Vitamin D (VD3) Intensifies the Effects of Exercise and Prevents Alterations of Behavior, Brain Oxidative Stress, and Neuroinflammation, in Hemiparkinsonian Rats.

In the present study, we investigated the effects of physical exercise in the presence of Vitamin D3 (VD3), on 6-hydroxydopamine (6-OHDA)-lesioned hemiparkinsonian rats. The animals were divided into sham-operated (SO), 6-OHDA-lesioned, and 6-OHDA-lesioned plus VD3 (1 µg/kg, 21 days), in the absence (no exercise, NE) and presence (with exercise, WE) of physical exercise on a treadmill (30 min, speed of 20 cm/s, once a day/21 days). This procedure started, 24 h after the stereotaxic surgery (injections of 6-OHDA into the right striatum). The animals were then subjected to behavioral (rotarod, open field, and apomorphine tests) and their brain areas were dissected for neurochemical, dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC) determinations, and immunohistochemical studies for tyrosine hydroxylase (TH), dopamine transporter (DAT), and vitamin D receptor (VD3R). The effects on the brain oxidative stress: nitrite/nitrate, glutathione (GSH), and malondialdehyde (MDA) measurements were also evaluated. Behavioral changes of the 6-OHDA lesioned group were improved by exercise plus VD3. Similar results were observed in dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) concentrations increased by exercise and VD3, compared with SO groups. Additionally, tyrosine hydroxylase (TH) and dopamine transporter (DAT) immunoexpressions were decreased in the 6-OHDA-lesioned groups, with values normalized after exercise and VD3. The VD3 receptor immunoexpression decreased in the 6-OHDA (NE) group, and this was attenuated by exercise, especially after VD3. While 6-OHDA lesions increased, VD3 supplementation decreased the oxidative stress, which was intensified by exercise. VD3 showed neuroprotective properties that were intensified by physical exercise. These VD3 actions on hemiparkinsonian rats are possibly related to its antioxidant and anti-inflammatory effects.

In vitro anthelmintic activity of Lippia alba essential oil chemotypes against Haemonchus contortus.

The aim of the present study was to evaluate the effects of the essential oils of Lippia alba chemotypes carvone and citral on H. contortus. Chemical characterization was performed by means of gas chromatography coupled to mass spectrometry (GC/MS). The anthelmintic effects of the essential oils were assessed through the egg hatch test (EHT) and the adult worm motility test (AWMT) using a multidrug-resistant H. contortus Kokstad isolate. Confocal laser scanning microscopy (CLSM) of eggs and adults of H. contortus and scanning electron microscopy (SEM) of adults were performed after treatment with oils for qualitative observations of their effects. The carvone chemotype of L. alba (LaCV) presented 70% carvone, and the citral chemotype of L. alba (LaCT) presented 29.4% geranial and 20.4% neral, respectively. In the EHT, the EC50 values of LaCV and LaCT were 0.2 and 0.3 mg/mL, respectively. In AWMT, after 12 h of exposure to 2 mg/mL LaCV and 2 mg/mL LaCT, 100% of adult nematodes were immobile. CLSM showed changes in larval motility inside the egg caused by LaCV, while LaCT promoted changes in larval formation. In adults exposed to both chemotypes, alterations in the anterior portion of the oesophagus were observed. In SEM, morphological changes were observed in the buccal capsule and in the medial portion of H. contortus adults. It is concluded that the two essential oils of L. alba, the chemotypes carvone and citral, caused morphological changes and inhibited the hatching of eggs and the motility of adult H. contortus nematodes.

Chemical Composition, Anthelmintic Activity, and Mechanism of Action of Lippia dominguensis Mold. Essential Oil on Haemonchus contortus.

Gastrointestinal nematode parasitism is a major burden to small ruminant production globally, compounded by increasing anthelmintic resistance. Previous studies have identified essential oils (EOs) from the Lippia genus with antiprotozoal and anthelmintic effects. Lippia dominguensis Moldenke (Ld), an endemic specie from the Dominican Republic, has similar popular uses, however, is chemically and pharmacologically yet uncharacterized. Here, we investigated the in vitro anthelmintic activity of LdEO and its ultrastructural effects on eggs and adult nematodes of Haemonchus contortus multidrug-resistant isolated. The GC/MS analysis showed linalool (33.85 %), 1,8-cineole (30.88 %), and δ-terpineol (10.61 %) as the main EO constituents. The LdEO showed an IC50 =0.523 mg/mL in the egg hatch test, and the motility in the adult worm motility test was 95.8 % at 1 mg/mL. The confocal scanning laser microscopy of eggs indicated permeabilization or disruption of egg cell membranes as the possible mechanism of action of LdEO. The scanning electron microscopy of adult worms showed wrinkling, undulations, and cuticular disruptions. The LdEO displayed significant in vitro anthelmintic activity on eggs and adult worms of H. contortus. Additionally, the LdEO showed low oral toxicity in mice at 2,000 mg/kg. Thus, additional in vivo studies are justified to determine its anthelmintic efficacy in small ruminants.

In vitro anthelmintic activity of an R-carvone nanoemulsions towards multiresistant Haemonchus contortus.

This work aimed to evaluate the in vitro anthelmintic effect of carvone nanoemulsions on Haemonchus contortus. Three R-carvone nanoemulsions were prepared: uncoated R-carvone nanoemulsions homogenized in a sonicator (UNAlg-son) and homogenized in an ultrahomogenizer (UNAlg-ultra) and sodium alginate-coated R-carvone (CNAlg-ultra). The physicochemical characterizations of the nanoemulsions were carried out. The anthelmintic activity was evaluated using egg hatch test (EHT), larval development test (LDT) and adult worm motility test (AWMT). Changes in cuticle induced in adult H. contortus were evaluated by scanning electron microscopy (SEM). The results were subjected to analysis of variance and compared using the Tukey test (P < 0.05). The effective concentration to inhibit 50% (EC50) of egg hatching and larval development was calculated. The particle sizes were 281.1 nm (UNAlg-son), 152.7 nm (UNAlg-ultra) and 557.8 nm (CNAlg-ultra), and the zeta potentials were −15 mV (UNAlg-son), −10.8 mV (UNAlg-ultra) and −24.2 mV (CNAlg-ultra). The encapsulation efficiency was 99.84 ± 0.01%. SEM of the nanoemulsions showed an increase in size. In EHT, the EC50 values of UNAlg-son, UNAlg-ultra and CNAlg-ultra were 0.19, 0.02 and 0.17 mg mL−1, respectively. In LDT, they were 0.29, 0.31 and 0.95 mg mL−1 for UNAlg-son, UNAlg-ultra and CNAlg-ultra, respectively. The adult motility inhibition was 100% after 12 h of exposure to UNAlg-ultra and CNAlg-ultra, while for UNAlg-son, it was 79.16%. SEM showed changes in the buccal capsule and cuticular damage. It was concluded that R-carvone nanoemulsions showed antiparasitic action demonstrating promise for the control of infections caused by gastrointestinal nematodes in small ruminants.

Long-term Environmental Enrichment Normalizes Schizophrenia-like Abnormalities and Promotes Hippocampal Slc6a4 Promoter Demethylation in Mice Submitted to a Two-hit Model.

Here, we explored the impact of prolonged environmental enrichment (EE) on behavioral, neurochemical, and epigenetic changes in the serotonin transporter gene in mice subjected to a two-hit schizophrenia model. The methodology involved administering the viral mimetic PolyI:C to neonatal Swiss mice as a first hit during postnatal days (PND) 5-7, or a sterile saline solution as a control. At PND21, mice were randomly assigned either to standard environment (SE) or EE housing conditions. Between PND35-44, the PolyI:C-treated group was submitted to various unpredictable stressors, constituting the second hit. Behavioral assessments were conducted on PND70, immediately after the final EE exposure. Following the completion of behavioral assessments, we evaluated the expression of proteins in the hippocampus that are indicative of microglial activation, such as Iba-1, as well as related to neurogenesis, including doublecortin (Dcx). We also performed methylation analysis on the serotonin transporter gene (Slc6a4) to investigate alterations in serotonin signaling. The findings revealed that EE for 50 days mitigated sensorimotor gating deficits and working memory impairments in two-hit mice and enhanced their locomotor and exploratory behaviors. EE also normalized the overexpression of hippocampal Iba-1 and increased the expression of hippocampal Dcx. Additionally, we observed hippocampal demethylation of the Slc6a4 gene in the EE-exposed two-hit group, indicating epigenetic reprogramming. These results contribute to the growing body of evidence supporting the protective effects of long-term EE in counteracting behavioral disruptions caused by the two-hit schizophrenia model, pointing to enhanced neurogenesis, diminished microglial activation, and epigenetic modifications of serotonergic pathways as underlying mechanisms.

Activity of chitosan-encapsulated Eucalyptus staigeriana essential oil on Haemonchus contortus.

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.

Early maternal separation enhances melanoma progression in adult female mice by immune mechanisms.

Maternal separation (MS) is a risk factor for major depressive disorder. Both cancer and depression seem to share a common biological link. Here, we evaluated the progression of melanoma and the underlying mechanisms related to this progression, namely cell proliferation and apoptosis, in adult female mice exposed to MS. Female C57BL/6 mice were exposed to MS for 60 min/day during the first 2 postnatal weeks (here called MS mice) or left undisturbed (here called non-MS mice). Melanoma cells were inoculated subcutaneously into the axillary region of adult animals, and tumor progression was evaluated for 25 days. Adult MS mice presented depressive-like behavior and working memory deficits. MS accelerated murine melanoma growth by mechanisms related to decreased apoptosis and increased cell proliferation rate, such as increased expression of IL-6 and mTOR. MS stimulated eukaryotic elongation factor 2 expression and increased the number of circulating monocytes and DNA damage in peripheral blood leukocytes, an effect associated with oxidative DNA damage. In conclusion, MS accelerated the progression of murine melanoma by mechanisms related to tumor proliferation and apoptosis, revealing a relationship between adverse childhood experiences and cancer progression, particularly melanoma.

Phenotypic and genotypic approaches for detection of anthelmintic resistant sheep gastrointestinal nematodes from Brazilian northeast.

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.

Anti-asthmatic effect of nitric oxide metallo-donor FOR811A [cis-[Ru(bpy)2(2-MIM)(NO)](PF6)3] in the respiratory mechanics of Swiss mice.

We aimed at evaluating the anti-asthmatic effect of cis-[Ru(bpy)2(2-MIM)(NO)](PF6)3 (FOR811A), a nitrosyl-ruthenium compound, in a murine model of allergic asthma. The anti-asthmatic effects were analyzed by measuring the mechanical lung and morphometrical parameters in female Swiss mice allocated in the following groups: untreated control (Ctl+Sal) and control treated with FOR811A (Ctl+FOR), along asthmatic groups untreated (Ast+Sal) and treated with FOR811A (Ast+FOR). The drug-protein interaction was evaluated by in-silico assay using molecular docking. The results showed that the use of FOR811A in experimental asthma (Ast+FOR) decreased the pressure-volume curve, hysteresis, tissue elastance, tissue resistance, and airway resistance, similar to the control groups (Ctl+Sal; Ctl+FOR). However, it differed from the untreated asthmatic group (Ast+Sal, p<0.05), indicating that FOR811A corrected the lung parenchyma and relaxed the smooth muscles of the bronchi. Similar to control groups (Ctl+Sal; Ctl+FOR), FOR811A increased the inspiratory capacity and static compliance in asthmatic animals (Ast+Sal, p<0.05), showing that this metallodrug improved the capacity of inspiration during asthma. The morphometric parameters showed that FOR811A decreased the alveolar collapse and kept the bronchoconstriction during asthma. Beyond that, the molecular docking using FOR811A showed a strong interaction in the distal portion of the heme group of the soluble guanylate cyclase, particularly with cysteine residue (Cys141). In summary, FOR811A relaxed bronchial smooth muscles and improved respiratory mechanics during asthma, providing a protective effect and promising use for the development of an anti-asthmatic drug.

Disseminated histoplasmosis in a cat rescued in Fortaleza, Brazil, and successfully treated with itraconazole - First case report identified molecularly.

An unneutered female cat of unknown age presented bloody lesions on the edematous face, and respiratory signs. Cytology and culture from the skin sample collected with fine-needle aspiration showed yeasts inside activated macrophages, and fungal growth characteristic of Histoplasma spp., which was molecularly confirmed that was Histoplasma capsulatum var. capsulatum. The cat was successfully treated with oral itraconazole (10 mg/kg/daily) for 120 days. This is the first case report of feline histoplasmosis confirmed molecularly in Brazil.

Anthelmintic activity of nanoencapsulated carvacryl acetate against gastrointestinal nematodes of sheep and its toxicity in rodents.

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.

Anthelmintic effect of Cymbopogon citratus essential oil and its nanoemulsion on sheep gastrointestinal nematodes.

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<0.05). In conclusion, nanoencapsulation reduced toxicity and increased efficacy on H. contortus.

Anthelmintic activity of Eucalyptus citriodora essential oil and its major component, citronellal, on sheep gastrointestinal nematodes.

This study aimed to evaluate the anthelmintic activity of Eucalyptus citriodora essential oil and citronellal on sheep gastrointestinal nematodes. Essential oil composition was determined by gas chromatography mass spectrometry. The substances were evaluated in vitro using adult worm motility test (AWMT) and transmission electron microscopy (TEM). The acute toxicity test in mice and the fecal egg count reduction test (FECRT) in sheep were performed. Citronellal was confirmed as the essential oil major constituent (63.9%). According to the AWMT, 2 mg/mL of essential oil and citronellal completely inhibited Haemonchus contortus motility at 6 h post exposure. H. contortus exposed to essential oil and citronellal exhibited internal ultrastructural modifications. The lethal dose 50 values in mice were 5,000 and 2,609 mg/kg for essential oil and citronellal, respectively. E. citriodora essential oil reduced sheep epg at 14 days post treatment by 69.5% (P<0.05). No significant differences were observed in epg between the citronellal and negative control groups (P>0.05). The interaction between citronellal and other constituents in the essential oil may be relevant for its in vivo anthelmintic activity. Thus, E. citriodora essential oil and citronellal pharmacokinetic studies may help elucidate the anthelmintic activity of these compounds.

Anthelmintic effect of thymol and thymol acetate on sheep gastrointestinal nematodes and their toxicity in mice.

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.

Haemonchus contortus ß-tubulin isotype 1 gene F200Y and F167Y SNPs are both selected by ivermectin and oxfendazole treatments with differing impacts on anthelmintic resistance.

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 ß-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 <0.05). In vitro results showed increased phenotypic resistance against both anthelmintic classes in groups IVM and IVMOXF while group OXF only developed resistance against BZ. Finally, we provide evidence that while isotype 1 ß-tubulin gene SNPs may have some involvement with ML resistance, the presence of these ß-tubulin SNPs alone are not sufficient to develop ML resistance.

Validação dos intervalos de referência hematológicos e bioquímicos estabelecidos para cães domiciliados na Amazônia Oriental, Pará, Brasil / Validation of hematological and biochemical reference intervals established for dogs housed in the Eastern Amazon, Pará, Brazil

O objetivo do presente estudo foi validar intervalos de referências (IRs) para hematologia e bioquímica sanguínea de cães domiciliados da Amazônia Oriental e estabelecer novos IRs para os parâmetros não validados. Foram utilizadas amostras de 44 cães adultos clinicamente saudáveis de diferentes raças e sexos. Na validação, foi utilizada a metodologia proposta pelo Clinical and Laboratory Standards Institute (CLSI) e na determinação dos IRs dos parâmetros não validados foi utilizada a metodologia estatística proposta pela American Society for Clinical Veterinary Pathology (ASCPV). Os IRs foram determinados por meio do programa Excel com o suplemento Reference Value Advisor (versão 2.1). O software realiza os cálculos de acordo com as recomendações CLSI, conforme sugerido pelas diretrizes da ASCVP. Um total de 25 parâmetros (13 hematológicos e 12 bioquímicos) foram submetidos ao processo de validação. Desse total, seis (24%) parâmetros não foram validados (Hemoglobina, CHGM, eosinófilos, linfócitos, albumina e GGT) e para estes foram estabelecidos novos intervalos. Para a maioria dos parâmetros hematológicos e bioquímicos (76%), os IRs estabelecidos previamente na literatura ainda são válidos para utilização em cães criados na Amazônia Oriental. Para os parâmetros não validados, apenas para eosinófilos e linfócitos recomendamos que outros trabalhos, com um número maior de animais, sejam realizados para confirmar os nossos resultados ou estabelecer novos IRs. Os IRs estabelecidos para hemoglobina, CHGM, albumina e GGT podem ser utilizados em substituição aos intervalos antigos.

The aim of the present study was to validate reference intervals (RIs) for hematology and blood biochemistry of dogs domiciled in the Eastern Amazon and to establish new RIs for parameters not yet validated. Samples from 44 clinically healthy adult dogs of different breeds and sexes were used. The methodology proposed by the Clinical and Laboratory Standards Institute (CLSI) was used for validation, and the statistical methodology proposed by the American Society for Clinical Veterinary Pathology (ASCPV) was used to determine the RIs of the non-validated parameters. The IRs were determined using the Excel program with the Reference Value Advisor add-in (version 2.1). The software performs calculations according to CLSI recommendations as suggested by the ASCVP guidelines. A total of 25 parameters (13 hematological and 12 biochemical) were submitted to the validation process. Of this total, six (24%) parameters were not validated (Hemoglobin, CHGM, eosinophils, lymphocytes, albumin and GGT) and new intervals were established for these. For most hematological and biochemical parameters (76%), the RIs previously established in the literature are still valid for use in dogs raised in the Eastern Amazon. For non-validated parameters, just for eosinophils and lymphocytes, we recommend that other studies, with a larger number of animals, be carried out to confirm our results or establish new IRs. The established IRs for hemoglobin, CHGM, albumin and GGT can be used in place of the old ranges.

In vitro effects of Eucalyptus staigeriana nanoemulsion on Haemonchus contortus and toxicity in rodents.

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.

Anthelmintic activity of Cymbopogon citratus against Haemonchus contortus.

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. citratus essential oil to control of H. contortus parasite of small ruminant.

Anthelmintic activity of nanoencapsulated carvacryl acetate against gastrointestinal nematodes of sheep and its toxicity in rodents / Atividade anti-helmíntica do acetato de carvacrila nanoencapsulado sobre nematoides gastrintestinais de ovinos e toxicidade em roedores

Abstract 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.

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.