ABSTRACT
Virus infection alters host gene expression, therefore ideal and stable reference housekeeping genes are required to normalise the expression of other expressed host genes in quantitative real-time PCR (qRT-PCR). The suitable reference gene may vary in response to different viral infections in different hosts or cells. In the present study, we cultured primary lamb testis cells (LTC) and assessed the expression stability of seven widely used housekeeping genes (B2M, HMBS, HPRT1, HSP-90, POLR2A, 18s_RNA, GAPDH) as reference genes in Sheeppox virus (SPPV) infected and control (uninfected-0h) LTC at 0.5h, 4.0h, 8.0h, and 12.0h post-infection) using NormFinder, Bestkeeper, geNorm, and the comparative ΔCT method in RefFinder based on their expression levels. Analysis revealed that HSP90, 18s_RNA, HPRT, POLR2A, and B2M were the most stable genes from the panel in the individual analysis group in 0h, 0.5h, 4.0h, 8.0h, and 12.0h, respectively. Furthermore, B2M was shown to be the most stable reference gene in the combined control with the respective and overall infected groups, except the control group of 4.0hpi of SPPV infection. In this study, we selected the most suitable reference genes in LTC for particular time points of SPPV infection. The identified most suitable housekeeping gene can be used during normalization of expression of other targeted genes at aspecific time point of SPPV infection.
Subject(s)
Capripoxvirus , Gene Expression Profiling , Animals , Gene Expression , Gene Expression Profiling/methods , Male , RNA, Ribosomal, 18S , Real-Time Polymerase Chain Reaction/methods , Reference Standards , Sheep/genetics , TestisABSTRACT
We examined 974 samples (304 coriander, 212 mint, 258 carrot, and 200 radish) collected from vegetable vendors in two cities, Bareilly (n = 832) and Kanpur (n = 142), in northern India during the early summer season in 2004. Salmonella was isolated from 35 samples (9 coriander, 5 mint, 10 radish, and 11 carrot) while Escherichia coli was detected in 181 samples (67 coriander, 44 mint, 36 carrot, and 34 radish). None of the E. coli belonged to the O:157 serogroup. Five Salmonella isolates from samples collected at Kanpur (3 coriander and 2 mint) belonged to 4 different serovars of S. enterica ssp. enterica-S. Mons, S. Rottenest, S. Saintpaul, and S. Weltevreden. Thirty Salmonella isolates from samples collected at Bareilly (11 carrot, 10 radish, 6 coriander, and 3 mint) belonged to 7 serovars-S. Anatum, S. Bsilla, S. Newport, S. Saintpaul, S. Teko, S. Virchow, and S. Weltevreden. The majority (82.9%) of Salmonella isolates were multidrug resistant. One quarter of the isolates were resistant to >or=10 antibiotics. Based on antibiotic resistance patterns, 35 isolates could be classified into 23 resistotypes. None of the 35 isolates was resistant to streptomycin and ceftriaxone, while >80% were resistant to sulfamethoxazole, nalidixic acid, and kanamycin. Resistance to imipenem (>20%) and amikacin (>30%) was also common. The correlation between presence of Salmonella and E. coli on raw vegetables was not significant (p = 0.13).
