Detection of antimicrobial resistance genes in extended spectrum beta-lactamase-producing Escherichia coli from milk of indigenous Beetal goats of Punjab.

Background: Antimicrobial resistance (AMR) is a burning issue in the present era. Mastitis in dairy animals is one of the most important causes of huge production loss to dairy farmers. Aims: The study aims to find the prevalence, antimicrobial resistance profile, and resistance genes in the extended-spectrum beta-lactamase-producing Escherichia coli in mastitic milk. Methods: A total of 125 milk samples were collected from Beetal goats suffering from clinical mastitis from different districts of Punjab and processed for bacterial isolation and further identification. The drug resistance profile of ESBL-producing E. coli and its associations with molecular markers was analyzed using statistical analysis. Results: The prevalence of ESBL-producing E. coli in dairy goats of Punjab was recorded as 6.4%. The isolates showed the highest resistance to the beta-lactam group of antibiotics. The resistance percentages of streptomycin, gentamicin, tetracycline, chloramphenicol, clotrimazole, and colistin were 50%, 37.5%, 50%, 25%, 25%, and 50%, respectively. The isolates showed intermediate resistance to imipenem (12.5%) and tetracycline (25%). The ESBL-producing E. coli isolates harbored the resistance genes blaCTXM (100%), blaTEM (62.5%), blaSHV (25%), blaOXA (37.5%), tetA (37.5%), tetB (25%), aadA (37.5%), sul1 (25%), MOXM (12.5%), DHAM (25%), and blaCMY-2 (50%). Tetracycline and sulphonamide resistances were statistically associated with their respective resistance genes (P<0.05). Streptomycin resistance was not statistically associated with the presence of the aadA gene (P>0.05). The genes blaIMP and blaNDM were not recorded in any of the isolates. In this study, 12.5% of the isolates showed co-resistance to colistin and carbapenem. Conclusion: Antimicrobial resistance is a hot topic and requires immediate attention.

Modelling the control of bovine brucellosis in India.

Brucellosis imposes substantial impacts on livestock production and public health worldwide. A stochastic, age-structured model incorporating herd demographics was developed describing within- and between-herd transmission of Brucella abortus in dairy cattle herds. The model was fitted to data from a cross-sectional study conducted in Punjab State of India and used to evaluate the effectiveness of control strategies under consideration. Based on model results, stakeholder acceptance and constraints regarding vaccine supply, vaccination of replacement calves in large farms should be prioritized. Test and removal applied at early stages of the control programme where seroprevalence is high would not constitute an effective or acceptable use of resources because significant numbers of animals would be 'removed' (culled or not used for breeding) based on false positive results. To achieve sustained reductions in brucellosis, policymakers must commit to maintaining vaccination in the long term, which may eventually reduce frequency of infection in the livestock reservoir to a low enough level for elimination to be a realistic objective. This work provides key strategic insights into the control of brucellosis in India, which has the largest cattle population globally, and a general modelling framework for evaluating control strategies in endemic settings.

Plasma Prolactin and Total Lipid Levels and Subsequent Risk of Breast Cancer in Pre- and Postmenopausal Women: Experience from an Indian Rural Centre.

AIMS AND OBJECTIVES: 1) To analyze serum lipid and prolactin levels in breast cancer patients and normal subjects; 2) to correlate those levels with risk and prognostic factors. MATERIAL AND METHODS: The present study was performed in the Department of Surgery, MMIMSR, Mullana, Ambala, from 2013 to 2014, at a rural centre. The study group comprised 40 patients with carcinoma of the breast who underwent surgery and the control group included 10 patients who underwent surgery for reasons other than carcinoma of the breast. Apart from routine tests, special investigations like estimation of serum lipids and prolactin levels were carried out in each patient to assess the general health status and detect any potential evidence of distance metastasis. RESULTS: Most patients were in the fourth and fifth decade of life. The mean value of serum total cholesterol in the study group (190.77 mg/dL) was higher than that of the control group (166.22 mg/dL), which was statistically significant. The mean value of LDL in the study group was 153.8 mg/dL, as compared to 118.4 mg/dL in the control group; therefore, the difference in LDL cholesterol levels between the two groups was statistically significant. The VLDL level was also higher in breast cancer patients, with a mean value of 35.25 mg/dL, as compared to 22.6 mg/dL in the control group. Serum triglycerides showed higher trends in the study group than in controls. The correlation coefficient of total lipids and prolactin was 0.428, which was significant (p value 0.002), and pointed to a positive relation between prolactin and total lipids, meaning that an elevation in total lipids would lead to an increase in prolactin levels. CONCLUSIONS: It was observed that significantly increased prolactin levels were found among patients with breast cancer. Serum lipids in carcinoma of the breast had higher levels of VLDL and LDL cholesterol and elevated triglyceride concentrations. Serum prolactin showed a statistically significant elevation in premenopausal patients as compared to postmenopausal subjects with breast cancer. Prolactin level may be also one of the risk factors for breast cancer, which points to its diagnostic significance.

Detection of Mycobacterium avium subsp. paratuberculosis in bovine faecal samples by Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) TaqMan Assay.

Mycobacterium avium subsp. paratuberculosis, the causative agent of Johne's disease is a degenerative chronic granulomatous disease of bovines. In the present study, quantitative real time polymerase chain reaction (qRT-PCR) using TaqMan chemistry targeting the IS900 sequence of Mycobacterium avium subsp. paratuberculosis (MAP) was employed for the molecular diagnosis of the disease in bovine faecal samples. Out of 200 bovine faecal samples processed, 7 samples were tested as positive by IS900 qRT-PCR. The sensitivity limit of detection of MAP DNA in faecal samples by qRT-PCR TaqMan assay was found to be 0.05pg. No amplification was observed in other Mycobacterial spp. viz. M. phlei, M. smegmatis, M. intracellulare and M. kansasii.

Detection of Mycobacterium avium subsp. Paratuberculosis in bovine faecal samples by Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) TaqMan Assay

Mycobacterium avium subsp. paratuberculosis, the causative agent of Johne’s disease is a degenerative chronic granulomatous disease of bovines. In the present study, quantitative real time polymerase chain reaction (qRT-PCR) using TaqMan chemistry targeting the IS900 sequence of Mycobacterium avium subsp. paratuberculosis (MAP) was employed for the molecular diagnosis of the disease in bovine faecal samples. Out of 200 bovine faecal samples processed, 7 samples were tested as positive by IS900 qRT-PCR. The sensitivity limit of detection of MAP DNA in faecal samples by qRT-PCR TaqMan assay was found to be 0.05pg. No amplification was observed in other Mycobacterial spp. viz. M. phlei, M. smegmatis, M. intracellulare and M. kansasii.

Isolation of Canine parvovirus with a view to identify the prevalent serotype on the basis of partial sequence analysis.

AIM: The aim of this study was to isolate Canine parvovirus (CPV) from suspected dogs on madin darby canine kidney (MDCK) cell line and its confirmation by polymerase chain reaction (PCR) and nested PCR (NPCR). Further, VP2 gene of the CPV isolates was amplified and sequenced to determine prevailing antigenic type. MATERIALS AND METHODS: A total of 60 rectal swabs were collected from dogs showing signs of gastroenteritis, processed and subjected to isolation in MDCK cell line. The samples showing cytopathic effects (CPE) were confirmed by PCR and NPCR. These samples were subjected to PCR for amplification of VP2 gene of CPV, sequenced and analyzed to study the prevailing antigenic types of CPV. RESULTS: Out of the 60 samples subjected to isolation in MDCK cell line five samples showed CPE in the form of rounding of cells, clumping of cells and finally detachment of the cells. When these samples and the two commercially available vaccines were subjected to PCR for amplification of VP2 gene, a 1710 bp product was amplified. The sequence analysis revealed that the vaccines belonged to the CPV-2 type and the samples were of CPV-2b type. CONCLUSION: It can be concluded from the present study that out of a total of 60 samples 5 samples exhibited CPE as observed in MDCK cell line. Sequence analysis of the VP2 gene among the samples and vaccine strains revealed that samples belonged to CPV-2b type and vaccines belonging to CPV-2.

Antigenic typing of canine parvovirus using differential PCR.

Canine parvovirus (CPV) is an enteric pathogen causing hemorrhagic enteritis in pups of 3-6 months of age and is mainly transmitted via feco-oral route. In the present study, a total of 85 animals rectal swabs suspected of CPV were tested using a PCR, nested PCR and a newly designed differential PCR. Using PCR 7 (8.23 %) animals were positive whereas 39 (45.88 %) were positive by using nested PCR and 40 (47.05 %) were positive for either one or more than one antigenic types of CPV using differential PCR. Using differential PCR it was found that CPV-2a and CPV-2b were the most prevailing antigenic types. Also it was found that dogs that were vaccinated too yielded positive CPV indicating a possible presence of additional CPV antigenic types. Thus, the primers used in differential PCR can be used in a single PCR reaction to detect various antigenic types of CPV.

The Lanindar test: a method of evaluating patient suitability for cataract surgery using assisted topical anaesthesia.

AIM: To evaluate an office-based Lanindar (light and nociceptive interaction noting distress and response) test to assist in the assessment of patient suitability for assisted topical anaesthesia (ATA) during phacoemulsification. METHODS: The Lanindar test was carried out at the preoperative assessment of 716 consecutive patients in the office of one of the authors (ICF). A standard desk lamp was shone in each patient's eye after pupillary dilation, while simultaneously elevating the upper eyelid digitally. A negative test indicated patient hypersensitivity to the light and aversion to digital pressure on the upper eyelid. A positive test was indicated by the patient feeling comfort and lack of blepharospasm and withdrawal in response to the light and digital pressure. chi (2) and Fisher's exact tests were used to assess the association between Lanindar results and suitability for ATA. The positive predictive value and specificity of the test as an indicator of patient suitability for ATA were calculated. Visual acuities at 1 and 4 weeks post-operative periods were compared between the ATA and ALA/GA (assisted local anaesthesia/general anaesthesia) group of patients. RESULTS: About 86.7% were Lanindar positive and 98.9% of these patients tolerated ATA. chi (2) and Fisher's exact tests demonstrated a significant association of a positive Lanindar test with successful ATA (chi (2)=660, P<0.001, Fisher's: P<0.001). The positive predictive value and specificity of the test were 98% (95% CI=98.04-99.7%) and 93.14% (95% CI=88.23-98.04%), respectively. Visual acuity outcomes were similar in the ATA and ALA/GA groups. CONCLUSION: The Lanindar is a simple, highly specific, office-based test to determine patient suitability for phacoemulsification under ATA.

Patient perceptions of second eye clear corneal cataract surgery using assisted topical anaesthesia.

AIM: To assess patient recall of intraoperative pain, anxiety, fear, and sensory (visual and auditory) perceptions during second eye clear corneal cataract surgery using assisted topical anaesthesia (ATA), in comparison with first eye cataract surgery using the same technique. METHODS: This prospective, consecutive, observational study was conducted in a free-standing dedicated ophthalmic day surgery centre. A voluntary questionnaire was distributed to 129 consecutive patients who underwent clear corneal cataract surgery using ATA. Two patients had to be converted to block anaesthesia, and were excluded. Patients were asked to rate intraoperative pain, anxiety, and fear using a visual analogue scale (VAS), and recollection of intraoperative visual and auditory perceptions. Results were analysed using the Mann-Whitney U and Spearman correlation tests. RESULTS: There were 70/127 (55%) patients undergoing first eye cataract surgery and 57/127 (45%) undergoing second eye surgery. There was no significant difference in mean pain, anxiety, and fear scores between those undergoing the second eye operation compared with those undergoing their first eye operation. Similarly, there was no significant difference in sensory perceptions between the two cohorts. Overall, there was a small but significant positive correlation between recall of visual and auditory perceptions and combined pain, fear, and anxiety scores (r=0.33, P=0.0002). CONCLUSION: There was no significant difference in levels of intraoperative pain, anxiety, fear, and sensory perceptions experienced by patients between the first eye and second eye surgeries. We recommend that preoperative counselling for a patient's second eye be as comprehensive as for the first eye surgery.

Sodium butyrate-treated embryonic stem cells yield hepatocyte-like cells expressing a glycolytic phenotype.

Embryonic stem cells serve as a promising technology to obtain specific cell types for a number of biomedical applications. Because traditional techniques, such as embryoid body formation result in a wide array of differentiated cells such as hepatic, neuronal, and cardiac lineages, strategies have been utilized which favor cell-specific differentiation to generate more uniformity. In the present study, we have investigated the use of sodium butyrate in a monolayer culture configuration to mediate hepatocyte differentiation of murine embryonic stem cells. Several functional assays used to characterize hepatocyte function (viz. urea secretion, intracellular albumin content, cytokeratin 18, and glycogen staining) were used to analyze the differentiating cell population, suggesting the presence of an enriched population of hepatocyte-like cells. Since mature hepatocytes mediate energy metabolism predominantly through oxidative means as opposed to hepatocyte precursors, which are primarily glycolytic, we have performed a kinetic analysis of glycolytic and functional capacity to characterize the differentiated cells. In conjunction with mitochondrial mass and activity measurements, we show that Na-butyrate-mediated differentiated cells mediate energy metabolism predominantly through glycolysis. This metabolic and mitochondrial characterization can assist in evaluating stem cell differentiation and may prove useful in identifying key regulatory molecules in mediating further differentiation.

Novel quantitative tools for engineering analysis of hepatocyte cultures in bioartificial liver systems.

Extracorporeal bioartificial liver devices (BAL) are perhaps among the most promising technologies for the treatment of liver failure, but significant technical challenges remain in order to develop systems with sufficient processing capacity and of manageable size. One key limitation is that during BAL operation, when the device is exposed to plasma from the patient, hepatocytes are prone to accumulate intracellular lipids and exhibit poor liver-specific functions. Based on hepatic intermediary metabolism, we have utilized mathematical programming techniques to optimize the biochemical environment of hepatocyte cultures towards the desired effect of increased albumin and urea synthesis. To investigate the feasible range of optimal hepatic function, we have obtained a Pareto optimal set of solutions corresponding to liver-specific functions of urea and albumin secretion in the metabolic framework using multiobjective optimization. The importance of amino acids in the supplementation and the criticality of the metabolic pathways have been investigated using logic-based programming techniques. Since the metabolite measurements are bound to be patient specific, and hence subject to variability, uncertainty has to be integrated with system analysis to improve the prediction of hepatic function. We have used the concept of two stage stochastic programming to obtain robust solutions by considering extracellular variability. The proposed analysis represents a new systematic approach to analyze behavior of hepatocyte cultures and optimize different operating parameters for an extracorporeal device based on real-time conditions.