rt-pcr

Canine Distemper have become a major concern within the veterinary clinical work. Thus, the appearance of many cases of canine distemper in adult animals with their vaccination plan up to date has alarmed veterinarians. Many cases of canine distemper have become a major concern within the clinical veterinary task. The purpose of this work was to detect the gene of hemagglutinin of the virus canine distemper, by using the Polymerase Chain Reaction associated with Reverse transcription (RT-PCR), as confirmation of the clinical diagnosis of disease. For this, peripheral blood samples of animals were used clinically patients and were grouped according to their date of extraction and using vaccines commercial as control. The results allow to demonstrate a high sensitivity of the technique, besides allowing the use of samples up to seven days of storage at 4°C, despite the fragility of RNA viral. Detection of canine distemper virus haemagglutinin gene in field samples and its high sensitivity, suggests studying its use as a diagnostic tool complementary to the clinical diagnosis of canine distemper in our country.

Worldwide, Canine Distemper Virus (CDV) infection is a highly prevalent disease with high morbidity and mortality. CDV causes a multisystemic disease in a wide range of hosts including 9 families of mammals among them some primates, cetaceans and numerous carnivores. It presents a high tropism for lymphoid, neurological and epithelial tissue, leading to an infection of almost all systems, so the clinical signs observed are very varied. The diagnosis is made based on the clinical presentation of the disease, which considers a variety of signs and must be confirmed by a laboratory diagnostic method. The molecular technique called Reverse Transcription Polymerase Chain Reaction (RT-PCR) has been used to characterize viral strains based on the basis of genetic differences on the hemagglutinin (H) gene of CDV has allowed the identification of 14 circulating lineages in the world. Two lineages, namely the America-1 and the Europe-1/South America-1 have been described in Chile. The goal of this work was to implement a multiplex RT-PCR protocol, which was built on the in silico design of primers based on the H gene nucleotide sequences stored in the Genbank® database. This method was capable of detecting the previously described two circulating genetic lineages of CDV in a differential way providing a supporting diagnostic tool for epidemiological studies in the country. These results suggest that the primers described here are extremely selective for the above-mentioned lineages. In addition, our initial screening indicated that most analyzed clinical samples corresponded to the America-1 lineage, stressing the need for a continuous surveillance in order to properly address the prevalence of both lineages in Chile.

Background: C-type natriuretic peptide (CNP) was isolated from porcine brain and is a 22-amino acid peptide which belongs to the natriuretic peptide (NP) family. Even though this peptide shares structural similarity to other endogenous NPs including atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) its receptor selectivity is different from other NPs. The present study was undertaken to investigate the expression of C-type natriuretic peptide (CNP) and its specific guanylyl cyclase (GC)-coupled receptor in the granulosa cells of the pig ovarian follicle. Results: Specific 125I-[Tyr0]-CNP(1-22) binding sites were localized in the granulosa cell layer of the ovarian follicle with an apparent dissociation constant (Kd>) and a maximal binding capacity (Bmax) of 1.41±0.39 nM and 2.75±0.65 fmol/mm2 respectively. Binding of 125I-[Tyr0]-CNP(1-22) to these sites was also prevented by atrial natriuretic peptide (ANP(1-28)), brain natriuretic peptide (BNP(1-26)) and des[Gln18,Ser19,Gly20, Leu21,Gly22] ANP(4-23) (C-ANP). Production of 3’,5’-cyclic guanosine monophosphate (cGMP) by particulate GC in the granulosa cell membranes was stimulated by natriuretic peptides (NPs) with a rank order of potency of CNP(1-22)>>BNP(1-26)>ANP(1-28). HS-142-1, a selective antagonist of the two recognized GC-coupled NPRs, inhibited CNP(1-22)-stimulated cGMP production in granulosa cell membranes in a dose-dependent manner. Also mRNAs for all three recognized NPRs were detected in granulosa cells using reverse transcriptase-polymerase chain reaction (RT-PCR). Serial dilution curves of granulosa cell extracts were parallel to the standard curve of synthetic CNP. Conclusion: These results indicate that CNP and its specific receptor are expressed in the granulosa cells of the pig ovary, and suggest that CNP may be a local autocrine and/or paracrine regulator via activation of its specific GC-coupled receptor, NPR-B.