Molecular mechanism of antimicrobial co-resistance Colistin (mcr-1) and ESBLs genes among Escherichia coli isolates from commercial chickens in Pakistan.

Emergence of plasmid mediated colistin and extended spectrum ß-lactamases (ESBL) resistant genes has been impacted the efficacy of colistin and ß-lactams drugs like 3rd, 4th generation cephalosporin. Current study was aimed to investigate antimicrobial resistance genes (ARGs) among Escherichia coli isolates from meat producing commercial broilers in Pakistan. Two hundred (n=200) fecal samples were collected during January-2018 to August-2019. For isolation of E. coli, pink colonies on MacConkey agar were transferred to EMB agar. Metallic sheen color colonies were tested biochemically using API-20E kit. The molecular identification of E. coli (n=153) was targeted by amplification of uid gene through polymerase chain reaction (PCR) and different ARGs i.e. gentamicin, streptomycin, tetracycline, colistin, ß-lactams drugs, quinolone and ampicillin followed by sequence analysis. Genotypically, followed by phenotypically of resistant ARGs of isolated PCR-confirmed E. coli (153) shoed resistant against gentamicin (aac(3)-IV), streptomycin (aadA1), tetracycline (tetA), colistine (mcr-1), ampicillin (bla-TEM) and bla-CTX-M were 86%, 88%, 86%, 88%, 83% & 77% respectively. 33/38 (86%) of the isolate was positive for quinolone resistance. Colistine (mcr-1), ESBLs (bla-TEM) and (bla-CTX-M) resistance genes were 88%, 83% and 77% respectively. About 33 isolated E. coli harbored the both mcr-1 and ESBLs genes. All of E. coli isolates were found sensitive to ceftriaxone (CTX-30) and imipenem (IMP-10). The Isolated E. coli showed single or multi clade decadency. The E. coli and ARGs sequences showed single or multi clade decadency. This is first comprehensive study from Pakistan that described the molecular evidences of ARGs and their co-existence in single isolates originated from commercial poultry. Commercial chicken (Broilers) can act as melting pot of antibiotic resistance genes for human being. It is alarming situation for surveillance of antibiotic resistance program because of more regulated prescription of antimicrobial agents in Pakistan.

Sero-epidemiology and risk factor analysis of human brucellosis in Punjab, Pakistan: a cross sectional study.

Human brucellosis is a neglected zoonotic problem worldwide with a high degree of morbidity in humans and is mostly overlooked due to other febrile conditions. The aim of this study was to evaluate the sero-prevalence and risk factors of human brucellosis among subjects living in Punjab, Pakistan. In this cross-sectional study, human blood samples were collected from seven districts of Punjab, Pakistan. Information regarding personal data, demographic data and potential risk factors was collected through a structured questionnaire. Detection of anti-Brucella antibodies was done through Rose Bengal Plate Test (RBPT) and Enzyme Linked Immunosorbent Assay (ELISA). Descriptive analysis, Chi square test and Odds ratio was applied using STATA software version 12. The sero-prevalence of human brucellosis was 13.13% with significantly higher percentage in males 17.23% and age group 25-40 years 16.50% (P=< 0.001). The demographic factors positively associated with human brucellosis were lack of education (P = 0.003; OR = 1.85) and farming as an occupation (P =<0.001; OR = 2.50) Similarly, among the risk factors studied, keeping animals at home (P =<0.001; OR = 2.03), slaughtering of animals (P =<0.001; OR = 15.87) and consuming raw milk (P =<0.001; OR = 5.42) were the factors strongly connected with human brucellosis. A massive awareness should be given to livestock farmers and individuals directly linked to animals regarding risk factors and transmission of brucellosis. Consumption of unpasteurized milk and its products should be condemned to curtail this neglected disease.

Prevalence and risk factors of Syphilis among blood donors of Punjab, Pakistan.

Syphilis is a sexually transmitted disease and its actual prevalence among Pakistani blood donors is currently unknown. A cross sectional study was conducted at different district healthcare hospitals of Punjab, Pakistan with an aim to evaluate the prevalence and risk factors associated with syphilis in blood donors using immunochromatographic test (ICT) and enzyme linked immunosorbent assay (ELISA). A total (n=1200) blood samples were collected from donors aged 18-65 years. All the information regarding personal data, demographic data and risk factors was collected via structured questionnaire. On the basis of ICT and ELISA, the overall prevalence of syphilis was 3.91% among blood donors. The demographic factors positively linked with syphilis were age (P= 0.000; Odds ratio, OR= 7.18; 95% confidence interval CI= 2.816-18.295) and education status (P= 0.000; Odds ratio, OR= 12.33; 95% confidence interval CI= 3.469-43.849) of donors. Similarly among the risk factors analyzed, marital status (P= 0.012; Odds ratio OR= 2.251; 95% confidence interval CI= 1.206- 4.202) and blood transfusion history (P= 0.030; Odds ratio OR= 1.981; 95% confidence interval CI= 1.083-3.623) were also strongly associated with syphilis. We emphasized the importance of promoting preventive measures for syphilis. The syphilis diagnosis should not be based on a single test. The present study indicates that higher prevalence is alarming for blood donors in Pakistan. Stringent donor screening is highly recommended to ensure maximum safe blood transfusion.

Sero-epidemiology and risk factor analysis of human brucellosis in Punjab, Pakistan: a cross sectional study

@#Human brucellosis is a neglected zoonotic problem worldwide with a high degree of morbidity in humans and is mostly overlooked due to other febrile conditions. The aim of this study was to evaluate the sero-prevalence and risk factors of human brucellosis among subjects living in Punjab, Pakistan. In this cross-sectional study, human blood samples were collected from seven districts of Punjab, Pakistan. Information regarding personal data, demographic data and potential risk factors was collected through a structured questionnaire. Detection of anti-Brucella antibodies was done through Rose Bengal Plate Test (RBPT) and Enzyme Linked Immunosorbent Assay (ELISA). Descriptive analysis, Chi square test and Odds ratio was applied using STATA software version 12. The sero-prevalence of human brucellosis was 13.13% with significantly higher percentage in males 17.23% and age group 25-40 years 16.50% (P=< 0.001). The demographic factors positively associated with human brucellosis were lack of education (P = 0.003; OR = 1.85) and farming as an occupation (P =<0.001; OR = 2.50) Similarly, among the risk factors studied, keeping animals at home (P =<0.001; OR = 2.03), slaughtering of animals (P =<0.001; OR = 15.87) and consuming raw milk (P =<0.001; OR = 5.42) were the factors strongly connected with human brucellosis. A massive awareness should be given to livestock farmers and individuals directly linked to animals regarding risk factors and transmission of brucellosis. Consumption of unpasteurized milk and its products should be condemned to curtail this neglected disease.

Prevalence and risk factors of Syphilis among blood donors of Punjab, Pakistan

@#Syphilis is a sexually transmitted disease and its actual prevalence among Pakistani blood donors is currently unknown. A cross sectional study was conducted at different district healthcare hospitals of Punjab, Pakistan with an aim to evaluate the prevalence and risk factors associated with syphilis in blood donors using immunochromatographic test (ICT) and enzyme linked immunosorbent assay (ELISA). A total (n=1200) blood samples were collected from donors aged 18–65 years. All the information regarding personal data, demographic data and risk factors was collected via structured questionnaire. On the basis of ICT and ELISA, the overall prevalence of syphilis was 3.91% among blood donors. The demographic factors positively linked with syphilis were age (P= 0.000; Odds ratio, OR= 7.18; 95% confidence interval CI= 2.816–18.295) and education status (P= 0.000; Odds ratio, OR= 12.33; 95% confidence interval CI= 3.469–43.849) of donors. Similarly among the risk factors analyzed, marital status (P= 0.012; Odds ratio OR= 2.251; 95% confidence interval CI= 1.206- 4.202) and blood transfusion history (P= 0.030; Odds ratio OR= 1.981; 95% confidence interval CI= 1.083-3.623) were also strongly associated with syphilis. We emphasized the importance of promoting preventive measures for syphilis. The syphilis diagnosis should not be based on a single test. The present study indicates that higher prevalence is alarming for blood donors in Pakistan. Stringent donor screening is highly recommended to ensure maximum safe blood transfusion.

KSHV vCyclin counters the senescence/G1 arrest response triggered by NF-κB hyperactivation.

Many oncogenic viruses activate nuclear factor-κB (NF-κB) as a part of their replicative cycles. We have shown recently that persistent and potentially oncogenic activation of NF-κB by the human T-lymphotropic virus 1 (HTLV-1) oncoprotein Tax immediately triggers a host senescence response mediated by cyclin-dependent kinase inhibitors: p21(CIP1/WAF1) (p21) and p27(Kip1) (p27) Here we demonstrate that RelA/NF-κB activation by Kaposi sarcoma herpesvirus (KSHV) latency protein vFLIP also leads to p21/p27 upregulation and G1 cell cycle arrest. Remarkably, KSHV vCyclin, another latency protein coexpressed with vFLIP from a bicistronic latency-specific mRNA, was found to prevent the senescence and G1 arrest induced by HTLV-1 Tax and vFLIP, respectively. This is because of the known ability of vCyclin/cyclin-dependent kinase 6 complex to resist p21 and p27 inhibition and cause p27 degradation. In KSHV-transformed BCBL-1 cells, sustained vFLIP expression with small hairpin RNAs-mediated vCyclin depletion resulted in G1 arrest. The functional interdependence of vFLIP and vCyclin explains why they are cotranslated from the same viral mRNA. Importantly, deregulation of the G1 cyclin-dependent kinase can facilitate chronic I-κB kinases/NF-κB activation.

Progression of histomonosis in commercial chickens following experimental infection with an in vitro propagated clonal culture of Histomonas meleagridis.

Four commercial strains of chickens, namely, ISA brown leghorn (ISA), TETRA-SL brown (TETRA-SL), Lohmann brown (LB), and Lohmann LSL (LSL), were infected with a well-defined clonal culture of Histomonas meleagridis (H. meleagridis/Turkey/Austria/2922-C6/04) to investigate their susceptibility to histomonosis. Each group included 16 chickens, which were housed under the same conditions in separate pens. All chickens were infected with 10(4) histomonads orally and intracloacally at 14 days of age. No mortality or clinical signs were observed during the experiment in all birds. Three birds of each chicken strain were euthanatized on days 4, 7, 10, 14, and 21 postinfection. Incidence of histomonosis on the basis of cecal lesions was found to be 64.00% in TETRA-SL, 62.50% in LB, 53.12% in LSL, and 43.75% in ISA chickens. Fewer lesions were noticed in livers than in ceca, with an incidence of 15.62% in TETRA-SL, 9.37% in LB, and 3.12% in ISA chickens. No liver lesions were found in the LSL chickens. Statistical analysis revealed that there was no significant difference (P > 0.05) in susceptibility to experimental H. meleagridis infection based on cecal and liver involvement. Polymerase chain reaction (PCR) and immunohistochemistry were found to be reliable tools to confirm the presence of histomonads and changes in the ceca. However, some negative PCR results were recorded from the livers despite the presence of macroscopic lesions. Additionally, DNA of H. meleagridis was detected by PCR in a few of the lungs, but immunohistochemistry was negative. Nucleic acid of the protozoan parasite was not detected in samples from kidney, brain, spleen, or bursa of Fabricius. Altogether, the high susceptibility of commercial chicken lines to histomonosis could be demonstrated and characterized by severe lesions in the ceca and insignificant involvement of the liver, approaching a maximum on days 7-14 postinfection.

Safety of avirulent histomonads to be used as a vaccine determined in turkeys and chickens.

In the present work, chickens and turkeys were infected with virulent or attenuated Histomonas meleagridis to investigate and compare the effect of both isolates on birds. Thereby, histomonads of a clonal culture were propagated in vitro either for a short period of time (21 passages) to preserve virulence or for 295 passages to achieve attenuation. On the first day of life birds of each species were infected with either virulent or attenuated parasites. Throughout the experiment, all birds were examined daily for clinical signs attributable to the infection. Furthermore, the excretion of viable parasites was determined after in vitro reisolation from cloacal swabs. For the investigation of pathological changes of organs a defined number of infected birds were killed on d 4, 7, 10, 14, and 21 postinfection (PI) and necropsy was performed. By this routine, changes in livers and ceca were classified by a scoring system to evaluate the severity of lesions. Samples of cecum, liver, and lung were generated and screened for the presence of parasites by PCR and immunohistochemistry. Turkeys infected with virulent histomonads showed first clinical manifestation of histomonosis on d 10 PI, whereas the remaining birds did not express clinical signs. Positive reisolations of virulent and attenuated histomonads were obtained intermittently from individual chickens and turkeys from d 2 PI until the end of the experiment. Both species of birds displayed lesions in the ceca and the liver following infection with virulent parasites, whereas no changes occurred in birds inoculated with attenuated histomonads. The PCR revealed the dissemination of virulent histomonads in ceca, livers, and lungs of some chickens and turkeys in contrast to attenuated parasites, which were exclusively found in cecal samples. The attenuated isolate of H. meleagridis did not induce clinical signs or pathological changes and offers high safety after infection of chickens and turkeys. Therefore, the in vitro attenuation and the use of avirulent histomonads represent a viable tool for vaccination against histomonosis.