INTRODUCTION
Helminth infection is a major limiting factor for sheep production around the world. The financial impact of these infections can be observed in the costs of veterinary services, reduced animal performance and even death. Haemonchus contortus is the main parasite responsible for this burden in many parts of the world (O'Connor et al. Reference O'Connor, Walkden-Brown and Kahn2006). Through the years, H. contortus has been controlled principally with chemicals anthelmintics; however, the incidence of anthelmintic resistance is increasing among the population (Gasser et al. Reference Gasser, Bott, Chilton, Hunt and Beveridge2008; Van den Brom et al. Reference Van den Brom, Moll, Kappert and Vellema2015). To date, this parasite has acquired resistance to practically every major drug class available on the market. Drug resistance is now threatening sheep production around the world (Van Wyk et al. Reference Van Wyk, Stenson, Van der Merwe, Vorster and Viljoen1999; Cezar et al. Reference Cezar, Toscan, Camillo, Sangioni, Ribas and Vogel2010; Medeiros et al. Reference Medeiros, Ramos and Banchero2014).
We have previously shown that various Bacillus isolates are highly toxic in vitro and in vivo to H. contortus larvae, suggesting that this group of bacteria can be used as a promising biological anthelmintic (Sinott et al. Reference Sinott, Cunha Filho, Castro, Lorenzon, Pinto, Capella and Leite2012, Reference Sinott, Dias de Castro, Leite, Gallina, De-Souza, Santos and Leite2014). Bacillus thuringiensis is a Gram-positive bacterium that produces crystal inclusions upon sporulation. These inclusions are comprised Cry and Cyt proteins, which are toxic to a wide range of insects species among the orders Lepidoptera, Diptera and Coleoptera (Bravo et al. Reference Bravo, Gill and Soberón2007; Pardo-López et al. Reference Pardo-López, Soberón and Bravo2013; Soberón et al. Reference Soberón, López-Díaz and Bravo2013), as well as other invertebrates of the phylum Nematoda (Marroquin et al. Reference Marroquin, Elyassnia, Griffits, Feiltelson and Aroian2000). Commercial formulations, which generally consist of a mixture of crystal proteins and bacterial spores, are widely used for insect control (Bravo et al. Reference Bravo, Likitvivatanavong, Gill and Soberón2011).
The use of B. thuringiensis as an anthelmintic presents certain advantages over traditional chemotherapeutic approaches, including an absence of chemical residues associated with its use, high target specificity and low mammalian toxicity (Schnepf et al. Reference Schnepf, Crickmore, Van Rie, Lereclus, Baum, Feitelson, Zeigler and Dean1998; Siegel, Reference Siegel2001). Bacillus thuringiensis var. israelensis is toxic to the larval stage of H. contortus (Sinott et al. Reference Sinott, Cunha Filho, Castro, Lorenzon, Pinto, Capella and Leite2012), and there is potential for its use as a biological anthelmintic that targets the free-living larval stages developing in pasture. Biological strategies have the potential to reduce the levels of larvae in the pasture, where higher parasite numbers are present. The current study aimed to examine the potential of B. thuringiensis var. israelensis Cry11Aa toxin to control the larval stage the nematode Haemonchus sp. in sheep livestock.
MATERIALS AND METHODS
Bacillus thuringiensis var. israelensis
Spores of the strain B. thuringiensis var. israelensis (from the collection of the Department of Microbiology and Parasitology, Federal University of Pelotas) were streaked onto a BHI (Brain Heart Infusion – Acumedia) agar plate and grown for 24 h at 28 °C. Single colonies from the plate were used to inoculate 50 mL of NYSM (Yousten, Reference Yousten1984) medium and were grown at 28 °C with rotary shaking at 150 rpm for 72 h. Starter cultures were used to inoculate a flask containing 500 mL of NYSM media and then cultured at 28 °C with rotary shaking at 150 rpm for 72 h to ensure complete sporulation.
Construction of the expression vector
Bacillus thuringiensis genomic DNA was extracted using the Illustra Bacteria Genomic Prep Mini Spin Kit (GE – Healthcare) following the manufacturer's protocol. The sequence that contained the gene cry11Aa was amplified by PCR using the set of primers Cry11Aa Forward 5′ – GGG GAT CCA TGG AAG ATA GTT CTT TAG – 3′ and Cry11Aa Reverse 5′ – CCG GTA CCC TAC TTT AGT AAC GG – 3′. The primers were constructed based on a sequence obtained from GenBank (access number: AL731825.1), using the program Vector NTI (Invitrogen). The Taq DNA polymerase (Invitrogen) was used in the PCR. The amplification reaction was performed in a final volume of 25 µL containing 0·3 µL of Taq DNA polymerase (5 U µL−1), 2·0 µL 10× PCR buffer, 1·0 µL 50 mm MgCl2, 0·5 µL dNTPs, 1·0 µL, 10 pm µL−1 of the forward and reverse primers, 2 µL template (solution containing 300 ng DNA) and sterile water to complete the volume of the reaction. Amplification was performed under the following conditions: 95 °C for 5 min (1 cycle), 94 °C for 50 s, 59 °C for 50 s, 72 °C for 2 min (32 cycles) and 72 °C for 3 min (1 cycle). The reaction was performed in a thermocycler (Eppendorf Mastercycle). The PCR products were cleaved with the BamHI and KpnI restriction enzymes before ligation of the fragment into the pAE vector, which had been digested with the same enzymes. The pAE plasmid containing the DNA sequence of the cry11Aa gene, without the stop codon, was extracted and sequenced to confirm the sequence and its frame with polyhistidine-tag. The recombinant clones obtained were named pAE-cry11Aa and were used for expression studies. Escherichia coli TOP10 cells were grown on Luria–Bertani (LB)-agar containing ampicillin (100 µg mL−1) for solid culture and were used for plasmid maintenance using standard transformation protocol heat shock (Sambrook et al. Reference Sambrook, Fritsch and Maniatis1989).
Culture growth conditions and protein purification
The B. thuringiensis bacteria were grown to sporulation on NYSM agar plates at 28 °C, harvested into water, and centrifuged (10 000 × g for 10 min, 4 °C). The pellet was washed twice in water to remove cell debris and secretory products. It was then suspended in water, and the volume adjusted to an OD600 of 0·5 and these suspensions were stored at −70 °C.
Escherichia coli BL21 (DE3) C43 was transformed with pAE-cry11Aa and grown in LB medium containing ampicillin (100 µg mL−1) at 37 °C for 16 h. An aliquot of the 5 mL culture was used to inoculate 500 mL of the same medium, and the E. coli BL21 were grown under the same conditions until an OD600 of 0·5–0·6 was reached. Expression of recombinant protein Cry11Aa was induced by the addition of 0·3 mm IPTG (Isopropyl β-D-1-thiogalactopyranoside) for 3 h at 37 °C. Cell aliquots were collected by centrifugation for analysis by SDS–PAGE, Western blot and larvicidal assays. Soluble recombinant proteins were purified by affinity chromatography, where each Akta Wash supernatant (approximately 15 mL) was applied through a 1 mL Ni–Sepharose Hi Trap chelating column (GE Healthcare) that was previously equilibrated with its respective solvation buffer, following the manufacturer's guidelines. The Ni+ column was washed with 5 mL of the respective Akta Wash, and the protein was eluted with 1 mL aliquots of Elution Buffer (NaH2PO4, 0·234% and NaCl, 2·92%) with differing concentrations of Imidazole (at 10, 20, 30, 40 and 100%). Fractions of the 1 mL elution were collected. All flow-through portions were saved and applied to 12% SDS–PAGE. The elution fractions containing the protein were confirmed by SDS–PAGE. The purified rCry11Aa was examined by SDS–PAGE and Western blotting and was quantified at A 280 according to a standard curve that was established using bovine serum albumin (BSA) on 12% SDS–PAGE.
For the SDS–PAGE, cell pellets from 1 mL of the induced culture were added to 100 µL of 2× protein loader buffer (4% sodium dodecyl sulphate, 20% glycerol, 120 mm Tris–HCL, pH 6·8, 10% 2-mercaptoethanol and 0·3% Bromophenol Blue). These were then incubated for 10 min at 99 °C for protein denaturation. Aliquots of 20 µL of each sample were loaded onto 12% SDS–PAGE. The gels were stained with Coomassie Brilliant Blue (CBB) or used to transfer the proteins to nitrocellulose membranes to perform the western blot. The nitrocellulose membranes were blocked with 5% skim milk powder in phosphate-buffered saline (PBS) at 37 °C agitating for 1 h, and then washed twice with PBS-T (PBS containing 0·1% Tween 20; pH 7·4) for 5 min. Membranes were incubated with the primary antibody—mouse monoclonal anti-His (C-term) (Invitrogen), 1:6000—for 1 h at 37 °C. The membrane was washed twice with PBS-T and incubated with peroxidase-conjugated rabbit anti-mouse IgG (1:6000) in PBS-T for 1 h at 37 °C. The membranes were then washed three times with PBS-T. The protein bands were visualized using a developing solution (5 mL of Milli-Q water; 5 mg of DAB – 3,3′-diaminobenzidinetetrahydrochloride; 0·75 mg of NH4). Incubation in this solution proceeded until well-defined bands appeared on the nitrocellulose membrane (5–10 min), which was then washed in sterile Milli-Q water and photo-documented.
To estimate the protein concentration in the bacterial suspension, 20 µL [108 colony-forming units (CFU) mL−1, B. thuringiensis and E. coli] was separated by SDS–PAGE (12%) and then visualized by CBB staining. Protein concentrations were than estimated by using a protein assay kit (Bio-Rad Laboratories) with BSA as the standard, using the image analysis software ImageJ (http://imagej.nih.gov/ij/).
Parasites
Feces from naturally gastrointestinal nematodes-infected male lambs (Corriedale, approximately 5 months old) were collected directly from the rectum. The number of nematodes eggs per gram (EPG) was determined using the Gordon and Whitlock (Reference Gordon and Whitlock1939) technique, and only those samples containing more than 1000 EPG were used. It was estimated that 70–80% of the incubated eggs would hatch under favourable conditions, and this was confirmed during the experiments. To obtain the larvae, coproculture was performed using the Roberts and O'Sullivan (Reference Roberts and O'Sullivan1950) technique using 4 g of fecal samples, held for 7 days in an incubator at 28 °C and relative humidity above 80%. The larvae (L3) were recovered, and then identification and counting performed. Collection of nematodes eggs from the donor sheep was conducted according to the World Association for the Advancement of Veterinary Parasitology (WAAVP) guidelines (Coles et al. Reference Coles, Bauer, Borgsteede, Geerts, Klei, Taylor and Waller1992). The Haemonchus sp. larvae were the most prevalent in the coproculture, accounting for approximately 80% of the genus present, identified using the guidelines from Ueno and Gonçalves (Reference Ueno and Gonçalves1998).
Larvicidal effect of Cry11Aa
Coproculture adapted from the Roberts and O'Sullivan (Reference Roberts and O'Sullivan1950) method was used. Briefly, 4 g of feces containing ⩾ 1000 EPG were inoculated with 2 mL of a bacterial suspension containing ~1 × 108 CFU mL−1 and treatments were as follows: (1) B. thuringiensis, (2) transformed E. coli expressing rCry11Aa, (3) non-transformed E. coli and (4) water as the control. Four containers for each treatment were used, and the experiment was repeated three times. After 7 days of incubation at 28 °C and relative humidity at 80%, larvae (L3) were recovered using the Roberts and O'Sullivan (Reference Roberts and O'Sullivan1950) technique, and counted under a microscope at 40 × magnification (Olympus, Cx21 model). To estimate the efficiency in larvae reduction we used the formula R = 100 (1−T/C), where R is the reduction (larvicidal effect), C is the number of larvae in the control, and T is the number of larvae in the bacteria treated group (Coles et al. Reference Coles, Bauer, Borgsteede, Geerts, Klei, Taylor and Waller1992).
Administration of Cry11Aa to sheep naturally infected with Haemonchus contortus
Sixteen male Corriedale sheep (~5 months old, maintained on native grazing, with a stool score of ⩾1000 EPG, positive for H. contortus) were divided in four groups with four animals each. All treatments were administered orally, and the groups were distributed as follows: A 30 mL bacterial suspension containing ~1 × 108 CFU mL−1 of: (1) B. thuringiensis, (2) transformed E. coli expressing rCry11Aa, (3) non-transformed E. coli and (4) water as the control. The fecal samples (8–10 g) were collected directly from the rectum at 12 h post-administration and larvicidal effects were evaluated by coprocultures as described above. Ethical approval for the use of sheep to provide feces for in vitro experiments was obtained from the Federal University of Pelotas, Animal Ethics Committee number CEEA 9118.
Statistical analysis
The percentage of total larval count reductions was determined using the method described by Coles et al. (Reference Coles, Bauer, Borgsteede, Geerts, Klei, Taylor and Waller1992), which uses the following formula: percentage reduction = 100 × (1−T/C), where T and C are the geometric means in the treatment and control groups, respectively, on day 7 post-treatment. The total larval counts were analysed by analysis of variance (ANOVA) and by the Tukey's test, and P-values of <0·05 were considered significant.
RESULTS
Expression of recombinant protein
The expression of Cry11Aa from B. thuringiensis var. israelensis by E. coli BL21 (DE3) C43 was confirmed by SDS–PAGE and Western blotting analysis using anti-6 × His Tag Mab. We observed a band at the expected size of ~72 kDa, which was similar to that of the B. thuringiensis var. israelensis spore/crystal suspension visualized by SDS–PAGE (Fig. 1).
Recombinant Cry11Aa characterization. Panel (A) rCry11Aa Western Blot. Lane 1: Protein marker Bio-Rad Dual colour; Lane 2: Recombinant Cry11Aa toxin expressed in Escherichia coli BL 21 (DE3) C43 probed with anti-histidine antibody (~72 kDa); Lane 3: Negative control (BSA 10%); Panel (B) 12% SDS–PAGE of bacterial suspensions. Lane 1: Protein marker Bio-Rad Dual colour; Lane 2: recombinant Cry11Aa toxin expressed in E. coli BL21 (DE3) C43; Lane 3: E. coli BL21 (DE3) C43 control; Lane 4: B. thuringiensis var. israelensis spore suspension with protein profile ranging from 10 to 120 kDa; Lane 5: BSA at a concentration of 0·25 µg µL−1; Line 6: BSA at a concentration 0·5 µg µL−1; Lane 7: BSA at a concentration of 1 µg µL−1.
Larvicidal effect of Cry11Aa
In vitro larvicidal effects against Haemonchus L2/L3 larvae were observed with both the native Cry11Aa expressed by B. thuringiensis and its recombinant form expressed in E. coli. We observed a larvicidal effect in vitro (P < 0·001) of 62 and 81%, respectively, compared with the control group, for the native Cry11Aa expressed in B. thuringiensis and the recombinant Cry11Aa expressed in the E. coli suspension when added to feces that were naturally infected with H. contortus. Notably, the recombinant Cry11Aa expressed as an inclusion body in E. coli expressing ~1·2 µg µL−1, had a significantly superior larvicidal effect of ~19% (P < 0·05) more than the native toxin present in the crystal/spore suspension of B. thuringiensis that expressed an approximately 3·9 µg µL−1 (Fig. 2). Additionally, there was no difference in egg counting among the groups.
Larvicidal effect of Cry11Aa in vitro. The data represent the mean (±s.d.) % larval reduction from three independent experiments, where B. thuringiensis var. israelensis (Bti, crystal/spore suspension 108 CFU mL−1), recombinant E. coli expressing Cry11Aa (EcCry, suspension 108 CFU mL−1), a non-recombinant E. coli (E. coli BL 21 (DE3) C43, suspension 108 CFU mL−1) and control (H20). Asterisks (**), represents statistical differences (P < 0·001) among the Control, E. coli control and Bti, and EcCry groups, where (*) represent statistical differences (P < 0·05) between Bti and EcCry groups.
Administration of B. thuringiensis and recombinant E. coli expressing Cry11Aa to sheep naturally infected with H. contortus
We observed that when the suspensions of B. thuringiensis and E. coli expressing recombinant Cry11Aa were administered orally to sheep naturally infected with H. contortus, both treatments demonstrated significant larvicidal effects (P < 0·001) compared with the control group, thus reducing the percentage of viable larvae. The native B. thuringiensis Cry11Aa reduced the viable larvae by ~79%, whereas the recombinant E. coli expressing Cry11Aa reached a ~90% reduction of viable larvae compared with the control group (Table 1).
Haemonchus larvae reduction. Range and arithmetic average of L2/L3 larvae was counted, and the efficiency of the treatment was calculated
EPG B, mean eggs counts before treatments; EPG A, mean eggs counts after treatments (±s.d.); AM, Arithmetic means.
The efficiency (% Reduction) was calculated using the formula R = 100 (1−T/C), where R is the reduction (larvicidal effect), C is the number of larvae in the control, and T is the number of larvae in the bacteria treated group.
DISCUSSION
Gastrointestinal parasites are a major cause of direct and indirect losses in sheep livestock. Different chemical drugs are increasingly being used in an ineffective endeavour to control these parasites, which have developed multidrug resistances to these traditional molecules (Kaplan, Reference Kaplan2004; Geary et al. Reference Geary, Woo, McCarthy, Mackenzie, Horton, Prichard, de Silva, Olliaro, Lazdins-Helds, Engels and Bundy2010; Medeiros et al. Reference Medeiros, Ramos and Banchero2014). To circumvent the emergence of resistance, studies have been conducted in an attempt to decontaminate the grazing environment (Barger, Reference Barger1999). Promising alternative options have included intercropping (rotational grazing of two or more different animal species) and recently, the potential use of biological control agents such as fungi (i.e. Duddingtonia flagrans) (Chandrawathani et al. Reference Chandrawathani, Jamnah, Waller, Höglund, Larsen and Zahari2002; Silva et al. Reference Silva, Braga, de Gives, Millán-Orozco, Uriostegui, Marcelino, Soares, Araújo, Vargas, Aguiar, Senna, Rodrigues, Froes and de Araújo2015). In agriculture, biological control has been used for decades either in combination with or as an alternative to traditional pesticides (Betz et al. Reference Betz, Hammond and Fuchs2000). However, the potential for using this type of control against gastrointestinal parasites in livestock is still being investigated. In this study, direct evidence is given for the use of the Cry11Aa toxin as a biological control strategy for haemonchosis in sheep.
Our previous findings suggested that B. thuringiensis has the potential to be used as a biological anthelmintic in sheep flocks. Recently, our group reported on the larvicidal effect of different strains of Bacillus against H. contortus larvae in naturally infected sheep feces (Sinott et al. Reference Sinott, Cunha Filho, Castro, Lorenzon, Pinto, Capella and Leite2012). However, the B. thuringiensis strain used in this study possesses more than one toxin (Fig. 1B) within the range of 10–120 kDa, which suggests its larvicidal effect is due to one or more toxins or their associations. There is reference (Bone et al. Reference Bone, Bottjer and Gill1988) that mentions a possible ovicidal effect by the B. thuringiensis toxins. However, like Wei et al. (Reference Wei, Hale, Carla, Platzer, Wong, Fang and Aroian2003) we did not observe any toxic effect in the egg hatching. Since the eggshell of nematodes are very resistant is unlike that the Cry11Aa can penetrated, so the effect observed by Bone et al. (Reference Bone, Bottjer and Gill1988), probably was mediated by non-Cry toxins.
In the present study, we observed that the principal larvicidal effect was mediated by the Cry11Aa toxin. Having been cloned and expressed in E. coli, it demonstrated an in vitro and in vivo larvicidal effect similar to, and even higher than the native B. thuringiensis crystal/spore suspension (Fig. 2 and Table 1).
Interestingly, when comparing the larvicidal effects using the same concentrations of microorganisms (2 × 108 CFU mL−1), the E. coli expressing Cry11Aa was significantly more toxic both in vitro and in vivo against Haemonchus larvae than the B. thuringiensis crystal/spore suspension. The larvicidal effect of the recombinant E. coli was higher than that of the B. thuringiensis, showing larval reductions of 81% in the in vitro assays and 90% in the in vivo assays, which was approximately 19% higher than the B. thuringiensis suspension in the in vitro and approximately 11% in vivo assay. It was more surprising that B. thuringiensis expressed a higher toxin concentration than E. coli ~3·9 and 1·2 µg µL−1, respectively, at the same bacterial concentration (Fig. 1B). In the free-living nematode, Caenorhabditis elegans has been suggested that this effect may be due to a defence mechanism that the nematode uses against several pathogens, thus preferentially moving towards an E. coli rather than a Bacillus and by doing so ingesting more E. coli than B. thuringiensis (Zhang et al. Reference Zhang, Lu and Bargmann2005; Hasshoff et al. Reference Hasshoff, Böhnisch, Tonn, Hasert and Schulenburg2007; Schulenburg and Ewbank, Reference Schulenburg and Ewbank2007). Also the toxin expressed as an inclusion body in E. coli might be easily solubilized in the larvae gut and by doing so might become more active (Höss et al. Reference Höss, Menzel, Gessler, Nguyen, Jehle and Traunspurger2013).
Wei et al. (Reference Wei, Hale, Carla, Platzer, Wong, Fang and Aroian2003) found a 50% reduction in brood size at considerably lower concentrations of Cry 5B2 (IC50: 0·00047 mm) when the protein was supplied by E. coli expressing it in intact cells. This suggests that the availability of the toxin at the intestinal receptors of the nematode may be higher when the protein is taken up as inclusions in E. coli cells (ingested as food) than as dissolved proteins. However, we did not pursue this hypothesis because it was not the objective of this study. Nevertheless, there are many reports showing that Cry toxin expressed in E. coli is more efficient than the native Cry toxin expressed in Bacillus (Capello et al. Reference Capello, Bungiro, Harrison, Bischof, Griffitts, Barrows and Aroian2006; Höss et al. Reference Höss, Menzel, Gessler, Nguyen, Jehle and Traunspurger2013).
An interesting feature of the Cry toxins is its larvae target specificity (Bravo et al. Reference Bravo, Gill and Soberón2007). Species specificity is largely determined by the binding of a specific toxin to surface proteins located on the microvilli of larvae mid-gut cells, where the toxic effects occur (de Maadg et al. Reference de Maadg, Bravo, Berry, Crick more and Schnepf2003). Factors such as intestinal pH, microvilli protein composition, cell type, and larvae diet may also affect toxin activity (Soberón and Bravo, Reference Soberón and Bravo2007). In this study, we demonstrated that B. thuringiensis Cry11Aa toxin acts against Haemonchus larvae when orally administered to sheep. Kotze et al. (Reference Kotze, O`Grady, Gough, Pearson, Bagnall, Kemp and Akhurst2005) reported that with a crystal/spore suspension of B. thuringiensis, the toxicity to nematode larvae was reduced when exposed to an acidic (pH ⩽3) environment, suggesting the necessity of some protection for the toxin. This might be important because the sheep abomasum has a pH of 2–3 and is rich in gastric proteases (Kotze et al. Reference Kotze, O`Grady, Gough, Pearson, Bagnall, Kemp and Akhurst2005). However, Lee et al. (Reference Lee, Machi and Ohba2002) reported the recovery of B. thuringiensis from the feces of different herbivores, and the recovered bacteria consisted of spores present in the consumed feed, suggesting that ingested spores/crystals are delivered intact to the feces of a feeding animal. We also reported that larvicidal Bacillus can multiply in sheep feces (Sinott et al. Reference Sinott, Cunha Filho, Castro, Lorenzon, Pinto, Capella and Leite2012), and it can be detected in the feces of the animal 4 h after its oral administration (Sinott et al. Reference Sinott, Dias de Castro, Leite, Gallina, De-Souza, Santos and Leite2014). In this study, we observed that orally administered native or recombinant Cry11Aa toxin that was delivered as an inclusion body in E. coli was efficient in the control of Haemonchus larvae, reaching a 90% reduction in larva survival. One might suggest that E. coli cells act as protectors of the recombinant Cry11Aa toxin, ensuring that little to no degradation (i.e. proteases, low pH, etc.) occurs in the ruminant stomach. This allows delivery of the toxin as an inclusion body, when it is ingested by Haemonchus larvae in the stool bulk. Other studies have similarly reported that when they are expressed in E. coli, recombinant Cry toxins are more toxic for the target parasite than the native toxin expressed as a crystal/spore suspension. Höss et al. (Reference Höss, Menzel, Gessler, Nguyen, Jehle and Traunspurger2013) observed that recombinant Cry5B as an inclusion body expressed in E. coli was more toxic to (or more available to intestinal receptors) of C. elegans, than native toxin as a crystal/spore suspension. Capello et al. (Reference Capello, Bungiro, Harrison, Bischof, Griffitts, Barrows and Aroian2006) investigated the toxic effects of Cry5B on Ancylostoma ceylanicum in vitro and found that it was highly toxic to early stage hookworm larvae; moreover, exposure of adult parasites to Cry5B was associated with a substantial reduction in adult female worm egg excretions.
Implementing biological parasite control programmes that ensure the health of both the livestock and the environment avoids the problems associated with parasitic resistance and ecological toxicity. Biological control using B. thuringiensis var. israelensis Cry11Aa may become an important tool for pasture decontamination because its larvicidal activity disrupts the nematode's life cycle.
In summary, the most important conclusion from our findings is that Cry11Aa from B. thuringiensis var. israelensis demonstrates a strong potential towards controlling an important nematode in veterinary medicine.
ACKNOWLEDGEMENTS
We like to thank Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, CAPES (Edital 032 – Parasitologia Básica), for de Lara, A.P.S.S scholarship.
FINANCIAL SUPPORT
This research was developed with funding received from the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, CAPES (Edital 032 – Parasitologia Básica), to Ana Paula de Souza Stori de Lara.
