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The aim of this study was to characterize the anthelmintic resistance (AR) of a sheep gastrointestinal nematode population, named Caucaia, from northeastern Brazil. Phenotypic tests performed were: egg hatch (EHT), larval development (LDT) and fecal egg count reduction (FECRT). Benzimidazoles (BZs) genotypic evaluation was by frequency of single nucleotide polymorphisms (SNPs) F200Y, F167Y and E198A, and for levamisole (LEV), by frequency of resistance alleles of Hco-acr-8 gene. The primers were designed specifically for Haemonchus contortus. Effective concentrations 50% (EC50) for BZs (EHT), and for macrocyclic lactones (MLs) and LEV (LDT) were 1.02 µg/mL, 1.81 ng/mL and 0.04 µg/mL, respectively. Resistance ratios for MLs and LEV were 0.91 and 3.07, respectively. FECRT efficacies of BZs, MLs, monepantel (MPTL) and LEV were 52.4; 87.0; 94.5 and 99.6%, respectively. qPCR for BZs demonstrated resistance allele frequencies of 0%, 26.24% and 69.08% for SNPs E198A, F200Y and F167Y, respectively. For LEV, 54.37% of resistance alleles were found. There was agreement between EHT, FECRT and qPCR for BZs, and agreement between LDT and qPCR for LEV. Thus, based on higher sensitivity of qPCR, and phenotypic evaluation, the Caucaia population was considered resistant to BZs, MLs, LEV and suspect for MPTL. Resumo O objetivo deste estudo foi caracterizar a resistência anti-helmíntica (RA) da população de nematoides gastrintestinais de ovinos, denominada Caucaia, do Nordeste brasileiro. Os testes fenotípicos foram: eclosão de ovos (TEO), desenvolvimento larvar (TDL) e redução da contagem de ovos nas fezes (TRCOF). A avaliação genotípica para benzimidazóis (BZs) foi por frequência de polimorfismos de nucleotídeo único (SNPs) F200Y, F167Y e E198A; e para levamisol (LEV), pela frequência alélica para resistência no gene Hco-acr-8. Os “primers” foram específicos para Haemonchus contortus. As concentrações efetivas 50% (CE50) para BZs (TEO) e para lactonas macrocíclicas (LMs) e LEV (TDL) foram 1,02 µg/mL, 1,81 ng/mL e 0,04 µg/mL, respectivamente. Os fatores de resistência para LMs e LEV foram 0,91 e 3,07, respectivamente. As eficácias para BZs, LMs, monepantel (MPTL) e LEV no TRCOF foram 52,4; 87,0; 94,5 e 99,6%, respectivamente. A qPCR para BZs demonstrou frequências de 0%, 26,24% e 69,08% para SNPs E198A, F200Y e F167Y, respectivamente. Para LEV foram encontrados 54,37% de alelos resistentes. Houve concordância entre TEO, TRCOF e qPCR para BZs, e entre TDL e qPCR para LEV. Baseada na maior sensibilidade da qPCR e avaliação fenotípica, a população Caucaia foi considerada resistente a BZs, LMs, LEV e suspeita para MPTL.

Parasitism by Haemonchus contortus is one of the main limiting factors in small ruminant production in tropical areas. Benzimidazoles (BZ) and macrocyclic lactones (ML) are the most used anthelmintic classes in gastrointestinal nematodes control. There is considerable scientific evidence of a possible relation between the anthelmintic resistance to BZ and ML. This study aimed to characterize the dynamics of anthelmintic resistance in an H. contortus susceptible isolate under selection pressure for BZ and ML alone or in combination and the role of isotype 1 β-tubulin gene SNPs in these situations. A total of 12 Somali sheep were infected with 5000 third stage larvae of H. contortus Inbred-Susceptible Edinburgh (ISE) isolate. Once infection was established, animals were distributed in three groups (n=4), each treated with crescent doses of oxfendazole (OXF), ivermectin (IVM) and oxfendazole plus ivermectin (IVMOXF). An additional control group with untreated animals was maintained during the entire experiment. After each treatment, eggs were collected and real-time PCR was performed to identify single nucleotide polymorphisms (SNPs) F167Y, F200Y and E198A, in addition to egg hatch test (EHT) for BZ and larval development test (LDT) for ivermectin resistance. All treatments led to increased resistance allelic frequencies at SNPs F200Y and F167Y (p

The use of herbal medicines either in combination with or instead of synthetic anthelmintics is an approach to reducing the exposure of parasites to synthetic chemicals. The present study aimed to assess the in vitro and in vivo effects of Spigelia anthelmia decoction (SaDec) on sheep gastrointestinal nematodes. SaDec was obtained by extracting active constituents of the plant in boiling water. The condensed tannins present in SaDec were quantified and subjected to phytochemical analysis. The egg hatch test (EHT), the larval development test (LDT), and an adult worm motility (AWM) assay were performed. Ultrastructural changes in the cuticle of the adult Haemonchus contortus were evaluated by scanning electron microscopy (SEM). Acute toxicity tests in mice were performed to define the safe dose of SaDec to be administered in the fecal egg count reduction test (FECRT) In the FECRT, fecal samples were collected at days 0, 7 and 14 post-treatment to estimate the eggs per gram (epg) and to identify the most prevalent nematode genera. The results of the EHT and LDT were analyzed using analysis of variance (ANOVA) and compared using Tukey’s test (P

The objective of this study was to evaluate the efficacy of carvacryl acetate (CVA) and nanoencapsulated CVA (nCVA) on gastrointestinal nematodes of sheep. The CVA was nanoencapsulated with chitosan/gum arabic and the efficacy of nanoencapsulation (EE), yield, zeta potential, nanoparticle morphology and release kinetics at pH 3 and 8 were analyzed. Acute and subchronic toxicity were evaluated in rodents and reduction of egg counts in the faeces (FECRT) of sheep. The sheep were divided into four groups (n = 10): G1, 250 mg/kg CVA; G2, 250 mg/kg nCVA; G3, polymer matrix and G4: 2.5 mg/kg monepantel. EE and nCVA yield were 65% and 57%, respectively. The morphology of the nanoparticles was spherical, size (810.6±286.7 nm), zeta potential in pH 3.2 (+18.3 mV) and the 50% release of CVA at pHs 3 and 8 occurred at 200 and 10 h, respectively. nCVA showed LD50 of 2,609 mg/kg. CVA, nCVA and monepantel reduced the number of eggs per gram of faeces (epg) by 57.7%, 51.1% and 97.7%, respectively. The epg of sheep treated with CVA and nCVA did not differ from the negative control (P>0.05). Nanoencapsulation reduced the toxicity of CVA; however, nCVA and CVA presented similar results in the FECRT. [Atividade anti-helmíntica do acetato de carvacrila nanoencapsulado sobre nematoides gastrintestinais de ovinos e toxicidade em roedores]. Resumo: O objetivo deste trabalho foi avaliar a eficácia do acetato de carvacrila (ACV) e do ACV nanoencapsulado (nACV) sobre nematóides gastrintestinais de ovinos. O ACV foi nanoencapsulado com quitosana/goma arábica e foi analisada a eficácia de nanoencapsulamento (EE), o rendimento, potencial zeta, morfologia das nanopartículas e cinética de liberação em pH 3 e 8. Foram avaliadas as toxicidades aguda e subcrônica em roedores e a redução da contagem de ovos nas fezes (RCOF) de ovinos. Os ovinos foram divididos em quatro grupos (n = 10): G1, 250 mg/kg ACV; G2, 250 mg/kg de nACV; G3, matriz polimérica e G4: 2,5 mg/kg de monepantel. A EE e o rendimento de nACV foram de 65% e 57%, respectivamente. A morfologia das nanopartículas foi esférica, tamanho (810,6±286,7 nm), potencial zeta no pH 3,2 (+18,3 mV) e a liberação de 50% de CVA nos pHs 3 e 8 ocorreu às 200 e 10 h, respectivamente. nACV apresentou DL50 de 2.609 mg/kg. ACV, nACV e o monepantel reduziram a contagem de ovos por grama de fezes (opg) em 57,7%, 51,1% e 97,7%, respectivamente. A contagem de opg de ovelhas tratadas com ACV e nCVA não diferiu do controle negativo (P>0,05). O nanoencapsulamento reduziu a toxicidade do AVC; no entanto, nACV e ACV apresentaram resultados semelhantes na RCOF. Made available in DSpace on 2020-10-01T09:12:11Z (GMT). No. of bitstreams: 1 CNPC-2020-Art-21.pdf: 1090808 bytes, checksum: b3f5b06793979fd2fe3daa1a072f8f3f (MD5) Previous issue date: 2020

Haemonchus contortus is the most prevalent parasitic nematode in tropical areas, and anthelmintic resistance is a global problem. Our objective was to characterize benzimidazole (BZ) resistance in gastrointestinal nematode populations in Ceara State, Brazil, using the egg hatch test (EHT) and in H. contortus populations using quantitative real-time polymerase chain reaction (qPCR). Twenty locations were surveyed, and fecal samples were collected from a minimum of 40 animals from each farm and pooled. Five thousand L3 from each farm were used to infect single animals at Embrapa (Brazilian Agricultural Research Company) to provide a source of eggs for both phenotypical and molecular tests. The mean EHT was 2.46 μg/ml (±0.58 μg/ml), and BZ resistance was detected at all surveyed locations. The mean resistant allelic frequencies at positions F200Y and F167Y were 34.16% (±12.13%) and 58.31% (±18.89%), respectively. The resistant allelic frequencies at F167Y were higher than those at F200Y in most studied locations. We also investigated the possibility that specific BZ utilization may influence resistant allelic frequencies. We selected three nematode populations based on the resistant SNP prevalence at F200Y and F167Y as follows: higher frequency at SNP F200Y, higher frequency at SNP F167Y and similar frequencies at both positions. Anthelmintic treatments included two BZs (oxfendazole and albendazole) and ivermectin. Three animals per population per treatment were infected with 5000 L3, and nematode eggs were collected for molecular test before and after anthelmintic treatments. The results showed preferential selection of SNP F167Y in response to oxfendazole, an increase in resistant SNP frequencies in general in response to albendazole and little change in relation to pre-treatment situations in response to ivermectin. Our results confirm that BZ resistance is common. The resistant allele at SNP F167Y in H. contortus prevails in Ceara State, and we provide evidence that this result may be due to the utilization of oxfendazole in recent years.

Carvacrol is a compound isolated from some essential oils. It has been reported to possess anthelmintic activity. Acetylation of this monoterpene has been proposed as a potential way to reduce the toxicity and enhance the pharmacological effects of carvacrol. This study aimed to evaluate the effect of carvacryl acetate (CA) using in vitro and in vivo assays with gastrointestinal nematodes of small ruminants. The egg hatching test (EHT), larval development test (LDT) and adult worm motility (AWM) assessment were conducted to evaluate the effect of the acetylated product and pure carvacrol on Haemonchus contortus eggs, larvae and adults. The structural changes induced in adult H. contortus were assessed using scanning electron microscopy (SEM). CA and carvacrol acute toxicity was evaluated in mice. Finally, the efficacy of 250mg/kg CA and 2.5mg/kg monepantel (positive control) were evaluated in 30 sheep naturally infected with gastrointestinal nematodes by the fecal egg count reduction test (FECRT). In vitro tests were analyzed by analysis of variance (ANOVA) followed by comparison with Tukey’s test. The efficacy was calculated by the Boot Street program using the arithmetic average. The number of eggs in feces (epg) of the groups were transformed to log (x+1) and subjected to ANOVA to compare differences among the groups by Tukey’s test. The level of significance was P

Parasitism by Haemonchus contortus is one of the main limiting factors in small ruminant production around the globe. Although several studies suggest the use of integrated management practices, these parasites have been controlled essentially with synthetic anthelmintic drugs. The resistance mechanism against the imidazothiazole derivative levamisole in Haemonchus contortus has not been fully described. Recently, resistance was associated with a 63bp deletion in the Hco-acr-8b gene that encodes a subunit for a nicotinic acetylcholine receptor. This study aimed to standardize a real time PCR (qPCR) protocol for levamisole resistance diagnosis in H. contortus populations based on this polymorphism and use it to characterize 23 field H. contortus populations obtained from different localities of Ceara State, Northeast Brazil. In addition, two populations of H. contortus were used as a standard of susceptibility and resistance, Inbred Strain Edinburgh (ISE) and Kokstad, respectively. Larval development tests (LDT) were performed on five field isolates and both EC50 and EC95 were estimated. LDT EC95 values provided a wider interval between susceptible and resistant populations than EC50 values (EC95 = 1.96–57.93 μM; EC50 = 0.05–0.39 μM), and were found to be more appropriate for differentiating them. Real time PCR results showed resistance allele frequencies ranged from 20.9 to 76.7%. Our results suggest that levamisole resistance may be present in field populations but it is not as widespread as benzimidazole resistance. This methodology may be useful to monitor levamisole resistance in field populations of H. contortus.

Strategies for controlling gastrointestinal nematodes have been developed based on the use of numerous alternative methods, including the use of phytotherapy. New formulations of essential oils with anthelmintic activity have been proposed as a means to optimize their biological effects. Thus, the objective of this study was to formulate a nanoemulsion to optimize the nematicide effect of Eucalyptus staigeriana essential oil (EsEO). Initially, physico-chemical analyses were performed to verify the stability of the E. staigeriana nanoemulsion (EsNano). In vitro tests were conducted to evaluate the ovicidal and larvicidal activities of both EsNano and EsEO against Haemonchus contortus, and toxicology tests were then performed on rodents. The EsEO content in the nanoemulsion was 36.4% (v/v), and the mean particle size was 274.3 nm. EsNano and EsEO inhibited larval hatching by 99% and 96.3% at 1 and 2mg/ml concentrations, respectively, and inhibited larval development by 96.3% and 97.3% at 8 mg/ml concentrations. The acute toxicity test revealed that the EsNano and EsEO doses required to kill 50% of the mice (LD50) were 1,603.9 and 3,495.9 mg/ml, respectively. EsNano did not alter the hematological parameters in the rats after treatment.

Thymol is a monoterpene and acetylation form of this compound can reduce the toxicity and enhance its biological effects. The objective of this study was to evaluate the effect of thymol and thymol acetate (TA) on egg, larva and adult Haemonchus contortus and the cuticular changes, acute toxicity in mice and the efficacy on sheep gastrointestinal nematodes. In vitro tests results were analyzed by analysis of variance (ANOVA) and followed by comparison with Tukey test or Bonferroni. The efficacy of in vivo test was calculated by the BootStreet program. In the egg hatch test (EHT), thymol (0.5 mg/mL) and TA (4 mg/mL) inhibited larval hatching by 98% and 67.1%, respectively. Thymol and TA (8 mg/mL) inhibited 100% of larval development. Thymol and TA (800 µg/mL) reduced the motility of adult worms, by 100% and 83.4%, respectively. Thymol caused cuticular changes in adult worm teguments. In the acute toxicity test, the LD50 of thymol and TA were 1,350.9 mg/kg and 4,144.4 mg/kg, respectively. Thymol and TA reduced sheep egg count per gram of faeces (epg) by 59.8% and 76.2%, respectively. In in vitro tests thymol presented better anthelmintic activity than TA. However TA was less toxic and in in vivo test efficacy was similar. Resumo Timol é um monoterpeno e a acetilação deste composto pode reduzir a toxicidade e potencializar os seus efeitos biológicos. O objetivo deste trabalho foi avaliar o efeito do timol e acetato de timolila (AT) sobre ovos, larvas e adultos de Haemonchus contortus e suas alterações cuticulares, toxicidade aguda em camundongos e a eficácia sobre nematoides gastrintestinais de ovinos. Os resultados dos testes in vitro foram analisados por análise de variância (ANOVA) e comparados pelo testes de Tukey ou Bonferroni. A eficácia do teste de redução da contagem de ovos nas fezes (RCOF) foi calculada pelo programa BootStreet. No teste de inibição da eclosão de ovos (TEO), timol (0,5 mg/mL) e AT (4 mg/mL) inibiram a eclosão das larvas em 98% e 67,1%, respectivamente. Timol e AT (8 mg/mL) inibiram 100% do desenvolvimento larval. Timol e AT (800 μg/mL) reduziram a motilidade dos nematoides adultos, em 100% e 83,4%, respectivamente. O timol provocou alterações cuticulares nos nematoides adultos. No teste de toxicidade aguda, a DL50 do timol e AT foi de 1.350,9 mg/kg e 4.144,4 mg/kg, respectivamente. Timol e AT reduziram a contagem de ovos por gramas de fezes (OPG) dos ovinos em 59,8% e 76,2%, respectivamente. Nos testes in vitro timol apresentou atividade melhor anti-helmíntica do que AT. Entretanto, AT foi menos tóxico do que o timol e no teste in vivo apresentaram eficácia semelhante.

Sustainable control of gastrointestinal nematodes (GIN) in small ruminants has been based on the use of alternative methods, including targeted selective treatment, such as FAMACHA. Another GIN control alternative is the use of herbal medicines, although in many cases their use is based on empirical knowledge. Biopolymer nanoformulations has been investigated to maximize the essential oil effects against sheep gastrointestinal nematodes. The aim of the present study was to combine a Eucalyptus staigeriana essential oil nanoemulsion (EsNano) with FAMACHA as an alternative control for sheep haemonchosis. The study was performed over six months at a commercial sheep farm located in a semiarid region of Northeast Brazil. Initially, a fecal egg count reduction test (FECRT) in sheep with levamisole, ivermectin and oxfendazole in sheep was performed used to determine the most effective anthelmintic to use as the positive control. Levamisole has been selected because it showed efficacy superior to 95%. EsNano was obtained and then its physicochemical properties were characterized. The average (±SE) size of the particles in the nanoemulsion was 276.8 (±12.3) nm with bimodal distribution and polydispersity. Nine visits were performed, from April to September 2013, with an interval of 17 days. One hundred sixty-two male and female sheep were divided into three groups (n=54 each) and were treated when FAMACHA score was 3, 4, or 5: G-EsNano 250mg kg-1 EsNano; G-Lev 7.5mg kg-1 levamisole (positive control), and G-Neg was not treated (negative control). Feces from sheep were collected to quantify the number of eggs per gram of feces (epg) and to identify nematode genera. Sheep weight gain was monitored. The epg data for each group and the average sheep weight gains were analyzed by variance analysis and compared with the Tukey’s test (P0.05). The epg variation was similar in the G-EsNano and G-Lev groups on visits (P>0.05), except the second and fifth evaluation in the epg groups were significantly different (P0,05). A variação opg foi semelhante para os grupos G-EsNano e G-Lev nas vistas (P>0,05), excetuando a segunda e quinta avaliação em que os opg dos grupos foram significativamente diferentes (P>0,05). O nematoide Haemonchus spp. foi o mais prevalente. Não houve aumento significativo de peso em qualquer um dos grupos tratados (P>0,05). Assim, a combinação de fitoterapia e FAMACHA pode ser uma alternativa para minimizar o uso anti-helmínticos sintéticos para controlar populações resistentes NGI em pequenos ruminantes.

INTRODUCTION: Considering the unknown situation regarding canine visceral leishmaniasis in Garanhuns, this study had the aim of investigating occurrences of anti-Leishmania spp antibodies in domesticated and partially domesticated dogs, and the possible risk factors involved. METHODS: In the first phase of the study, 256 blood samples were collected from dogs and subjected to the indirect fluorescent antibody test (IFAT) reaction at a dilution of 1:40. Additionally, 23 IFAT-positive samples were tested using an immunochromatographic dipstick test. In the second phase, new blood samples were collected from 18 dogs that were IFAT-positive in the first phase. These animals were retested using IFAT (1:40 and 1:80) and, additionally, by means of the polymerase chain reaction to investigate the Leishmania infantum DNA. Furthermore, 16 of these samples were retested using the immunochromatographic dipstick test. RESULTS: In the first phase of the study, 16% of the samples were IFAT-positive (1:40) and only three (13%) were positive in the immunochromatographic dipstick test. In the second phase, 12 samples were IFAT-positive at the dilution of 1:40, and seven were also positive at 1:80. None of the samples were positive in the polymerase chain reaction testing or in the immunochromatographic dipstick test. Clinical signs suggestive of visceral leishmaniasis were observed in 4.9% of the IFAT-positive dogs. There were no statistical differences in relation to age, sex or clinical status of the dogs, but there was a difference in relation to place of origin. CONCLUSIONS: The domesticated and partially domesticated dogs living in Garanhuns present anti-Leishmania spp antibodies, but are mostly asymptomatic.

Parasitic nematodes are of major economic importance in livestock. An alternative for the control of parasites is phytotherapy. This study evaluated the efficacy of Cymbopogon citratus decoction (CcD), C. citratus essential oil (CcEo) and citral against Haemonchus contortus using in vitro egg hatch test (EHT) and larval development test (LDT) and an in vivo test using a Meriones unguiculatus (gerbil) model. The effect of 800 mg/kg CcEo was evaluated in gerbils that had been artificially infected with 5,000 third-stage H. contortus larvae. The effective concentrations required to inhibit 50% (EC50) of egg hatching were 0.46, 0.14 and 0.13 mg/mL for CcD, CcEo and citral, respectively. The EC50 values in the LDT were 5.04, 1.92 and 1.37 mg/mL for CcD, CcEo and citral, respectively. H. contortus population in the group treated with C. citratus essential oil was reduced by 38.5% (P< 0.05) in comparison to the control group. These results suggest that it may be possible to use C. citratusessential oil to control of H. contortus parasite of small ruminant. O parasitismo por nematoides tem grande importância econômica no rebanho. Uma alternativa para o controle de parasitas é a fitoterapia. Este estudo avaliou a eficácia do decocto de Cymbopogon citratus (DCc), do óleo essencial de C. citratus (OECc) e do citral contra Haemonchus contortus utilizando o teste in vitro de eclosão dos ovos (TEO) e o teste de desenvolvimento larval (TDL) e um teste in vivo com modelo Meriones unguiculatus (gerbil). O efeito de 800 mg/kg de OECc foi avaliado em gerbils infectados artificialmente com 5000 larvas de terceira fase de H. contortus. As concentrações efetivas necessárias para inibir 50% (CE50) da eclosão dos ovos foram 0,46; 0,14 e 0,13 mg/mL para DCc, OECc e citral, respectivamente. Os valores da CE50 no TDL foram de 5,04; 1,92 e 1,37 mg/mL para DCc, OECc e citral, respectivamente. No grupo tratado com óleo de C. citratus a população H. contortus foi reduzida em 38,5% (P < 0,05) em comparação com o grupo controle. Estes resultados sugerem que pode ser possível a utilização de óleo essencial de C. citratus para controle de H. contortus, parasita de pequenos ruminantes.

Haemonchus contortus is the most prevalent nematode in Brazil. The objective of this study was to select 6 populations of H. contortus of known or suspected benzimidazole resistance status and characterize these using quantitative real-time polymerase chain reaction (qPCR) for single nucleotide polymorphisms (SNPs) F200Y, F167Y and E198A in the β-tubulin isotype 1 gene. qPCR was performed using DNA from a pool of 10 adult male H. contortus from a single animal per farm. Faecal egg count reduction test (FECRT) and egg hatch test (EHT) were used to determine the resistance status. Samples were obtained from 6 farms located in 5 counties in the Ceara State: Taua, Boa Viagem, Quixada, Santa Quiteria and Solonopole. The inbred-susceptible-Edinburgh (ISE) isolate was used as reference for comparative purposes in the qPCR. Benzimidazole resistance was detected by FECRT on all farms with efficacy values ranging from 0 to 51%. EC50 values as determined by EHT were all above 1.49μg/ml. High frequencies of the resistant SNPs F200Y and F167Y alleles were detected but no resistance was detected at SNP E198A. Our results suggest that the SNPs F167Y and F200Y are both important for benzimidazole resistance in the studied populations.

Phytotherapy can be an alternative for the control of gastrointestinal parasites of small ruminants. This study evaluated the efficacy of Alpinia zerumbet, Coriandrum sativum, Tagetes minuta and Lantana camaraessential oils by two in vitro assays on Haemonchus contortus, an egg hatch test (EHT) and larval development test (LDT). No effect was observed for L. camara in the EHT. A. zerumbet, C. sativum and T. minuta essential oils exhibited a dose-dependent effect in the EHT, inhibiting 81.2, 99 and 98.1% of H. contortus larvae hatching, respectively, at a concentration of 2.5 mg mL-1. The effective concentration to inhibit 50% (EC50) of egg hatching was 0.94, 0.63 and 0.53 mg mL-1 for A. zerumbet, C. sativum and T. minuta essential oils, respectively. In LDT, L. camara, A. zerumbet, C. sativum and T. minuta at concentration of 10 mg mL-1 inhibited 54.9, 94.2, 97.8 and 99.5% of H. contortus larval development, presenting EC50 values of 6.32, 3.88, 2.89 and 1.67 mg mL-1, respectively. Based on the promising results presented in this in vitro model, it may be possible use of these essential oils to control gastrointestinal nematodes. However, their anthelmintic activity should be confirmed in vivo. Fitoterapia pode ser uma alternativa para o controle de parasitas gastrintestinais de pequenos ruminantes. Este estudo avaliou a eficácia dos óleos essenciais de Alpinia zerumbet, Coriandrum sativum, Tagetes minuta e Lantana camara sobre Haemonchus contortus através de dois testes in vitro, teste de eclosão dos ovos (TEO) e teste de desenvolvimento larval (TDL). Nenhum efeito foi observado para L. camara no TEO. Os óleos essenciais de A. zerumbet, C. sativum e T. minuta exibiram um efeito dose dependente no TEO inibindo a eclosão das larvas de H. contortusem 81,2, 99 e 98,1%, respectivamente, na concentração de 2,5 mg mL-1. A concentração efetiva para inibir 50% (CE50) da eclosão dos ovos foi de 0,94, 0,63 e 0,53 mg mL-1 para os óleos essenciais de A. zerumbet, C. sativum e T. minuta, respectivamente. No TDL, 10 mg mL-1 de L. camara, A. zerumbet, C. sativum e T. minuta inibiram em 54,9, 94,2, 97,8 e 99,5% do desenvolvimento larvar, apresentando valores de CE50 de 6,32, 3,88, 2,89 e 1,67 mg mL-1, respectivamente. Com base nos resultados promissores apresentados neste modelo in vitro, pode ser possível a utilização destes óleos essenciais para controlar os nematoides gastrintestinais. No entanto, a sua atividade anti-helmíntica deve ser confirmada in vivo.

Considering the unknown situation regarding canine visceral leishmaniasis in Garanhuns, this study had the aim of investigating occurrences of anti-Leishmania spp antibodies in domesticated and partially domesticated dogs, and the possible risk factors involved.In the first phase of the study, 256 blood samples were collected from dogs and subjected to the indirect fluorescent antibody test (IFAT) reaction at a dilution of 1:40. Additionally, 23 IFAT-positive samples were tested using an immunochromatographic dipstick test. In the second phase, new blood samples were collected from 18 dogs that were IFAT-positive in the first phase. These animals were retested using IFAT (1:40 and 1:80) and, additionally, by means of the polymerase chain reaction to investigate the Leishmania infantum DNA. Furthermore, 16 of these samples were retested using the immunochromatographic dipstick test.In the first phase of the study, 16% of the samples were IFAT-positive (1:40) and only three (13%) were positive in the immunochromatographic dipstick test. In the second phase, 12 samples were IFAT-positive at the dilution of 1:40, and seven were also positive at 1:80. None of the samples were positive in the polymerase chain reaction testing or in the immunochromatographic dipstick test. Clinical signs suggestive of visceral leishmaniasis were observed in 4.9% of the IFAT-positive dogs. There were no statistical differences in relation to age, sex or clinical status of the dogs, but there was a difference in relation to place of origin.The domesticated and partially domesticated dogs living in Garanhuns present anti-Leishmania spp antibodies, but are mostly asymptomatic.

Phytotherapy has been described as an alternative method for the control of gastrointestinal nematodes in small ruminants. Goal of the encapsulation of essential oils in biopolymer matrices is to optimize the biological effects of these oils. The aim of the present study was to evaluate the in vitro and in vivo anthelmintic activity of encapsulated Eucalyptus staigeriana essential oil (EncEs) on the eggs and larvae of Haemonchus contortus. Therefore, the egg hatching test (EHT), larval development test (LDT) and worm load evaluation were performed to evaluate Meriones unguiculatus experimentally infected with H. contortus. The chemical constituents of E. staigeriana essential oil (EsEO) and the in vitro oil release profile from the chitosan matrix at a pH of 1.2 and 7.0 were also characterized. EncEs and EsEO inhibited larval hatching by 97.19% and 99.96% at doses of 1.5 and 1.0 mg ml(-1), respectively. In the LDT, EncEs and EsEO induced a larvicidal effect greater than 95% at concentrations of 5.8 and 8 mg ml(-1), respectively. EncEs and EsEO decreased H. contortus load in M. unguiculatus by 40.51% and 46.44%, respectively. The major chemical constituents of EsEO were (+)-Limonene (72.9%), 1,8-Cineole (9.5%) and o-Cimene (4.6%). The release profile of EsEO was 30% in acid and 25% at neutral pH. The similar efficacy of EncEs and EsEO demonstrates that there was no optimization of anthelmintic action following the encapsulation process. Therefore, the use of new encapsulation matrices with controlled release in the pH of the abomasum should be investigated.

The anthelmintic resistance stimulated the search for strategies for controlling gastrointestinal nematodes, including the use of free essential oils or its nanoemulsion. This study evaluated the anthelmintic efficacy of Cymbopogon citratus essential oil (CcEO) and C. citratus essential oil nanoemulsion (CcEOn). Pysicochemical analyses were performed. The in vitro effect was determined using the egg hatch test (EHT) on Haemonchus contortus and in vivo effect was evaluated in sheep infected with gastrointestinal nematodes. The animals were treated with CcEO (500 mg/kg) or CcEOn (450 mg/kg) for the fecal egg count (FEC) and the determination of worm burden. The main component of CcEO was citral. The CcEO content in the nanoemulsion was 20% (v/v), and the mean particle size was 248 nm. In EHT, CcEO and CcEOn (1.25 mg/mL) inhibited larval hatching by 98.4 and 97.1%, respectively. Three animals treated with CcEO died whereas in the group treated with CcEOn one animal died. The FEC and total worm burden of the treated groups did not differ from the negative control (p>0.05). The CcEOn showed efficacy only on H. contortus (p

Resumo: Os nematoides gastrintestinais são um dos principais fatores limitantes na criação de pequenos ruminantes no mundo. Os benzimidazóis (BZ), as lactonas macrocíclicas (LM) e os imidazotiazóis são os anti-helmínticos mais utilizados para o controle desses parasitas. Contudo, o uso de fármacos leva inevitavelmente ao desenvolvimento de resistência anti-helmíntica (RAH). O diagnóstico de RAH é realizado principalmente por meio de métodos fenotípicos com baixa sensibilidade. Dessa forma, métodos moleculares, são necessários para melhorar a capacidade de detecção da RAH. Os objetivos desse trabalho foram avaliar o estado da RAH em nematoides gastrintestinais de ovinos no Ceará, investigar a relação entre a resistência a BZ e LM e padronizar técnica de diagnóstico de resistência molecular a levamisol (LEV) em H. contortus. Para tanto, o trabalho foi dividido em três experimentos. No experimento I foi realizado um levantamento da situação da resistência a BZ por meio do teste de eclosão de ovos (TEO) e da PCR em tempo real (qPCR) para identificar os polimorfismos de nucleotídeo único (SNPs) F167Y, F200Y e E198A. No experimento II foi realizada a seleção para resistência do isolado de H. contortus Inbred-Susceptible Edinburgh (ISE) com doses crescentes de oxifendazol (OXF), ivermectina (IVM) e oxifendazol mais ivermectina (IVMOXF). O desenvolvimento da resistência foi avaliado por qPCR, TEO para BZ e teste de desenvolvimento larva (TDL) para ivermectina. No terceiro experimento foi realizado a qPCR para o diagnóstico de resistência a LEV, baseando-se na deleção de 63pb no gene Hco-acr-8b codificante para os receptores nicotínicos de acetilcolina. Foram testadas 26 populações de H. contortus: Kokstad (LEV resistente), ISE (LEV susceptível) e 24 populações obtidas de rebanhos no estado do Ceará. Foi realizado o TDL para LEV em cinco isolados de campo. No experimento I a concentração efetiva média para inibir 50% da eclosão de ovos (CE50) no TEO foi de 2,46 ?g/mL (± 0,58 ?g/mL) e a resistência a BZ foi detectada em todas as fazendas pesquisadas. As frequências médias de alelos resistentes nos códons F200Y, F167Y e E198A foram 34,16% (± 12,13%), 58,31% (± 18,89%) e 0,016 (±0,012), respectivamente. No experimento II todos os tratamentos levaram ao aumento da frequência de alelos resistentes para os SNP F200Y e F167Y (p

Resumo: Haemonchus contortus é o nematóide mais prevalente na região Nordeste do Brasil. O seu controle é baseado na utilização de benzimidazóis de forma generalizada e o uso inadequado acelera a seleção de parasitos resistentes. Métodos fenotípicos, tais como, o teste de redução na contagem de ovos nas fezes (FECRT) e o teste de eclosão de ovos (TEO) são amplamente utilizados para caracterizar a resistência anti-helmíntica (RA), embora com baixa sensibilidade. Os métodos baseados na reação em cadeia pela polimerase (PCR) são também utilizados para diagnosticar a resistência a benzimidazóis em H. contortus. Estes métodos detectam polimorfismos de nucleotídeo único (SNP) F200Y, F167Y e E198A no gene codificante para o isotipo 1 da ?-tubulina, que são conhecidos por estarem associados à resistência a benzimidazóis. O objetivo deste estudo foi investigar a presença de polimorfismos responsáveis pela resistência a benzimidazóis em populações de Haemonchus contortus isoladas no estado do Ceará. A PCR em tempo real (qPCR) foi realizada para identificação de cada SNP conhecido no gene codificante para o isotipo 1 da ?-tubulina, para tanto foi utilizado DNA de um pool de 10 adultos de H. contortus do sexo masculino obtidos de um animal por rebanho. O FECRT e o TEO foram usados para determinar a resistência original presente na população. As amostras testadas foram obtidas a partir de 6 fazendas localizadas em 5 municípios do estado do Ceará (Nordeste do Brasil): Tauá (TA1 e TA2), Boa Viagem (BV), Quixadá (QX), Santa Quitéria (SQ) e Solonópole (SO). O isolado inbredsusceptible-Edinburgh (ISE), previamente caracterizado como susceptível a benzimidazóis, foi utilizado como referência para fins de comparação na qPCR. A resistência a benzimidazóis foi detectada no FECRT em todas as fazendas com valores que variam de 0 a 50% de redução de OPG. Os valores das concentrações inibitórias de 50% da eclosão das larvas (CE50) determinados no TEO foram todos superiores a 1,62 µg/mL. Nos resultados da qPCR foram observadas altas frequências dos SNP F200Y e F167Y. As maiores frequências de alelos resistentes nas populações estudadas foram para o SNP F167Y e não foram detectados alelos resistentes para o SNP E198A. As frequências de alelos resistentes para o isolado ISE foram 1,7%, 0% e 0% para os SNP F200Y, F167Y e E198A, respectivamente. Os resultados sugerem que os SNP F167Y e F200Y são importantes para a resistência a benzimidazóis nas populações estudadas e devem ser incluídos no diagnóstico molecular de resistência anti-helmíntica no Ceará. Abstract: Haemonchus contortus is the most prevalent nematode in Northeast Brazil. Control based on benzimidazole utilization is widespread and its inadequate use accelerates the selection of resistant parasites. Phenotypic methods such as faecal egg count reduction test (FECRT) and egg hatch assay (EHA) are widely used to characterize anthelmintic resistance (AR) albeit with low sensitivity. PCR based methods are also used to diagnose resistance to benzimidazoles in H. contortus. These methods detect single nucleotide polymorphisms (SNPs) F200Y, F167Y and E198A in the -tubulin isotype 1 gene that are known to be associated with benzimidazole resistance. The objective of this study was to investigate the presence of polymorphisms responsible for resistance to benzimidazoles in Haemonchus contortus populations isolated in the state of Ceará. Real time (qPCR) was performed to identify each known SNP in the ?-tubulin isotype 1 gene. DNA was extracted from a pool of 10 adult male H. contortus from a single animal per farm. FECRT and EHA were used to determine the original resistance present in the population. Tested samples were obtained from 6 farms located in 5 counties in the Ceará State (Northeast Brazil): Tauá (TA1 and TA2), Boa Viagem (BV), Quixadá (QX), Santa Quitéria (SQ) and Solonópole (SO). The inbred-susceptible-Edinburgh (ISE) isolate, previously reported as benzimidazole susceptible, was used as reference for comparison purposes in the qPCR assay. Benzimidazole resistance was detected by FECRT in all farms with values ranging from 0 to 50%. Half maximal effective concentration (EC50) values as determined by EHA were all above 1.62 µg/mL. Real-time PCR results showed high frequencies of the SNPs F200Y and F167Y. The most frequent resistant allele in the studied populations was SNP F167Y and resistance for SNP E198A was not detected. Resistant allele frequencies for the ISE isolate were 1.7%, 0% and 0% for SNPs F200Y, F167Y and E198A, respectively. Our results suggest that the SNPs F167Y and F200Y are both important for benzimidazole resistance in the studied populations and should be included in the molecular diagnosis of anthelmintic resistance in the Ceará. Made available in DSpace on 2017-08-22T09:28:30Z (GMT). No. of bitstreams: 1 TSPolimorfismosresponsaveis.pdf: 1115617 bytes, checksum: 9ff090ac61e9f80519c36426b8383fbb (MD5) Previous issue date: 2013-09-10 Dissertação (Mestrado em Ciências Veterinárias) - Universidade Estadual do Ceará, Fortaleza. Orientação: Claudia Maria Leal Bevilaqua; Co-orientação: Jomar Patrício Monteiro (Embrapa Caprinos e Ovinos).