Fungi extract in the inhibition of the egg hatching and larval development of the Haemonchus contortus

1 State University of Montes Claros, Brazil. 2 Center for Biological Sciences and Health, Campus Universitário Professor Darcy Ribeiro Vila Mauricéia Montes Claros Minas Gerais, Brasil. 3 Federal of São Paulo, Brazil. 4 Faculty of Animal Science and Food Engineering, Av. Duque de Caxias Norte, 225-Campus Fernando Costa USPPirassununga, São Paulo, Brasil. 5 Federal University of Minas Gerais, Brazil. 6 Institute of Agricultural Sciences, Avenida Universitária, 1000 Bairro Universitário Montes Claros Minas Gerais. 39400-006, Brasil.

hemonchosis may show anemia and submandibula swelling, with high mortality in young lambs and peripartal ewes.Both sexes at all age levels may be infected, decreasing weight gain and feed conversion efficiency; reduced meat, wool, and milk production; and reproductive disorders (Sczesny-Moraes et al., 2010;Taylor et al., 2010).
The frequent treatment with synthetic antihelminthics is the main method of control.However, it has promoted rapid selection of resistant helminthes populations (Duarte et al., 2012;Adiele et al., 2013;Soro et al., 2013).Resistance has been observed to the main classes of antihelminthics in different continents, impeding the animal breeding (Taylor et al., 2009).Thus, the search for alternatives is being widely done in order to decrease animal infection, the contamination of animal products, the environment and the human beings by chemical antihelminthics residues.
Researches using fungi as a biological control have shown that this alternative consists of a promising control method of gastrointestinal nematodes (Araujo et al., 2004).Nevertheless, the development of fungal formulations economically practicable and easily applicable is a challenge in the implementation of the fungal species in programs of biological control (Campos et al., 2007).
Paecilomyces lilacinus can infect eggs and adult females of Meloidogyne spp.nematode and causes death of embryos in 5 to 7 days.This fungus has given excellent results under varying conditions (Mukhtar et al., 2013).Similarly, Trichoderma longibrachiatum has been found to be an effective biocontrol agent for the management nemateded of root-knot and other plant nematodes.The mechanisms of T. longibrachiatum against nematodes are essentially unknown (Zhang et al., 2014).
Little is known about the effect of these fungi extracts in the ruminant gastrointestinal nematodes cycle.In this paper, it was assessed the efficacy of the extracts of the fungi P. lilacenus and T. longibrachiatum in the inhibition of egg hatching and of the larval development of H. contortus.

Study area
The research was conducted in a rural area in city of Montes Claros in the north of Minas Gerais state, Brazil (W 43°50'33.56";S 16°41'10.05").The climate of the region is tropical wet with dry summer (As) according to the Köppen classification (Alvares et al., 2013), is marked by a long dry season from May to October and a rainy period in January and February.

Molecular identification
Isolated fungi were obtained from the rectum of the sheep matrix of the breed Santa Inês raised in tropical field (Freitas et al., 2012).Extraction of total DNA of microorganisms was performed according to Rosa et al. (2009).The primers ITS1 (TCCGTAGGTGAACCTGCGG) and ITS4 (TCCTCCGCTTATTGATATGC) were used for amplification of the rDNA region of aerobic mycelia fungi, as described by White et al. (1990).The primers used for yeasts were NL1 (5'-GCA TAT CAA AAG GAA GAG TAA GCC-3') and NL4 (5'-GGT AAG CTT GCA TGT CCG G-3').The Polymerase Chain Reaction (PCR) was performed and amplicons produced were purified and assayed in NanoDrop ND 1000 (NanoDrop Technologies) as reported in Abrão et al. (2014).The sequencing reactions were performed in DYEnamicTM (Amersham Biosciences, USA) associated with automated sequencing system MegaBACETM 1000 (Lachance et al., 1999).The sequences of the rDNA fragments were analyzed according to Altschul et al. (1997), in the BLASTN program (Basic Local Alignment Search Tool -2215 BLAST version 2.0) from the National Center for Biotechnology.Isolates with similarity ≥99% to a previously deposited sequence were considered to belong to the same species.After identification, the cultures were preserved as Castellani (1967).

Sample collection and aqueous and ethanolic extracts
The fungi were previously cultivated in plates containing solid Agar Sabouraud (Sabouraud Dextrose Agar, Prodimol Biotechnology, MG, Brazil) during seven days at 28°C.It was used as inoculum five discs of about 5 mm of diameter of the fungal cultures transferred to Erlenmeyers containing liquid Sabouraud, with no yeast extract.It was incubated in termoshaker at 30°C and 150 rpm during seven days.After this period, the cultivations were filtered twice in Whatman n° 1 paper and in Millipore membrane with 0.22 μm of porosity, using vacuum pump.The liquid obtained of each culture, called aqueous extract, was used immediately, and the fungal mass was frozen at -4°C.
To obtain the ethanolic extracts, an adapted method from Nery et al. (2010) was used.100 mL of ethyl alcohol P.A. (99.5ºGL) was added to each 50 g of fungal mass.The mixture was homogenized and transferred to a beaker tighten with plastic wrap and incubated in bain-marie at 40°C for 90 min.Later, it was filtrated through two Whatman nº 1 papers.The extracts obtained were placed in Petri plates for dehydration in an incubator with forced air circulation at 40 ± 5°C for three days and stored in freezer at -4°C.
The dry matter (DM) of the extract subsamples was determined at 105°C according to Association of Official Analytical Chemists (AOAC, 1990), to the tested concentrations calculus (Cunniff, 1995).

Egg hatching inhibition
Flotation, sedimentation, and filtration in saturated NaCl solution were conducted to obtain nematode eggs from feces (Coles et al., 1992) of two Santa Inês lambs infected with H. contortus and with an average fecal egg count of >1000 g -1 , determined using the modified McMaster technique (Gordon and Whitlock, 1939).The procedures were done in accordance with the ethical principles of the animal experimentation, approved in the 42/2008 protocol of the Ethics Committee on the Animal Experimental of the Federal University of Minas Gerais.
The treatments were conducted with five replicates: Positive control with levamisole phosphate (0.3 mg mL -1 ); negative control with sterile purified water; and experimental treatments comprising four concentrations of each extracts (Tables 1 and 2).
Experimental mixtures comprised 100 μL of fecal suspension with an average of 80 fresh eggs and 100 μL of the extracts.The samples were homogenized and incubated in a BOD incubator at 28°C for 48 h.Subsequently, 15 μL of Lugol's solution was added to each tube, which were then stored at 4°C for counting of unembryonated and embryonated eggs, and stage one larvae (L1) Different letters in columns indicates significant differences by Duncan test (P < 0.05).*% EHI = 100 × (1 -L1 of treatment / L1 of control).(Coles et al., 1992).
The egg count and L1 was transformed into relative values for the initial number of eggs for replicate.The means were compared using Tukey's test (P < 0.05).Probit regression was employed to determine the concentrations sufficient to inhibit 50% (LC50) and 90% (LC90) of egg hatching with SAEG software (SAEG, 2007).The percent of egg hatching inhibition (EHI) and larval development was calculated using the formula modified after Coles et al. (1992): *% EHI = 100 × (1 -L1 of treatment / L1 of control).

Inhibition of larval development
To evaluate the effectiveness of aqueous extract of P. lilacinus and T. longibrachiatum on larval development inhibition (LDI), the quantitative coproculture method (Borges, 2003;Nery et al., 2010; Treatments were conducted, each with five replicates, including positive controls with 2 mL of 16 μg g -1 ivermectin solution (LA Ranger, Vallée, Minas Gerais, Brazil) added to 2 g of feces, and negative control of 2 mL of sterile purified water (negative control).The experimental treatments consisted of aqueous extract of P. lilacinus and T. longibrachiatum standardized at 0.28 and 1.13 mg g -1 and 0.20 and 0.79 mg dried weight (dw) g -1 , respectively.
The cultures were incubated in a BOD incubator at 28°C for seven days and assessed for presence of infective larvae (L3).The following formula, adapted from Borges (2003), was used to determine the percent reduction of larvae (L3) per gram of feces (LPGF): % LDI = 100 × (1 -LPGF of the treated group/LPGF of the untreated group).
The data was log transformed, log (x+1), and submitted to variance analysis.The means were compared by the Duncan's test (P < 0.05).The LC50 and LC90 were determined by probit analysis with SAEG software (SAEG, 2007).

RESULTS AND DISCUSSION
Sequence analysis of rDNA revealed the presence of P. lilacinus and T. longibrachiatum with 99.9% of similarity.These species have been used largely in the control of plants nematodes and they are producers of lytic enzymes, which are able to destroy parasitical structures (Ferreira et al., 2008;Shuwu et al., 2015;Huang et al., 2016).
For P. lilacinus, it verified efficacy of 100% in the EHI in all concentrations of aqueous extract (1.96 to 7.85 mg mL -1 ) and efficacy superior to 99% in the concentrations ≥ 16.8 mg mL -1 of the ethanolic extract (Table 1).The LC 50 and LC 90 obtained in the egg hatching inhibition, using ethanolic extract, were 4.77 and 10.31 mg mL -1 , respectively (Figure 1).This way, it is suggested that these fungus metabolites can have effect in the inhibition of the embryonic development and in the egg hatching inhibition of H. contortus (Zhang et al., 2014).
It was not possible to estimate the LC 50 and LC 90 for EHI experiments with a aqueous extract of the P. lilacinus because the low tested concentration showed 100% of efficacy.For the aqueous extract and ethanol from T. longibrachiatum it was observed a dose-dependent response.
For the ethanolic extract of T. longibrachiatum, all evaluated concentrations presented significant reduction in the number of L1 when related to the distilled water control with efficacy superior to 95% (Table 2).The aqueous extract at 0.59 mg mℓ -1 presented significant reduction in the average of L1 larvae hatched, and all concentrations reduced the total number of present eggs and larvae after 48 h of incubation, when compared to the distilled water control (Table 2).Thus, it is suggested that enzymes or the fungus metabolites could have caused degradation in these structures, destroying them completely and impeding the microscopic examination.
In the inhibition of larval development, the concentration of 0.79 mg g -1 of the aqueous extract of T. longibrachiatum promoted a reduction (p<0.05) in the average of the infective larvae with efficacy ≥ 92% (Table 3), showing that, even in fecal material, the metabolites presented lethal effect for the eggs and the nematode larvae.Due to the low productivity in dry matter to the aqueous extract of P. lilacinus used in the quantitative coproculture, the concentrations used were too low, not obtaining relevant efficacy (Table 3).
To all experiments using the aqueous and ethanolic extracts of P. lilacinus and T. longibrachiatum, it was Probability of hatching (%) Concentrations (mg/mL) verified eggs and larvae L1 with structural deformity in the barks and cuticles.The distilled water controls performed embryonic and normal larval development to the nematode.Some fungi species can destroy nematodes with toxin action (Nordbring-Hertz, 1988) and others can produce metabolites with ovicidal effects (Waller and Faedo, 1993).Nematophagous fungi can penetrate the cuticle structure of the nematode larvae because they have proteases, chitinases and collagenases involved directly in the nematode infection process (Gryndler et al., 2003).According to Braga et al. (2011), the fungus Pochonia clamydosporia produces a protease that destroys the eggs of fitonematodes and of Ancylostoma spp.
Fungi are known to have huge metabolic diversity providing a wide range of commercial compounds, including many antibiotics used in medicine.Furthermore, metabolites produced by fungi may have potential nematicides, being targets of research in biotechnology (Smedsgaard and Nielsen, 2005).
A new peptide extracted of Omphalotus olearius fungus has demonstrated similar action to commercial nematicide Ivermectin (Mayer et al., 1997).Review by Li et al. (2007) showed 179 compounds isolated from fungi that have demonstrated activity as nematicide groups of alkaloids, peptides terpenoids, macrolide compounds, oxygen heterocycle and benzo compounds, aliphatic compounds, simple aromatic compounds.
According to Ferreira et al. (2008), the species P. lilacinus is a parasite of eggs and of female adults of fitonematodes and present great relevance to the biological control.Devrajan and Seenivasan (2002) observed that P. lilacinus presented toxic effect to the adult fitonematodes from the genus Melodoidyne.This fungal species also performed efficacy in vitro on Taenia saginata eggs in conditions at the end of ten days (Braga, 2008) and on Toxocara canis eggs after seven days of treatment (Araújo et al. 2004).
On the commercialized bioproducts in relation to the control of pathogenic agents causers of diseases in plants, there are three bioproducts based on A. flavus for the fungi control, 11 based on Paecilomyce ssp.for the nematodes control and 55 based on T. longibrachiatum for the fungi and bacteria control (Sosa-Gomez, 2002).As regards the commercialized bioproducts in relation to the control of pathogenic agents that causes diseases in plants, there are three bioproducts based on A. flavus for fungi control, 11 based on Paecilomyce ssp.for nematodes control, and 55 based on T. longibrachiatum for fungi and bacteria control (Sosa-Gomez, 2002).

Conclusion
The aqueous extracts of P. lilacinus and ethanolic of T. longibrachiatum at low concentrations inhibited completely egg hatching.The aqueous extract of T. longibrachiatum ≥ 0.79 mg mL -1 showed efficacy ≥ 92% to LDI of H. contortus.The isolated ones from these fungi were obtained naturally from the digestive tract of sheep and in future studies the probiotic effect of these species must be evaluated on the in vive inhibition of the cycle of gastrointestinal nematodes.

Figure 1 .
Figure 1.Probability of hatching inhibition of H. contortus at different concentrations of the ethanolic extract of P. lilacenus.

Table 1 .
Effects of aqueous extract and ethanolic extract of P. lilacenus on hatchability of Haemonchus contortus.

Table 2 .
Effects of aqueous extract and ethanolic extract of T. longibrachiatum on hatchability of H. contortus.

Table 3 .
Effectiveness of the aqueous extract of P. lilacenus and T. longibrachiatum on the larval development of H. contortus in quantitative fecal cultures.