Comparison of multiplex and ordinary PCR for diagnosis of paratuberculosis and tuberculosis in blood samples (buffy coat) of cattle and buffaloes.

BACKGROUND: Paratuberculosis and tuberculosis (TB) caused by Mycobacterium avium paratuberculosis (MAP) and Mycobacterium tuberculosis complex (MTC), respectively are economically important, chronic debilitating diseases affecting the dairy herds and are also potential zoonotic threats. AIMS: Differential diagnosis of paratuberculosis and TB in blood samples of cattle and buffaloes. METHODS: In this study, an in-house developed multiplex polymerase chain reaction (PCR) targeting MAP, Mycobacterium bovis and Mycobacterium smegmatis was used in blood samples (buffy coat) parallel with IS900 PCR and esxB PCR for diagnosis of paratuberculosis and TB, respectively; in a total of 202 cattle and buffaloes. RESULTS: Out of 202 animals, 12 (5.9%) and 17 (8.4%) animals were positive for MAP by multiplex PCR and IS900 PCR, respectively; from which only 8 (4%) animals were positive by both tests; whereas 4 and 9 animals were exclusively positive by multiplex PCR and IS900 PCR, respectively. None of the animals were found to be positive for M. bovis and M. smegmatis by the multiplex PCR. However, the esxB PCR detected 13 (6.4%) animals positive for TB. In fact, 3 (1.5%) animals were found to be co-infected by both paratuberculosis and TB. CONCLUSION: The in-house multiplex PCR detected MAP in buffy coat and there was a fair degree of agreement between the multiplex PCR and IS900 PCR in detection of MAP DNA though the latter detected more number of animals to be positive for MAP. Besides, esxB PCR showed a high diagnostic potential and can be used for diagnosis of TB from blood.

Predicting intracranial progression following stereotactic radiosurgery for brain metastases: Implications for post SRS imaging.

PURPOSE: Follow-up imaging after stereotactic radiosurgery (SRS) is crucial to identify salvageable brain metastases (BM) recurrence. As optimal imaging intervals are poorly understood, we sought to build a predictive model for time to intracranial progression. METHODS: Consecutive patients treated with SRS for BM at three institutions from January 1, 2002 to June 30, 2017 were retrospectively reviewed. We developed a model using stepwise regression that identified four prognostic factors and built a predictive nomogram. RESULTS: We identified 755 patients with primarily non-small cell lung, breast, and melanoma BMs. Factors such as number of BMs, histology, history of prior whole-brain radiation, and time interval from initial cancer diagnosis to metastases were prognostic for intracranial progression. Per our nomogram, risk of intracranial progression by 3 months post-SRS in the high-risk group was 21% compared to 11% in the low-risk group; at 6 months, it was 43% versus 27%. CONCLUSION: We present a nomogram estimating time to BM progression following SRS to potentially personalize surveillance imaging.

Diagnosis of Mycobacterial infections (Tuberculosis and Paratuberculosis) in tissue samples using molecular (inhouse multiplex PCR, PCR and TaqMan real-time PCR), histopathology and immunohistochemical techniques.

An in-house multiplex PCR designed for differential detection of M. bovis, M. avium paratuberculosis (MAP) and M. smegmatis in tissue samples, besides PCR and TaqMan real-time PCR targeting esxA (ESAT-6) and esxB (CFP-10) gene of M. tuberculosis complex (MTC) and IS900 gene targeting MAP were carried out in the present study. Tissue samples comprising of lung, mediastinal lymph node, intestine and mesenteric lymph node from cattle and buffaloes (n=16) suspected to have died of tuberculosis (TB) or paratuberculosis (JD) were collected at post-mortem. Out of 16 animals, only 4 (25%) cases were positive for M. bovis by in-house designed multiplex PCR, where as 9 (56.25%) cases were positive for MTC by both conventional and real-time PCR targeting esxA (ESAT-6) and esxB (CFP-10) genes. Out of 9 TB positive animals, 2 (22.22%) had co-infection with MAP as confirmed by IS900 PCR, real-time PCR and also by in-house multiplex PCR. No samples were found positive for M. smegmatis by the in-house multiplex PCR. Analytical sensitivity of in-house multiplex primers targeting M. bovis, MAP and M. smegmatis were 170 fg/µl, 300 fg/µl and 51 fg/µl of genomic DNA respectively. Analytical sensitivity of primers for both conventional and real time PCR tareting esxA were 8 pg/µl and 800 fg/µl, esxB were 800 fg/µl and 80 fg/µl and IS900 were 30 fg/µl and 3 fg/µl of M. tuberculosis and MAP genomic DNA respectively. The tissue samples were also subjected to histopathology and Immunohistochemical (IHC) staining using ESAT-6 and CFP-10 antibodies for TB and anti-JD anti-serum (raised in rabbit) for JD. All the animals positive for TB (9 of 9) and JD (2 of 2) in PCR and real-time PCR were also positive in histopathology and IHC. Thus, combination of molecular, histopathology and IHC techniques increased the accuracy of TB and JD diagnosis, all of which had same sensitivity and specificity except the in-house multiplex PCR which was comparatively less sensitive in M. bovis detection. However, the molecular methods were found to be more convenient and rapid, capable of making the diagnosis within 6 hours. The study indicated that Real-time PCR was ten times more sensitive than the conventional PCR.

Midgut microbial community of Culex quinquefasciatus mosquito populations from India.

The mosquito Culex quinquefasciatus is a ubiquitous species that serves as a major vector for west nile virus and lymphatic filariasis. Ingestion of bloodmeal by females triggers a series of physiological processes in the midgut and also exposes them to infection by these pathogens. The bacteria normally harbored in the midgut are known to influence physiology and can also alter the response to various pathogens. The midgut bacteria in female Cx. quinquefasciatus mosquitoes collected over a large geographical area from India was studied. Examination of 16S ribosomal DNA amplicons from culturable microflora revealed the presence of 83 bacterial species belonging to 31 bacterial genera. All of these species belong to three phyla i.e. Proteobacteria, Firmicutes and Actinobacteria. Phylum Proteobacteria was the most dominant phylum (37 species), followed by Firmicutes (33 species) and Actinobacteria (13 species). Phylum Proteobacteria, was dominated by members of γ-proteobacteria class. The genus Staphylococcus was the largest genus represented by 11 species whereas Enterobacter was the most prevalent genus and recovered from all the field stations except Leh. Highest bacterial prevalence was observed from Bhuj (22 species) followed by Nagrota (18 species), Masimpur (18 species) and Hathigarh (16 species). Whereas, least species were observed from Leh (8 species). It has been observed that individual mosquito harbor extremely diverse gut bacteria and have very small overlap bacterial taxa in their gut. This variation in midgut microbiota may be one of the factors responsible for variation in disease transmission rates or vector competence within mosquito population. The present data strongly encourage further investigations to verify the potential role of the detected bacteria in mosquito for the transmission of lymphatic filariasis and west nile virus. To the best of our knowledge this is the first study on midgut microbiota of wild Cx. quinquefasciatus from over a large geographical area.

Cyanobacterial diversity in the rhizosphere of rice and its ecological significance.

This investigation was undertaken to characterize the abundance and genera-wise diversity of cyanobacteria in the rice rhizosphere and nitrogen-fixing ability of the isolated strains. The cyanobacterial strains belonging to the genera Nostoc and Anabaena comprised 80% of the rhizosphere isolates, which were also efficient in enhancing the germination and growth of wheat seeds and exhibited significantly high protein accumulation and IAA production. Distinct profiles for the cyanobacterial strains were obtained on amplification with extended Hip 1 primer - HipTG, indicative of the diversity among these strains. Our investigation helped in identifying promising cyanobacterial isolates from the rhizosphere of rice, which can be utilized in developing efficient plant growth promoting cyanobacterial inoculants.

Evaluation of fungicidal activity of extracellular filtrates of cyanobacteria--possible role of hydrolytic enzymes.

A set of seventy axenised and unicyanobacterial isolates belonging to the genus Anabaena were evaluated for biocidal activity against a set of phytopathogenic fungi. Among them, 35 Anabaena strains showed zone of inhibition against one or more fungi. The extracellular filtrates from 4 and 8 weeks old cultures of these Anabaena strains were further evaluated in terms of hydrolytic enzymes, proteins and IAA employing standard methods. Significant differences were also observed among the strains in terms of their FPase, chitosanase and xylanase activity, while low and relatively similar values of CMCase, cellobiase and protease activity were recorded in the strains analyzed. IAA production was also observed in all the strains. Comparative evaluation of activity of hydrolytic enzymes and antifungal activity revealed that such enzymes may contribute to the fungicidal activity of the cyanobacterial strains, besides other bioactive compounds, including IAA, which are established promising traits for biocontrol agents. This study is a first time report on the production of hydrolytic enzymes by these oxygenic photosynthetic prokaryotes, which can be potential candidates for the development of biocontrol agent(s) against selected phytopathogenic fungi.

Dynamics of traffic noise in a tropical city Varanasi and its abatement through vegetation.

Noise level monitoring and its reduction with different width and height of vegetation belt were studied in the Varanasi city. Noise level monitoring of the Varanasi city revealed the fact that area category A (without vegetation) was highly polluted as compare to area category B (with vegetation) having less fluctuation of traffic load. Four plant species Putranjeva roxburghi, Cestrum nocturnum, Hibiscus rosasinensis and Murraya peniculata were tested for noise reduction study at different frequencies. Experiment revealed the fact that H. rosasinensis reduced noise highest at both low and high frequencies (100-500 Hz, 22 dB and 2.5-6.3 KHz 26 dB), followed by M. peniculata (100-500 Hz, 18 dB and 2.5-6.3 KHz 20 dB), P. roxburghi (100-500 Hz 15 dB and 2.5-6.3 KHz 17 dB) and C. nocturnum (100-500 Hz 9 dB and 2.5-6.3 KHz 14 dB). Significance of vegetation belt in noise reduction was established with multiple regression models.

Survival of bio-inoculants on fungicides-treated seeds of wheat, pea and chickpea and subsequent effect on chickpea yield.

Survival of Mesorhizobium ciceri (SP(4)) and Azotobacter chroococcum (CBD-15 and M(4)) was tested on chickpea (Cicer arietinum) seeds treated with fungicides bavistin [methyl N-(1H-benzimidazol-2yl) carbamate] and thiram (tetramethyl-thiuram disulfide), whereas survival of phosphate solubilizing bacteria (PSB), Pseudomonas striata (27) and Bacillus polymyxa (H(5)) was examined on two cultivars (Arkel and BV) of pea (Pisum sativum) seeds treated with thiram. Viability of Azotobacter chroococcum (W(5)) was also examined on wheat (Triticum aestivum) seeds treated with bavistin, captan (cis-N-trichloromethyl thio-4 cyclohexane-1, 2-dicarboximide) and thiram under laboratory conditions using standard dilution and the plate count technique. All the tested strains of diazotrophs and PSB showed decline in their viable population on prolonged contact with fungicides. However, PSB showed variation in their viable population even with the cultivar. BV cultivar of pea seeds showed better recovery of viable P. striata (10.75 to 10.61 log no. of viable cells with in 0-24 hrs) in the presence of thiram, whereas the Arkel cultivar of pea resulted in better recovery of viable B. polymyxa. Azotobacter chroococcum (W(5)), a potential strain for wheat, showed better survival in the presence of bavistin, compared to thiram and captan. Higher viable population of Mesorhizobium ciceri (SP(4)) and Azotobacter chroococcum (M(4)) was recovered from chickpea seeds treated with bavistin compared to thiram. However, thiram-treated seeds resulted in a greater number of extractable Azotobacter chroococcum (CBD-15). Under field conditions, adverse effect of thiram was reflected on the performance of Mesorhizobium ciceri (SP(4)) and A. chroococcum (M(4)) strains, resulting in reduced root and shoot biomass and grain yield, compared to bavistin treated and culture inoculated treatment. CBD-15 showed better performance in the presence of thiram compared to bavistin.

Palmo-plantar lichen sclerosus et atrophicus.

A case of lichen sclerosus et atrophicus (LSA) in a male patient who presented with multiple hypopigmented to depigmented macules, polygonal in shape, distributed in a bilaterally, symmetrical manner over the hands, feet and flexor aspect of wrists is being reported. Histopathology confirmed the diagnosis of LSA.