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Use of Fungicide and Nematicide \ud Tolerant Strains of Biocontrol Agents for the Control of \ud Macrophomina Phaseolina and Root-Knot Nematode
No full textMOLECULAR PHYLOGENY AND DIVERSITY ANALYSIS OF BOVIDAE (BOSELAPHUS TRAGOCAMELUS, ANTILOPE CERVICAPRA) AND CERVIDAE (AXIS AXIS, AXIS PORCINUS) IN PAKISTAN
No full textN/
MOLECULAR AND SEROLOGICAL CHARACTERIZATION OF SOILBORNE FRANCISELLA TULARENSIS
No full textTularemia, caused by Francisella tularensis (F. tularensis), is a zoonotic disease \ud
transmitted through contact with infected animals and contaminated environment. The disease has \ud
been reported from many countries of the world but no study has been done in Pakistan. In the \ud
current project, a total of 2280 soil samples representing 456 villages of eight districts of Punjab \ud
province were collected from way-points having human-animal interaction, processed for genomic \ud
extraction and tested through real time PCR for presence or absence of F. tularensis. Association \ud
of risk factors was determined from data such as gender and age of animals, plough method, \ud
irrigation system, fertilizer type used, availability of veterinary services, level of farmer education, \ud
physical and chemical composition of the soil. Moreover serum samples (n=707) collected \ud
randomly from goat (n = 200), sheep (n = 175), cattle (n = 179), and buffalo (n = 153) were \ud
analyzed for antibodies against F. tularensis by using enzyme-linked immunosorbent assay. \ud
Seventy four soil samples (3.24 percent) were found positive for F. tularensis. Phylogenetic \ud
analysis showed 100 percent similarity index with F. tularensis sub specie holarctica reported \ud
from other regions like USA, Sweden, Spain, Turkey and Germany. Presence of F. tularensis in \ud
soil showed negative association with increase in number of human density (0.7159; 0.3834\ud
0.2054). Prevalence of anti- Ft ELISA antibodies were significantly higher (p<0.05) in large \ud
ruminants (cattle and buffalo) as compared to small ruminants (goat and sheep). Age and gender\ud
wise analyses showed non-significant differences (p>0.05) between small and large ruminants. \ud
Whereas, rain-irrigation system (2.96: 1.35- 6.48), lack of veterinary services (4.77:1.26-18.03) \ud
and use of organic fertilizer (5.3: 11.38- 20.39) have positive association with prevalence of anti- \ud
Ft ELISA antibodies in the serum. Sero-prevalence of F. tularensis in the animals has significant \ud
association with quantity of clay in soil (p<0.05). A conventional PCR based test has also been \ud
optimized for detection of F. tularensis using tul4 gene specific primers. Specificity of primer \ud
showed Ft detection in soil DNA in the presence of other cross-reactive organism. Sensitivity was \ud
determined in two fold dilutions with detection limit of up to 320 pg/µL. Utilizing pET28a vector, \ud
a construct was prepared containing transformed tul4 gene (450bp) showing 100 percent sequence \ud
homology to query gene sequence. For manufacturing diagnostic assays especially in developing \ud
countries where availability of BSL-3 facilities and positive control reagents is an issue, provision \ud
of tul4 gene based constructs in vector can act as positive control and safe to use
Studies on Urinary Electrolytes, Renal and Liver Functions and Serum Lipid Profile in Non Obese Patients of Essential Hypertension in Relation to Age and Sex in Southern Punjab, Pakistan.
No full textEssential hypertension has the characteristic of persistent rise in blood pressure. \ud
Hypertension is characterized by multi factorial disease that involves derangements in the \ud
functions of heart, blood vessels and the renal system. Short and long-term control of \ud
blood pressure is affected by alterations in heart function, the peripheral arteriolar \ud
resistance and other related factors. The relationship of arterial blood pressure and \ud
sodium consumption has long been under investigation. Hypertension is frequently \ud
encountered in societies with raised mean sodium chloride intake and is rare in \ud
populations with low sodium intake. Physical exercise can affect sodium balance due to \ud
increased loss in sweat. Sodium loss in sweat depends on total diet, salt intake, sweating \ud
volume, hydration condition and level of acclimatization to the hot ambiance. \ud
The present study was conducted to assess the role of urinary sodium, LDL/HDL, and \ud
high salt and fat intake in non-obese hypertensives in different age groups. \ud
The study was conducted on 500 cases of non-obese hypertensives with equal number of \ud
controls in Southern Punjab, Pakistan. The cases of secondary hypertension were \ud
excluded from the study. The urinary sodium, serum lipid profile (TC, LDL, HDL, TG, \ud
LDL/HDL) renal functions (urea, creatinine, uric acid) and liver functions (SGOT, \ud
SGPT, bilirubin) were measured by specific kits. \ud
There was a statistically significant difference between normotensives and essential \ud
hypertensives as regards urinary sodium, bilirubin and lipid profile. The study revealed \ud
that essential hypertensives had higher values of sodium, potassium, urea, creatinine and \ud
uric acid. Lipid profile TC, LDL-C, HDL-C, and TG were also found higher in essential \ud
hypertensives. Bilirubin was also higher in essential hypertensives
Establishment of Early and Sensitive Diagnosis and Molecular Characterization of Peste des Petits Ruminants (PPR) Virus
No full textPeste des petits ruminants (PPR) is a highly contagious transboundary animal disease of economic importance in the developing world and infects small ruminants like sheep and goat. It is caused by a single stranded RNA virus namely peste des petits ruminants virus (PPRV) belonging to the genus Morbillivirus of the family Paramyxoviridae. Since initial reports in1942, PPR has infected a large population of small ruminants across Asia and Africa causing huge economic losses, therefore, Office International des Epizooties (OIE) has declared it as list-A disease. In Pakistan, a loss of more than US$ 342.15 million occurs annually due to this disease. Lack of sensitive diagnosis and control of free animal movement are major reasons for the unnoticed spread of disease in the country which may lead to further evolution of the underlying virus. For sensitive diagnosis, molecular methods are preferred over conventional serological assays as the former have higher sensitivity and additional benefit of molecular characterization. In this context, this bi-partite study on PPRV was designed: the first part included establishment of sensitive and efficient molecular detection methods; the second parts focused on molecular characterization of PPRV in Pakistan and its comparison with other lineages of the virus. In the first part, two molecular methods of PPRV detection including reverse-transcription PCR (RTPCR), real-time RT-PCR (rRT-PCR) were established for sensitive laboratory diagnosis while RT-loop mediated isothermal amplification (RT-LAMP) assay for sensitive on-site diagnosis. \ud
During the study, a total of 448 clinical samples were collected from 25 places of 11 districts in Pakistan and subjected to molecular detection and analysis of the suspected underlying virus. A newly developed cell line designated as CHS-20 that express sheep signaling lymphocyte activation molecule (SLAM) were used for isolation of PPRV in clinical samples. For comparative analysis of PPRVs from Pakistan with those of lineages, 192 clinical samples from two Asian (Pakistan and Bangladesh) and seven African countries (Benin, Côte d´Ivoire, Burkina Faso,\ud
Nigeria, Cameroon, Kenya and South Sudan) were processed for phylogenetic investigation. \ud
Clinically, the mortality rate was more than 30% in eight out of nineteen (42.1%) outbreaks that were studied in Pakistan. High proportion of affected animals was 1-18 months old. Adults (>18 months) did not suffer from high mortality rates. In these outbreaks attended during the study, a total of 1364 animals were at risk, 77.27% contracted the infection of PPR and 33.58% died due to the clinical signs /symptoms of the disease. RT-PCR detected PPRV in 42.41% out of 448 samples. 53.68% of 231 swabs, 44.20% of 198 blood and 57.89% of 19 tissues were positive for PPR virus. For real-time RT-PCR, a maximum threshold cycle (Ct) value of 37.5 was set as cut-off value to declare a field sample positive for PPRV. Out of 157 samples selected for comparative detection, real-time RT-PCR (rRT-PCR) detected all samples which were positive in regular RT-PCR, however, detected 17 more samples as positive that were negative by regular RT-PCR. The positive percent agreement between the two tests was found 100% while negative percent agreement was 78.48% whereas the overall agreement of 89.17% was observed. For onsite diagnosis of PPRV in field conditions, RT-LAMP assay was found ideal for the detection of virus directly in clinical sample within one hour. Spatio-temporal curve generated during the assay in ESE Quant tube scanner software allowed the direct monitoring of amplification excluding the need for end-point gel electrophoresis or the risk of UV exposure. All the samples which were detected by RT-LAMP assay were confirmed by conventional RT-PCR. \ud
CHS-20 cells (kidney fibroblast cells of African Green Monkey origin (CV-1 cell line) expressing sheep signaling lymphocyte activation molecule (SLAM)) were highly efficient for isolation (success rate of 74.2%) of PPRV in clinical samples. The sequencing and phylogenetic analysis of PPRV isolates from Pakistan identified lineage IV which was subdivided into three subgroups viz. SG-D, SG-T and SG-I based on highly close relation to the PPRV from neighbouring countries of Dubai, Tajikistan and Iran, respectively. In comparative study, the analysis of PPRV from African and Asian countries revealed that lineage IV has spread across many areas in Africa which were previously known to harbour only lineage III or II. Simultaneously, lineage I was not detected in areas which were historically known for it. Our study highly advocated the application of molecular diagnostic methods for\ud
sensitive diagnosis of PPR especially its mild form and the importance of continuous monitoring of the evolution of PPRV. It accentuates the need to develop new evolutionary markers that can compare virulence of these viruses. It emphasizes in a timely manner to contain the spread of virus before it becomes more looming threat for global livestock and the poor economy. the decision support system