Characterizations of Larval Gut Bacteria of Anopheles Subpictus Grassi and Their Role in Mosquito Development in Hooghly, West Bengal, India.

Malaria is a serious vector borne disease transmitted by different species of Anopheles mosquitoes. The present study was aimed to isolate & characterize the bacterial flora from the gut of An. subpictus larvae prevalent in Hooghly and explore their roles in host survival and development. Mosquito larvae and adults were collected from field and were maintained in laboratory. Bacterial load in the larval midgut was determined, predominant strains were isolated and characterized by polyphasic approach. Role of these bacteria in larval survival & development were assayed. Bacterial load in the gut of larvae was found to vary in field collected and lab reared mosquitoes in different seasons. Morphological, bio-chemical and molecular analysis explored four common bacterial isolates namely Bacillus subtilis, Bacillus pumilus, Bacillus cereus & Proteus vulgaris in the larval gut throughout the year. Larval survival rate was greatly reduced (0.06) & time of pupation was prolonged (17.8±0.57) in absence of their gut bacteria. Total tissue protein (7.78±0.56), lipid (2.25±0.19) & carbohydrate (16.5±0.79) contents of larvae and body weight & wing length of adult male (0.17±0.02 & 1.74±0.43) & female (0.19±0.02 & 1.99±0.46) mosquitoes were also found to be greatly reduced in the absence of gut bacteria. Developmental characteristics were restored with the introduction of culture suspension of all four resident gut bacterial isolates. Present study indicates that the mosquitoes solely depend on their gut bacteria for their survival & development. So, manipulation or control of this gut bacterial communities might inhibit survival & development of vector mosquitoes.

Prolonged Laboratory Maintenance of Toxoplasma gondii Tachyzoites in Co-cultivation with the Bovine Theileria annulata-Infected Lymphoblastoids

Background and Aims: In most of the studies, Toxoplasma gondii is maintained in laboratory mice or studied in vitro using non-lymphoid cell lines or primary mouse macrophages. The target of our research was to design a new axenic culture of Toxoplasma gondii tachyzoites to providing a sufficient quantity of them. Material and Methods: Theileria annulata-infected lymphoblastoids, which had been maintained up to 260 sub-cultures to attenuate the Theileria annulata, were evaluated for their suitability to the cultivation of Toxoplasma gondii tachyzoites. This cultivation process was carried out continuously for up to 10 passages, and after each 5 sub-culture, 0.1 ml of culture suspension (1×106 tachyzoites) was inoculated to each BALB/c mouse. Results: It was observed that the tachyzoites have attacked the lympho blastoids, multiplied inside them, and many fresh tachyzoites with typical shape and gliding movement were present in the culture suspension. In all processes of cultivation, the pathogenesis of parasites remained stable, and they were able to proliferate in mice and eventually lead to the death of the animals. Conclusions: We describe here a new protocol for prolonged maintenance of tachyzoites of Toxoplasma gondii, which is more efficient (both in terms of yield and cost (it does not need fetal calf serum)) than other traditional methods for maintenance of the parasite.

Determination of FMDV 146S particle concentration by spectrometric method during viral RNA quantification

During FMD vaccine production, special attention is paid to the concentration of 146S particles bearing the critical biological features of FMDV and being the main components that have an effect on vaccine immunogenicity. For this reason, each batch of vaccine raw material is tested for 146S component concentration. The paper presents the results of the use of a spectrometric method for whole particle concentration determination during quantification of FMDV RNA extracted after immune capture. It is an inexpensive, easy-to-perform method allowing for determination of FMDV 146S particle concentration in the non-inactivated culture suspension. 146S particle concentration was found to depend on the number of RNA molecules extracted from virions after their strain-specific immune capture and quantitatively detected by the spectrometric method. The presented method allows for determination of 146S component concentration in the non-inactivated vaccine raw material using the proposed linear model. The spectrometric method showed 94.5–99.5% correlation with real time reverse transcription polymerase chain reaction and complement fixation test based on the results of tests of 360 non-inactivated suspensions of FMDV of all types. Tests of the positive control demonstrated 99.0–99.6% compatibility of actual and expected results. FMDV genome and 146S particles were not detected in the negative control, and that was in line with expectations.

Short Communication: Callus induction in purple and white-purple varieties of Orthosiphon aristatus (Blume) Miq.

Faramayuda F, Mariani TS, Elfahmi, Sukrasno. 2020. Short Communication: Callus induction in purple and white-purple varieties of Orthosiphon aristatus (Blume) Miq. Biodiversitas 21: 4967-4972. Orthosiphon aristatus (Blume) Miq. are known to have many benefits, including stimulating urine expenditure (diuretics) and dissolving kidney stones. O. aristatus widely planted in Indonesia are purple and white-purple. The main secondary metabolite components of O. aristatus are sinensetin, rosmarinic acid, and eupatorin. One of the initial steps to increase secondary metabolites in O. aristatus is by induction of callus using plant tissue, which later can be developed into a culture suspension for secondary metabolites. The materials used are the leaf of two varieties of O. aristatus that have been sterilized and grown on Murashige and Skoog media with growth regulatory 2,4-dichlorophenoxyacetic acid at a concentration of 0.4;1.0; 2.0 mg/L. The identification of secondary metabolites of callus was carried out by thin-layer chromatography. The best growth regulating agent for callus induction on the leaves of purple and white-purple varieties of O. aristatus is 2,4-D 0.4 mg/L on Murashige and Skoog media. These media can grow callus at a faster time, friable, and slightly white-yellow color. The identification of secondary metabolites in callus acetone extract showed the presence of sinensetin and rosmarinic acid.

Antimicrobial efficacy of neem and liquorice with chlorhexidine on Streptococcus sanguis, Streptococcus mutans, Lactobacillus and Actinomyces naeslundii – An In Vitro Study

The study was to formulate 2% neem and 2% liquorice mouthwashes and to compare the antimicrobial efficacy of these mouthwashes with the standard 0.2% chlorhexidine mouthwash. Alcoholic solution was prepared and added to neem mixture and liquorice mixture separately and made up to a volume of 16000 ml with purified water. Nine dilutions of each drug were done with Brain heart infusion broth (BHI) for MIC. Culture suspension was added in each serially diluted tube of 200 μl. The tubes were incubated for 24 hours and observed for turbidity. Minimum inhibitory concentration (MIC) of 2% neem, 2% liquorice and 0.2% chlorhexidine against Lactobacillus, Actinomyces naeslundii, Streptococcus sanguis, Streptococcus mutans is determined by serial dilution analysis. Streptococcus mutans shows sensitivity to all three mouthwashes at a concentration starting from 0.2 μg/ml. Lactobacillus shows sensitivity to neem and chlorhexidine mouthwashes at a concentration starting from 1.6 μg/ml, whereas liquorice is effective at a concentration starting from 3.125 μg/ml. Streptococcus sanguis shows sensitivity to chlorhexidine and liquorice mouthwashes at a concentration starting from 25 μg/ml, whereas it shows sensitivity to neem at a concentration starting from 50 μg/ml. Actinomyces naeslundii shows sensitivity to chlorhexidine and neem mouthwashes at a concentration starting from 1.6 μg/ml, whereas it shows sensitivity to liquorice at a concentration starting from 3.125μg/ml. Analysis showed an inhibition of all the four strains by the mouthwashes. The MIC for the studied mouthwashes was found to be similar to that of 0.2% chlorhexidine.

Biotic and abiotic stress roles in drugs production through in vitro approaches in plants – a review

Plant metabolic engineering is a modern discipline that promises to create opportunities in pharmaceutical industries to produce and biomedicine. Over the long period natural and synthetic plant hormones have had tremendous implications in callus/cell culture /suspension/ for secondary metabolites production (SMs). Generally, SMs plays a vital fundamental role in protecting the plant from biotic and abiotic attacks to which it may be subjected. This review article focused on the relationship between various factors related to the drug production. In medicinal plants, in vitro studies, based on biotic factors such as fungal/endo-phytic fungal elicitors/microbe-derived exogenous elicitor yeast extract (YE) were cross checked with the abiotic six factor groups, including auxins and cytokinins, gamma radiation, lights, temperature, carbon sources, photoperiods, precursor chemicals and plant metabolic enzymes. Moreover, key enzymes and gene networks can serve as a resource to selected potential targets for specific SMs production. This is the first review to describe the light factors needed for the SM production, which has favorable role for SMs. We envisage that the researcher can design how to modulate the stress factors in terms of drug improvement from medicinal plants.

Experimental Staphylococcus aureus Mastitis Infection Model by Teat Dipping in Bacterial Culture Suspension in Dairy Cows

Mastitis is inflammation of mammary glands usually caused by bacteria such as Staphylococcus aureus. Dairy cows are susceptible to mastitis during early dry and transition periods. Effective vaccine is needed during these periods. One of the limitations to develop an effective vaccine against S. aureus is the absence of good infection model. Intramammary infusion (IMIF) with S. aureus has been used as an infection model to test vaccine efficacy. IMIF is reliable in causing mastitis, but it bypasses physical barriers, non-specific natural defenses, and immunity in the teat canal. IMIF also transfers a large number of bacteria into the intramammary area at once. The objective of this study was to develop S. aureus IMIF model that mimics natural infection. Eight Holstein dairy cows were randomly divided into two groups of experimental (n = 5) and control (n = 3) cows. All teats of experimental cows were dipped in S. aureus culture suspension, whereas that of control cows were dipped in phosphate-buffered saline. Results showed that four of five cows were infected with challenge strain by day 3 of the challenge. The remaining cow was infected with Staphylococcus chromogenes. In conclusion, an experimental S. aureus intramammary infection can be induced by teat dipping into bacterial suspension.

Seed Biopriming with Microbial Inoculant Triggers Local and Systemic Defense Responses against Rhizoctonia solani Causing Banded Leaf and Sheath Blight in Maize (Zea mays L.)

Plant growth promoting rhizobacteria Pseudomonas aeruginosa strain MF-30 isolated from maize rhizosphere was characterized for several plant growth stimulating attributes. The strain MF-30 was also evaluated for antifungal properties against Rhizoctonia solani causing banded leaf and sheath blight in maize (Zea mays L.) under in vitro conditions and was found to have higher mycelial growth suppression in the culture suspension (67.41%) followed by volatile organic compounds (62.66%) and crude extract (51.20%) in a dual plate assay. The endophytic and epiphytic colonization ability was tested using Green Fluorescent Protein (GFP)-tagging. Visualization through confocal scanning laser microscope clearly indicated that strain MF-30 colonizes the root and foliar parts of the plants. Further, the effects of seed bio-priming with P. aeruginosa MF-30 was evaluated in the induction and bioaccumulation of defense-related biomolecules, enzymes, natural antioxidants, and other changes in maize under pot trial. This not only provided protection from R. solani but also ensured growth promotion under pathogenic stress conditions in maize. The maximum concentration of hydrogen peroxide (H2O2) was reported in the root and shoot of the plants treated with R. solani alone (8.47 and 17.50 mmol mg−1 protein, respectively) compared to bioagent, P. aeruginosa MF-30 bio-primed plants (3.49 and 7.50 mmol mg−1 protein, respectively). Effects on total soluble sugar content, total protein, and total proline were also found to enhanced significantly due to inoculation of P. aeruginosa MF-30. The activities of anti-oxidative defense enzymes phenylalanine ammonia lyase (PAL), ascorbate peroxidase, peroxidase, superoxide dismutase, and catalase increased significantly in the plants bio-primed with P. aeruginosa MF-30 and subsequent foliar spray of culture suspension of MF-30 compared to pathogen alone inoculated plants. qRT-PCR analysis revealed that seed bio-priming and foliar application of P. aeruginosa MF-30 significantly increased the expression of PR-1 and PR-10 genes with the simultaneous decrease in the disease severity and lesion length in the maize plants under pathogenic stress conditions. A significant enhancement of shoot and root biomass was recorded in MF-30 bio-primed plants as compared to untreated control (p < 0.05). Significant increase in plant growth and antioxidant content, as well as decreased disease severity in the P. aeruginosa MF-30 bio-primed plants, suggested the possibility of an eco-friendly and economical means of achieving antioxidants-rich, healthier maize plants.

THE ABILITY OF Lactobacillus plantarum BSL IN REDUCING THE TISSUE DAMAGE OF LIVER AND SPLEEN IN RATS INFECTED BY Listeria monocytogenes ATCC 7644

Lactobacillus plantarum BSL, previously isolated from Indonesian sauerkraut. In this study, we investigated the ability of L. plantarum BSL in reducing the tissue damage of liver and spleen in rats infected by Listeria monocytogenes ATCC 7644. Treatment group of rats received 0.5 mL culture suspension (109 CFU/mL) of L. plantarum BSL and control group received 0.5 mL of 0.85% w/v NaCl daily for nine days of experiment. Both groups were infected at 3rd day with 0.5 mL of suspension of L. monocytogenes (109CFU/mL). At the 2nd (before infection), 5th, 7th, and 9th day (after infection), the rats were sacrificed and then, liver and spleen were assessed for histopathological. Our study revealed that the administration of L. plantarum BSL could be able to reduce the liver and spleen damage of the experimental rats.

In vitro corrosion studies of stainless-steel dental substrates during Porphyromonas gingivalis biofilm growth in artificial saliva solutions: providing insights into the role of resident oral bacterium

A stainless-steel 321 dental substrate significantly corroded within Porphyromonas gingivalis growth culture in artificial saliva culture suspension, with and without NaF additive.