First report of bovine viral diarrhea virus subgenotypes 1d and 1e in southern Chile.

Bovine viral diarrhea virus (BVDV) affects cattle worldwide causing severe productive and economic loss. In this study, we investigated the subgenotypes of BVDV circulating in cattle samples from the Aysén region, an active cattle breeding area located in southern Chile. Partial amplification of the 5' untranslated region (UTR) was performed by polymerase chain reaction (PCR), and twelve samples were analyzed by Sanger sequencing and phylogenetic analysis. Eight samples were identified as belonging to Pestivirus bovis subgenotype 1e, three to 1-b, and one to 1-d. The phylogenetic analyses performed revealed a marked distance between these now-identified strains and those previously reported in the country. These findings support the need to continually expand the analysis of the variability of the viral phylogeny for the currently circulating BVDV strains and to update the vaccines recommended for this livestock area and surrounding areas.

Effects of recombinant FSH (bscrFSH) and pituitary FSH (FSH-p) on embryo production in superovulated dairy heifers inseminated with unsorted and sex-sorted semen.

Superovulation is a drug-based method used in cattle to stimulate the ovarian folliculogenesis and the number of oocytes and transferable embryos. The present study aimed to test the effects of recombinant FSH (bscrFSH) and pituitary FSH (FSH-p) on ovarian response and in vivo embryo production in superovulated dairy heifers inseminated with unsorted and sex-sorted semen. Forty healthy Holstein heifers subjected to a superovulation (SOV) protocol by using FSH-p or bscrFSH were divided randomly into four groups: a) FSH-p inseminated with unsorted semen (USP; n = 10), b) FSH-p inseminated with sex-sorted semen (SSP; n = 10), c) bscrFSH inseminated with unsorted semen (USR; n = 10), and d) bscrFSH inseminated with sex-sorted semen (SSR; n = 10). Ultrasonography was carried out on Day 8 (estrus) and Day 15 (embryo collection) to evaluate the ovarian structures [follicles (FL), corpora lutea (CL), and non-ovulated follicles (NOFL)]. Embryonic-derived parameters were scored on Day 15 [total structures collected (TS), unfertilised oocytes (UFOs), total embryos (TEs), transferable embryos (TFEs), freezable embryos (FEs), and degenerated embryos (DEs)]. No differences were observed regarding ovarian structures (FL and NOFL) irrespective of SOV protocol or group assessed (P > 0.05). CL increased in bscrFSH-derived SOV protocol (P < 0.05). On Day 15, the embryonic-derived parameters TEs, TFEs, and FEs decreased in SSP/SSR compared to USP/USR (P < 0.05). Differences were observed regarding UFOs, with a greater number in SSP and SSR (P = 0.01). In conclusion, the bscrFSH-derived SOV protocol showed improved results compared to FSH-p-derived SOV protocol regarding ovarian (CL) and embryo-derived (TFE) parameters irrespective of the type of semen used.

Effects of Extra-Long-Acting Recombinant Bovine FSH (bscrFSH) on Cattle Superovulation.

Over the last few years, several commercial FSH products have been developed for cattle superovulation (SOV) purposes in Multiple Ovulation and Embryo Transfer (MOET) programs. The SOV response is highly variable among individuals and remains one of the main limiting factors in obtaining a profitable number of transferable embryos. In this study, follicle stimulating hormone (FSH) from different origins was included in two SOV protocols, (a) FSH from purified pig pituitary extract (NIH-FSH-p; two doses/day, 12 h apart, four consecutive days); and (b) extra-long-acting bovine recombinant FSH (bscrFSH; a single dose/day, four consecutive days), to test the effects of bscrFSH on the ovarian response, hormone profile levels, in vivo embryo production and the pluripotency gene expression of the obtained embryos. A total of 68 healthy primiparous red Angus cows (Bos taurus) were randomly distributed into two experimental groups (n = 34 each). Blood sample collection for progesterone (P4) and cortisol (C) level determination was performed together with ultrasonographic assessment for ovarian size, follicles (FL) and corpora lutea (CL) quantification in each SOV protocol (Day 0, 4, 8, and 15). Moreover, FSH profiles were monitorised throughout both protocols (Day 0, 4, 5, 6, 7, 8, 9, 10, and 15). In vivo embryo quantity and quality (total structures, morulae, blastocysts, viable, degenerated and blocked embryos) were recorded in each SOV protocol. Finally, embryo quality in both protocols was assessed by the analysis of the expression level of crucial genes for early embryo development (OCT4, IFNt, CDX2, BCL2, and BAX). P4 and cortisol concentration peaks in both SOV protocols were obtained on Day 15 and Day 8, respectively, which were statistically different compared to the other time-points (p < 0.05). Ovarian dimensions increased from Day 0 to Day 15 irrespective of the SOV protocol considered (p < 0.05). Significant changes in CL number were observed over time till Day 15 irrespective of the SOV protocol applied (p < 0.05), being non- significantly different between SOV protocols within each time-point (p > 0.05). The number of CL was higher on Day 15 in the bscrFSH group compared to the NIH-FSH-p group (p < 0.05). The number of embryonic structures recovered was higher in the bscrFSH group (p = 0.025), probably as a result of a tendency towards a greater number of follicles developed compared to the NIH-FSH-p group. IFNt and BAX were overexpressed in embryos from the bscrFSH group (p < 0.05), with a fold change of 16 and 1.3, respectively. However, no statistical differences were detected regarding the OCT4, CDX2, BCL2, and BCL2/BAX expression ratio (p > 0.05). In conclusion, including bscrFSH in SOV protocols could be an important alternative by reducing the number of applications and offering an improved ovarian response together with better embryo quality and superior performance in embryo production compared to NIH-FSH-p SOV protocols.

Distribución y abundancia de piojos (Werneckiella equi) y eficacia del tratamiento con Triclorfón en caballos mestizos / Distribution and Abundance of Lice (Werneckiella equi) and Efficacy of Treatment Using Trichlorfon in Crossbred Horses

El piojo masticador Werneckiella equi es un ectoparásito comúnmente encontrado en equinos; puede causar irritación, llevando a hiperqueratosis, prurito intenso y alopecia. Existen diversos productos en el mercado utilizados para el tratamiento de ectoparásitos; sin embargo, la información disponible sobre su efectividad es escasa. El objetivo de este estudio fue determinar la abundancia y distribución de W. equi y la eficacia de triclorfón en el control de los phthirapteras en caballos mestizos, utilizando una dosis única, aplicada mediante aspersión. Se utilizaron 34 caballos mestizos naturalmente infestados con piojos, que se distribuyeron al azar en dos grupos: Control (sin tratamiento; n=17) y Tratado con triclorfón diluido al 0,15% (n=17). La distribución y abundancia de los piojos fue estimada por medio de recuento directo de los phthirapteras en diferentes zonas del cuerpo del caballo, observándose solo diferencias significativas (P≤0,05) en la región de la mano con respecto a zona del abdomen y dorso lateral del tronco. La efectividad fue evaluada a los 28 días posttratamiento, observándose diferencias significativas entre el grupo Control y Tratado, resultando en un porcentaje de eficacia del 93,5% y demostrándose que triclorfón aplicado al 0,15% muestra un efectivo tratamiento para piojos en equinos.

The chewing louse, Werneckiella equi, is an ectoparasite commonly found in equine. This parasite may cause irritation, leading to hyperkeratosis, intense pruritus, and alopecia. There are several products in the market used in the treatment of ectoparasites; however, available information regarding their effectiveness is scarce. A study was undertaken to determine both the abundance and distribution of W. equi and the efficacy of trichlorfon (an organophosphate) in controlling the horse lice, using a single dose, employing the spray method. A total of 34 crossbred horses, naturally infested with lice were used. The animals were randomly distributed into two groups: Control group (without treatment; n=17); and Treatment group (n=17): animals treated with 0.15% w/v trichlorfon. The distribution and abundance of lice were determined by direct counting of phthiraptera in different areas of the horse body. The results show only minor significant differences (P≤0.05) in the hand region, when compared to the abdomen and the dorso-lateral trunk. The efficacy was evaluated 28 d posttreatment, showing significant differences between the two groups, being the treatment efficacy of 93.5%. It is concluded that trichlorfon, when applied as a 0.15% w/v spray, results in an effective treatment for equine lice.

The influence of gastrointestinal parasitism on fecal elimination of doramectin, in lambs.

A study was done to investigate the effect of parasitism on patterns of doramectin (DRM) fecal elimination in lambs. Fourteen Suffolk Down parasitized lambs (26.9 ± 1.5 kg body weight: bw) were purposely selected for the study. Seven pairs of lambs were allocated into two experimental groups. Group I (non-parasitized) was pre-treated with 3 repeated administrations of 5mg/kg bw of fenbendazole to maintain a non-parasitized condition. In Group II (parasitized), the lambs did not receive any anthelmintic treatment. After 85 d of the pre-treatment period, both groups were treated with a subcutaneous injection of 200 µg/kg bw of DRM. Fecal samples were collected at different times between -85 d before and 60 d after the DRM treatment, for both parasitological and chromatographic analysis. Samples were analyzed by high-performance liquid chromatography (HPLC) with fluorescence detection. Data of DRM concentrations were expressed as wet weight. A non-linear pharmacokinetic analysis was performed and results were compared using the Mann Whitney test. Fecal maximum concentrations (C(max)) of DRM were 1.37 ± 0.19 µg/g (parasitized group) and 0.86 ± 0.15 µg/g (non-parasitized group) observed at the time of the maximum concentration (T(max)) of 2.1 ± 0.4 and 3.1 ± 0.3d, respectively. Differences in C(max) values were significant (P<0.05). The accumulated elimination of DRM in feces, expressed as the percentage of DRM total dose, was 67.1% in the parasitized group, whereas in the non-parasitized group it was 56.5%. Our results showed that gastrointestinal parasitic diseases can modify the patterns of DRM fecal elimination, when the drug is administered by subcutaneous route in lambs.

Patterns of doramectin tissue residue depletion in parasitized vs nonparasitized lambs.

The effect of gastrointestinal parasitism on patterns of edible tissue depletion of doramectin was studied in greyface Suffolk lambs. Twelve weight-matched pairs of lambs were allocated into group I (nonparasitized, pretreated with three administrations of 5 mg/kg fenbendazole) and group II (parasitized, did not receive anthelmintic treatment). Both groups were maintained together under similar conditions for 70 days, when they were treated with a subcutaneous dose of 0.2 mg/kg bw doramectin. At 7, 14, 21, and 28 days after doramectin administration, three lambs from each group were slaughtered and samples of liver, kidney, muscle, and fat were obtained. Pre-treatment with fenbendazole significantly reduced the nematode fecal egg count and significantly increased lamb body weight compared to the parasitized group. Doramectin was detected in all of the tissues up to 28 days post-treatment. Significantly higher and more persistent doramectin concentrations were found in the nonparasitized lambs compared to the parasitized animals. Considering the EMEA maximum residue limits for doramectin in fat, the calculated withdrawal period for the healthy lambs (43 days) was significantly higher than that for the parasitized animals (26 days).

Validación de un método analítico y determinación de residuos de invermectina en tejidos de ovino / Validation of an analytical method and determination of invermectin residues in sheep tissues

Se realizó un estudio con el objetivo de validar un método analítico sensible y confiable para la detección de residuos de ivermectina (IVM) en muestras de hígados, riñón, músculo y grasa, junto con determinar las concentraciones del fármaco en tejidos de ovinos tratados por vía subcutánea. Muestras de tejidos libres de fármaco fueron sobrecargadas con concentraciones de IVM entre 1 y 50 ng/g (hígado, riñón y músculo); 5 a 200 ng/g (grasa), luego fueron sometidas a extracción en fase sólida y analizados por cromatografía líquida de alta eficiencia (HPLC). Para el estudio de residuos se utilizaron 12 ovinos Suffolk Down de 27,8 ± 1,3 kg de peso, los que fueron tratados con 0,2 mg/kg de IVM vía subcutánea, luego se sacrificaron grupos de 3 animales a los 1,5; 7; 14 y 21 días post tratamiento. La ausencia de interferencias y una adecuada simetría de los cromatogramas indica una buena especificidad del método analítico empleado para la detección de IVM en los tejidos analizados. Los porcentajes de recuperación fluctuaron entre 70 a 93,2 por ciento. El límite de cuantificación se estableció en hígado: 0,48 ng/g; riñón: 1,02 ng/g; músculo:0,18ng/g y grasa: 2,65ng/g. La validación de la metodología analítica demostró adecuados valores de sensibilidad, presición y axactitud que permiten obtener resultados confiables para la detección y cuantificación de residuos de IVM en tejidos de ovinos. En los ovinos tratados con IVM, las mayores concentraciones de residuos fueron observadas a los 1,5 días post tratamiento en hígado (281,7 ± 116,95 ng/g) y grasa (248,67 ± 90,85 ng/g), los que persistieron hasta el día 21 con concentraciones de 0,63 ± 0,2 ng/g y 4,07 ± 2,25 ng/g, respectivamente. Las menores concentraciones de residuos de IVM fueron observadas en las muestras de músculo.

A study was undertaken in order to validate a precise and reliable analytical method for the detection of ivermectin’s (IVM) tissue residues in sheep, and to know the patterns of the drug concentrations depletion in edible tissues such as liver, kidney, muscle and fat, from treated animals by subcutaneous route. Drug free tissue samples were fortified with increasing concentrations of IVM (1 to 50 ng IVM/g for liver, kidney and muscle; and 5 to 200 ng IVM/g for adipose tissue) and then were subjected to solid phase extraction and analyzed by high performance liquid chromatography (HPLC). Twelve sheep weighing 27.8 ± 1.3 kg, were treated with 0.2 mg/kg of IVM by subcutaneous route, and then were slaughtered in groups of three animals at 1.5, 7.0, 14.0, and 21.0 days post treatment. The specificity of the method was demonstrated by the absence of interferences and the adequate symmetry of chromatograms. The percentage of recovery ranged from 70 to 93.2% for all tissues analyzed and different drug concentrations. The limit of quantification of the method was established in 0.48 ng/g for liver; 1.02 ng/g for kidney; 0.18 ng/g for muscle and 2.65 ng/g for adipose tissue. The validated analytical methodology showed satisfactory results of sensitivity, precision and accuracy that allow it use for the detection and quantification of tissue residues of IVM in sheep. From the tissues samples of sheep treated with IVM, the higher concentrations were found in liver (281.7 ± 116.95 ng/g) and adipose tissue (248.67 ± 90.85 ng/g) at 1.5 days, and the drug concentrations in both tissues were maintained for a period of 21 days post treatment with 0.63 ± 0.2 ng/g and 4.07 ± 2.25 ng/g respectively. The lowest concentrations of IVM in tissues were observed in muscle samples.

Gastrointestinal parasitism reduces the plasma availability of doramectin in lambs.

A study was undertaken to investigate the effect of parasitism on plasma availability and pharmacokinetic behaviour of doramectin (DRM) in lambs. Fourteen parasitised grey face Suffolk lambs (26.9 +/- 1.5 kg bodyweight) were selected for the study. Seven pairs of lambs were allocated to two groups to obtain an approximately even weight distribution. Group I (non-parasitised) was pre-treated with three repeated administrations of 5 mg/kg fenbendazole to maintain a parasite free condition. In group II (parasitised), the lambs did not receive any anthelmintic treatment. After the 85-day pre-treatment period, both groups of animals were treated with DRM by subcutaneous (SC) injection in the shoulder area at 200 microg/kg. Throughout the experimental period, both groups were maintained together under similar feeding and management conditions. Blood samples were collected by jugular venepuncture at different set times between 0.5 h and 60 days post-treatment. After plasma extraction and derivatisation, samples were analysed by high performance liquid chromatography (HPLC) with fluorescence detection. A computerised kinetic analysis was performed and the data were compared using the Student's paired t test. The parent molecule was detected in plasma between 30 min and either day 20 (parasitised) or day 35 (non-parasitised) post-DRM treatment. The AUC values of the parasitised group (143.0 +/- 18 ng d/mL) were significantly lower (P<0.05) than those observed in the parasitically naïve animals (229.6 +/- 21.7 ng d/mL). The mean residence time (MRT) in the parasitised group (3.4 +/- 0.3 days) was significantly shorter (P<0.05) than in the healthy group (6.6 +/- 0.6 days). Study results have shown that parasitic disease, through alteration in the body condition, can produce significant changes in the plasma disposition of DRM when administered SC to parasitised lambs.