Successful application of modified crude DNA extraction from muscle tissues for various types of PCR amplifications.

BACKGROUND: One of the most challenging aspects of nucleic acid amplification tests is the extraction of genomic DNA. However, achieving satisfactory quality and quantity of genomic DNA is not always easy, while the demand for rapid, low-cost and less laborious DNA isolation methods is ever-increasing. METHODS AND RESULTS: We have developed a rapid (⁓2 min) crude DNA extraction method leading to direct-PCR that requires minimum reagents and laboratory equipment. It was developed by eliminating the time-consuming purification steps of DNA extraction, by processing the sample in optimized amounts of Taq KCl PCR buffer and DNARelease Additive/Proteinase K in only two minutes and carrying out amplification using conventional Taq DNA polymerase. The DNA preparation method was validated on muscle tissue samples from 12 different species as well as 48 cooked meat samples. Its compatibility was also successfully tested with different types of PCR amplification platforms extensively used for genetic analysis, such as simplex PCR, PCR-RFLP (Restriction Fragment Length Polymorphism), multiplex PCR, isothermal amplification, real-time PCR and DNA sequencing. CONCLUSIONS: The developed protocol provides sufficient amount of crude DNA from muscle tissues of different species for PCR amplifications to identify species-of-origin via different techniques coupled with PCR. The simplicity and robustness of this protocol make nucleic acid amplification assays more accessible and affordable to researchers and authorities for both laboratory and point-of-care tests.

Development and validation of a universal primer pair for the taxonomic and phylogenetic studies of vertebrates.

BACKGROUND: Recent studies in the field of molecular identification have described 16S rRNA gene as a highly informative fragment of mitochondrial DNA for species discrimination. This study presents a newly developed universal primer pair yielding an approximately 350 bp fragment of mitochondrial 16S rRNA, variable enough to encompass and identify all vertebrate classes. METHODS AND RESULTS: The primers were designed by aligning and analyzing over 1500 16S rRNA sequences downloaded from the NCBI nucleotide database. A total of 93 vertebrate species, spanning 27 orders and 55 families, were PCR-amplified to validate the primers. All the target species were successfully amplified and identified when aligned with reference sequences from the NCBI nucleotide database. Using the Kimura 2-parameter model, low intra-species genetic divergence of the target region was observed - from 0 to 4.63%, whereas relatively higher inter-species genetic divergence was observed, ranging from 4.88% to 69.81%. Moreover, the newly developed primers were successfully applied to a direct PCR protocol, making the workflow very cost-effective, time-saving and less laborious in comparison to conventional PCR. CONCLUSIONS: The short length, high variability and conserved priming sites of the target fragment across all vertebrate species make it a highly desirable marker for species identification and discrimination.

A biochemical and histological evaluation of in vivo exposure of bisphenol P for multi-organ toxicity and pathology in rats.

Bisphenol P (BPP) is a structural analog of bisphenol A (BPA) and is increasingly used as a substitute of BPA in commercial and household applications. In recent years, BPP has been frequently detected in terrestrial and aquatic ecosystems. Very little epidemiological and experimental information are available on the toxicity potential of BPP in human and animal systems, which is very concerning in view of its increasing use. The current study evaluated the biochemical and histopathological effects of BPP in rats. The seven experimental groups (n = 5 rats/group) included BPA5 (5 mg), BPA50 (50 mg), BPA100 (100 mg), BPP5 (5 mg), BPP50 (50 mg), and BPP100 (100 mg) while the remaining one group served as untreated control. At the end of treatment, the organs (liver, kidney, heart, and lung) of rats were harvested for oxidative stress and histopathological analyses. A significant (p < .05) decrease was observed in the weight of the liver, lungs, and kidneys in the BPP100 group similar to the BPA100 group compared with the control group. Further, a significant (p < .05) decrease was also observed for concentrations of antioxidant enzymes (catalase, peroxidase, superoxide dismutase, and glutathione peroxidase) in the liver, lungs, kidneys, and heart at the highest two doses of BPP similar to the respective BPA groups compared with the control group. The two highest doses of BPP induced histopathological changes in the liver such as nuclei distortion, excessive necrosis of hepatocytes, nuclei shrinkage and pyknosis of cells with disrupted cell structure (BPP100), and cellular congestion and degeneration of hepatocytes (BPP50) similar to the two respective doses of BPA. The BPP treated groups also showed varying histopathological changes in kidney tissue, heart tissue, and lung tissue similar to BPA treated rats. In conclusion, the present study indicated that BPP has the potential to induce oxidative stress and alter the histomorphological architecture of different organs and is as deleterious as BPA.

Molecular detection of Coxiella burnetii in raw meat samples collected from different abattoirs in districts Kasur and Lahore of Punjab, Pakistan.

Coxiella burnetii is the zoonotic pathogen that causes Q fever; it is widespread globally. Livestock animals are its main reservoir, and infected animals shed C. burnetii in their birth products, feces, vaginal mucus, urine, tissues, and food obtained from them, i.e., milk and meat. There were previously very few reports on the prevalence of C. burnetii in raw meat. This study aimed to determine the prevalence of C.burnetii and its molecular characterization in raw ruminant meat from the Kasur and Lahore districts in Punjab, Pakistan, as this has not been reported so far. In this study, 200 meat samples, 50 from each species of cattle, buffalo, goat, and sheep, were collected from the slaughterhouses in each district, Kasur and Lahore in 2021 and 2022. PCR was used for the detection of the IS1111 element of C. burnetii. The data were recorded and univariate analysis was performed to determine the frequency of C. burnetii DNA in raw meat samples obtained from different ruminant species using the SAS 9.4 statistical package. Of the total of 200 raw meat samples, C. burnetii DNA was present in 40 (20%) of them, tested by PCR using the IS1111 sequence. The prevalence of C.burnetii differed among the studied species of ruminants. When species were compared pairwise, the prevalence in cattle was statistically significantly lower than in sheep (P = 0.005). The sequence alignment based on origin implied that the strains are genetically diverse in different districts of Punjab, Pakistan. The findings demonstrated that the prevalence of C. burnetii, especially in raw meat samples, deserves more attention from the health care system and professionals from Punjab, Pakistan, i.e., abattoir workers and veterinarians.

Description of novel variants in consanguineous Pakistani families affected with intellectual disability.

BACKGROUND: Intellectual disability (ID) is a neurodevelopmental condition, affecting 1-3% of the population. Genetic factors play a key role causing the limitation in intellectual functioning and adaptive behavior. The heterogeneity of ID makes it more difficult for genetic and clinical diagnosis. Mapping of variants through next generation DNA sequencing in consanguineous families would help to understand the molecular parthenogenesis of ID. OBJECTIVE: The aim of this study was to describe the genetic variants of ID in consanguineous Pakistani families. METHODS: We analyzed four unrelated consanguineous Pakistani families having an intellectual disability through whole exome sequencing (WES). Data was analyzed using different bioinformatics tools and software. RESULTS: We mapped four novel variants in different ID genes. Each variant is found in different family, co-segregating with a recessive pattern of inheritance. The variants found are; c.1437delG:p.Asn480Thrfs*10, mapped in FKRP, c.2041 C>A:p.Leu681Met in HIRA, c.382 C>T:p.Arg128Cys in BDH1 and c.267+1G>A:p.? identified in TRAPPC6B. CONCLUSIONS: These variants help in demonstration of status and molecular basis of intellectual disability in Pakistani population leading to provision of genetic counseling services and a contribution in disease variant database.

Effect of storage temperature and duration on direct PCR amplification of various feather types and DBS matrices.

The use of direct PCR has been pioneered over the last decade for DNA analysis of biological specimens of distinct origins. The information on how longer these specimens can be stored and amplified by direct PCR is however scanty. Such a piece of information could expedite research and diagnostic studies without compromising the reliability of results. The current study was therefore designed to analyze the effect of storage temperature and duration on direct PCR amplification of biological specimens having either low quantity or high quantity of DNA. Whole blood, dried blood spots (DBS), and feathers from chicken were stored for five years at three different temperatures, viz. room temperature (∼25 °C), 4 °C, and -20 °C. These samples were subjected to crude DNA extraction by diluting them in PBS buffer and heating at 98 °C after 1 day, 7 days, 15 days, 1 month, 3 months, 6 months, 1 year, 3 years and 5 years of storage. The crude DNA was PCR-amplified with the use of DNA sexing primers as well as DNA barcoding primers. Incubation at 98 °C for 10 min of any type of sample in PBS buffer was sufficient for crude DNA extraction. There was irrelevant impact of feather type, DBS matrix nature and storage temperature on amplification success over the period of analysis. It was possible to successfully accomplish the amplification of 96 samples with the use of routine PCR reagents within 3.5-6.0 hrs. In short, economical and fast genetic analysis of commonly used avian samples is feasible after their storage for longer time at room temperature.

Genomic and Computational Analysis of Novel SNPs in TNP1 Gene Promoter Region of Bos indicus Breeding Bulls.

Transition nuclear proteins (TNPs), the principal proteins identified in the condensing spermatids chromatin, have been found to play a key role in histone displacement and chromatin condensation during mammalian spermatogenesis. One such gene belonging to the TNP family called TNP1 gene is abundantly expressed in the regulation of spermatogenesis, and its sequence is remarkably well conserved among mammals. Genomic analysis, by sequencing and computational approach, was used to identify the novel polymorphisms and to evaluate the molecular regulation of TNP1 gene expression in Sahiwal cattle breeding bulls. DNA samples were sequenced to identify novel single nucleotide polymorphisms (SNPs) in the TNP1 gene. Modern computational tools were used to predict putative transcription factor binding in the TNP1 promoter and CpG islands in the TNP1 promoter region. In the TNP1 gene, four SNPs, three TATA boxes, and one CAAT box were identified. One CAAT box was discovered at 89 bp upstream of start site ATG. The computational analyses indicated that the polymorphisms inside the promoter sequence results in an added HNF-1 transcription factor binding site. In contrast, the other variations may remove the naturally occurring SRF transcription factor binding site. The CpG islands in the TNP1 promoter region were predicted to be absent by the MethPrimer program before and after SNP site mutations. These findings pave the way for more research into the TNP1 gene's promoter activity and the links between these SNPs and reproductive attributes in the Sahiwal breeding bulls.

Association study of CLDN14 variations in patients with kidney stones.

Claudin-14 protein plays an essential role in regulating calcium ions in the kidney and ear. Two phenotypes, hearing loss and kidney stones, were reportedly associated with variations in the CLDN14 gene. This study aimed to understand CLDN14 mutations' contribution to hearing loss and renal stone formation in a Pakistani cohort. We analyzed CLDN14 sequence variations in 100 patients, along with healthy individuals, to assess whether specific polymorphisms were associated with the disease. Also, we performed an in silico analysis using a mutation database and protein annotation. The rs219779's genotype CT (p = 0.0020) and rs219780's genotype AG (p = 0.0012) were significantly associated with kidney stones. We also found that a novel haplotype, "TA" associated with kidney stone formation, has moderate linkage disequilibrium. The TA haplotype was significantly correlated with a kidney stone risk formation of 3.76-fold (OR (CI 95%) = 3.76 (1.83-7.72)) and p = 0.0016 compared to other haplotypes. In silico analysis revealed that mutations associated with hearing loss were not correlated with renal stone formation but affected claudin-14 protein stability. We structurally mapped a novel TA haplotype of CLDN14 that, based on our analysis, likely contributes to the pathogenesis of renal stones.

Sequencing the CaSR locus in Pakistani stone formers reveals a novel loss-of-function variant atypically associated with nephrolithiasis.

BACKGROUND: Nephrolithiasis (NL) affects 1 in 11 individuals worldwide and causes significant morbidity and cost. Common variants in the calcium sensing receptor gene (CaSR) have been associated with NL. Rare inactivating CaSR variants classically cause hyperparathyroidism, hypercalcemia and hypocalciuria. However, NL and familial hypercalciuria have been paradoxically associated with select inactivating CaSR variants in three kindreds from Europe and Australia. METHODS: To discover novel NL-associated CaSR variants from a geographically distinct cohort, 57 Pakistani families presenting with pediatric onset NL were recruited. The CaSR locus was analyzed by directed or exome sequencing. RESULTS: We detected a heterozygous and likely pathogenic splice variant (GRCh37 Chr3:122000958A>G; GRCh38 Chr3:12228211A>G; NM_000388:c.1609-2A>G) in CaSR in one family with recurrent calcium oxalate stones. This variant would be predicted to cause exon skipping and premature termination (p.Val537Metfs*49). Moreover, a splice variant of unknown significance in an alternative CaSR transcript (GRCh37 Chr3:122000929G>C; GRCh38 Chr3:122282082G >C NM_000388:c.1609-31G >C NM_001178065:c.1609-1G >C) was identified in two additional families. CONCLUSIONS: Sequencing of the CaSR locus in Pakistani stone formers reveals a novel loss-of-function variant, expanding the connection between the CaSR locus and nephrolithiasis.

Exploring the Potential of Interferon Gamma Gene as Major Immune Responder for Bovine Tuberculosis in River Buffalo.

Bovine tuberculosis (bTB) is a widespread zoonotic infection targeting the livestock sector, especially in developing countries, and posing a risk to humans and animal populations. Its recent prevalence in river buffaloes has been estimated as higher as 33.7%. In emergent countries like Pakistan, there is likeliness of human-livestock interfaces extensively and lacking of effective preventive measures that illustrate the risk of spreading the infection at a remarkable rate. The river buffalo (Bubalus bubalis) is an upkeep host of Mycobacterium bovis and is responsible for disease transmission among buffaloes and other livestock species. In this study, potential molecular biomarkers in the Interferon-gamma gene (IFNg) were identified after genomic screening of river buffaloes. Unique genomic loci in river buffalo proved the novelty of the genomic structure of this phenomenal animal but also highlighted its significance in natural immunity against the Mycobacterium. A total of eight single nucleotide polymorphisms were identified in the coding region of IFNg. The SNPs in the exonic region were all transitions, i.e., the conversion of purines to purines. These SNPs were analyzed for Hardy Weinberg Equilibrium, chi2 test, gene diversity, and protein structural conformation. Pathway analysis in tuberculosis revealed that IFNg inhibits the antigen-presenting cells (APC) through JAK and STAT pathways. Network analysis of IFNg proteins in both species showed strong associations among the immunity-related proteins (interleukins, tissue necrosis factors) and receptors of interferons. The identified polymorphic sites might be novel-potentiated markers for the selection of animals with superior immune response against bTB and can be exploited as promising genomic sites for breeding the resistant animal herds to combat Mycobacterium infection in a long run.

Whole exome sequencing revealed novel variants in consanguineous Pakistani families with intellectual disability.

BACKGROUND: Intellectual disability (ID) is a heterogeneous disorder affecting 1-3% of the population. Elucidation of monogenic variants for ID is a current challenge. These variants can be better demonstrated in consanguineous affected families. OBJECTIVE: The study was designed to find the genetic variants of ID in consanguineous families. METHODS: We analyzed five unrelated consanguineous Pakistani families affected with ID using whole exome sequencing (WES). Data was analyzed using different bioinformatics tools and software. RESULTS: We mapped four variants including three novels in four different ID known genes. Each variant is found in a different family, co-segregating with a recessive pattern of inheritance. The novel variants found are; c. 2_4del (p.?) mapped in ROS1 and c. 718G>A (p.Gly240Arg) in GRM1. Another novel causative variant, c.2673del (p.Gly892Aspfs*17) identified in COL18A1 in a recessive form, a gene reported for Knobloch syndrome that manifests ID along with typical retinal abnormalities, and this phenotype was confirmed on reverse phenotyping. A mutation c.2134C>T (p.Arg712*) in TRAPPC9 has been found first time in the homozygous recessive form in our enrolled three affected siblings while it was previously reported in compound heterozygous form in a Caucasian descent. While fifth family remained unsolved. CONCLUSION: These mutations in four different genes with a recessive inheritance would be a contribution to the disease variant database of this devastating disorder.

Seroprevalence and Molecular Detection of Brucellosis in Hospitalized Patients in Lahore Hospitals, Pakistan.

Brucellosis is one of the most notorious zoonoses worldwide. The disease is common and endemic in humans and animals of Pakistan, but lack of awareness and lack of research have resulted in an increased incidence in the human population. The present study aimed to determine the seroprevalence and at molecular detection of brucellosis in patients with clinical symptoms in six different hospitals from Lahore, which is the capital city of Punjab province. A total of 218 blood samples were collected from hospitalized patients. The samples were initially screened by the Rose Bengal Plate Test (RBPT), and then quantitative real-time PCR (qRT-PCR) was applied. An overall seroprevalence of 17% (37/218) was found. The highest prevalence was found at the Lady Health center (36.53%), which was followed by the Lady Willingdon Hospital (28.6%). Female patients showed a higher seroprevalence than males and peaked at 34% (n = 32) for women who suffered from abortion. In total, 16.8% of patients younger than 30 years showed seropositive reactions, while the prevalence was 19% in patients between 31 and 50. Thirty-three DNA samples from 24 seropositive and nine seronegative patients tested positive, 32 samples were found positive for B. abortus DNA, and one sample failed to be identified at the species level. Almost all positive cases had direct contact with animals and consumed unpasteurized dairy products. Research on human brucellosis is still scarce in Pakistan. For the diagnosis of brucellosis, serology and molecular tools should be combined if isolation by culture is not possible. Nationwide control activities and increasing awareness for zoonotic brucellosis are needed.

Molecular characterization and phylogenetic analyses of Fasciola gigantica of buffaloes and goats in Punjab, Pakistan.

Fasciola gigantica is considered to be a major pathogen causing fasciolosis in the Indian subcontinent, resulting in production losses of millions of dollars in the livestock industry. Understading the dispersal origin and the patterns of spread of F. gigantica is important. A total of 53 Fasciola flukes collected from buffaloes and goats in Punjab, Pakistan between 2017 and 2018 were identified as F. gigantica based on the multiplex PCR for the phosphoenolpyruvate carboxykinase (pepck) and the PCR-restriction fragment length polymorphism (RFLP) for DNA polymerase delta (pold). A significant genetic difference between F. gigantica from buffaloes and goats was indicated by the genetic analyses of mitochondrial markers, NADH dehydrogenase subunit 1 (nad1) and cytochrome C oxidase subunit 1 (cox1). Phylogenetic analysis of the seventeen nad1 haplotypes of F. gigantica from Pakistan with those in neighbouring countries of the Indian subcontinent revealed that all the haplotypes identified in Pakistan were clustered in haplogroup A. fasciola gigantica with the eight haplotypes might be expanded in Pakistan from Indian origin, along with the migration of the domestic animals, since they were related to Indian haplotypes. In contrast, the remaining nine haplotypes were not shared with any neighbouring countries, suggesting independent origin, probably from neighbouring Middle East countries. However, cautious interpretation is required due to the very limited samples size of this study. Our study provides a proof of concept for a method that could be used to investigate the epidemiology of F. gigantica.

Population demographic history and population structure for Pakistani Nili-Ravi breeding bulls based on SNP genotyping to identify genomic regions associated with male effects for milk yield and body weight.

The domestic Nili-Ravi water buffalo (Bubalus bubalis) is the best dairy animal contributing 68% to total milk production in Pakistan. In this study, we identified genome-wide single nucleotide polymorphisms (SNPs) to estimate various population genetic parameters such as diversity, pairwise population differentiation, linkage disequilibrium (LD) distribution and for genome-wide association study for milk yield and body weight traits in the Nili-Ravi dairy bulls that they may pass on to their daughters who are retained for milking purposes. The genotyping by sequencing approach revealed 13,039 reference genome-anchored SNPs with minor allele frequency of 0.05 among 167 buffalos. Population structure analysis revealed that the bulls were grouped into two clusters (K = 2), which indicates the presence of two different lineages in the Pakistani Nili-Ravi water buffalo population, and we showed the extent of admixture of these two lineages in our bull collection. LD analysis revealed 4169 significant SNP associations, with an average LD decay of 90 kb for these buffalo genome. Genome-wide association study involved a multi-locus mixed linear model for milk yield and body weight to identify genome-wide male effects. Our study further illustrates the utility of the genotyping by sequencing approach for identifying genomic regions to uncover additional demographic complexity and to improve the complex dairy traits of the Pakistani Nili-Ravi water buffalo population that would provide the lot of economic benefits to dairy industry.

Single tube multiplex PCR assay for the identification of banned meat species.

Food adulteration has a direct impact on public health, religious faith, fair-trades, and wildlife. In the present study, a reliable and sensitive assay has been developed for verifying meat adulteration in food chain. The multiplex PCR system was optimised for identification of chicken, cow/buffalo, sheep/goat, horse/donkey, pork, and dog DNAs in a single reaction mixture simultaneously. The primers were designed using 12 S rRNA gene sequences with fragment size in the range of 113 bp to 800 bp, which can be easily visualised on agarose gel electrophoresis making the technique economical. After validation of accuracy, specificity, and sensitivity, commercially available meat products (n = 190) were screened, comprising both raw and cooked meat samples. The results demonstrated a high rate of adulteration (54.5%) in meat products. The technique developed here can be easily used for screening of different meat products for export and import purposes as well as for food inspection and livestock diagnostic laboratories.

Genetic diversity and multiplicity of infection in Fasciola gigantica isolates of Pakistani livestock.

Fasciola spp. are responsible for over 3 billion US dollars of production loss annually in livestock and cause widespread zoonotic disease. Nevertheless, understating of the emergence and spread of the trematode species is poor. The multiplicity of F. gigantica infection and its spread is potentially influenced by multiple factors, including the abundance of suitable intermediate hosts, climatic conditions favouring the completion of the parasite's lifecycle, and translocation of infected animals, or free-living parasite stages between regions. Here we describe the development of a 'tremabiome' metabarcoding sequencing method to explore the numbers of F. gigantica genotypes per infection and patterns of parasite spread, based on genetic characteristics of the mitochondrial NADH dehydrogenase 1 (mt-ND-1) locus. We collected F. gigantica from three abattoirs in the Punjab and Balochistan provinces of Pakistan, and our results show a high level of genetic diversity in 20 F. gigantica populations derived from small and large ruminants consigned to slaughter in both provinces. This implies that F. gigantica can reproduce in its definitive hosts through meiosis involving cross- and self-breeding, as described in the closely related species, Fasciola hepatica. The genetic diversity between the 20 populations derived from different locations also illustrates the impact of animal movements on gene flow. Our results demonstrate the predominance of single haplotypes, consistent with a single introduction of F. gigantica infection in 85% of the hosts from which the parasite populations were derived. This is consistent with clonal reproduction in the intermediate snail hosts.

Evaluation of transmission potential and pathobiological characteristics of mallard originated Avian orthoavulavirus 1 (sub-genotype VII.2) in commercial broilers.

Newcastle disease (ND), caused by Avian orthoavulavirus 1 (AOAV-1), affects multiple avian species around the globe. Frequent disease outbreaks are not uncommon even in vaccinates despite routine vaccination and, in this regards, viruses of diverse genotypes originating from natural reservoirs (migratory waterfowls) play an important role in a disease endemic setting. Though genomic characterization of waterfowl originated viruses has been well-elucidated previously, there is a paucity of data on clinico-pathological assessment of mallard-originated sub-genotype VII.2 in commercial chickens. Hence, the current study was designed to evaluate its transmission potential, tissue tropism and micro- and macroscopic lesions in commercial broilers. Based on complete genome and complete F gene, phylogenetic analysis clustered the study isolate within genotype VII and sub-genotype VII.2 in close association with those reported previously from multiple avian species worldwide. The study strain was found to be velogenic on the basis of typical residue pattern in the F-protein cleavage site (112R-RQ-K-R↓F117), sever disease induction in chicken, tissue tropism and subsequent clinico-pathological characteristics. Giving a clear evidence of horizontal transmission, a 100% mortality was observed by 4th and 6th day post infection (dpi) in chickens challenged with the virus and those kept with the challenged birds (contact birds), respectively. The observed clinical signs, particularly the greenish diarrhea, and macroscopic lesions such as pinpoint hemorrhages in proventriculus and caecal tonsils were typical of the infection caused by an AOAV-1 in chickens. The virus exhibited a broad tissue tropism where genomic RNA corresponding to study virus was detected in all of the tissues collected from recently mortile and necropsied birds. The study concludes that mallard-originated Avian orthoavulavirus 1 is highly velogenic to commercial chicken and therefore ascertain continuous disease monitoring and surveillance of migratory/aquatic fowls to better elucidate infection epidemiology and subsequent potential impacts on commercial poultry.

Consanguinity: A blessing or menace at population level?

Consanguinity has highly complex and multifaceted aspects with sociocultural as well as biological debates on its pros and cons. The biological upshot of consanguinity includes the increased homozygosity, which results in manifold increased risk of genetic disorders at family and population levels. On the other hand, in addition to social, cultural, political, and economic benefits, consanguineous marriages have biological advantages at the population level. The consequence of consanguineous marriages is an upsurge in the number of homozygous diseased individuals with fewer chances of mating and reduced chances of survival, therefore evolutionarily confining the transmission of disease alleles to future generations and encouraging its elimination from a population. Protective effects of consanguinity have also been observed in a few diseases in different populations. Although attractive for many reasons, nonconsanguineous marriages will cause risk alleles to spread throughout the population, making most individuals carriers, and ultimately will resume the production of recessive diseases in subsequent generations. Although consanguinity, from an evolutionary point of view, is beneficial at the population level, it increases the risk of diseases in the very next generation. Presently, there is no treatment for most of the genetic disorders; we cannot opt for consanguinity for long-term benefits. Nonconsanguineous marriages are a better strategy by which we may delay disease manifestation for some generations until science offers a viable solution.

Low genetic diversity of Ehrlichia canis associated with high co-infection rates in Rhipicephalus sanguineus (s.l.).

BACKGROUND: Rhipicephalus sanguineus sensu lato (s.l.) is the most widely distributed ixodid tick and is a vector of major canine and human pathogens. High-throughput technologies have revealed that individual ticks carry a high diversity of pathogens, including bacteria, protozoa and viruses. Currently, it is accepted that co-infections (multiple pathogen species within an individual) are very common in ticks and influence pathogen acquisition and transmission as well as host infection risk. However, little is known on the impact of the genetic diversity of pathogens on the incidence of co-infections. Herein, we studied the frequency of co-infections in R. sanguineus (s.l.) and their association with the genetic diversity of Ehrlichia canis. METHODS: Rhipicephalus sanguineus (s.l.) female ticks (n = 235) were collected from healthy farm dogs in three districts of Pakistan. Microfluidic real-time PCR, a powerful nanotechnology for high-throughput molecular detection of pathogens, was used to test the presence of 25 bacterial and seven parasitic species in individual ticks. The genetic diversity of E. canis was evaluated by characterizing the trp36 gene. RESULTS: A total of 204 ticks were infected with at least one pathogen and 109 co-infected with two (80%) or three (20%) pathogens. Rickettsia massiliae (human pathogen) and E. canis (zoonotic dog pathogen) were the most common pathogens co-infecting (30.4%) ticks. Furthermore, all identified co-infections included R. massiliae and/or E. canis. Multiple correspondence analysis (MCA) revealed that single infections did not show clear regional association whereas some co-infections were restricted to certain geographical regions. The sequence analysis of trp36 in representative samples allowed the identification of three E. canis strains with low genetic diversity, and the strain found in Muzaffargarh district appeared to be more adapted to co-infection with R. massiliae. CONCLUSIONS: Rhipicephalus sanguineus (s.l.) harbors multiple co-infections with human and dog pathogens of zoonotic potential. Findings of this study suggest that genetic diversity of E. canis may favor co-infections with different pathogens.

SE33 locus as a reliable genetic marker for forensic DNA analysis systems

Background/aim: Genetic variation, an authentic tool of individual discrimination, is being used for forensic investigations worldwide. A missing result for even one out of 13-17 markers leads to an inconclusive report. Additional reliable markers are required to compensate such deficiencies. The SE33 locus has high genetic variability in different populations and is being used in forensic investigation systems in some countries. The purpose of the study was to assess the viability of use of the SE33 locus as a supportive marker for forensic DNA profiling. Materials and methods: Amplification of the SE33 locus was performed using the PowerPlex ES Monoplex System SE33 (Promega). After genotyping 204 Pakistani individuals, different genetic and forensic parameters for the SE33 locus were studied. Results: Genotyping of the SE33 locus revealed a total of 43 alleles including 3 novel alleles. Significant values of different forensic and genetic parameters including power of discrimination, power of exclusion, and polymorphism information content were observed. Conclusions: Addition of the SE33 locus in forensic DNA profiling may help to produce conclusive reports where results are inconclusive due to degraded evidence samples. The SE33 locus can confidently be used for Pakistani and neighboring populations having common ancestors from Iran to Central Asia, the Middle East, India and Turkey.