Ovine Toll-like Receptor 9 (TLR9) Gene Variation and Its Association with Flystrike Susceptibility

Toll-like receptors (TLRs) are a family of proteins that play a role in innate immune responses by recognising pathogen-associated molecular patterns derived from various microbes. Of these receptors, TLR9 recognises bacterial and viral DNA containing unmethylated cytosine-phosphate-guanine (CpG) motifs, and variation in TLR9 has been associated with resistance to various infectious diseases. Flystrike is a problem affecting the sheep industry globally and the immune response of the sheep has been suggested as one factor that influences the response to the disease. In this study, variation in ovine TLR9 from 178 sheep with flystrike and 134 sheep without flystrike was investigated using a polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) approach. These sheep were collected from both commercial and stud farms throughout New Zealand and they were of 13 different breeds, cross-breds and composites. Four alleles of TLR9 were detected, including three previously identified alleles (*01, *02 and *03) and a new allele (*04). In total six single nucleotide polymorphisms (SNPs) were found. Of the three common alleles in the sheep studied, the presence of *03 was found to be associated with a reduced likelihood of flystrike being present (OR = 0.499, p = 0.024). This suggests that variation in ovine TLR9 may affect a sheep’s response to flystrike, and thus the gene may have value as a genetic marker for improving resistance to the disease.

The relationship between mitochondrial ND5 gene polymorphisms and in vitro embryo production in Sanjabi sheep

The aim of this study was to investigate mitochondrial ND5 gene polymorphisms and their relationship with in vitro maturation (IVM) and in vitro culture (IVC) of Sanjabi sheep. Blood and ovarian samples of adult ewes were obtained from a local slaughterhouse. For each ovarian sample, cumulus–oocyte complexes larger than 3 mm in diameter were aspirated from follicles, and their IVM and IVC rates were recorded. A 666-bp fragment of the ND5 gene was amplified using the polymerase chain reaction. The samples were genotyped using a modified single-stranded conformation polymorphism (SSCP) method, and an association study was conducted with IVM and IVC rates. Six different SSCP patterns, designated A, B, C, D, E and F with respective frequencies of 8, 47, 4, 4, 32 and 5%, respectively, were observed. According to the results of association analysis, there was no significant association between the ND1 gene polymorphisms and the IVM and IVC rates (P > 0.05).

Single-Strand Conformation Polymorphism Fingerprint Method for Dictyostelids

Dictyostelid social amoebae are a highly diverse group of eukaryotic soil microbes that are valuable resources for biological research. Genetic diversity study of these organisms solely relies on molecular phylogenetics of the SSU rDNA gene, which is not ideal for large-scale genetic diversity study. Here, we designed a set of PCR–single-strand conformation polymorphism (SSCP) primers and optimized the SSCP fingerprint method for the screening of dictyostelids. The optimized SSCP condition required gel purification of the SSCP amplicons followed by electrophoresis using a 9% polyacrylamide gel under 4°C. We also tested the optimized SSCP procedure with 73 Thai isolates of dictyostelid that had the SSU rDNA gene sequences published. The SSCP fingerprint patterns were related to the genus-level taxonomy of dictyostelids, but the fingerprint dendrogram did not reflect the deep phylogeny. This method is rapid, cost-effective, and suitable for large-scale sample screening as compared with the phylogenetic analysis of the SSU rDNA gene sequences.

Short Communication: A highly polymorphic caprine keratin-associated protein gene identified and its effect on cashmere traits

Five keratin-associated protein 6 genes (KRTAP6) have been identified in sheep and variation in some KRTAP6 has been associated with wool fibre diameter-related traits, but none of these homologues has been identified in goats. In this study, we reported the identification of the sheep KRTAP6-5 homologue on goat chromosome 1 and PCR-single strand conformation polymorphism analysis in 300 Longdong cashmere goats revealed the existence of twelve variant sequences. Both coding region and 3’UTR of the putative caprine KRTAP6-5 displayed a biggest sequence similarity to ovine KRTAP6-5 gene. This suggested that the gene represents caprine KRTAP6-5 sequences, and these sequences composed twenty three genotypes which was the most polymorphism gene in KRTAPs that have been studied. Among these sequences, fifteen nucleotide substitutions and a 24-bp insertion/detection were identified. Variation in goat KRTAP6-5 was associated with variation in mean fibre diameter, suggesting that KRTAP6-5 is worthy of further study in the context of variation in cashmere traits.

Cytomegalovirus Genotype and Virulence in Infants with Congenital Infection

Objective Cytomegalovirus (CMV) virulence may depend on genetic variability in several regions of the genome. This study aimed to assess specific CMV genotypes' association with the severity of symptomatic congenital CMV disease at birth. Methods CMV glycoprotein B (gB), glycoprotein N (gN), glycoprotein H (gH), and UL144 strains were identified by nested polymerase chain reaction, restriction fragment length polymorphism, and heteroduplex mobility assay single-stranded conformation polymorphism in 50 infants infected congenitally and 25 asymptomatic infants. Results gN1 (p = 0.010) and UL144-B (p = 0.034) genotypes were associated, by logistic regression, with reduced risk of developing symptomatic congenital CMV infection. gN1 (p = 0.020) and gN3 (p = 0.022) genotypes were associated with reduced risk of severe symptomatic disease. Conversely, gB1 (p = 0.018) was the most virulent genotype and was associated with severe symptoms. Conclusion An association among gB1, gN1, gN3, and UL144-B genotypes of CMV and severity of congenital CMV disease might exist. gB, gN, and UL144 genotypes could be important virological markers of infant infection.

Genetic Variability Analysis of Early Growth Response 2 (EGR2) Gene in Native Goat Breeds of Kerala

Background: India is fortunate to have a vast livestock resource with the availability of 28 well defined goat breeds. Kerala is the home for two breeds of goats namely Malabari and Attapady Black. Due to high prolificacy and income through lower input goat rearing had attracted numerous farmers. Selection with the aid of molecular markers associated with production traits plays an essential role in goat breeding programmes. The EGR2 gene is a part of multigene family which encodes Cys2His2 type zinc-finger proteins which is responsible for DNA binding. This gene has a major role in cellular prolification, reproduction, proper growth and development ovarian follicles. Thus present study was conducted with an objective to detect single nucleotide variations of Early Growth Response 2 gene in native goat breeds of Kerala.Methods: This research was conducted in 153 Malabari goats and 129 Attappady Black goats from six centers viz., University Goat and Sheep farm Mannuthy and 5 field centers of ICAR- All India Coordinated Research Project on Goat Improvement. Genomic DNA was isolated and PCR was performed to amplify Exon 1, Exon 2 and 5 fragments of Exon 3 regions of Early Growth Response 2 gene. Single stranded conformation polymorphism (SSCP) technique was performed to detect Single Nucleotide Polymorphism (SNPs).Result: The SSCP revealed similar banding patterns and sequencing did not indicate any nucleotide variations in all the exons screened suggesting that EGR2 gene is highly conserved in native goat breeds of Kerala. This is the first study conducted to characterize EGR2 gene in goats making the current study a novel one.

Molecular Differentiation of Four Species of Oropsylla (Siphonaptera: Ceratophyllidae) Using PCR-Based Single Strand Conformation Polymorphism Analyses and DNA Sequencing

It is often difficult to distinguish morphologically between closely related species of fleas (Siphonaptera). Morphological identification of fleas often requires microscopic examination of internal structures in specimens cleared using caustic solutions. This process degrades DNA and/or inhibits DNA extraction from specimens, which limits molecular-based studies on individual fleas and their microbiomes. Our objective was to distinguish between Oropsylla rupestris (Jordan), Oropsylla tuberculata (Baker), Oropsylla bruneri (Baker), and Oropsylla labis (Jordan & Rothschild) (Ceratophyllidae) using PCR-based single strand conformation polymorphism (SSCP) analyses and DNA sequencing. A 446 bp region of the nuclear 28S ribosomal RNA (rRNA) gene was used as the genetic marker. The results obtained for 36 reference specimens (i.e., fleas that were morphologically identified to species) revealed no intraspecific variation in DNA sequence, whereas the DNA sequences of the four species of Oropsylla differed from one another at two to six nucleotide positions. Each flea species also had a unique SSCP banding pattern. SSCP analyses were then used to identify another 84 fleas that had not been identified morphologically. DNA sequencing data confirmed the species identity of fleas subjected to SSCP. This demonstrates that PCR-SSCP combined with DNA sequencing of the 28S rRNA gene is a very effective approach for the delineation of four closely related species of flea.