RESUMO
Animal welfare and economic implications of infectious diseases in cattle demand an efficient surveillance as the foundation for control and eradication programmes. Bovine respiratory syncytial virus (BRSV), Parainfluenza virus type 3 (PI3V), Bovine herpes virus-1 (BoHV-1), Bovine viral diarrhoea virus (BVDV), and Enzootic bovine leukosis virus (EBLV) cause common and often underdiagnosed diseases in cattle that are endemic in most countries [1]. A hallmark of individual exposure to a viral pathogen is the presence of antibodies directed towards that virus. The aim of this study was to develop and validate a pentaplex assay to simultaneously detect and quantify antibodies against BRSV, PI3V, BoHV-1, BVDV and EBLV in serum, as an efficient tool to yield epidemiological data. Monoplex assays were initially developed using either complete BRSV or BoHV-1 viral lysates, or recombinant proteins for BVDV, EBLV or PI3V as capture antigens. In addition, 125 serum samples from unvaccinated cattle, which were classified as positive or negative for each of the viruses by commercial ELISA kits, were used for validation. Conditions established for the Luminex monoplex assays were adopted for the pentaplex assay. The accuracy, determined by the area under the ROC curve, was greater than 0.97, and assay diagnostic sensitivities and specificities were over 95 and 90%, respectively, for all antigens. Intra (r) and interassay (R) coefficients of variation were under 10 and 20â%, respectively. Selectivity towards target viruses was shown by binding inhibition assays where unbound viruses reduced fluorescence intensities. Diagnostic agreement for samples analysed simultaneously in the monoplex and multiplex assays was almost perfect. In conclusion, a highly sensitive pentaplex assay was validated for the simultaneous identification of antibodies directed against BVDV, BoHV-1, PI3V, BRSV and EBLV in serum. The developed pentaplex assay complies with performance characteristics established by international guidelines for diagnostic tests and may be used as a tool for the implementation of epidemiological surveillance.
RESUMO
Background: Nutritional supplementation before breeding (Flushing) has become a common practice and is a reliable method to improve lambing and twining rates in sheep. The improvement of the body condition of a ewe is reflected in a higher number of ovulatory follicles and is termed "static effect of nutrition". Shorter periods of nutritional supplementation can also affect follicular development in the absence of changes in the body condition and weight of the animal, which is known as "acute effect of nutrition". Studies of follicular development in small ruminants have shown that 4 to 5 follicular waves occur during the estrous cycle, and that waves emerge every 5 to 7 days. However, the selection phase of the follicular wave occurs within 3 to 4 days, thus the length of flushing could last merely the time needed to push follicles over the selection threshold. Review: Here we examined the evidence produced by our research on the minimum length and appropriate timing of nutrient supplementation needed to enhance ovulation rate and prolificacy in sheep. Ewes have follicles ready to reach ovulatory size at any time of the estrous cycle and, when a follicular phase is induced, most show estrus and ovulate within 60 to 80 h. Hence, ovulatory follicles should commit for ovulation shortly after the decline in progesterone if they are to achieve ovulatory competence. We showed that an ultrashort flushing (USF) given as a single administration of a glycogenic substance at the time of prostaglandin-induced luteolysis (Control =1.6 ± 0.06 vs. USF= 2.08± 0.06) or progestin withdrawal (Control =1.64 ± 0.07 vs. USF= 2.41± 0.09) increased ovulation rate (P < 0.01). This increase was associated with elevated glucose and insulin concentrations for 12 h after USF (P < 0.01). However, the diameter of the three largest follicles did not change between the day of flushing and the day of estrus and did not differ between the control and the USF (P > 0.10). The USF could act either by advancing follicle maturation, or by affecting the feedback loop between the ovaries and gonadotrophin secretion. Therefore, we measured mRNA abundance for LH receptor (LHr), 3ß-hydroxysteroid dehydrogenase (3ß-HSD) and P450 aromatase at 0, 12, 24 and 48 h after the start of luteolysis in ewes treated or not with USF. Aromatase mRNA decreased in large follicles 12 h after USF (P < 0.01), with no changes on mRNA for LHr or 3ß-HSD. Further, we observed that the in vitro rumen fermentation and the in vivo glucose plasma concentrations in response to an isoenergetic (1470.83 kcal) single oral drench of glycerol, propylene glycol or molasses differed. The longer time to begin fermentation of glycerol allows for its absorption and its direct use for glucose production in the liver. Molasses was preferably fermented to butyrate, whereas propylene glycol was preferably fermented to propionic acid thus serving as glycogenic substrate. Glycerol and propylene glycol increased glucose and insulin concentrations in vivo, whereas molasses did not, thus the later may not be suitable for the USF. An increase in ovulation rate and prolificacy was also obtained with treatments that cause nutrient redistribution in the animal such as beta adrenergic receptor agonists and bovine somatotropin. Conclusions: We have developed an ultrashort flushing with glycogenic solutions that when applied at luteolysis will increase ovulation rate in sheep. This hyper-acute effect of nutrition caused a reduction in mRNA for P450 aromatase 12 hours after the glycogenic drench. In addition, from our results we can infer that ovine follicles can develop ovulatory capacity within 48 h after being selected when coinciding with the follicular phase.