Wasim Shehzad

Direct sequencing of FAH gene in Pakistani tyrosinemia type 1 families reveals a novel mutation.

Abstract Background: Hereditary tyrosinemia type 1 (HT1) is a rare inborn error of tyrosine catabolism with a worldwide prevalence of one out of 100,000 live births. HT1 is clinically characterized by hepatic and renal dysfunction resulting from the deficiency of fumarylacetoacetate hydrolase (FAH) enzyme, caused by recessive mutations in the FAH gene. We present here the first report on identification of FAH mutations in HT1 patients from Pakistan with a novel one. Methods: Three Pakistani families, each having one child affected with HT1, were enrolled over a period of 1.5 years. Two of the affected children had died as they were presented late with acute form. All regions of the FAH gene spanning exons and splicing sites were amplified by polymerase chain reaction (PCR) and mutation analysis was carried out by direct sequencing. Results of sequencing were confirmed by restriction fragment length polymorphism (PCR-RFLP) analysis. Results: Three different FAH mutations, one in each family, were found to co-segregate with the disease phenotype. Two of these FAH mutations have been known (c.192G>T and c.1062+5G>A [IVS12+5G>A]), while c.67T>C (p.Ser23Pro) was a novel mutation. The novel variant was not detected in any of 120 chromosomes from normal ethnically matched individuals. Conclusions: Most of the HT1 patients die before they present to hospitals in Pakistan, as is indicated by enrollment of only three families in 1.5 years. Most of those with late clinical presentation do not survive due to delayed diagnosis followed by untimely treatment. This tragic condition advocates the establishment of expanded newborn screening program for HT1 within Pakistan.

Economic Significance of Tropical Theileriosis on a Holstein Friesian Dairy Farm in Pakistan

Abstract The dairy industry in Pakistan is booming, and investors are anxious to fund dairy farms that are using high-milk-producing (exotic) cattle breeds such as Holstein Friesians that are not native to the country. Unfortunately, the benefits of increased milk production do not provide resistance to pathogens present in regions where the exotic breeds are introduced. Therefore, the current study was conducted to evaluate the economic impact of Theileria annulata on a commercial Holstein Friesian dairy farm in the District of Ranjanpur, in the Province of Punjab, Pakistan. The economic impact of T. annulata infection was calculated for cattle with subclinical and clinical theileriosis. Losses were estimated based on milk production, morbidity, mortality, and tick control costs (organophosphate sprays). Animals were classified into groups after screening for mastitis, teat abnormality, abnormal parturition, intestinal parasites, and hemoparasites (T. annulata, Babesia spp., and Anaplasma spp.). Microscopy was done for hemoparasites and intestinal parasites. PCR was used to confirm microscopic identification of T. annulata. Animals were classified into 3 groups: group A (normal), group B (subclinical theileriosis), and group C (acute theileriosis). Hemoparasites were observed microscopically in 28.7% of cows. Theileria annulata was found in 8%, and the herd incidence (new cases) of T. annulata was 2.8%. Milk production, animal rectal temperature, and body condition scores between group A and groups B and C were significantly different (P < 0.05). But the enlargement of sub-scapular lymph node and interval of body condition score of the 3 groups were not significant (P > 0.05). The total expenditure incurred due to theileriosis was US

Genetic analysis of fructose-1,6-bisphosphatase (FBPase) deficiency in nine consanguineous Pakistani families.

74.98 per animal and 13.83% of total farm costs. Hence theileriosis caused significant economic loss of US

Development and validation of a loop-mediated isothermal amplification assay for the detection of Mycoplasma bovis in mastitic milk

18,743.76 (0.02 million) on this Holstein Friesian dairy.

Molecular characterization of Hepatozoon canis from farm dogs in Pakistan

Abstract Background: Fructose-1,6-bisphosphatase (FBPase) deficiency is a rare inherited metabolic disorder characterized by recurrent episodes of hypoglycemia, ketosis and lactic acidosis. FBPase is encoded by FBP1 gene and catalyzes the hydrolysis of fructose-1,6-bisphosphate to fructose-6-phosphate in the last step of gluconeogenesis. We report here FBP1 mutations in nine consanguineous Pakistani families affected with FBPase deficiency. Methods: Nine families having one or two individuals affected with FBPase deficiency were enrolled over a period of 3 years. All FBP1 exonic regions including splicing sites were PCR-amplified and sequenced bidirectionally. Familial cosegregation of mutations with disease was confirmed by direct sequencing and PCR-RFLP analysis. Results: Three different FBP1 mutations were identified. Each of two previously reported mutations (c.472C>T (p.Arg158Trp) and c.841G>A (p.Glu281Lys)) was carried by four different families. The ninth family carried a novel 4-bp deletion (c.609_612delAAAA), which is predicted to result in frameshift (p.Lys204Argfs*72) and loss of FBPase function. The novel variant was not detected in any of 120 chromosomes from normal ethnically matched individuals. Conclusions: FBPase deficiency is often fatal in the infancy and early childhood. Early diagnosis and prompt treatment is therefore crucial to preventing early mortality. We recommend the use of c.472C>T and c.841G>A mutations as first choice genetic markers for molecular diagnosis of FBPase deficiency in Pakistan.

Induction of Th1 type-oriented humoral response through intranasal immunization of mice with SAG1-Toxoplasma gondii polymeric nanospheres

Mycoplasma mastitis is often difficult to control due to a lack of rapid and accurate diagnostic tools. The aim of the current study was to develop a loop-mediated isothermal amplification (LAMP) assay for the detection of Mycoplasma bovis (M. bovis) in mastitic milk. The assay was developed using primers designed for three different target genes: uvrC, 16S rRNA, and gyrB, and validated using mastitic milk samples previously found positive for the target pathogen. Specificity of the developed assay was determined by testing cross-reactivity of LAMP primers against closely related bovine mastitis bacterial pathogens. The sensitivity was found to be higher compared to conventional polymerase chain reaction (PCR). The LAMP assay was also capable of detecting M. bovis in PCR-negative milk samples of cows with clinical mastitis. The uvrC primers were found to be more sensitive, while gyrB primers were more specific; however, 16S rRNA primers were less specific and sensitive compared to either uvrC or gyrB primers. Cohen’s kappa values for uvrC, gyrB, and 16S rRNA primers used in the LAMP assays were 0.940, 0.970, and 0.807, respectively. There was a high level of agreement between the test results and the true-disease status as indicated by the receiver operating characteristic (ROC) curve. Our findings suggest that the newly developed LAMP assays targeting the uvrC and gyrB genes could be a useful tool for rapid and accurate diagnosis of mastitis caused by M. bovis.

Occurrence of Campylobacter in retail meat in Lahore, Pakistan

Hepatozoon canis is a tick-borne pathogen of canids, which is distributed worldwide. However, very little is known about this protozoan parasite in Pakistan. This study provides the first molecular evidence of H. canis from farm dogs from three agro-ecological zones of Punjab, Pakistan. A conventional PCR targeting the 18S rRNA gene was used to characterize H. canis from farm dogs from three districts, namely Kasur, Rawalpindi, and Muzaffargarh, in Punjab. Of 341 blood samples tested, 155 (45.5%) were positive for H. canis, 73 (61.3%) from Kasur, 46 (42.5%) from Rawalpindi, and 36 (31.5%) from Muzaffargarh. Phylogenetic analyses revealed that 18S rRNA sequences of H. canis from this study clustered in three clades with those of H. canis from previously published studies to the exclusion of all other Hepatozoon spp. included in the analysis. This study provides the first insight into H. canis from farm dogs in Pakistan. Furthermore, it lays a foundation for future studies of the parasite to assess the impact of canine hepatozoonosis in dogs from various agro-ecological zones in Pakistan where pet ownership of dogs is increasing.

First report of the molecular detection of Ancylostoma caninum in Lahore, Pakistan: the threat from pets

Abstract About one-third of the world population is prone to have infection with T. gondii, which can cause toxoplasmosis in the developing fetus and in people whose immune system is compromised through disease or chemotherapy. Surface antigen-1 (SAG1) is the candidate of vaccine against toxoplasmosis. Recent advances in biotechnology and nano-pharmaceuticals have made possible to formulate nanospheres of recombinant protein, which are suitable for sub-unit vaccine delivery. In current study, the local strain was obtained from cat feces as toxoplasma oocysts. Amplified 957 bp of SAG1 was cloned into pGEM-T and further sub-cloned into pET28-SAG1. BL21 bacteria were induced at different concentrations of isopropyl β-d-1-thiogalactopyranoside for the expression of rSAG1 protein. An immunoblot was developed for the confirmation of recombinant protein expression at 35 kDa that was actually recognized by anti-HIS antibodies and sera were collected from infected mice. PLGA encapsulated nanospheres of recombinant SAG1 were characterized through scanning electron microscopy. Experimental mice were intraperitoneally immunized with rSAG1 protein and intra-nasally immunized with nanosphere. The immune response was evaluated by indirect ELISA. In results intra-nasally administered rSAG1 in nanospheres appeared to elicit elevated responses of specific IgA and IgG2a than in control. Nanospheres of rSAG1 are found to be a bio-compatible candidate for the development of vaccine against T. gondii.

FIRST REPORT OF TRYPANOSOMA EVANSI INFECTION (SURRA) IN A PUMA (FELIS CONCOLOR) OF LAHORE ZOO, PAKISTAN

Campylobacter, one of the emerging zoonotic pathogens, is worldwide in distribution. This thermo-tolerant pathogen is one of the leading causes of diarrhea and gastroenteritis in humans. The main sources of infection are contaminated meat and meat products. A cross-sectional study was conducted to estimate the prevalence of Campylobacter species in retail meat in the Lahore district of Pakistan from September 2014 to January 2015. A total of 600 samples (200 samples each of beef, mutton, and chicken) were collected from retail shops through convenience sampling and preceded for Campylobacter contamination using the ISO 10272-1:2006 (E) method. Campylobacter was present in all three types of meat; the highest prevalence being recorded in chicken meat (29%) followed by mutton (18%) and beef (15.5%). Campylobacters were isolated from 125 (20.8%) samples out of the 600 meat samples. Campylobacter jejuni was more common (74.4%) than C. coli (25.6%). The highest number of Campylobacters were isolated in September (25/125) and November (23/125) while low numbers were isolated in October and December with isolates rate of (17/125) and (19/125), respectively. The highest prevalence was seen in the oldest and overpopulated town of Data Gunj Bakhsh 16% (20/125) while lowest prevalence was seen in a newer and least populated town of Gulburg (7/125). These results indicate that Campylobacter species are circulating in various meat sources in Lahore and that it may pose a threat to public health.

Induction of specific humoral immune response in mice immunized with ROP18 nanospheres from Toxoplasma gondii

The molecular prevalence of Ancylostoma caninum was determined in stray and pet dogs in Lahore, Pakistan from July 2014 to August 2015. A total of 500 dog faecal samples were first evaluated using a sedimentation technique and further through a PCR assay targeting the ITS-2 region of the A. caninum genome. Overall, 130 (26%) samples were positive for hookworm infestation by microscopic examination. Only microscopically positive samples were processed for PCR and 89 (17.6%) were positive for A. caninum. Sequence analysis of amplicons showed 100% homology with A. caninum and the genotypes clustered in one clade with Brazilian A. caninum hookworms. There was a significantly (P < 0.05) increased prevalence in male dogs younger than six months old. Labrador retrievers had higher A. caninum incidence compared to German shepherds and other breeds. To the best of our knowledge, this is the first report of the molecular prevalence of A. caninum in dogs in Pakistan. The novel results of the present study allow us to conclude that A. caninum is more prevalent in pet dogs, especially puppies, and this can be a potential threat for humans that come into contact with such animals. Therefore, the routine monitoring of pets, especially the more susceptible breeds, is essential for disease control.