Development of multiplex RT-PCR assay for simultaneous detection of four viruses infecting apple (Malus domestica).

The major viruses infecting apple cultivars throughout the world including India are apple mosaic virus (ApMV), apple stem pitting virus (ASPV), apple stem grooving virus (ASGV), apple chlorotic leaf spot virus (ACLSV), and recently, a new virus, apple necrotic mosaic virus (ApNMV), was reported from mosaic-infected apple cultivars in India. The aim of this study was to detect the ApNMV virus along with the other three viruses (ApMV, ASPV and ASGV) simultaneously by multiplex RT-PCR. Four primer-pair-produced amplicons of 670, 550, 350 and 210 bp corresponding to ApNMV, ApMV, ASPV and ASGV, respectively, were found to be specific for these viruses when tested individually. The annealing temperature (55°C), primer concentration (0·8 µl) and other components of the master mix were standardized for the development of one-step m-RT-PCR assay. The m-RT-PCR protocol developed was further validated with 30 samples from seven symptomatic or asymptomatic apple cultivars, which revealed the presence of more than one virus in these cultivars. Most of the viruses were found to be present either alone or in mixed infection; however, ASPV was more common in tested cultivars. An easy, cost-effective and rapid multiplex RT-RCR protocol was developed to detect the four viruses, which infect apple plants either in individually or together in the field. This assay will help in the surveying and indexing of apple germplasm and the distribution of all four viruses in the apple growing regions of India.

Role of biometals in activation of immune cum inflammatory response in ovine ageing eye: a potential model for understanding human geriatric eye diseases.

The present study was designed to evaluate the age-related changes in biometal and antimicrobial peptide (cathelicidin) concentration and their role in oxidative cum pro-inflammatory cascade in an ovine animal model. Clinically healthy ovine (n = 126) were grouped as Group I (n = 55, age = up to 3 years), Group II (n = 52, age = above 3-below 6 years) and Group III (n = 19, age = 6 years above). Samples (aqueous humour and lens of the eye) were collected stored at - 80 °C till further analysis. In aqueous humour, the concentration of zinc (p < 0.001 in group III), copper (p < 0.05 in group II and p < 0.001 group III) and iron (p < 0.05 in group III) were significantly increased compared to group I. While as the concentration of magnesium were significantly decreased in group II (p < 0.001) and group III (p < 0.05) compared to group I. Similarly in eye lens the level of copper remained uniform as no significant change was observed across different age groups, while as significantly elevated levels of iron were observed in group III (p < 0.001) compared to group I. whereas, levels of lens Zinc (p < 0.05 in group II) and magnesium (p < 0.05 in group III and p < 0.001 in group II) were significantly decreased compared to group I. Age-dependent increase in levels of oxidation products which include advanced oxidation protein products (AOPP) in aqueous humour and lenses of group II and group III (p < 0.001) and MDA in aqueous humour of group III (p < 0.05) were found compared to levels recorded in group I. In contrast, levels of antioxidants which include lens vitamin C in group II and group III (p < 0.01) and lens superoxide dismutase (SOD) in group III (p < 0.001) were significantly increased compared to group I. Levels of pro-inflammatory cytokines in aqueous humour revealed significantly (p < 0.001) age-dependent increase in IL-1, IL-6 and TNF-α elevated in group III, and group II as compared to group I, However, cathelicidin level in aqueous humour of group III and group II were significantly (p < 0.001) lower as compared to groups I. Furthermore,the present study observed significant (p < 0.05) metal-metal positive interaction between copper levels in lens with levels of (iron and magnesium) in aqueous humour, levels of Zn in lens with levels of Zn in aqueous humour, levels of Mg in lens with levels of (Cu, Zn and Mg) in aqueous humour. In addition,the present study reports significantly negative interaction between levels of lens Fe with levels of lens magnesium level, aqueous humour magnesium level and levels of copper in aqueous humour. A significantly positive correlation was observed between oxidative markers and pro-inflammatory cytokine levels, while a significant negative correlation was observed between antioxidant defence markers and pro-inflammatory cytokine. These results suggest the essential role of age-related changes in biometal levels, oxidative stress and pro-inflammatory cytokines. These changes might help understand age-related changes in pathogenesis and effective targeting of pathogenetic pathways in ocular diseases.

Bilateral follicular cysts in a water buffalo.

The present short communication puts on record a case of bilateral, multiple follicular cysts in a water buffalo along with a detailed description of its ovarian biometry and follicular fluid composition. The ovarian weight and biometrical parameters were much higher than in normal cycling buffaloes. A total of three follicular cysts were observed, two on the right ovary and one on the left ovary, measuring 4.9, 3.0 and 2.6 cm yielding 21, 9 and 5 ml of follicular fluid, respectively. The cystic fluid was deep yellow in colour with a viscous consistency. The follicular fluid concentrations of glucose, total protein, cholesterol, acid phosphatase, calcium, phosphorus and progesterone in all the cysts were within the range reported previously in normal buffalo follicular fluid; however, the alkaline phosphatase concentration in cyst 1 and total bilirubin concentration in cysts 1 and 2 were higher than the values in normal follicular fluid. In contrast, the levels of urea nitrogen in cysts 1 and 3, and oestradiol in cyst 3 were lower than the normal values. All the three follicles had an oestradiol to progesterone ratio less than 1. The results of our study suggest that follicular cysts in buffalo are oestrogenically inactive and have an altered concentration of certain biochemical and hormonal constituents.