Functional characterization of GhNAC2 promoter conferring hormone- and stress-induced expression: a potential tool to improve growth and stress tolerance in cotton.

The GhNAC2 transcription factor identified from G. herbaceum improves root growth and drought tolerance through transcriptional reprogramming of phytohormone signaling. The promoter of such a versatile gene could serve as an important genetic engineering tool for biotechnological application. In this study, we identified and characterized the promoter of GhNAC2 to understand its regulatory mechanism. GhNAC2 transcription factor increased in root tissues in response to GA, ethylene, auxin, ABA, mannitol, and NaCl. In silico analysis revealed an overrepresentation of cis-regulatory elements associated with hormone signaling, stress responses and root-, pollen-, and seed-specific promoter activity. To validate their role in GhNAC2 function/regulation, an 870-bp upstream regulatory sequence was fused with the GUS reporter gene (uidA) and expressed in Arabidopsis and cotton hairy roots for in planta characterization. Histochemical GUS staining indicated localized expression in root tips, root elongation zone, root primordia, and reproductive tissues under optimal growth conditions. Mannitol, NaCl, auxin, GA, and ABA, induced the promoter-driven GUS expression in all tissues while ethylene suppressed the promoter activity. The results show that the 870 nt fragment of the GhNAC2 promoter drives root-preferential expression and responds to phytohormonal and stress signals. In corroboration with promoter regulation, GA and ethylene pathways differentially regulated root growth in GhNAC2-expressing Arabidopsis. The findings suggest that differential promoter activity governs the expression of GhNAC2 in root growth and stress-related functions independently through specific promoter elements. This multifarious promoter can be utilized to develop yield and climate resilience in cotton by expanding the options to control gene regulation. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-024-01411-2.

Modeling the impact of biolarvicides on malaria transmission.

Biolarvicides are in use in several parts of the world for malaria vector control. We propose a five compartment dynamical systems model to study malaria transmission when biolarvicides are administered, to study the impact of this environmentally safe method on malaria spread. A comprehensive analysis of the model is presented. Model analysis shows that the basic reproductive rate R is larger in the absence of biolarvicides as compared to their presence. Theoretical analysis is corroborated by data from field studies. We show that there exist intermediate parameter regimes that separate disease-free and endemic states, which can in turn be modulated by biolarvicide use. Using Latin hypercube sampling we study the sensitivity of the model to parameter value changes. Calibration of our model to mosquito population and biolarvicide data for indoor and outdoors scenarios, yield parameter values hitherto not available or measurable. We validate our model with malaria incidence data from a region in India and provide predictions for malaria incidence in the presence and absence of biolarvicide. This model provides a prognostic tool to field work involving biolarvicide use in control of malaria.

Prevalence of Strongyle infections in goat of Maha Koushal region, Madhya Pradesh, India.

The coproculture study on Strongyle infection of goats was carried out in small holder farmers kept under semi-intensive management system in Balaghat, Narsinghpur and Chhindwara district, Madhya Pradesh, during the period from July 2011 to February 2012. Copro-culture of the samples positive for Strongyle infection revealed Haemonchus sp., Trichostrongylus sp., Oesophagostomum sp., Strongyloides sp., and Bunostomum sp. in a decreasing order in goats. The larvae of Haemonchus sp. (61.63 %) and Strongyloides sp. (7.50 %) were highest in Balaghat, Trichostrongylus (18.13 %) in Narsinghpur, while Oesophagostomum sp. (10.50 %) and Bunostomum sp. (5.75 %) were in Chhindwara district. The finding of this study indicates that, even though subclinical in nature, Strongyle infection are one of the major problems that could hamper health and productivity and there is need for design a programme to minimize and control Strongyle infection in goats in the study area.

Expression of GhNAC2 from G. herbaceum, improves root growth and imparts tolerance to drought in transgenic cotton and Arabidopsis.

NAC proteins are plant-specific transcription factors that play essential roles in regulating development and responses to abiotic and biotic stresses. We show that over-expression of the cotton GhNAC2 under the CaMV35S promoter increases root growth in both Arabidopsis and cotton under unstressed conditions. Transgenic Arabidopsis plants also show improved root growth in presence of mannitol and NaCl while transgenic cotton expressing GhNAC2 show reduced leaf abscission and wilting upon water stress compared to control plants. Transgenic Arabidopsis plants also have larger leaves, higher seed number and size under well watered conditions, reduced transpiration and higher relative leaf water content. Micro-array analysis of transgenic plants over-expressing GhNAC2 reveals activation of the ABA/JA pathways and a suppression of the ethylene pathway at several levels to reduce expression of ERF6/ERF1/WRKY33/ MPK3/MKK9/ACS6 and their targets. This probably suppresses the ethylene-mediated inhibition of organ expansion, leading to larger leaves, better root growth and higher yields under unstressed conditions. Suppression of the ethylene pathway and activation of the ABA/JA pathways also primes the plant for improved stress tolerance by reduction in transpiration, greater stomatal control and suppression of growth retarding factors.

Prevalence of gastro-intestinal parasitic infections in goat of Madhya Pradesh, India.

Gastrointestinal (GI) parasitism in animals is one of the major problems in India causing emaciation, anaemia, oedema, weakness, diarrhoea and death. Present study was designed to generate epidemiological data on GI parasitism of goats of Madhya Pradesh, India. During 8 months study period, a total of 960 samples were collected and examined by sedimentation and floatation methods followed by egg per gram out of 960 samples, 907 (94.48 %) were positive for one or more gastrointestinal parasite, wherein coccidia was predominant (82.4 %) followed by strongyles (69.27 %), amphistomes (22.71 %), Strongyloides sp. (9.17 %), Trichuris sp. (3.85 %), Moniezia sp. (3.02 %), Schistosomes sp. (2.29 %) and Fasciola sp. (1.77 %). The seasonal incidence was found highest in monsoon (98.06 %) and lowest in winter (91.67 %). The incidence of gastrointestinal parasitism was found higher in kids (96.25 %) in comparison with adult goats (93.89 %).

Exposure to norepinephrine enhances Brachyspira pilosicoli growth, attraction to mucin and attachment to Caco-2 cells.

Brachyspira pilosicoli is an anaerobic intestinal spirochaete that colonizes the large intestine of a variety of species of birds and mammals, including human beings. Colonization may result in a mild colitis and diarrhoea in a condition known as 'intestinal spirochaetosis'. The catecholamine norepinephrine (NE), which is known to influence the behaviour of many bacterial species, may be present in the colon. The purpose of the current study was to determine whether exposure of B. pilosicoli to NE would influence its in vitro behaviour in assays that may reflect in vivo colonization potential. B. pilosicoli strain 95/1000 was used in all the assays. Addition of NE at a concentration of 0.05 mM to B. pilosicoli growing in anaerobic broth significantly increased spirochaete numbers after 4 days incubation. The effect of higher concentrations of NE was not significant. Exposure to 0.05 mM NE, but not to higher concentrations, also resulted in significantly more spirochaete cells entering capillary tubes containing 4 % porcine gastric mucin than occurred with untreated cultures. When NE was added to chemotaxis buffer in capillary tubes, significantly more spirochaetes were attracted to the buffer containing NE at 0.1, 0.5 and 1.0 mM than to buffer containing 0.05 mM NE, or when no NE was added. Exposure of B. pilosicoli cultures to 0.05 mM NE prior to incubation with Caco-2 monolayers resulted in more attachment to the monolayer than occurred with non-exposed cultures. These results show that at higher concentrations, NE acts as a chemoattractant for B. pilosicoli, and at 0.05 mM it increases the spirochaete's growth rate, attraction to mucin and rate of attachment to cultured enterocytes. These activities are likely to enhance the ability of B. pilosicoli to colonize, and may be induced by conditions that increase NE concentrations in the intestinal tract, such as the stresses associated with crowding.

Attraction of Brachyspira pilosicoli to mucin.

The anaerobic intestinal spirochaete Brachyspira pilosicoli colonizes the large intestine of various species, including humans. In the colon this spirochaete can penetrate the overlying mucus layer, attach by one cell end to the underlying enterocytes, and initiate localized colitis and diarrhoea. The aim of this study was to investigate whether, as part of the colonization process, B. pilosicoli is attracted to mucin. Fifteen B. pilosicoli strains isolated from humans, pigs, chickens and dogs, and a control strain of Brachyspira hyodysenteriae, were analysed for their ability to enter solutions of hog gastric mucin in an in vitro capillary tube assay. No significant attraction was detected with 1 % mucin, but some strains started to enter a 2 % solution, and attraction then increased with increasing concentrations to peak at around 6-8 % mucin. A similar increase was seen with B. hyodysenteriae, although this activity peaked at 6 % mucin and then declined, suggesting that the two species have different affinities for mucin. These mucin concentrations were much higher than those used in previous experimental studies with Brachyspira species. The viscosities of the 6-8 % mucin solutions were around 7-12 mPa s, which were similar to the measured viscosities of the mucus layer overlying the epithelium in the caecum and colon of experimental pigs. The strains varied in their motility, as assessed by their ability to enter tubes containing chemotaxis buffer, but there was no significant relationship between this motility and the extent of their ability to enter the mucin solutions. Different strains also had different propensities to enter the mucin solutions, but there were no consistent differences according to the host species of origin. B. pilosicoli strain 95/1000 was attracted towards a solution of d-serine, suggesting that chemotaxis was involved in the attraction to mucin; however, 95/1000 was also attracted to viscous solutions of polyvinylpyrrolidone (PVP), in a manner mirroring the response to mucin, and hence suggesting the involvement of viscotaxis in the attraction to mucin. B. hyodysenteriae B204 showed a similar viscotaxis to PVP. Further studies are required to determine whether the in vitro interaction of a given strain with mucin is a useful indicator of its in vivo colonization ability, and hence could be used as a potential marker for virulence.

The intestinal spirochete Brachyspira pilosicoli attaches to cultured Caco-2 cells and induces pathological changes.

BACKGROUND: Brachyspira pilosicoli is an anaerobic spirochete that has received relatively little study, partly due to its specialized culture requirements and slow growth. This bacterium colonizes the large intestine of various species, including humans; typically, a dense layer of spirochete cells may be found intimately attached by one cell end to the surface of colonic enterocytes. Colonized individuals may develop colitis, but the mechanisms involved are not understood. The current study aimed to develop an in vitro model to investigate this process. METHODOLOGY/PRINCIPAL FINDINGS: Four strains of B. pilosicoli were incubated at a high multiplicity of infection with monolayers of a human colonic adenocarcinoma cell line (Caco-2 cells). One strain isolated from a pig (95/1000) and one from a human (WesB) attached to the monolayers. Colonization increased with time, with the Caco-2 cell junctions being the initial targets of attachment. By electron microscopy, individual spirochete cells could be seen to have one cell end invaginated into the Caco-2 cell membranes, with the rest of the spirochete draped over the Caco-2 cell surface. After 6 h incubation, the monolayer was covered with a layer of spirochetes. Colonized monolayers demonstrated a time-dependent series of changes: staining with labelled phalloidin identified accumulation of actin at the cell junctions; ZO-1 staining revealed a loss of Caco-2 tight junction integrity; and Hoechst staining showed condensation and fragmentation of nuclear material consistent with apoptosis. Using quantitative reverse transcription PCR, the colonized monolayers demonstrated a significant up-regulation of interleukin-1beta (IL-1beta) and IL-8 expression. B. pilosicoli sonicates caused significant up-regulation of IL-1beta, TNF-alpha, and IL-6, but culture supernatants and non-pathogenic Brachyspira innocens did not alter cytokine expression. CONCLUSIONS/SIGNIFICANCE: The changes induced in the Caco-2 cells provide evidence that B. pilosicoli has pathogenic potential, and give insights into the likely in vivo pathogenesis.

Penicillin resistance in the intestinal spirochaete Brachyspira pilosicoli associated with OXA-136 and OXA-137, two new variants of the class D beta-lactamase OXA-63.

Penicillin resistance mediated by beta-lactamase activity has been reported previously in the anaerobic intestinal spirochaete Brachyspira pilosicoli, and a novel class D beta-lactamase (OXA-63) hydrolysing oxacillin was described recently in a resistant human strain from France. In the current study, 18 B. pilosicoli strains from Australia and Papua New Guinea were tested for ampicillin and oxacillin susceptibility, and investigated for the presence of the class D beta-lactamase gene blaOXA-63 using PCR. PCR products were amplified from seven human and four porcine strains that were penicillin resistant, but not from seven penicillin-sensitive strains. Sequence analysis of the whole gene amplified from seven of the resistant strains from humans and pigs revealed only minor nucleotide differences among them, but there were significant differences compared with blaOXA-63. The predicted amino acid sequence of the enzyme from all seven strains had the same key structural motifs as the previously reported OXA-63, but two variants with 94-95% identity with OXA-63 were identified. OXA-136 had an additional amino acid and 12 other consistent amino acid substitutions compared with OXA-63. OXA-137 had the same differences compared with OXA-63 as OXA-136, but had an additional amino acid substitution at position 16. No structures consistent with integrons or transposons were found in the nucleotide sequences in the vicinity of blaOXA-136 in partially sequenced B. pilosicoli strain 95/1000, and the GC content (25.2 mol%) of the gene was similar to that of the whole genome. The gene encoding OXA-136 from B. pilosicoli strain Cof-10 conferred penicillin resistance on Escherichia coli. This study shows that penicillin resistance in human and porcine B. pilosicoli strains from Australia is associated with the production of two variants of OXA-63, and that susceptible strains lack the genes encoding OXA-63 or the variants.

In vitro antiviral activity of plant extracts on goatpox virus replication.

Four plants having known medicinal properties were screened for inhibition of goatpox virus (GTPV) replication in vitro. Of the 4 plants, extract of Acacia arabica (Babul) and Eugenia jambolana (Jamun) leaves had inhibition (%) 99.70 and 99.92 at their maximum non toxic concentrations, 99.93 +/- 0.38 and 1999.73 +/- 0.50 microg/ml, respectively in all cytopathic effect (CPE) inhibition assays. Inhibition of GTPV virus replication was further confirmed by PCR and SYBR Green based quantitative real-time QPCR assays specific for GTPV. Results indicated that the extract of Acacia arabica and Eugenia jambolana leaves inhibited GTPV replication in vitro.

Tail hair as an indicator of environmental exposure of cows to lead and cadmium in different industrial areas.

This study examines the use of tail hair from cows as a possible biomarker of environmental exposure to lead and cadmium around different industrial areas. Respective blood and tail hair samples were collected from a total of 317 apparently healthy cows above 3 years of age. This includes 287 cows reared in industrial and urban areas and 30 cows from areas free from polluting sources. Significantly (P<0.05) higher lead and cadmium residues were recorded in hair from cows reared around lead-zinc smelter and closed lead cum operational zinc smelter. However, cows from those areas had significantly (P<0.05) higher blood lead but not cadmium concentration as compared to respective control value. Although mean blood lead concentration in cows around aluminum processing plant and urban cum small industrial areas and that of cadmium around steel processing plant were significantly (P<0.05) higher than respective control, the mean hair lead and cadmium content remained statistically (P>0.05) comparable to that of respective control values. The blood lead was significantly correlated with hair lead (r = 0.672, P<0.01) and cadmium (r = 0.309, P<0.05). There was a significant correlation between lead and cadmium concentration (r = 0.610, P<0.01) in hair and a nonsignificant correlation between blood and hair cadmium suggesting that cadmium accumulation in hair was influenced by blood and hair lead concentrations in cows environmentally exposed to lead.

Changes in plasma hormones profile and liver function in cows naturally exposed to lead and cadmium around different industrial areas.

The present study was carried out to assess the endocrine status and liver function in adult cows reared in polluted environment around different industrial units in India. The effect on endocrine system was examined by determination of plasma level of thyroid hormones, thyroxin (T4) (n=269) and triidothyronin (T3) (n=269), stress hormone cortisol (n=266), and reproductive hormones such as estradiol (n=84) and progesterone (n=84) in cows (>3 years) reared around different polluted industrial and non-polluted areas. The respective blood lead and cadmium concentration was also determined in all the cows. The mean plasma levels of both T3 and T4 were significantly (P<0.05) higher around lead zinc smelter (2.43+/-0.26 and 41.1+/-2.9nmol/L) and closed lead cum operational zinc smelter (1.81+/-0.16 and 42.4+/-6.2nmol/L), where the mean blood lead level (0.86+/-0.06 and 0.51+/-0.09mug/ml) was also significantly higher than that of cows (0.07+/-0.01mug/ml) from unpolluted areas. Regression analysis of data from 269 cows revealed a significant (P<0.01) positive correlation between the blood lead and plasma T3 (r=0.287) and T4 (r=0.173). The correlation between thyroidal hormones and the blood cadmium concentration (r=-0.079 and -0.48; P>0.05) was not significant. Plasma cortisol level had also a non-significant (P>0.05) correlation (r=-0.092) with blood lead level.However, the mean cortisol level (4.02+/-1.96nmol/L) of cows in phosphate rock mining areas was significantly (P<0.05) higher than that of controls (1.98+/-0.70nmol/L). The mean plasma estradiol level was significantly (P<0.05) higher in cows around closed lead cum operational zinc smelter (47.1+/-19.5pg/ml) than that of the control animals (21.8+/-3.9pg/ml) and in rest of the areas, the difference did not reach the statistical significance (P>0.05). The serum biochemical analysis in 36 cows around lead-zinc smelter with the highest mean blood lead level (0.86+/-0.06mug/ml) amongst all the industrial/urban areas surveyed, and in 15 animals from non-polluted areas revealed a significant positive correlation between blood lead and serum ALT (alanine transaminase) (r=0.688, P<0.01) and AST (aspartate transaminase) (r=0.390, P<0.01) and a negative correlation with serum total lipids (r=-0.337, P<0.05), total protein (r=-0.449, P<0.01) and albumin(r=-0.662, P<0.01). It is concluded from the study that the natural exposure to lead in polluted environments disturbs the endocrine profile and the higher blood lead level alters serum biochemical parameters indicative of liver functions.

Blood lead levels in lactating cows reared around polluted localities; transfer of lead into milk.

Lead is pervasive environmental pollutant with potential public health hazard as a contaminant of food from animal origin. The present study examines the blood and milk lead level in animals reared in areas around different industrial activities and to find out correlation between blood and milk lead levels in lactating cows. Blood and milk samples (n=149) were collected from animals reared around steel processing unit (n=22), lead-zinc smelter (n=21), aluminum processing plant (n=25), rock phosphate mining area cum phosphate fertilizer plant (n=21), coal mining areas (n=46) and closed lead but functional zinc smelter (n=14). Samples were also collected from randomly chosen 52 lactating cows reared in non-polluted areas to serve as controls. Significantly (P<0.05) higher blood lead level was recorded in animals reared around lead-zinc smelting factories followed by closed lead but functional zinc smelter, aluminum processing unit and steel manufacturing plant, as compared to values recorded for control animals. The highest milk lead level (0.84+/-0.11 microg/ml) was detected in animals reared in the vicinity of lead-zinc smelting unit followed by aluminum processing plant and steel processing unit. Analysis of correlation between blood lead levels and lead excretion in milk through sorting the blood lead values into nine different ranges irrespective of site of collection of samples (n=201) revealed significant correlation (r=0.469 at P<0.01) between blood and milk lead concentrations. The lactating cows with blood lead levels above 0.20 microg/ml (Groups 5 to 9) had significantly (P<0.05) higher milk lead excretion than those with blood lead levels from non-detectable to 0.20 microg/ml (Groups 1 to 4). Pearson correlation analysis between blood and milk lead concentrations in 122 animals with blood lead 0.20 microg/ml indicating that the excretion of lead through milk increases with the increase in blood lead level above 0.20 microg/ml.

Blood lead levels in lactating cows reared around polluted localities; transfer of lead into milk.

Lead is pervasive environmental pollutant with potential public health hazard as a contaminant of food from animal origin. The present study examines the blood and milk lead level in animals reared in areas around different industrial activities and to find out correlation between blood and milk lead levels in lactating cows. Blood and milk samples (n = 149) were collected from animals reared around steel processing unit (n = 22), lead-zinc smelter (n = 21), aluminum processing plant (n = 25), rock phosphate mining area cum phosphate fertilizer plant (n = 21), coal mining areas (n = 46) and closed lead but functional zinc smelter (n = 14). Samples were also collected from randomly chosen 52 lactating cows reared in non-polluted areas to serve as controls. Significantly (P < 0.05) higher blood lead level was recorded in animals reared around lead-zinc smelting factories followed by closed lead but functional zinc smelter, aluminum processing unit and steel manufacturing plant, as compared to values recorded for control animals. The highest milk lead level (0.84 +/- 0.11 microg/ ml) was detected in animals reared in the vicinity of lead-zinc smelting unit followed by aluminum processing plant and steel processing unit. Analysis of correlation between blood lead levels and lead excretion in milk through sorting the blood lead values into 9 different ranges irrespective of site of collection of samples (n = 201) revealed significant correlation (r = 0.469 at P < 0.01) between blood and milk lead concentrations. The lactating cows with blood lead levels above 0.20 microg/ml (groups 5-9) had significantly (P < 0.05) higher milk lead excretion than those with blood lead levels from non detectable to 0.20 microg/ml (groups 1-4). Pearson correlation analysis between blood and milk lead concentrations in 122 animals with blood lead <0.20 microg/ml showed non-significant correlation (r = 0.030 at P < 0.05) but a significant correlation was observed between these two parameters with blood lead level above >0.02 microg/ml indicating that the excretion of lead through milk increases with the increased in blood lead level above 0.20 microg/ml.

Evaluation of a homeopathic complex in the clinical management of udder diseases of riverine buffaloes.

We report an uncontrolled observational study of the treatment of udder diseases of buffalo, using a homeopathic complex medicine. Mastitis is an economically important disease of buffaloes. In India economic losses due to mastitis are estimated at 526 million US dollars annually. Conventional veterinary treatment relies on costly antibiotics; cure rate is only 60% in field conditions with a problem of milk residues. The present investigation was undertaken to evaluate the effectiveness of a homeopathic complex in the management of clinical udder health problems of riverine buffaloes. Cases of subclinical mastitis were excluded from the study. A total of 102 mastitic quarters (fibrosed--40, nonfibrosed--62) and five cases each of blood in milk and udder oedema in lactating buffaloes were treated with a homeopathic complex consisting of Phytolacca 200c, Calcarea fluorica 200c, Silicea 30c, Belladona 30c, Bryonia 30c, Arnica 30c, Conium 30c and Ipecacuanha 30c. The diagnosis of udder diseases and recovery criterion was based on physical examination of udder and milk and CMT/WST score. Bacteriological analysis and somatic cell count were not performed. Treatment was 80 and 96.72% effective in cases of fibrotic mastitis and nonfibrosed mastitis respectively. Recovery period was 21-42 days (fibrosed) and 4-15 days (nonfibrosed). Udder oedema and blood in milk responded favourably in 2-5 days. Cost of treatment was 0.07 US dollars per day. The homeopathic complex medicine may be effective and economical in the management of udder health problems of buffaloes. Definitive conclusions are premature due to the limited number of observations and lack of control group.

Evaluation of a homeopathic complex in the clinical management of udder diseases of riverine buffaloes

We report an uncontrolled observational study of the treatment of udder diseases of buffalo, using a homeopathic complex medicine. Mastitis is an economically important disease of buffaloes. In India economic losses due to mastitis...AU