Antimicrobial-resistant non-typhoidal Salmonella enterica prevalence among poultry farms and slaughterhouses in Chitwan, Nepal.

BACKGROUND AND AIM: Antibiotic-resistant infections are one of the leading threats to public health globally. Indiscriminate use of antibiotics in food animal production is an important driver of resistance, particularly among foodborne pathogens such as non-typhoidal Salmonella enterica (NTS). While there has been extensive research on antimicrobial-resistant (AMR) S. enterica in India and China, there have been few studies in countries in South Asia, including Nepal. This is particularly important with the rise of commercial poultry farming in Nepal as a means of economic development and nutritional subsistence. This descriptive study seeks to identify the prevalence and resistance patterns of NTS serotypes focusing on Chitwan, Nepal's leading poultry producing district. MATERIALS AND METHODS: A mixture of purposive and judgment sampling of 18 poultry farms and 20 slaughterhouses representing a broad geographic distribution across multiple municipalities in Chitwan was conducted in May 2019. Environmental samples taken from poultry farms included: Water, litter, feces, feed, farm swabs, and eggshell swabs. Biological samples taken from nearby slaughterhouses included: Muscle, heart, liver, skin, cecum, crop, and spleen. Samples were cultured and tested for the presence of NTS. Positive isolates were serotyped and tested for antimicrobial susceptibility to seven antibiotics known to be important to both human and animal health regionally. Farm practices were also characterized through a survey, the results of which are detailed in the accompanying paper. RESULTS: Out of 708 samples (288 environmental and 420 biological), 103 (15%) tested positive for NTS (9% of environmental; n=26, 18% of biological; n=77). The percentage of positive environmental and biological samples varied by source. Environmental sample positive rates were water (27.5%), feces (10.6%), litter (8.6%), farm swabs (5%), feed (1.8%), and eggshells (0%). Biological sample positive rates were skin (28%), heart (23%), crop (20%), muscle (15%), liver (15%), spleen (15%), and cecum (12%). Out of 103 positive S. enterica isolates, 48.5% were identified as Salmonella Typhimurium, 35% Salmonella Enteritidis, 7.8% Salmonella Gallinarum, 4.9% Salmonella Virchow, and 3.9% were Salmonella Agona. Of the 103 positive isolates, 80 (78%) were resistant to at least one antibiotic, and 21 (20%) were multidrug-resistant (MDR). CONCLUSION: NTS is highly prevalent among Chitwan's growing poultry industry with higher rates of positivity found in slaughterhouse samples compared with environmental samples from farms. In addition, a high rate of AMR (78%) was revealed, and an extremely concerning number of those were shown to be MDR (20%). This baseline data has important implications for poultry production and consumption in the region. Further research will elucidate the extent to which this contamination and drug resistance is impacting the health of the local population and help inform treatment and management strategies.Note: The characterization of the poultry industry and practices that might be linked to NTS contamination in the Chitwan district are detailed in the previous paper in this series (www.veterinaryworld.org/Vol.14/February-2021/14.pdf).

Prevalence of non-typhoidal Salmonella and risk factors on poultry farms in Chitwan, Nepal.

BACKGROUND AND AIM: Poultry is becoming an increasingly important source of protein in the Nepalese diet. The Chitwan region of Nepal is the hub of the emerging poultry industry. Little is known about the prevalence of non-typhoidal Salmonella (NTS) on poultry farms or the role of farm management practices that may contribute to the presence of NTS on farms. The role of poultry in the transmission of Salmonella enterica to humans is also poorly defined. This descriptive study seeks establish baseline data through estimation of the prevalence of NTS on broiler and layer operations in various farms of the Chitwan district of Nepal. MATERIALS AND METHODS: Based on district documents on poultry production and meat marketing, a purposive sampling of 18 commercial poultry farms comprising ten broilers farms and eight layers farms was conducted. Environmental samples including water, litter, feces, feed, farm, and eggshell swabs were randomly collected from each farm. Samples were cultured and tested for the presence of NTS; positives were serotyped, and antimicrobial susceptibility determined. A comprehensive farm and practice questionnaire was administered to each farm manager. RESULTS: The farm level point prevalence rate was 55% (10 of 18 farms) for S. enterica. Of the total 288 farm environmental samples collected, 26 samples (9%) were positive. The rate of isolation varied according to the origin of samples: Water (27.5%), feces (10.6%), litter (8.6%), farm swabs (5%), feed (1.8%), and eggshells (0%). Farm management variables/risk factors are summarized and categorized as non-modifiable and modifiable for analysis. Broiler operations were more likely to be positive than layer operations as were poultry houses with two or less open sides. All-in/all-out management style was found to be protective. Due to the small sample size (18 farms), no associations reached statistical significance. CONCLUSION: Based on environmental sampling results, NTS is highly prevalent on the poultry farms in the Chitwan district of Nepal. Certain risk factors are associated with finding NTS on farms. Our findings are generally in agreement with other studies in similar countries with rapidly emerging poultry industries. The identification of risk factors provides owners, technicians, and veterinarians with some guidance to help reduce the prevalence of NTS on farms. This baseline data are critical to understanding the epidemiology of zoonotic strain of NTS in the region and are necessary for the design of future studies and mitigation plans and underlines the need for a one-health approach to protect public health-related to Salmonella spp. from poultry farms.

Erratum to "Sero-prevalence of Toxoplasma gondii in sheep in different geographical regions of Nepal" [5C (June 2018) 7-9].

[This corrects the article DOI: 10.1016/j.vas.2018.01.001.].

Lrig2 and Hpse2, mutated in urofacial syndrome, pattern nerves in the urinary bladder.

Mutations in leucine-rich-repeats and immunoglobulin-like-domains 2 (LRIG2) or in heparanase 2 (HPSE2) cause urofacial syndrome, a devastating autosomal recessive disease of functional bladder outlet obstruction. It has been speculated that urofacial syndrome has a neural basis, but it is unknown whether defects in urinary bladder innervation are present. We hypothesized that urofacial syndrome features a peripheral neuropathy of the bladder. Mice with homozygous targeted Lrig2 mutations had urinary defects resembling those found in urofacial syndrome. There was no anatomical blockage of the outflow tract, consistent with a functional bladder outlet obstruction. Transcriptome analysis revealed differential expression of 12 known transcripts in addition to Lrig2, including 8 with established roles in neurobiology. Mice with homozygous mutations in either Lrig2 or Hpse2 had increased nerve density within the body of the urinary bladder and decreased nerve density around the urinary outflow tract. In a sample of 155 children with chronic kidney disease and urinary symptoms, we discovered novel homozygous missense LRIG2 variants that were predicted to be pathogenic in 2 individuals with non-syndromic bladder outlet obstruction. These observations provide evidence that a peripheral neuropathy is central to the pathobiology of functional bladder outlet obstruction in urofacial syndrome, and emphasize the importance of LRIG2 and heparanase 2 for nerve patterning in the urinary tract.

Sero-prevalence of Toxoplasma gondii in sheep in different geographical regions of Nepal.

The present study was conducted to investigate the prevalence of Toxoplasma gondii in sheep in Nepal. Blood samples were collected from 235 sheep from three districts of three different eco-zones namely, mountainous Jumla (88), hilly Pokhara (62) and plain/terai Chitwan (85). The samples were tested by using commercial ELISA kit. The overall prevalence of T. gondii infection in sheep was 36.17% (CI: 30.29-42.49%). The region wise prevalence showed highest in Chitwan (57.65%; CI: 47.04-67.60%), followed by Pokhara (32.94%; CI: 23.88-43.48%) and Jumla (9.41%; CI: 4.85-17.49%). Prevalence of T. gondii in Jumla was significantly lower than Pokhara and Chitwan (p < 0.05) but no significant difference in seroprevalence was encountered between Pokhara and Chitwan (p > 0.05). Similarly, no significant difference (p > 0.05) in prevalence of T. gondii was found in sex and age groups (p > 0.05). The result showed that T. gondii parasite is widely spread in the studied geographical regions of Nepal.

Cytochrome P450 2E1 (CYP2E1) regulates the response to oxidative stress and migration of breast cancer cells.

INTRODUCTION: The cytochrome P450 (CYP) enzymes are a class of heme-containing enzymes involved in phase I metabolism of a large number of xenobiotics. The CYP family member CYP2E1 metabolises many xenobiotics and pro-carcinogens, it is not just expressed in the liver but also in many other tissues such as the kidney, the lung, the brain, the gastrointestinal tract and the breast tissue. It is induced in several pathological conditions including cancer, obesity, and type II diabetes implying that this enzyme is implicated in other biological processes beyond its role in phase I metabolism. Despite the detailed description of the role of CYP2E1 in the liver, its functions in other tissues have not been extensively studied. In this study, we investigated the functional significance of CYP2E1 in breast carcinogenesis. METHODS: Cellular levels of reactive oxygen species (ROS) were measured by H2DCFDA (2 2.9.2 2',7'-dichlorodihydrofluorescein diacetate) staining and autophagy was assessed by tracing the cellular levels of autophagy markers using western blot assays. The endoplasmic reticulum stress and the unfolded protein response (UPR) were detected by luciferase assays reflecting the splicing of mRNA encoding the X-box binding protein 1 (XBP1) transcription factor and cell migration was evaluated using the scratch wound assay. Gene expression was recorded with standard transcription assays including luciferase reporter and chromatin immunoprecipitation. RESULTS: Ectopic expression of CYP2E1 induced ROS generation, affected autophagy, stimulated endoplasmic reticulum stress and inhibited migration in breast cancer cells with different metastatic potential and p53 status. Furthermore, evidence is presented indicating that CYP2E1 gene expression is under the transcriptional control of the p53 tumor suppressor. CONCLUSIONS: These results support the notion that CYP2E1 exerts an important role in mammary carcinogenesis, provide a potential link between ethanol metabolism and breast cancer and suggest that progression, and metastasis, of advanced stages of breast cancer can be modulated by induction of CYP2E1 activity.

Potent dual inhibitors of TORC1 and TORC2 complexes (KU-0063794 and KU-0068650) demonstrate in vitro and ex vivo anti-keloid scar activity.

Mammalian target of rapamycin (mTOR) is essential in controlling several cellular functions. This pathway is dysregulated in keloid disease (KD). KD is a common fibroproliferative dermal lesion with an ill-defined treatment strategy. KD demonstrates excessive matrix deposition, angiogenesis, and inflammatory cell infiltration. In KD, both total and phosphorylated forms of mTOR and p70(S6K)(Thr421/Ser424) are upregulated. Therefore, the aim of this study was to investigate adenosine triphosphate-competitive inhibitors of mTOR kinase previously unreported in keloid and their comparative efficacy with Rapamycin. Here, we present two mTOR kinase inhibitors, KU-0063794 and KU-0068650, that target both mTORC1 and mTORC2 signaling. Treatment with either KU-0063794 or KU-0068650 resulted in complete suppression of Akt, mTORC1, and mTORC2, and inhibition of keloid cell spreading, proliferation, migration, and invasive properties at a very low concentration (2.5 µmol l(-1)). Both KU-0063794 and KU-0068650 significantly (P<0.05) inhibited cell cycle regulation and HIF1-α expression compared with that achieved with Rapamycin alone. In addition, both compounds induced shrinkage and growth arrest in KD, associated with the inhibition of angiogenesis, induction of apoptosis, and reduction in keloid phenotype-associated markers. In contrast, Rapamycin induced minimal antitumor activity. In conclusion, potent dual mTORC1 and mTORC2 inhibitors display therapeutic potential for the treatment of KD.

Superior effect of combination vs. single steroid therapy in keloid disease: a comparative in vitro analysis of glucocorticoids.

Keloid disease (KD) is a fibroproliferative disorder of unknown etiology. Current use of corticosteroid injection is partially beneficial with 80% recurrence rate. Additionally, the efficacy of different steroids, alone or in combination as opposed to monotherapy, in treating KD remains unclear. Here, we compared the single and combined efficacy of glucocorticoids-dexamethasone (Dex), triamcinolone (TAC), and methylprednisolone (Medrol)-on primary keloid fibroblasts (KFs) (n = 27) and normal skin (n = 19) fibroblasts at cellular, protein, and messenger RNA levels in vitro. Our results demonstrated that cytotoxicity to steroids was dose dependent. Cell spreading, attachment, and proliferation were significantly (p < 0.05) reduced by Medrol and TAC. Migration and invasion properties of KF were inhibited significantly (p < 0.05) by Medrol and TAC compared with Dex. At both protein and messenger RNA levels, keloid-associated fibrotic markers were significantly (p < 0.05) decreased by Medrol and TAC compared with Dex. However, vascular endothelial growth factor expression was significantly (p = 0.01) decreased by Dex compared with TAC and Medrol. Medrol and TAC caused significant (p < 0.04) apoptosis, whereas Dex inhibited the UV-induced apoptosis and up-regulated survivin. Blocking of glucocorticoid receptor by RU486 inhibited cytoprotective property of Dex and apoptotic properties of TAC and Medrol. Double treatment with Dex + TAC and Dex + Medrol significantly (p < 0.05) induced apoptosis. In conclusion, this is the first study to report the efficacy of three well-known steroids on KF and suggest that combination may be superior than using a single steroid in treating KD.

Keloid disease can be inhibited by antagonizing excessive mTOR signaling with a novel dual TORC1/2 inhibitor.

Keloid disease (KD) is a fibroproliferative lesion of unknown etiopathogenesis that possibly targets the PI3K/Akt/mTOR pathway. We investigated whether PI3K/Akt/mTOR inhibitor, Palomid 529 (P529), which targets both mammalian target of rapamycin complex 1 (mTORC-1) and mTORC-2 signaling, could exert anti-KD effects in a novel KD organ culture assay and in keloid fibroblasts (KF). Treatment of KF with P529 significantly (P < 0.05) inhibited cell spreading, attachment, proliferation, migration, and invasive properties at a low concentration (5 ng/mL) and induced substantial KF apoptosis when compared with normal dermal fibroblasts. P529 also inhibited hypoxia-inducible factor-1α expression and completely suppressed Akt, GSK3ß, mTOR, eukaryotic initiation factor 4E-binding protein 1, and S6 phosphorylation. P529 significantly (P < 0.05) inhibited proliferating cell nuclear antigen and cyclin D and caused considerable apoptosis. Compared with rapamycin and wortmannin, P529 also significantly (P < 0.05) reduced keloid-associated phenotypic markers in KF. P529 caused tissue shrinkage, growth arrest, and apoptosis in keloid organ cultures and substantially inhibited angiogenesis. pS6, pAkt-Ser473, and mTOR phosphorylation were also suppressed in situ. P529 reduced cellularity and expression of collagen, fibronectin, and α-smooth muscle actin (substantially more than rapamycin). These pre-clinical in vitro and ex vivo observations are evidence that the mTOR pathway is a promising target for future KD therapy and that the dual PI3K/Akt/mTOR inhibitor P529 deserves systematic exploration as a candidate agent for the future treatment of KD.

In vitro study of novel collagenase (XIAFLEX®) on Dupuytren's disease fibroblasts displays unique drug related properties.

Dupuytren's disease (DD) is a benign, fibroproliferative disease of the palmar fascia, with excessive extracellular matrix (ECM) deposition and over-production of cytokines and growth factors, resulting in digital fixed flexion contractures limiting hand function and patient quality of life. Surgical fasciectomy is the gold standard treatment but is invasive and has associated morbidity without limiting disease recurrence. Injectable Collagenase Clostridium histolyticum (CCH)--Xiaflex®--is a novel, nonsurgical option with clinically proven in vivo reduction of DD contractures but with limited in vitro data demonstrating its cellular and molecular effects. The aim of this study was to delineate the effects of CCH on primary fibroblasts isolated from DD and non-DD anatomical sites (using RTCA, LDH, WST-1, FACS, qRT-PCR, ELISA and In-Cell Quantitative Western Blotting) to compare the efficacy of varying concentrations of Xiaflex® against a reagent grade Collagenase, Collagenase A. Results demonstrated that DD nodule and cord fibroblasts had greater proliferation than those from fat and skin. Xiaflex® exposure resulted in dose- and time-dependent inhibition of cellular spreading, attachment and proliferation, with cellular recovery after enzyme removal. Unlike Collagenase A, Xiaflex® did not cause apoptosis. Collagen expression patterns were significantly (p<0.05) different in DD fibroblasts across anatomical sites - the highest levels of collagen I and III were detected in DD nodule, with DD cord and fat fibroblasts demonstrating a smaller increase in both collagen expression relative to DD skin. Xiaflex® significantly (p<0.05) down-regulated ECM components, cytokines and growth factors in a dose-dependent manner. An in vitro scratch wound assay model demonstrated that, at low concentrations, Xiaflex® enabled a faster fibroblast reparatory migration into the wound, whereas, at high concentrations, this process was significantly (p<0.05) inhibited. This is the first report elucidating potential mechanisms of action of Xiaflex® on Dupuytren fibroblasts, offering a greater insight and a better understanding of its effect in DD.