C Assis ldquoControl of bovine gastrointestinalnematode parasites using pellets of the nematode-trappingfungus Monacrosporium thaumasiumrdquo Ciencia Rural vol 34no 2 pp 457ndash463 2004

[11] P A Ferreira S Ferraz E A Lopes and L G Freitas ldquoPara-sitismo de ovos deMeloidogyne exigua por fungos nematofagose estudo da compatibilidade entre os isolados fungicosrdquo RevistaTropical de Ciencias Agrarias e Biologicas vol 2 no 21 2008

[12] M Dalla-Pria and S Ferraz ldquoControle biologico de Meloidog-yne incognita por seis especies deMonacrosporium isoladas oucombinadas comVerticillium chlamydosporiumrdquo FitopatologiaBrasileira vol 21 pp 30ndash34 1996

[13] T de Hollanda Ayupe T S A Monteiro F R Braga etal ldquoAssessment of compatibility between the nematophagousfungi Arthrobotrys robusta and Duddingtonia flagrans underlaboratory conditionsrdquo Revista Iberoamericana de Micologıavol 33 no 2 pp 129-130 2016

[14] A D O Tavela J V de Araujo F R Braga et al ldquoCoad-ministration of sodium alginate pellets containing the fungiDuddingtonia flagrans and Monacrosporium thaumasium oncyathostomin infective larvae after passing through the gas-trointestinal tract of horsesrdquo Research in Veterinary Science vol94 no 3 pp 568ndash572 2013

[15] HMGordon andHVWhitlock ldquoNew technique for countingnematode eggs in sheep faecesrdquo Journal of the CSIR vol 12 pp50ndash52 1939

[16] R K Keith ldquoThe differentiation of the infective larvae of somecommon nematode parasites of cattlerdquo Australian Journal ofZoology vol 1 no 2 pp 223ndash235 1953

[17] P M De Gives K G Davies S J Clark and J M BehnkeldquoPredatory behaviour of trapping fungi against srf mutants ofCaenorhabditis elegans and different plant and animal parasiticnematodesrdquo Parasitology vol 119 no 1 pp 95ndash104 1999

[18] S-O Dimander J Hoglund and P J Waller ldquoSeasonal trans-lation of infective larvae of gastrointestinal nematodes of cattleand the effect of Duddingtonia flagrans A 3-year plot studyrdquoVeterinary Parasitology vol 117 no 1-2 pp 99ndash116 2003

[19] M E Fontenot J E Miller M T Pena M Larsen and AGillespie ldquoEfficiency of feeding Duddingtonia flagrans chlamy-dospores to grazing ewes on reducing availability of parasiticnematode larvae on pasturerdquo Veterinary Parasitology vol 118no 3-4 pp 203ndash213 2003

[20] A R Silva J V Araujo F R Braga et al ldquoBiological controlof sheep gastrointestinal nematodiasis in a tropical region ofthe southeast of Brazil with the nematode predatory fungiDuddingtonia flagrans and Monacrosporium thaumasiumrdquoParasitology Research vol 105 no 6 pp 1707ndash1713 2009

[21] V Longo Ribeiro Vilela T F Feitosa F R Braga et al ldquoCoad-ministration of nematophagous fungi for biological control overgastrointestinal helminths in sheep in the semiarid region ofnortheastern Brazilrdquo Veterinary Parasitology vol 221 pp 139ndash143 2016

[22] F R Braga A R e Silva J M Araujo R O Carvalho JV de Araujo and L N Frassy ldquoPredatory activity of thenematophagous fungi duddingtonia flagrans monacrosporiumthaumasium and arthrobotrys robusta on strongyloides sterco-ralis infective larvaerdquo Journal of the Brazilian Society of TropicalMedicine vol 43 no 5 pp 588ndash590 2010

[23] M Mota C Bevilacqua and J V Araujo ldquoAtividade predatoriados fungos Arthrobotrys conoides e Monacrosporium thau-masium sobre larvas infectantes de Haemonchus contortus decaprinosrdquo Ciencia Animal vol 10 pp 37ndash41 2000

[24] MMorgan J M Behnke J A Lucas and J F Peberdy ldquoIn vitroassessment of the influence of nutrition temperature and larvaldensity on trapping of the infective larvae of Heligmosomoidespolygyrus by Arthrobotrys oligospora Duddingtonia flagransand Monacrosporium megalosporumrdquo Parasitology vol 115no 3 pp 303ndash310 1997

[25] A A Castro C R C Oliveira D H S Anjos et al ldquoPotencialdos fungos nematofagos Arthrobotrys sp e Monacrosporiumthaumasium para o controle de larvas de ciatostomıneos deequinos (Nematoda Cyathostominae)rdquo Revista Brasileira deParasitologia Veterinaria vol 12 no 2 pp 53ndash57 2003

[26] P Mendoza-de Gives and V M Vazquez-Prats ldquoReduction ofHaemonchus contortus infective larvae by three nematophagousfungi in sheep faecal culturesrdquo Veterinary Parasitology vol 55no 3 pp 197ndash203 1994

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

4 BioMed Research International

May Jun Jul Aug Sep Mean

Group 1Group 2Group 3

Group 4Group 5

0100200300400500600700800900

Mea

n w

eigh

t gai

n (g

day

)

lowast

Figure 3 Mean weight gains (gday) from each group Measure-ments were taken from April to September 2012 in Ouro BrancoMG Brazil Significant differences between each treated group andthe control group are indicated by an asterisk (Tukeyrsquos test)

Analysis of the culture plates confirmed the fungalgrowth the specific conidia of each isolate and the abilityof D flagrans A robusta and M thaumasium to predate L3in all treated groups confirming the passage of the isolatesthrough the animalsrsquo gastrointestinal tracts The presence ofnematophagous fungi was not detected in the feces of thecontrol group animals during the experiment

Figure 3 shows the mean weight gains for the animalsin the five groups The weight gains of the animals in thetreated groups (G1 G2 G3 and G4) differed from those ofthe animals in the control group (G5) (119875 lt 005) in the lastmonth of the study

4 Discussion

Studies evaluating the coadministration of nematophagousfungi are scarce and this work was the first to evaluatethe combined use of the fungi A robusta D flagrans andM thaumasium in bovines In this study the heifers fromthe group treated with D flagrans alone exhibited an FECreduction of 964 when compared with the heifers in thecontrol group Several studies using the fungus D flagrans inruminants also reported smaller monthly mean FEC countsamong the treated animals in relation to the control group[3 18ndash20] In studies using the same D flagrans isolate inBrazil others researchers also obtained significant reductionsin FEC in treated crossbred Holstein-Zebu and Nellore bulls31 and 57

There was a 938 reduction in the FEC among thegroup treated with D flagrans and M thaumasium whencompared with the FEC of the control group Studies usingM thaumasium in bovines reported that the monthly meanFECwere lower in treated animals In studieswhere crossbredHolstein-Zebu heifers and Nellore bulls were treated withthis same Monacrosporium thaumasium isolate the authorsobtained FEC reductions of 888 and 478 respectively[3 9]

Furthermore the coadministration of pellets containingD flagrans and M thaumasium employing the same formu-lation as that used in this experiment was tested in sheepand the author demonstrated that this treatment was effectivein controlling gastrointestinal helminths in young and adultsheep in the semiarid region of northeastern Brazil [21] Inaddition the researchers found that the FEC rates remainedstatistically significantly lower throughout the study withoutthe administration of salvage deworming reaching a 76reduction in the FEC of treated animals when compared withthe FEC of controls which still required to be dewormedseven times [21]

In this study the group treated with D flagrans and Arobusta showed an FEC reduction of 853 in relation to thatof the control group Other study reported a 519 reductionin the FEC of crossbred Holstein-Zebu calves treated with anisolate of A robusta

Moreover the compatibility between isolates A robustaand D flagrans was evaluated under laboratory conditionsWith the aid of direct confrontation and antibiosis andvolatile metabolite tests the authors verified that the Arobusta isolate colonized approximately two-thirds of theplate suggesting that there was competition (and subsequentantagonism) between these two fungi Specifically A robustareduced the growth of D flagrans suggesting the actionof volatile antibiotics in inhibiting mycelial growth Theseresults corroborate the findings of the in vivo experimentdescribed herein since the groups of animals that receivedthe coadministration treatments containing the A robustaisolate demonstrated lower nematode reduction results whencompared to the other treatments with statistical significance(119875 lt 005)

The heifers in the group treated with the combinationof the three fungi had an FEC reduction of 827 whencompared with the animals in the control group There areno previous records in the literature describing the coadmin-istration of the three nematophagous fungi in the biologicalcontrol of nematode parasites in vivo This is the first reportof its kind the fungal combinations that were tested wereeffective in reducing the FEC in cattle Furthermore thecoadministration of these three isolates was less effective thanthe coadministration of D flagrans and M thaumasium orthe administration of theD flagrans isolate alone at reducingthe FEC

Trap formation and L3 predation by fungal isolates wereconfirmed by in vitro assays Braga et al [1] reported that Dflagrans showed greater predatory activity in vitro (803)on L1 Angiostrongylus vasorum when compared with Mthaumasium (745) and A robusta (718) In another invitro study Braga et al [22] compared the predatory abilityof the same isolates used in this study on L3 Strongyloidesstercoralis The L3 reductions were 837 (D flagrans) 755(M thaumasium) and 732 (A robusta) A study conductedto examine the interaction between L3 H contortus in goatand the fungi M thaumasium and A conoides showed thatboth strains were able to reduce the larval population butMthaumasium proved to be more efficient [23] These resultsare in agreement with the findings of this work the greatestreduction in L3 observed at the end of the experiment was

BioMed Research International 5

due to D flagrans while combinations featuring A robustawere less efficient at reducing L3 and the FEC counts

In the present study the percent reduction of L3 achievedby the M thaumasium isolate alone was 923 SimilarlyAraujo et al [10] recorded larval reduction in the coprocul-ture of animals treated with M thaumasium in the Braziliansemiarid region In their study the combination ofD flagransand M thaumasium resulted in a 907 decrease in thenumber of larvae in the coproculture

Furthermore the in vitro action of fungal isolates Arobusta and M thaumasium on L3 cyathostome in horseswas compared and the percent reduction obtained with Mthaumasium 313 (934) was higher than that obtained withthe A robusta isolate (863) at 25∘C The combination ofthe same isolates (A robusta +M thaumasium) in this studyreduced the L3 recovered in the coproculture by 815 whilea combination of isolates A robusta + M thaumasium + thefungus D flagrans decreased L3 by 783

It is important to note that climate plays an important rolein the ability of fungi to trap nematodes particularly sincethe optimum growth temperature varies with each fungalspeciesMorgan et al observed that temperatures in the rangeof 20∘Cndash33∘C influenced the larvae trapping percentage indifferent species of fungi [24] Thus optimal rainfalls andpredation temperatures that is specific environmental cli-matic conditions may be directly related to the results of thisstudy To support this Castro et al reported that A robustaexhibited the best larval trapping results at temperaturesranging from 25∘C to 28∘C while M thaumasium was notaffected by temperatures of 25∘Cndash30∘C which confirms thattemperature influences the degree of trapping depending onthe cyathostome species or genus [25]

In addition Castro et al obtained a 9336 reductionin the number of cyathostome larvae after administering Mthaumasium at 25∘CThe authors found that temperatures of25∘C 28∘C and 30∘C did not affect the performance of Mthaumasium which demonstrated an mean efficiency of 94[25] The results obtained in our study corroborate the find-ings of these previous works and suggest that the efficiencywith which larvae are controlled by nematophagous fungiessentially depends on the choice of the fungal species as wellas on their suitability for specific temperature conditions

The fungus Monacrosporium demonstrated unvaryingperformance in vitro at a temperature range of 15∘Cndash30∘Cas reported by Mendoza-de Gives and Vazquez-Prats [26]and Castro et al [25] even at 30∘C a common temperaturein the tropics thus this fungi would be better adapted to theconditions of the Brazilian climateThese results may supportand explain the higher degree of efficiency exhibited by thecombinationM thaumasium and D flagrans in reducing theFEC and L3 in this study particularly when compared withthe administration of A robusta and D flagrans Moreoverthe results also corroborate the reduced percentage of L3observed in the coproculture after administering combina-tions featuring A robusta In this study the regionrsquos tempera-tures ranged from 243∘C to 305∘C whichmay be consideredunsuitable for optimal predation by A robusta

Of note all of the treated groups showed a similar patternof weight gain during the study Even though notable weight

gain differences were found between heifers in the treatedgroups there was a significant difference (119875 lt 001) in weightgain between the treated groups which varied in an inverselyproportional manner to both the number of larvae recoveredin pasture and the FEC Specifically the heifers treated withD flagrans and D flagrans + M thaumasium showed greatermean weights This reinforces the fact that administeringpellets containing fungi was favored when the animals werepretreated Moreover greater weight gains in the treatedanimals (as compared to those in the control group) werealso observed by Araujo et al when testing the fungusM thaumasium in goats in the Brazilian semiarid regionFurthermore Braga et al [1] studied horses in the fieldthe researchers administered the nematophagous fungus Dflagrans and they observed significantweight gain differencesbetween the groups treated with the fungus and the controlanimals The animals that were treated with the fungusdemonstrated greater weight gains than those in the controlgroup

5 Conclusion

Treating dairy cattle with alginate pellets containing andcoadministering the nematophagous fungi D flagrans Arobusta andM thaumasium resulted in the biological controlof gastrointestinal nematodes in bovines although the levelof control was not increased when compared with the useof the fungus D flagrans alone Coadministration with theA robusta isolate was not considered a good alternative Theadministration of D flagrans alone was found to be morepromising than coadministration for continuous use in dairycattle in this tropical region in Brazil

Ethical Approval

The authors assert that all procedures contributing to thiswork comply with the ethical standards of the relevantnational and institutional committees on human experimen-tation and with the Helsinki Declaration of 1975 as revised in2008

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

The authors would like to thank the author of the thesisldquoViability of Predatory Fungi M 421 thaumasium and Dflagrans and Moxidectin on Gastrointestinal Nematodes ofBeef 422 Cattlerdquo a member of this research group whosemethodology forms the basis of this paper 423 and theyacknowledge Fundacao de Amparo a Pesquisa de MinasGerais (FAPEMIG) for the support in 424 project financingand in scientific dissemination for the publication of thispaperThis work was financially supported by FAPEMIG andCAPES

6 BioMed Research International

References

[1] F R Braga J V Araujo A R Silva et al ldquoBiological controlof horse cyathostomin (Nematoda Cyathostominae) usingthe nematophagous fungus Duddingtonia flagrans in tropicalsoutheastern Brazilrdquo Veterinary Parasitology vol 163 no 4 pp335ndash340 2009

[2] F R Braga J V Araujo A K Campos et al ldquoIn vitro evaluationof the action of the nematophagous fungi Duddingtonia fla-grans Monacrosporium sinense and Pochonia chlamydosporiaon Fasciola hepatica eggsrdquo World Journal of Microbiology andBiotechnology vol 24 no 8 pp 1559ndash1564 2008

[3] R C L Assis A Viabilidade dos fungos predadoresMonacrosporium thaumasiume Duddingtonia flagrans eMoxidectina sobre nematoides gastrintestinais de bovinos decorte [Tese de Doutorado] UFV Vicosa Brazil 2013

[4] R O Carvalho J V Araujo F R Braga et al ldquoBiologicalcontrol ofAncylostomosis in dogs using the nematode-trappingfungus Monacrosporium thaumasium in southeastern BrazilrdquoVeterinary Parasitology vol 165 no 1-2 pp 179ndash183 2009

[5] J V Araujo A P S Gomes and M P Guimaraes ldquoBiolog-ical control of bovine gastrointestinal nematode parasites insouthern Brazil by the nematode-trapping fungus Arthrobotrysrobustardquo Revista Brasileira de Parasitologia Veterinaria vol 7pp 117ndash122 1998

[6] M Larsen P Nansen J Wolstrup J Groslashnvold S A Henriksenand A Zorn ldquoBiological control of trichostrongyles in calves bythe fungus Duddingtonia flagrans fed to animals under naturalgrazing conditionsrdquoVeterinary Parasitology vol 60 no 3-4 pp321ndash330 1995

[7] A S Dias J V Araujo A K Campos F R Braga and T AFonseca ldquoApplication of a formulation of the nematophagousfungus Duddingtonia flagrans in the control of cattle gas-trointestinal nematodiosisrdquo World Journal of Microbiology andBiotechnology vol 23 no 9 pp 1245ndash1252 2007

[8] J VAraujo andWM Sampaio ldquoEffects of temperaturemineralsalt and passage through gastrointestinal tract of calves on algi-nate formulation of Arthrobotrys robustardquo Revista Brasileira deParasitologia Veterinaria vol 9 pp 55ndash59 2000

[9] P Alves J Araujo M Guimaraes R L Assis P Sarti andA Campos ldquoAplicacao de formulacao do fungo predador denematoides Monacrosporium thaumasium (Drechsler 1937)no controle de nematoides de bovinosrdquo Arquivo Brasileiro deMedicina Veterinaria e Zootecnia vol 55 no 5 pp 568ndash5732003

[10] J V Araujo M P Guimaraes A K Campos N C Sa PSarti and R C Assis ldquoControl of bovine gastrointestinalnematode parasites using pellets of the nematode-trappingfungus Monacrosporium thaumasiumrdquo Ciencia Rural vol 34no 2 pp 457ndash463 2004

[11] P A Ferreira S Ferraz E A Lopes and L G Freitas ldquoPara-sitismo de ovos deMeloidogyne exigua por fungos nematofagose estudo da compatibilidade entre os isolados fungicosrdquo RevistaTropical de Ciencias Agrarias e Biologicas vol 2 no 21 2008

[12] M Dalla-Pria and S Ferraz ldquoControle biologico de Meloidog-yne incognita por seis especies deMonacrosporium isoladas oucombinadas comVerticillium chlamydosporiumrdquo FitopatologiaBrasileira vol 21 pp 30ndash34 1996

[13] T de Hollanda Ayupe T S A Monteiro F R Braga etal ldquoAssessment of compatibility between the nematophagousfungi Arthrobotrys robusta and Duddingtonia flagrans underlaboratory conditionsrdquo Revista Iberoamericana de Micologıavol 33 no 2 pp 129-130 2016

[14] A D O Tavela J V de Araujo F R Braga et al ldquoCoad-ministration of sodium alginate pellets containing the fungiDuddingtonia flagrans and Monacrosporium thaumasium oncyathostomin infective larvae after passing through the gas-trointestinal tract of horsesrdquo Research in Veterinary Science vol94 no 3 pp 568ndash572 2013

[15] HMGordon andHVWhitlock ldquoNew technique for countingnematode eggs in sheep faecesrdquo Journal of the CSIR vol 12 pp50ndash52 1939

[16] R K Keith ldquoThe differentiation of the infective larvae of somecommon nematode parasites of cattlerdquo Australian Journal ofZoology vol 1 no 2 pp 223ndash235 1953

[17] P M De Gives K G Davies S J Clark and J M BehnkeldquoPredatory behaviour of trapping fungi against srf mutants ofCaenorhabditis elegans and different plant and animal parasiticnematodesrdquo Parasitology vol 119 no 1 pp 95ndash104 1999

[18] S-O Dimander J Hoglund and P J Waller ldquoSeasonal trans-lation of infective larvae of gastrointestinal nematodes of cattleand the effect of Duddingtonia flagrans A 3-year plot studyrdquoVeterinary Parasitology vol 117 no 1-2 pp 99ndash116 2003

[19] M E Fontenot J E Miller M T Pena M Larsen and AGillespie ldquoEfficiency of feeding Duddingtonia flagrans chlamy-dospores to grazing ewes on reducing availability of parasiticnematode larvae on pasturerdquo Veterinary Parasitology vol 118no 3-4 pp 203ndash213 2003

[20] A R Silva J V Araujo F R Braga et al ldquoBiological controlof sheep gastrointestinal nematodiasis in a tropical region ofthe southeast of Brazil with the nematode predatory fungiDuddingtonia flagrans and Monacrosporium thaumasiumrdquoParasitology Research vol 105 no 6 pp 1707ndash1713 2009

[21] V Longo Ribeiro Vilela T F Feitosa F R Braga et al ldquoCoad-ministration of nematophagous fungi for biological control overgastrointestinal helminths in sheep in the semiarid region ofnortheastern Brazilrdquo Veterinary Parasitology vol 221 pp 139ndash143 2016

[22] F R Braga A R e Silva J M Araujo R O Carvalho JV de Araujo and L N Frassy ldquoPredatory activity of thenematophagous fungi duddingtonia flagrans monacrosporiumthaumasium and arthrobotrys robusta on strongyloides sterco-ralis infective larvaerdquo Journal of the Brazilian Society of TropicalMedicine vol 43 no 5 pp 588ndash590 2010

[23] M Mota C Bevilacqua and J V Araujo ldquoAtividade predatoriados fungos Arthrobotrys conoides e Monacrosporium thau-masium sobre larvas infectantes de Haemonchus contortus decaprinosrdquo Ciencia Animal vol 10 pp 37ndash41 2000

[24] MMorgan J M Behnke J A Lucas and J F Peberdy ldquoIn vitroassessment of the influence of nutrition temperature and larvaldensity on trapping of the infective larvae of Heligmosomoidespolygyrus by Arthrobotrys oligospora Duddingtonia flagransand Monacrosporium megalosporumrdquo Parasitology vol 115no 3 pp 303ndash310 1997

[25] A A Castro C R C Oliveira D H S Anjos et al ldquoPotencialdos fungos nematofagos Arthrobotrys sp e Monacrosporiumthaumasium para o controle de larvas de ciatostomıneos deequinos (Nematoda Cyathostominae)rdquo Revista Brasileira deParasitologia Veterinaria vol 12 no 2 pp 53ndash57 2003

[26] P Mendoza-de Gives and V M Vazquez-Prats ldquoReduction ofHaemonchus contortus infective larvae by three nematophagousfungi in sheep faecal culturesrdquo Veterinary Parasitology vol 55no 3 pp 197ndash203 1994

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

BioMed Research International 5

due to D flagrans while combinations featuring A robustawere less efficient at reducing L3 and the FEC counts

In the present study the percent reduction of L3 achievedby the M thaumasium isolate alone was 923 SimilarlyAraujo et al [10] recorded larval reduction in the coprocul-ture of animals treated with M thaumasium in the Braziliansemiarid region In their study the combination ofD flagransand M thaumasium resulted in a 907 decrease in thenumber of larvae in the coproculture

Furthermore the in vitro action of fungal isolates Arobusta and M thaumasium on L3 cyathostome in horseswas compared and the percent reduction obtained with Mthaumasium 313 (934) was higher than that obtained withthe A robusta isolate (863) at 25∘C The combination ofthe same isolates (A robusta +M thaumasium) in this studyreduced the L3 recovered in the coproculture by 815 whilea combination of isolates A robusta + M thaumasium + thefungus D flagrans decreased L3 by 783

It is important to note that climate plays an important rolein the ability of fungi to trap nematodes particularly sincethe optimum growth temperature varies with each fungalspeciesMorgan et al observed that temperatures in the rangeof 20∘Cndash33∘C influenced the larvae trapping percentage indifferent species of fungi [24] Thus optimal rainfalls andpredation temperatures that is specific environmental cli-matic conditions may be directly related to the results of thisstudy To support this Castro et al reported that A robustaexhibited the best larval trapping results at temperaturesranging from 25∘C to 28∘C while M thaumasium was notaffected by temperatures of 25∘Cndash30∘C which confirms thattemperature influences the degree of trapping depending onthe cyathostome species or genus [25]

In addition Castro et al obtained a 9336 reductionin the number of cyathostome larvae after administering Mthaumasium at 25∘CThe authors found that temperatures of25∘C 28∘C and 30∘C did not affect the performance of Mthaumasium which demonstrated an mean efficiency of 94[25] The results obtained in our study corroborate the find-ings of these previous works and suggest that the efficiencywith which larvae are controlled by nematophagous fungiessentially depends on the choice of the fungal species as wellas on their suitability for specific temperature conditions

The fungus Monacrosporium demonstrated unvaryingperformance in vitro at a temperature range of 15∘Cndash30∘Cas reported by Mendoza-de Gives and Vazquez-Prats [26]and Castro et al [25] even at 30∘C a common temperaturein the tropics thus this fungi would be better adapted to theconditions of the Brazilian climateThese results may supportand explain the higher degree of efficiency exhibited by thecombinationM thaumasium and D flagrans in reducing theFEC and L3 in this study particularly when compared withthe administration of A robusta and D flagrans Moreoverthe results also corroborate the reduced percentage of L3observed in the coproculture after administering combina-tions featuring A robusta In this study the regionrsquos tempera-tures ranged from 243∘C to 305∘C whichmay be consideredunsuitable for optimal predation by A robusta

Of note all of the treated groups showed a similar patternof weight gain during the study Even though notable weight

gain differences were found between heifers in the treatedgroups there was a significant difference (119875 lt 001) in weightgain between the treated groups which varied in an inverselyproportional manner to both the number of larvae recoveredin pasture and the FEC Specifically the heifers treated withD flagrans and D flagrans + M thaumasium showed greatermean weights This reinforces the fact that administeringpellets containing fungi was favored when the animals werepretreated Moreover greater weight gains in the treatedanimals (as compared to those in the control group) werealso observed by Araujo et al when testing the fungusM thaumasium in goats in the Brazilian semiarid regionFurthermore Braga et al [1] studied horses in the fieldthe researchers administered the nematophagous fungus Dflagrans and they observed significantweight gain differencesbetween the groups treated with the fungus and the controlanimals The animals that were treated with the fungusdemonstrated greater weight gains than those in the controlgroup

5 Conclusion

Treating dairy cattle with alginate pellets containing andcoadministering the nematophagous fungi D flagrans Arobusta andM thaumasium resulted in the biological controlof gastrointestinal nematodes in bovines although the levelof control was not increased when compared with the useof the fungus D flagrans alone Coadministration with theA robusta isolate was not considered a good alternative Theadministration of D flagrans alone was found to be morepromising than coadministration for continuous use in dairycattle in this tropical region in Brazil

Ethical Approval

The authors assert that all procedures contributing to thiswork comply with the ethical standards of the relevantnational and institutional committees on human experimen-tation and with the Helsinki Declaration of 1975 as revised in2008

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

The authors would like to thank the author of the thesisldquoViability of Predatory Fungi M 421 thaumasium and Dflagrans and Moxidectin on Gastrointestinal Nematodes ofBeef 422 Cattlerdquo a member of this research group whosemethodology forms the basis of this paper 423 and theyacknowledge Fundacao de Amparo a Pesquisa de MinasGerais (FAPEMIG) for the support in 424 project financingand in scientific dissemination for the publication of thispaperThis work was financially supported by FAPEMIG andCAPES

6 BioMed Research International

References

[1] F R Braga J V Araujo A R Silva et al ldquoBiological controlof horse cyathostomin (Nematoda Cyathostominae) usingthe nematophagous fungus Duddingtonia flagrans in tropicalsoutheastern Brazilrdquo Veterinary Parasitology vol 163 no 4 pp335ndash340 2009

[2] F R Braga J V Araujo A K Campos et al ldquoIn vitro evaluationof the action of the nematophagous fungi Duddingtonia fla-grans Monacrosporium sinense and Pochonia chlamydosporiaon Fasciola hepatica eggsrdquo World Journal of Microbiology andBiotechnology vol 24 no 8 pp 1559ndash1564 2008

[3] R C L Assis A Viabilidade dos fungos predadoresMonacrosporium thaumasiume Duddingtonia flagrans eMoxidectina sobre nematoides gastrintestinais de bovinos decorte [Tese de Doutorado] UFV Vicosa Brazil 2013

[4] R O Carvalho J V Araujo F R Braga et al ldquoBiologicalcontrol ofAncylostomosis in dogs using the nematode-trappingfungus Monacrosporium thaumasium in southeastern BrazilrdquoVeterinary Parasitology vol 165 no 1-2 pp 179ndash183 2009

[5] J V Araujo A P S Gomes and M P Guimaraes ldquoBiolog-ical control of bovine gastrointestinal nematode parasites insouthern Brazil by the nematode-trapping fungus Arthrobotrysrobustardquo Revista Brasileira de Parasitologia Veterinaria vol 7pp 117ndash122 1998

[6] M Larsen P Nansen J Wolstrup J Groslashnvold S A Henriksenand A Zorn ldquoBiological control of trichostrongyles in calves bythe fungus Duddingtonia flagrans fed to animals under naturalgrazing conditionsrdquoVeterinary Parasitology vol 60 no 3-4 pp321ndash330 1995

[7] A S Dias J V Araujo A K Campos F R Braga and T AFonseca ldquoApplication of a formulation of the nematophagousfungus Duddingtonia flagrans in the control of cattle gas-trointestinal nematodiosisrdquo World Journal of Microbiology andBiotechnology vol 23 no 9 pp 1245ndash1252 2007

[8] J VAraujo andWM Sampaio ldquoEffects of temperaturemineralsalt and passage through gastrointestinal tract of calves on algi-nate formulation of Arthrobotrys robustardquo Revista Brasileira deParasitologia Veterinaria vol 9 pp 55ndash59 2000

[9] P Alves J Araujo M Guimaraes R L Assis P Sarti andA Campos ldquoAplicacao de formulacao do fungo predador denematoides Monacrosporium thaumasium (Drechsler 1937)no controle de nematoides de bovinosrdquo Arquivo Brasileiro deMedicina Veterinaria e Zootecnia vol 55 no 5 pp 568ndash5732003

[10] J V Araujo M P Guimaraes A K Campos N C Sa PSarti and R C Assis ldquoControl of bovine gastrointestinalnematode parasites using pellets of the nematode-trappingfungus Monacrosporium thaumasiumrdquo Ciencia Rural vol 34no 2 pp 457ndash463 2004

[11] P A Ferreira S Ferraz E A Lopes and L G Freitas ldquoPara-sitismo de ovos deMeloidogyne exigua por fungos nematofagose estudo da compatibilidade entre os isolados fungicosrdquo RevistaTropical de Ciencias Agrarias e Biologicas vol 2 no 21 2008

[12] M Dalla-Pria and S Ferraz ldquoControle biologico de Meloidog-yne incognita por seis especies deMonacrosporium isoladas oucombinadas comVerticillium chlamydosporiumrdquo FitopatologiaBrasileira vol 21 pp 30ndash34 1996

[13] T de Hollanda Ayupe T S A Monteiro F R Braga etal ldquoAssessment of compatibility between the nematophagousfungi Arthrobotrys robusta and Duddingtonia flagrans underlaboratory conditionsrdquo Revista Iberoamericana de Micologıavol 33 no 2 pp 129-130 2016

[14] A D O Tavela J V de Araujo F R Braga et al ldquoCoad-ministration of sodium alginate pellets containing the fungiDuddingtonia flagrans and Monacrosporium thaumasium oncyathostomin infective larvae after passing through the gas-trointestinal tract of horsesrdquo Research in Veterinary Science vol94 no 3 pp 568ndash572 2013

[15] HMGordon andHVWhitlock ldquoNew technique for countingnematode eggs in sheep faecesrdquo Journal of the CSIR vol 12 pp50ndash52 1939

[16] R K Keith ldquoThe differentiation of the infective larvae of somecommon nematode parasites of cattlerdquo Australian Journal ofZoology vol 1 no 2 pp 223ndash235 1953

[17] P M De Gives K G Davies S J Clark and J M BehnkeldquoPredatory behaviour of trapping fungi against srf mutants ofCaenorhabditis elegans and different plant and animal parasiticnematodesrdquo Parasitology vol 119 no 1 pp 95ndash104 1999

[18] S-O Dimander J Hoglund and P J Waller ldquoSeasonal trans-lation of infective larvae of gastrointestinal nematodes of cattleand the effect of Duddingtonia flagrans A 3-year plot studyrdquoVeterinary Parasitology vol 117 no 1-2 pp 99ndash116 2003

[19] M E Fontenot J E Miller M T Pena M Larsen and AGillespie ldquoEfficiency of feeding Duddingtonia flagrans chlamy-dospores to grazing ewes on reducing availability of parasiticnematode larvae on pasturerdquo Veterinary Parasitology vol 118no 3-4 pp 203ndash213 2003

[20] A R Silva J V Araujo F R Braga et al ldquoBiological controlof sheep gastrointestinal nematodiasis in a tropical region ofthe southeast of Brazil with the nematode predatory fungiDuddingtonia flagrans and Monacrosporium thaumasiumrdquoParasitology Research vol 105 no 6 pp 1707ndash1713 2009

[21] V Longo Ribeiro Vilela T F Feitosa F R Braga et al ldquoCoad-ministration of nematophagous fungi for biological control overgastrointestinal helminths in sheep in the semiarid region ofnortheastern Brazilrdquo Veterinary Parasitology vol 221 pp 139ndash143 2016

[22] F R Braga A R e Silva J M Araujo R O Carvalho JV de Araujo and L N Frassy ldquoPredatory activity of thenematophagous fungi duddingtonia flagrans monacrosporiumthaumasium and arthrobotrys robusta on strongyloides sterco-ralis infective larvaerdquo Journal of the Brazilian Society of TropicalMedicine vol 43 no 5 pp 588ndash590 2010

[23] M Mota C Bevilacqua and J V Araujo ldquoAtividade predatoriados fungos Arthrobotrys conoides e Monacrosporium thau-masium sobre larvas infectantes de Haemonchus contortus decaprinosrdquo Ciencia Animal vol 10 pp 37ndash41 2000

[24] MMorgan J M Behnke J A Lucas and J F Peberdy ldquoIn vitroassessment of the influence of nutrition temperature and larvaldensity on trapping of the infective larvae of Heligmosomoidespolygyrus by Arthrobotrys oligospora Duddingtonia flagransand Monacrosporium megalosporumrdquo Parasitology vol 115no 3 pp 303ndash310 1997

[25] A A Castro C R C Oliveira D H S Anjos et al ldquoPotencialdos fungos nematofagos Arthrobotrys sp e Monacrosporiumthaumasium para o controle de larvas de ciatostomıneos deequinos (Nematoda Cyathostominae)rdquo Revista Brasileira deParasitologia Veterinaria vol 12 no 2 pp 53ndash57 2003

[26] P Mendoza-de Gives and V M Vazquez-Prats ldquoReduction ofHaemonchus contortus infective larvae by three nematophagousfungi in sheep faecal culturesrdquo Veterinary Parasitology vol 55no 3 pp 197ndash203 1994

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

6 BioMed Research International

References

[1] F R Braga J V Araujo A R Silva et al ldquoBiological controlof horse cyathostomin (Nematoda Cyathostominae) usingthe nematophagous fungus Duddingtonia flagrans in tropicalsoutheastern Brazilrdquo Veterinary Parasitology vol 163 no 4 pp335ndash340 2009

[2] F R Braga J V Araujo A K Campos et al ldquoIn vitro evaluationof the action of the nematophagous fungi Duddingtonia fla-grans Monacrosporium sinense and Pochonia chlamydosporiaon Fasciola hepatica eggsrdquo World Journal of Microbiology andBiotechnology vol 24 no 8 pp 1559ndash1564 2008

[3] R C L Assis A Viabilidade dos fungos predadoresMonacrosporium thaumasiume Duddingtonia flagrans eMoxidectina sobre nematoides gastrintestinais de bovinos decorte [Tese de Doutorado] UFV Vicosa Brazil 2013

[4] R O Carvalho J V Araujo F R Braga et al ldquoBiologicalcontrol ofAncylostomosis in dogs using the nematode-trappingfungus Monacrosporium thaumasium in southeastern BrazilrdquoVeterinary Parasitology vol 165 no 1-2 pp 179ndash183 2009

[5] J V Araujo A P S Gomes and M P Guimaraes ldquoBiolog-ical control of bovine gastrointestinal nematode parasites insouthern Brazil by the nematode-trapping fungus Arthrobotrysrobustardquo Revista Brasileira de Parasitologia Veterinaria vol 7pp 117ndash122 1998

[6] M Larsen P Nansen J Wolstrup J Groslashnvold S A Henriksenand A Zorn ldquoBiological control of trichostrongyles in calves bythe fungus Duddingtonia flagrans fed to animals under naturalgrazing conditionsrdquoVeterinary Parasitology vol 60 no 3-4 pp321ndash330 1995

[7] A S Dias J V Araujo A K Campos F R Braga and T AFonseca ldquoApplication of a formulation of the nematophagousfungus Duddingtonia flagrans in the control of cattle gas-trointestinal nematodiosisrdquo World Journal of Microbiology andBiotechnology vol 23 no 9 pp 1245ndash1252 2007

[8] J VAraujo andWM Sampaio ldquoEffects of temperaturemineralsalt and passage through gastrointestinal tract of calves on algi-nate formulation of Arthrobotrys robustardquo Revista Brasileira deParasitologia Veterinaria vol 9 pp 55ndash59 2000

[9] P Alves J Araujo M Guimaraes R L Assis P Sarti andA Campos ldquoAplicacao de formulacao do fungo predador denematoides Monacrosporium thaumasium (Drechsler 1937)no controle de nematoides de bovinosrdquo Arquivo Brasileiro deMedicina Veterinaria e Zootecnia vol 55 no 5 pp 568ndash5732003

[10] J V Araujo M P Guimaraes A K Campos N C Sa PSarti and R C Assis ldquoControl of bovine gastrointestinalnematode parasites using pellets of the nematode-trappingfungus Monacrosporium thaumasiumrdquo Ciencia Rural vol 34no 2 pp 457ndash463 2004

[11] P A Ferreira S Ferraz E A Lopes and L G Freitas ldquoPara-sitismo de ovos deMeloidogyne exigua por fungos nematofagose estudo da compatibilidade entre os isolados fungicosrdquo RevistaTropical de Ciencias Agrarias e Biologicas vol 2 no 21 2008

[12] M Dalla-Pria and S Ferraz ldquoControle biologico de Meloidog-yne incognita por seis especies deMonacrosporium isoladas oucombinadas comVerticillium chlamydosporiumrdquo FitopatologiaBrasileira vol 21 pp 30ndash34 1996

[13] T de Hollanda Ayupe T S A Monteiro F R Braga etal ldquoAssessment of compatibility between the nematophagousfungi Arthrobotrys robusta and Duddingtonia flagrans underlaboratory conditionsrdquo Revista Iberoamericana de Micologıavol 33 no 2 pp 129-130 2016

[14] A D O Tavela J V de Araujo F R Braga et al ldquoCoad-ministration of sodium alginate pellets containing the fungiDuddingtonia flagrans and Monacrosporium thaumasium oncyathostomin infective larvae after passing through the gas-trointestinal tract of horsesrdquo Research in Veterinary Science vol94 no 3 pp 568ndash572 2013

[15] HMGordon andHVWhitlock ldquoNew technique for countingnematode eggs in sheep faecesrdquo Journal of the CSIR vol 12 pp50ndash52 1939

[16] R K Keith ldquoThe differentiation of the infective larvae of somecommon nematode parasites of cattlerdquo Australian Journal ofZoology vol 1 no 2 pp 223ndash235 1953

[17] P M De Gives K G Davies S J Clark and J M BehnkeldquoPredatory behaviour of trapping fungi against srf mutants ofCaenorhabditis elegans and different plant and animal parasiticnematodesrdquo Parasitology vol 119 no 1 pp 95ndash104 1999

[18] S-O Dimander J Hoglund and P J Waller ldquoSeasonal trans-lation of infective larvae of gastrointestinal nematodes of cattleand the effect of Duddingtonia flagrans A 3-year plot studyrdquoVeterinary Parasitology vol 117 no 1-2 pp 99ndash116 2003

[19] M E Fontenot J E Miller M T Pena M Larsen and AGillespie ldquoEfficiency of feeding Duddingtonia flagrans chlamy-dospores to grazing ewes on reducing availability of parasiticnematode larvae on pasturerdquo Veterinary Parasitology vol 118no 3-4 pp 203ndash213 2003

[20] A R Silva J V Araujo F R Braga et al ldquoBiological controlof sheep gastrointestinal nematodiasis in a tropical region ofthe southeast of Brazil with the nematode predatory fungiDuddingtonia flagrans and Monacrosporium thaumasiumrdquoParasitology Research vol 105 no 6 pp 1707ndash1713 2009

[21] V Longo Ribeiro Vilela T F Feitosa F R Braga et al ldquoCoad-ministration of nematophagous fungi for biological control overgastrointestinal helminths in sheep in the semiarid region ofnortheastern Brazilrdquo Veterinary Parasitology vol 221 pp 139ndash143 2016

[22] F R Braga A R e Silva J M Araujo R O Carvalho JV de Araujo and L N Frassy ldquoPredatory activity of thenematophagous fungi duddingtonia flagrans monacrosporiumthaumasium and arthrobotrys robusta on strongyloides sterco-ralis infective larvaerdquo Journal of the Brazilian Society of TropicalMedicine vol 43 no 5 pp 588ndash590 2010

[23] M Mota C Bevilacqua and J V Araujo ldquoAtividade predatoriados fungos Arthrobotrys conoides e Monacrosporium thau-masium sobre larvas infectantes de Haemonchus contortus decaprinosrdquo Ciencia Animal vol 10 pp 37ndash41 2000

[24] MMorgan J M Behnke J A Lucas and J F Peberdy ldquoIn vitroassessment of the influence of nutrition temperature and larvaldensity on trapping of the infective larvae of Heligmosomoidespolygyrus by Arthrobotrys oligospora Duddingtonia flagransand Monacrosporium megalosporumrdquo Parasitology vol 115no 3 pp 303ndash310 1997

[25] A A Castro C R C Oliveira D H S Anjos et al ldquoPotencialdos fungos nematofagos Arthrobotrys sp e Monacrosporiumthaumasium para o controle de larvas de ciatostomıneos deequinos (Nematoda Cyathostominae)rdquo Revista Brasileira deParasitologia Veterinaria vol 12 no 2 pp 53ndash57 2003

[26] P Mendoza-de Gives and V M Vazquez-Prats ldquoReduction ofHaemonchus contortus infective larvae by three nematophagousfungi in sheep faecal culturesrdquo Veterinary Parasitology vol 55no 3 pp 197ndash203 1994

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom