Restriction Enzymes HaeIII Research Articles

Adaptive capability of slow-growing backyard poultry as indicated by physiological and molecular responses in a hot and humid coastal climate

Assessing the adaptability of slow-growing rural chickens for improving thermotolerance to suit the global climate change is a major research need. This work was aimed to evaluate the adaptability of CARI-Debendra chickens and to identify the polymorphism as well as expression profiling of thermotolerant genes (HSP70 and GRP78) under prevailing temperature-humidity indices and thermal stress in a coastal environment. One hundred sixty straight run chicks were reared at THI≥75 (control) and THI>80 under coastal climate till 12 weeks. Polymorphism of HSP70 and GRP78 candidate genes were explored using restriction enzymes TaqI and HaeIII to identify possible thermotolerance markers. Expression profiling of both the genes in liver, intestine and pectoralis muscle was determined through quantitative real-time PCR. Rectal and body surface temperature recorded in the neck and back showed significant differences (P < 0.01) with higher temperature in THI>80 group. Comparatively lower live weights (P < 0.05) and poor FCR were recorded in THI>80 group. The villi height in all intestinal segments was significantly lower (P < 0.01), but deeper crypt depth was observed in THI>80 than control group. A lowest thymus weight (P < 0.05) was noted with no significant differences in immune response in treatment group. Serum levels of cholesterol, activities of lactate dehydrogenase, creatinine kinase and concentration of potassium, sodium and thyroxine hormone were not different between the 2 groups. The concentration of triiodothyronine and chloride ion was lower in THI>80 group indicating adaptive changes for thermoregulation. HSP70 gene expressions in the three tissues were differentially increased (P < 0.01) by temperature-humidity indices, but the expression of GRP78 was not different between the 2 groups. The results concluded that the environmental factors interact with genetics on adaptability towards thermotolerance in slow-growing chickens.

Detection of Phytoplasma belonging to the 16SrXXI group in Scott pine and mountain pine

One of the reasons for the deterioration of coniferous stands condition in a number of European countries is associated with their infection with phytoplasmas (obligate intracellular pathogens) — bacteria lacking a cell wall. The aim of the work is to identify the presence of phytoplasma infection in samples of Scots pine (Pinus sylvestris L.) and mountain pine (Pinus mugo Turra) collected in the Moscow and Samara regions, which had characteristic symptoms of the disease, and to determine the taxonomic affiliation of the phytoplasm. Phytoplasma was detected using direct and nested PCR with primer pairs P1/16S-Sr and R16F2n/R16R2 respectively. Phytoplasma DNA was found in six of the seven pine specimens, including an asymptomatic one. Analysis of restriction fragment length’s polymorphism after digestion of DNA amplicons with restriction endonucleases AluI, MseI, HhaI, HpaII, HaeIII, RsaI, and TaqI indicated the similarity of Russian strains of Scotch pine and mountain pine phytoplasmas to Lithuanian strains of PineLRN and PineBLD of mountain pine phytoplasma (GenBank Accession Number MK089821 and MK089819, respectively) identified as ‘Candidatus Phytoplasma pini’ (subgroup 16SrXXI-A). A phytoplasma related to this species has been registered on the territory of the Russian Federation for the first time.

Polymorphism of growth hormone (GH/HaeIII) gene and its association with the performance of merino cross rams

Merino cross sheep (75% Merino × 25% Garut) are introduced by the government of Indonesia for meat production purposes. The present study aimed to determine the polymorphism in the exon 2 region of the growth hormone (GH) gene (422 bp) in Merino cross rams using the PCR-RFLP technique and to analyze its relationship with body weight and body measurements of the rams. A total of 145 rams aged one-year-old with an average body weight of 29.08 ± 7.96 kg from the breeding station in West Java, Indonesia were considered as the experimental animals. It was indicated that a missense mutation of c.55G &gt; A (p.G19S) was detected in the target sequence of the GH gene in Merino cross rams. The PCR-RFLP analysis in the GH gene of Merino cross with HaeIII restriction enzyme (GH/HaeIII) was observed in a moderate category with a polymorphic informative content (PIC) value of 0.22. Therefore, the G allele was more frequent than the A allele (0.85 versus 0.15). Furthermore, the genotype AA was not present among the sheep that were part of the study. However, the polymorphism of p.G19S was found to have no significant association with birth weight and body measurements in one-year-old Merino cross sheep. However, the GH/HaeIII gene in Merino cross rams exhibited polymorphism, primarily with two genotypes: GG (wildtype) and GA (carrier). The G allele was identified as the dominant allele in the ovine GH gene, occurring with a frequency of 0.85. Importantly, the polymorphism of the GH/HaeIII gene was significantly linked to birth weight and chest depth in one-year-old Merino cross rams. These findings provide preliminary insights that could potentially aid in the early stages of molecular selection for Indonesian Merino cross sheep.

Molecular Characterization and Gene Expression of MHC II DRB3 Gene Associated with Subclinical Mastitis in Goats

The Major Histocompatibility Complex (MHC) genes have a vital role in the immune response of vertebrates by encoding molecules involved in self/non-self-discrimination and antigen presentation. This study focused on the MHC II DRB3 gene in goats, specifically comparing its nucleotide and amino acid sequences with those of other ruminant species. Four different goat breeds (Native, Upgraded, Anglo-Nubian, and Saanen) were analyzed, and their MHC II DRB3 sequences were aligned with sequences from NCBI GenBank. The nucleotide sequences exhibited high similarity between the different goat breeds and other ruminant species, indicating a conserved structure across mammalian species. Results from sequence comparisons revealed varying degrees of similarity between goat MHC II DRB3 sequences and those of Capra hircus, Ovis aries, Bos taurus, Bos grunniens, and Bubalus bubalis. Phylogenetic analysis further supported the close relationship between goats and other ruminants. Functional domain analysis identified conserved regions in the MHC II DRB3 gene across different breeds of goats and other ruminants. Moreover, polymorphism analysis using the HaeIII restriction enzyme highlighted genetic variability within the MHC II DRB3 gene in goats. This study enhances our understanding of the MHC II DRB3 gene in goats, its similarities to other ruminant species, and its potential role in immune responsiveness.

Polymorphism of growth hormone (GH) gene and its association with performance and body conformation of Harnali sheep.

The present study was carried out to study the polymorphism in the GH gene and its association with various performance and body conformation traits, viz., birth weight (B-WT), weaning weight (W-WT), six-month body weight (6M-WT), one-year body weight (Y-WT), annual greasy fleece weight (AGFW), body length (BL), body height (BH), heart girth (HG) and paunch girth (PG) in 138 Harnali sheep. PCR-RFLP was performed to identify polymorphism in the targeted region of the GH gene. The PCR product of 422bp size of the GH gene was amplified encompassing partial exon 2 and inton 3 in Harnali sheep. The PCR product was digested with HaeIII restriction enzyme for the detection of Single nucleotide polymorphism (SNP). The digested products revealed the presence of two genotypes, i.e. AA and AB in the studied population. A > G mutation (A781G) was observed in our resource population. The AA genotype was found to be the predominant genotype (0.62). Chi square value revealed that resource population was not under Hardy-Weinberg equilibrium with respect to target locus. Period of birth was found to have significant effect on W-WT, Y-WT, BL, BH and PG. Sex of animal was found to have significant (P < 0.05) effect on W-WT and highly significant (P < 0.01) effect on 6M-WT, Y-WT and AGFW in Harnali sheep. The effect of genotype was found to be significant (P < 0.05) on annual greasy fleece weight. AB genotype was found to be associated with higher annual greasy fleece weight and can be used as a potential candidate marker in selection criteria for improving greasy fleece weight in Harnali sheep.

Milk Protein Genes Polymorphism in Indigenous and Crossbred Cattle from a private Dairy Farm in Ethiopia

Kappa casein and beta lactoglobulin genes are major milk proteins which have a direct effect on protein content in dairy cattle. Molecular-based selection through the identification of genetic polymorphism of major protein genes can be used to gain genetic improvement of milk protein yield. The objective of this study was to identify kappa casein (CSN3) and beta lactoglobulin (LGB) genes polymorphisms in indigenous and crossbreed cattle. A total of 90 whole blood samples were collected from individual animal in a private dairy farm. DNA extraction and quality assessment were done salting out procedure and gel electrophoresis, respectively. Polymerase chain reaction (PCR) was performed with gene specific primers. For genotyping, PCR products of CSN3 gene was digested with HinfI and HindIII while LGB gene was digested with HaeIII restriction enzymes. Two haplotypes A and B; three genotypes, AA, AB and BB were observed at CSN3 of HinfI site and LGB HaeIII site, but only AA and AB were observed at CSN3 of HindIII site in crossbred and indigenous cattle populations. However, at CSN3 locus, A allele was found to be more common (0.65) in indigenous cattle than the B allele (0.35), while B (0.51) allele and AB genotype (0.81) were more frequent in crossbred cattle. But, LGB B allele was higher in indigenous cattle (0.67) compared to Allele A (0.33). LGB BB genotype (0.57) is higher followed by AA genotype in indigenous cattle while both allele and genotypic frequencies are equal in crossbred cattle. Both CSN3 and LGB loci were polymorphic in studied populations. Expected heterozygosity was higher in crossbred (0.49, 0.50) than in indigenous (0.38, 0.33) cattle at CSN3 and LGB locus, respectively which might be due to breed variation. The current findings showed that both CSN3 and LGB genes could be promising diagnostic markers in selecting dairy cattle breed. However, further investigations with large sample size and association study with milk composition is required to substantiate the current result.

Exploring the Potential in Combining the Two Molecular Approaches, COI Barcoding and PCR-RFLP (COIBar-RFLP) in Identification of Selected Species of the Family Carangidae

The family Carangidae is considered a markedly diverse, widespread taxon. Due to the characteristic “cryptic diversity” and “hybrid speciation” within the family, there is an exigency for taxonomic approaches that go beyond traditional phenotypic modus in identifying species. Mitochondrial cytochrome c oxidase subunit - I (COI) barcoding gene region plays a significant role over phenotypic characters in identifying species. This study evaluates the combination of the two molecular approaches: the COI DNA barcoding and PCR-RFLP for judicious species discrimination. The partial mitochondrial COI gene region of the selected Carangid fish species was amplified, sequenced and confirmed sequences with a mean length of 655 bp were submitted to the main databases; NCBI and BOLD. Intraspecific and interspecific nucleotide divergences were computed by the Kimura-2-Parameter (K2P) and they ranged between (0.00–2.96 %) and (6.46–21.83 %), respectively. The possibility of acquiring the same RFLP profiles given by the restriction enzymes HaeIII and MbOII was observed by the theoretical cleavage of 250 reference sequences of the corresponding gene. Hence, major and minority composite haplotypes of each species were obtained based on the fragment types derived by both HaeIII and MbOII. The resulting divergence values were compatible with the previously reported values for marine fish species. All the species were clearly differentiated by both RFLP banding patterns and the highest probability assumption of getting the same RFLP profile was compatible with the most abundant composite haplotype of each species. This reveals the practicability of the combination of two consolidated molecular approaches, COI barcoding and PCR-RFLP (COIBar-RFLP).

Polymorphism of the myostatin gene exon 1 using PCR-RFLP technique in five beef cattle in Indonesia

This study aims to analyze the polymorphism of the myostatin (MSTN) gene in exon 1 at SNPc.111G>C (rs523392653) and SNPc.267G>A (rs383271508) on various beef cattle in Indonesia. A total of 136 DNA samples were analyzed in this study, consisting of 33 heads of Madura, 31 heads of PO, 36 heads of Bali, 18 heads of Simmental, and 18 heads of Limousin cattle. The DNA sample was collected from the Animal Molecular Genetic Laboratory, Faculty of Animal Science, IPB University. Along 608 bp of DNA sequence was amplified using PCR with forward primer was 5′-CAA GTT GTC TCT CAG ACT GG-3′ and reverse primer was 5′-CTC CTC CTT ACA TAC AAG CC-3′ and annealing temperature was 59°C. The amplified DNA was then digested with HaeIII and AluI restriction enzymes using the RFLP method to determine polymorphism. Genotype frequencies, allele frequencies, PIC, and heterozygosity values were calculated with the PopGen32 program. The HaeIII enzyme produces two genotypes, GG and CG, while the AluI enzyme produces three: GG, AG, and AA. SNPc.111G>C polymorphism was found in Madura and PO cattle, while SNPc.267G>A polymorphism was found in Madura, PO, and Bali cattle. Both Simmental and Limousin cattle were monomorphic.

Study of polymorphism of toll like receptor-4 gene in crossbred cattleand its association with somatic cell count

The present study was undertaken to study the TLR4 gene polymorphism in exon 3 and its association with somatic cell count in HF×Gir cows using PCR-RFLP. Sixty crossbred cows (40 affected and 20 normal) between 3rd to 5th lactation were selected from Bombay Gowrakshak Mandali, Betegaon, Maharashtra. The confirmation of mastitis was done with the help of California MastitisTest (CMT) and the blood samples of 40 affected animals were collected aseptically along with 20 normal animals. The mean of somatic cell score in normal cows ranged between 2.86 to 2.99 × 105 cells/ml, whereas for affected animal the range of somatic cell score was 3.45 to 5.72 × 105 cells/ml. The genomic DNA was isolated and exon 3 of TLR4 gene was successfully amplified by PCR using a specific primer. The PCR product was digested with HaeIII restriction enzyme. The PCR-RFLP of exon 3 with HaeIII enzyme revealed two genotypes ‘BB’ and ‘AB’ with band pattern 49 bp, 98 bp &amp; 196 bp (BB) and 49 bp, 98 bp, 196 bp &amp; 245 bp (AB), respectively. The mean of somatic cell score estimated for animals with genotypes ‘BB’ and ‘AB’ was 3.99±0.13 and 3.66±0.03, respectively. The twomean ‘t’ test analysis showed non-significant difference between somatic cell count with genotypes ‘BB’ and ‘AB’. It indicated that there was no association between the genotypes of exon 3 of TLR4 gene with somatic cell count in crossbred cows under present study.

Polymorphism of the growth hormone (GH) gene and its association with growth traits in the Kilakarsal breed of sheep

The goal of this study was to investigate the single nucleotide polymorphism of the growth hormone gene and its association with growth traits in the Kilakarsal breed of sheep. Polymerase Chain Reaction – Restriction Fragment Length Polymorphism (PCR-RFLP) of the growth hormone gene (422 bp) with HaeIII restriction enzyme revealed an A781G transition mutation at the exon 2 and the existence of two genotypes, viz., AA (366 bp and 56 bp) and AB (422 bp, 366 bp and 56 bp). The occurrence of the B allele is due to the A781G transition in the exon 2 of the growth hormone gene, which causes an amino acid change from Serine to Glycine. The genotype BB was absent in the Kilakarsal sheep population. The absence of the BB genotype at the A781G locus of the growth hormone gene in the Kilakarsal sheep population indicates that the natural selection process might be acting against this particular genotype through reduced viability or early embryonic death. Further, the statistical analysis revealed the significantly higher yearling weight of the AB than the AA genotype (P=0.038), and an appreciable difference of 1.40 kg in 9-month weight. The highly significant (P&lt;0.001) Chi-square value (29.94) showed that the population is not the under Hardy-Weinberg equilibrium. This indicates that molecular markers associated with body weight should be explored for their use in marker assisted selection in Kilakarsal population.

Three Previously Unseen Genotypes Detected in IGFBP - 3 Gene in Buffalo Breed

Anatolian water buffalo breed is Turkey’s sole water buffalo breed, and their numbers steadily increased with the national “Water Buffalo Breeding by Breeders Project”. This study aimed to investigate of the gene region polymorphisms (Intron-2, Exon-2/Intron-3, Exon-3) of the meat-yield-related Insulin-like Growth Factor Binding Protein-3 (IGFBP-3) gene in Anatolian water buffaloes by TaqI, HaeIII, and MspI restriction endonucleases. The phenol/chloroform method was used for DNA isolation from 151 blood samples, and extracted DNAs were amplified by touchdown PCR using specific primers. Amplified PCR products were digested with restriction endonucleases (REs) and separated in 3% agarose gel electrophoresis (AGE), then genotypes were determined. Results revealed two genotypes [AA (98.68%) and AC (1.32%)] and two alleles [A (0.99) and C (0.01)] for the Exon-2 to Intron-3 region from HaeIII digestion. TaqI digestion of the Intron-2 region revealed three genotypes [AA (7.94%), AB (3.97%), and BB (88.10%)] and two alleles [A (0.10) and B (0.90)]. MspI digestion of the Exon-3 region revealed only the AA genotype and A allele, thus revealing monomorphism. Overall, HaeIII digestion revealed insignificant polymorphism (P&amp;gt;0.05), and TaqI digestion revealed significant polymorphism (P&amp;lt;0.001) for their respective regions. Gene polymorphisms of these regions were investigated for the first time in Anatolian water buffaloes. Additionally, three novel genotypes for the IGFBP-3 gene (one from HaeIII and two from TaqI) were determined for the first time. The novel B allele from TaqI digestion was observed to have a substantial frequency.

Mapping gut parasitism patterns in a cohort of Egyptians

In developing countries, the prevalence of intestinal parasitic infection is still significant, particularly due to geographical and socioeconomic variables. The objective of this study was to map the distribution pattern of intestinal parasitic infection in a cohort of the Egyptian population, as well as to assess associated risk factors. A cross-sectional hospital-based study was conducted on 386 patients. A single fecal specimen was collected from the study individual and examined microscopically for the detection of parasitic infection. DNA was extracted from all samples and utilized to amplify Entamoeba histolytica complex species, Cryptosporidium species, Giardia intestinalis assemblages, and Blastocystis species using PCRs. Typing of Cryptosporidium species and Giardia intestinalis assemblages was performed using restriction enzymes RasI and HaeIII respectively. While Blastocystis spp. subtypes (ST) were identified through sequencing of PCR products and phylogenetic analysis. 59.6% (230/386) of the study patients were infected with one or more intestinal parasites, 87.4%; 201/230 of patients had mono-parasitic infections, and 12.6%; 29/230 had multiple-parasitic infections (P < 0.0001). The predominant protozoa were Blastocystis, followed by Entamoeba histolytica complex, and Giardia intestinalis both as mono-parasites and as part of multiple parasites. Molecular assays showed that Blastocystis ST3, Entamoeba dispar, Giardia intestinalis assemblage B, and Cryptosporidium hominis were the most prevalent species. Intestinal parasitic infection was significantly associated with age, gender, residence, and water source. Multi-parasitism showed that residency in a rural area was a risk factor (OR 4.49; 95% CI 1.51–13.37; P = 0.007). Egyptians residing in rural areas have a high prevalence of intestinal multi-parasitism. Therefore, to lessen the prevalence and effects of these infections in this group, effective and sustainable control methods, providing health education focusing on good personal hygiene habits, and providing a safe drinking water supply should be implemented.

Development of a New Rapid Identification Method for &lt;i&gt;Bifidobacterium globosum&lt;/i&gt; and &lt;i&gt;B. pseudolongum&lt;/i&gt;Using Published Genome Data

Bifidobacterium globosum is expected to be applied to foods as a probiotic, and a method to rapidly distinguish it from the related Bifidobacterium pseudolongum is necessary for product development and quality control. In this study, we evaluated existing methods of bacterial species classification for B. globosum/pseudolongum with the published whole-genomes. The results showed that the Polymerase Chain Reaction (PCR) - Restriction Fragment Length Polymorphism (RFLP) method by the restriction enzyme HaeIII for hsp60 genes and the sequence analysis of hsp60 and rpoB gene sequences could not accurately distinguish B. globosum/pseudolongum, and both species could be accurately distinguished by the sequence analysis of clpC gene sequences. The clpC gene sequence similarity was at least 97.9% in the same species and no more than 95.4% in different species. Therefore, we developed a novel method of RFLP by restriction enzyme BspT104I on PCR products of clpC genes to identify B. globosum/pseudolongum. This method was considered to be faster and more accurate than previously proposed methods for identification of B. globosum/pseudolongum. Thus, the method presented in this study enables efficient species classification and it is very useful in utilization of B. globosum/pseudolongum for foods.

The field inoculation of Acacia saligna with efficient rhizobia strains for sand quarry restoration in Algeria

Sand quarries constituting very specific environments where native plants and surface soil are destroyed. The resulting substrates are practically sterile from an organic and biological point of view due to the absence of humus, microfauna and flora. planting legumes like Acacia saligna inoculated with his microbial partner rhizobia is strategic key to enhance the substrate fertility and lead to an ecological restoration of sandpits. Here we have selected the most efficient couple A. saligna-rhizobia with the goal to rehabilitate Sidi Lakhdar sandpit in the end of exploitation and to follow the filed persistence of introduced rhizobia into A. saligna nodules. The in vitro efficiency test permitted the selection of three inocula which performed differently once transferred onto natural soil and in the field. The ASB13 (Ensifer sp.) were found to be better inoculum for sandpit rehabilitation and A. saligna-rhizobia couple introduction for above and underground degraded sites. The introduced rhizobia didn’t persist in nodules of A. saligna in different treated blocks which were revealed by the PCR-RFLP profiles after the digestion by restriction enzyme HaeIII. These results support the literature data on failure/success of inoculants pertain to the persistence of rhizobia (inoculants) in soil, inoculant success is affected not only by environmental conditions such as temperature, pH, salinity but also by competitivity of indigenous rhizobia. We conclude that the rhizobia-based inocula selection for field application necessitates testing selected strains under the environmental conditions prevailing in the site intended to be rehabilitated.

First Molecular Identification of Bulinus africanus in Lake Malawi Implicated in Transmitting Schistosoma Parasites.

The freshwater snail genus Bulinus plays a vital role in transmitting parasites of the Schistosoma haematobium group. A hybrid schistosome between S. haematobium and S. mattheei has been recently detected using DNA-based identification methods in school children along the Lake Malawi shoreline in Mangochi District. This finding raised the need for contemporary revaluation of local interactions between schistosomes and snails, with a particular focus on snail species within the Bulinus africanus group. In 2017 and 2018, malacological surveys sampled several freshwater sites in Mangochi District. Collected snails (n = 250) were characterised using cytochrome oxidase subunit 1 gene (cox1), with DNA barcoding of the ‘Folmer’ region and a rapid PCR-RFLP typing assay with double digestion with HaeIII and SacI restriction enzymes. DNA cox1 sequence analysis, with phylogenetic tree construction, suggested the presence of at least three B. africanus group taxa in Lake Malawi, B. globosus, alongside first reports of B. africanus and B. angolensis, which can be differentiated by PCR-RFLP methods. In addition, a total of 30 of the 106 B. africanus group snails (28.30%) were positive to the Schistosoma-specific screen using real-time PCR methods. This study provides new insight into the recent changes in the epidemiology of urogenital schistosomiasis as likely driven by a new diversity of B. africanus group snails within the Lake.

Molecular characterization and association of lactoferrin gene to subclinical mastitis in goats (Capra hircus)

The study characterized the lactoferrin (Lf) mRNA gene in different goat breeds in the Philippines and determined its association with subclinical mastitis (SCM). The study involved collection of milk at second week of lactation (n=75) and blood samples (n=5) to obtain extracted RNA and using cDNA to amplify Lf gene through polymerase chain reaction. The nucleotide and amino acid sequences were determined and used as reference in the evaluation of phylogenetic relationship. Amplified products were utilized for RFLP analysis before determining the association of the gene with SCM. Results of the study demonstrated that Lf gene in goats registered a molecular weight of 2135. Nucleotide and amino acid sequence of Lf gene revealed high similarity (99%) in Saanen, Anglo-Nubian and Philippine native goats with that of Capra hircus (U53857) Lf gene submitted to GenBank. Phylogenetic studies showed that Lf gene of Anglo-Nubian, Saanen and Native goats clade together with Lf gene of C. hircus (U53857). Three genotypes in goats were documented using the restriction enzymes AluI and HaeIII. Based on the Statistical analysis, association (comp 5.65, p = 0.0308) has been established between the Lf genes of goats with genotype BB to SCM using HaeIII restriction enzyme.

The role of p.Val444Ala variant in the ABCB11 gene and susceptibility to biliary atresia in Vietnamese patients.

Biliary atresia (BA) is the most serious type of obstructive cholangiopathy that occurs in infants. BA can be the cause of death in children under 2 years if untreated early. However, the etiology of the disease is not known. BA is considered to be the result of the destruction of the bile duct system including the accumulation of bile acids. The bile salt export pump, a transporter protein encoded by the ABCB11 gene, plays the main role in the exportation and accumulation of bile acids. The p.Val444Ala variant in this gene is known to be associated with many cholestatic diseases. However, to date no study have been performed to evaluate the association of this variant with susceptibility to the risk of BA. In this study, we aimed to identify the frequency of p.Val444Ala variant and the risk of BA in Vietnamese patients.The polymerase chain reaction (PCR)– restriction fragment length polymorphism method was used to determine the frequency of alleles c.1331T>C (p.Val444Ala, rs2287622) in the ABCB11 gene in 266 Vietnamese patients with BA and 150 healthy people. The gene segment containing the variant was amplified by PCR with specific primers, after that the PCR products were cut by HaeIII restriction enzyme and analyzed on agarose gel to determine the genotypes. The frequency of alleles was assessed statistically to determine the association between these alleles and the risk of disease in patients.In our study, the frequency of alleles c.1331T>C (p.Val444Ala, rs2287622) in the ABCB11 gene was investigated the first time in the patients with BA. The results showed that CC and TC genotypes were significantly different between BA patients and healthy people (P < .01), and the C allele was associated with an increased risk of BA (odds ratio = 2.47; 95% confidence interval: 1.84–3.32; P < .01). The initial results of clinical, biochemical, and genetic analysis in our study suggested that the p.Val444Ala variant in the ABCB11 gene may be a susceptibility factor for the disease in Vietnamese patients with BA. These results provided new insights into the role of this ABCB11 variant in the pathogenesis of BA.

Characterization of BoLA class II DQA and DQB by PCR-RFLP, cloning, and sequencing reveals sequence diversity in crossbred cattle

The BoLA class II DQA and DQB genes in crossbred cattle were studied using PCR-RFLP, cloning, and sequencing techniques. Seventy-two crossbred cattle (Vrindavani) were used in the current study. HaeIII and XbaI restriction enzymes digested DQA exon 2-3, revealing seven (HaeIII-A-G) and three (XbaI A-C) motifs, respectively. The BoLA-DQB gene was analyzed using PCR-RFLP with PstI and TaqI restriction enzymes, yielding five restriction motifs for each restriction enzyme (PstI-A-E and TaqI-A-E). In crossbred cattle, addition, deletion, and substitutions were observed in distinct sequences, resulting in variations in overall gene length. Changes in nucleotides at positions 64–80, 110–200, and 207–264 were largely responsible for polymorphism in DQA exon 2. The phylogenetic analysis predicted a high degree of nucleotide and amino acid changes in DQA exon 2-3 and DQB exon 2. DQA genes had a nucleotide dissimilarity of 0.3–25.4 percent, while DQB genes had a nucleotide dissimilarity of 1.5–14.3 percent. We cloned and sequenced 20 genotypes based on PCR-RFLP of the DQA and DQB genes. The current study observed variation in the DQA and DQB genes and will serve as a foundation for future research on the BoLA DQA and DQB genes.

Variation of prolactin and β-Lactoglobulin genes in the Indonesian FH Cattle

Prolactin is a polypeptide hormone, encoded by the prolactin (PRL) gene, synthesized and secreted by anterior pituitary, and affecting milk yield and composition. β-Lactoglobulin (BLG) is the major whey proteinin the milk of ruminants. This study was conducted to identify the PRL and LGB genes polymorphism in the Indonesian FH cattle. A total of 139 individual cattle blood samples from West Java were used to obtain DNA samples through the DNA extraction process. Identification of the PRL and LGB genes was performed using PCR-RFLP method with RsaI (PRL gene) and HaeIII (BLG gene) restriction enzymes. The PRL gene was amplified using forward primer 5’-ccaaatccactgaattatgctt-3’ and reverse primer 5’-acagaaatcacctctctcattca-3’. The BLG gene was amplified using forward primer 5’-tgtgctggacaccgactacaaaaag-3’ and reverse primer 5’-gctcccggtatatgaccaccctct-3’. The PRL and BLG genes in the Indonesia FH cattle were polymorphic based on the PCR-RFLP analysis but the heterozygosity value was low. There were two alleles (G and A) and three genotypes (GG, GA, and AA) identified in the PRL gene of the Indonesian FH cattle with genotype frequencies were 0.914, 0.079, and 0,007 for GG, GA, and AA genotypes respectively. There were two genotypes (CC and CG) identified in the BLG gene with genotype frequencies were 0.91 (CC), and 0.09 (CG). Information about the PRL and BLG genes polymorphism in this study can be considered for further study to analyse its association with milk yield trait.

Molecular Identification of Leishmania Species in an Outbreak of Re-Emerged Cutaneous Leishmaniasis in Southwestern Iran During 2015 - 2016

Background: Dezful and its suburbs, as the second city in Khuzestan Province, southwest of Iran, has been an endemic area of cutaneous leishmaniasis (CL) with a low incidence rate since the last decades. However, the disease incidence has rapidly increased, and now is considered as a re-emerging parasitic disease in the area. Objectives: The aim of this study was to identify the most prevalent CL species in Dezful Region. Methods: A total of 196 microscopically confirmed slides from CL patients referred to Dezful Health Center were randomly collected in the period of 2015 - 2016. After DNA extraction from microscopically positive slides, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was carried out on 61 eligible specimens using ribosomal internal transcribed spacer 1 (ITS1) gene. The HaeIII restriction enzyme was used for the identification of species. Results: Samples were randomly collected from 196 acute CL cases, including 110 (56.2%) males and 86 (43.8%) females. Most infections were seen in the age range of 2 - 7 years (65/196, 33.1%). Totally, 60.1% of the cases had only one lesion, and half of the lesions appeared on hands. Furthermore, 162 (82.7%) cases were referred to Dezful Health Center in the cold seasons (autumn and winter). Results of PCR-RFLP on 61 eligible isolates showed that 60 (98.4%) isolates were Leishmania major, and only 1 (1.6%) isolate was Leishmania tropica. Conclusions: Our findings indicated that L. major is the main agent of re-emerged CL in Dezful and its suburbs, and the disease is a zoonosis.