Monthly Progress report (AGB) November, 2012
 Genetic analysis of genes controlling growth, milk production and disease resistance etc.
in Pakistani food animals were done; 150 Nili Ravi buffalo and almost 100 animals of
Sahiwal breed are screened for the prolactin gene polymorphism. Results showed that
animals of Nili-Ravi buffalo were found to be monomorphic for GG genotype, while in
case of Sahiwal and Achai cattle the prolactin gene present in three polymorphic form
having three genotypes GG, GA & AA. The frequency of these genotypes varies
according to the breeds. In Sahiwal cattle the frequency of genotypes GG was found to be
higher. More blood samples collection and molecular analysis is in process.
 Optimization for the Beta-Defensen gene amplification; Beta Defensen an important milk
production gene its amplification has been optimized after many PCRs at 550C annealing
temperature and screening using this method is in process.
 Arrangements were made for the Friesian pure and cross bred blood sample collection
from different Military livestock farms.
 Genetic analysis of inherited disorders in cattle. PCR is optimized for the amplification of
gene encoding bovine CD18 using S2 primers
 Screening for the Citrullinaemia and BLAD is in process presently working on Achai and
Sahiwal breed. Screened 200 samples and seen some mutations trying to confirm through
sequencing.
 Test for the diagnosis of the CVM disease is optimized through ARMS-PCR while this
test is tricky and difficult to handle so now trying to optimize a comparatively easy
method Polymerase chain reaction primer introduced restriction analysis (PCR-PIRA) for
this method primers and other enzymes have been ordered and chemicals are prepared.
 Test for the diagnosis of bovine hereditary Zinc deficiency is optimized and now waiting
for the samples of Friesian and its cross bred present in Paksitan.
 A project on epigenetic is optimized, in Nilli-Ravi breed epigentic profiling of prolactin,
leptin and BoLA promoters is going on. In this DNA samples of Nilli ravi were treated
with bisulfate treatment using Sodium bisulfate, Hydroquinone and NaOH and treated
DNA was purified using Easy DNA kit of Invitrogen Co. Bisulfite treated DNA samples
were optimized for Prolactin and Leptin pomotor primers and few samples have been
polymerized for the promoters. Waiting to order the sequencing of the products for
further analysis.
 Preparation and maintenance of primary liver cell culture of chicken embryos.
 Isolation and molecular characterization of AAV-4. Liver samples were collected from
different outbreak regions of AAV-4. AAV was detected by Agar gel precipitation test.
AAV-4 was isolated in chicken embryo liver cell culture. PCR was standardized using
hexon gene specific primers.
 Production of Hyperimmune sera against AAV-4.
 Hyperimmune sera was raised in chicks by using cell culture propagated AAV-4.
Formalin inactivated vaccine was prepared and injected in 10 day old chicks. Sera was
collected three weeks post vaccination.
 Detection of Avian adeno virus-4 from field samples by AGPT and two were found
positive.
 Isolation of two samples of Avian adeno virus-4 in cell culture.
 Maintenance of LFBK cell line.
 Isolation of 3 FMD viruses.
 Standardization of virus neutralization test for FMDv.