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Background: Anemia is said to be present when the hemoglobin level is more than two standard deviations below the mean for the child’s age and sex. Anemia is the most common global nutritional disorder and may lead to psychomotor retardation, low intelligence, decreased learning capability, weakening of immune system and predisposes to serious infections in children. Methods: The study was an observational study. Physical signs of severe anemia were looked in patients attending outpatient department or admitted in hospital. Children with hemoglobin levels

Background: Anemia is one of the leading causes of mortality and morbidity in children. The etiology of anemia is multifactorial including nutritional anemia, hemolytic anemia, aplastic anemia, storage disorders, hematological malignancies, anemia secondary to some chronic diseases. Methods: The study is an observational study. Physical signs of severe anemia were looked in patients attending outpatient department or admitted in hospital. Children with hemoglobin levels

Epidemiological data about determinants of influenza A virus (IAV) in the Pakistani population is scarce. We aimed to conduct a prospective hospital-based active surveillance study from October 2015 to May 2016 to identify potential risk factors associated with IAV infection among patients with influenza-like illness (ILI) and severe acute respiratory illness (SARI). Surveillance was conducted in Lahore General Hospital, selected as a sentinel site in Lahore District, Pakistan. Nasal/throat samples were collected along with epidemiological and clinical data from enrolled patients. Real-time reverse-transcription polymerase chain reaction (rRT-PCR) was performed to identify IAV and its subtypes (H1N1pdm09, H3N2). Data were analyzed to determine risk factors and risk markers associated with IAV infections. A total of 311 suspected ILI and SARI cases were enrolled in the study, and among these 50 were IAV-positive. Of these 50 confirmed cases of IAV, 14 were subtyped as H1N1pdm09 and 15 were H3N2; the remaining 21 were untyped. A final multivariable model identified four independent risk factors/markers for IAV infection: exposure history to ILI patients within last 7 days and gender being male were identified as risk factors of IAV infection, while use of antibiotics prior to hospital consultation and presence of fever were identified as risk markers. We concluded that adopting nonpharmaceutical interventions like hand hygiene, masks, social distancing, and where possible, avoiding identified risk factors could decrease the risk of IAV infection and may prevent imminent outbreaks of IAV in the community.

Sphingomonas sp. Shah is a bacterium that was first isolated from mammalian cell cultures. According to ribotyping data it is very much homologous to the clinically important pathogen Sphingomonas paucimobilis, which has generated pseudo-outbreaks. Using a tissue culture system, Sphingomonas sp. Shah was discovered to induce apoptosis in human lung epithelial carcinoma. Apoptosis of infected cells was determined by numerous criteria including (1) visual alterations in cellular morphology, (2) initiation of nuclear marginalization and chromatin compaction condensation, (3) the attendance of a high percentage of cells with subG1 DNA content, and (4) caspase-3 activation. In the current study we demonstrate the induction of apoptosis in mammalian lung epithelial cells upon infection with Sphingomonas sp. Shah and provide insight into the molecular processes triggering apoptosis.

Background Influenza A virus (IAV) remains an important global public health threat with limited epidemiological information available from low-and-middle-income countries. The major objective of this study was to describe the proportions, temporal and spatial distribution, and demographic and clinical characteristics of IAV positive patients with influenza like illness (ILI) and severe acute respiratory illness (SARI) in Lahore, Pakistan. Methods Prospective surveillance was established in a sentinel hospital from October 2015 to May 2016. All eligible outpatients and inpatients with ILI or SARI were enrolled in the study. Nasal and/or throat swabs were collected along with clinico-epidemiological data. Samples were tested by real-time RT-PCR (rRT-PCR) to identify IAV and subtype. The descriptive analysis of data was done in R software. Results Out of 311 enrolled patients, 284 (91.3%) were ILI and 27 (8.7%) were SARI cases. A distinct peak of ILI and SARI activity was observed in February. Fifty individuals (16%) were positive for IAV with peak positivity observed in December. Of 50 IAV, 15 were seasonal H3N2, 14 were H1N1pdm09 and 21 were unable to be typed. The majority of IAV positive cases (98%) presented with current or history of fever, 88% reported cough and 82% reported sore throat. The most common comorbidities in IAV positive cases were hepatitis C (4%), obesity (4%) and tuberculosis (6%). The highest incidence of patients reporting to the hospital was seen three days post symptoms onset (66/311) with 14 of these (14/66) positive for IAV. Conclusion Distinct trends of ILI, SARI and IAV positive cases were observed which can be used to inform public health interventions (vaccinations, hand and respiratory hygiene) at appropriate times among high-risk groups. We suggest sampling from both ILI and SARI patients in routine surveillance as recommended by WHO.

Background:Congenital heart disease (CHD) is the commonest of all congenital lesions and is the most common type of heart disease among children. Congenital heart disease, in a definition proposed by Mitchell et al is a gross structural abnormality of the heart or intrathoracic great vessels that is actually or potentially of functional significance. It is the most common cause of major congenital anomalies, representing a major global health problem. Twenty-eight percent of all major congenital anomalies consist of cardiac anamolies. In India, 10% of the present infant mortality may be accounted for by Congenital Heart Disease as reported by Saxena et al. Aim:To study the Spectrum of Neonatal Congenital Cardiac Disorders Material and methods:It was an observational prospective study carried out in the Neonatology Section, Department of Pediatrics, GB Panth Childrens hospital Srinagarfrom November 2017 to August 2019. Results:During the two year study, 471 neonates were diagnosed with congenital heart disease in which 335 (71.1%) were acyanotic and 136 (28.9%) were cyanotic. Males and females comprised of 253 and 218 respectively with a male to female ratio of 1.2:1.The most common cyanotic CHD was d-transposition of great arteries (d-TGA) 39 (8.3%), followed by tetralogy of Fallot (TOF) 23 (4.9%) and single ventricle (double inlet left ventricle [DILV] with pulmonary arterial hypertension/pulmonary stenosis [PAH/PS]) 17 (3.6%).In our study the most common acyanotic CHD was ventricular septal defect (VSD) 103 (32.5%) followed by atrial septal defect (ASD) 98 (20.8%) and PDA 47 (10.0%). Conclusion:Congenital heart disease (CHD) is a common congenital disorder of the neonatal population. Early diagnosis and timely management are key factors for optimal outcome of this problem.

Patients with self-reported penicillin allergies frequently receive unnecessary broad-spectrum antibiotics, which are associated with poor outcomes for patients and healthcare systems. The objective of this study was to determine the significance of broad-spectrum antibiotic prescription among patients with a documented penicillin allergy.Retrospective chart review identified a cohort of penicillin allergic patients admitted to the primary medical-surgical floors at Avera McKennan that received intravenous or oral antibiotics. We recorded the allergy manifestation and severity, and all antibiotics administered within 24 hours of admission. The cohort was further divided into various subgroups and analyzed using Chi-Square or a Fischer's exact test.190 patients with documented penicillin allergies received antibiotics between Dec. 1, 2018, and March 31, 2019. A severe penicillin allergy was documented in 86.3 percent of cases. Cephalosporins, vancomycin, and fluoroquinolones represented 34.1 percent, 18.2 percent, and 12.7 percent of initial antibiotics, respectively. No significance was noted in the comparison of antibiotic choice between patients with a specified versus an unspecified penicillin allergy. The number of cephalosporin prescriptions was significantly lower in a surgical prophylaxis subgroup of patients compared to a non-surgical prophylaxis subgroup.Our study supports literature suggesting patients with documented penicillin allergies are at risk of unwarranted broad-spectrum antibiotic use. We noted an alarming number of unverified penicillin allergies. Prescription patterns did not appear to be altered based on verification or type of recorded allergic reaction. Surgical patients may be at greater risk. Our findings call for heightened antibiotic stewardship especially regarding patients with a documented penicillin allergy.

We aim to describe the basic demographics, clinical course and outcomes of critically ill patients with Covid-19 admitted to Avera McKennan Hospital and University Health Center Intensive Care Unit (ICU) between March 20 and May 4, 2020.In this single centered, retrospective, observational study, we enrolled 37 critically ill adults with COVID-19 pneumonia admitted to the (ICU) between March 20 and May 4, 2020. Demographic data, admitting symptoms, laboratory values, co-morbidities, treatments and clinical outcomes were collected. Data was compared between survivors and non-survivors. We aim to describe our data and report the 28-day mortality as of June 1, 2020.Of 154 patients admitted with COVID-19 pneumonia during our study period, 37 (24 percent) were critically ill and required an ICU stay. The mean age was 58 years and 76 percent were men. Of these 37 patients, 28 (78 percent) had a chronic illness (diabetes in 43 percent, hypertension in 47 percent). In addition, 54 percent were associated with a local meat packing plant. Most common presenting symptoms were dyspnea (92 percent), cough (70 percent) and fever (68 percent). The mean PaO2/ FiO2 ratio was 143 (67-362). Significant lab findings include the following: 54 percent of patients had lymphocytopenia, the mean ferritin was 850 ng/mL (10-3528), the mean D-Dimer was 4.09 FEU ug/mL and the mean IL-6 was 96.5 pg/mL. At 28 days, 24 percent (nine) had died. Twenty-five (68 percent) patients required mechanical ventilation, with 10 (27 percent) of those patients requiring initiation of neuromuscular blocking agents for ventilator compliance. Of those four (40 percent) did not survive. In addition, 20 patients (54 percent) were proned. Pneumomediastinum or pneumothorax occurred in five of the 37 (14 percent). Renal replacement therapy was required in 6 of the 37 patients, 4 of whom (66 percent) died. Steroids were used in 70 percent of patients, tocilizumab in 59 percent, and hydroxychloroquine in 27 percent. All patients received antibiotics. Convalescent plasma became available for our 5th patient. A total of 29 (78 percent) received convalescent plasma, (86 percent of survivors and 56 percent non-survivors). Median ICU length of stay was 11 days for both survivors (1-49) and non-survivors (1-21). There were no differences in age, body mass index (BMI), or initial PaO2/FiO2 (P/F) among those two groups. Non-survivors (nine) included the two immune compromised patients in our cohort, two patients with pre-existing DNR/DNI status, and one death within two hours of admit. Compared with survivors, more of the non-survivors received vasopressors (78 percent vs 46 percent), dialysis (44 percent vs 7 percent) and hydroxychloroquine (44 percent vs 21 percent). The first 5 patients treated in the ICU did not survive. One month after the initial case was reported in South Dakota, our ICU experienced a six-week surge. At its highest, COVID-19-related census reached 63 percent of the ICU capacity (15/24).Mortality of critically ill patients with COVID-19 is high. Multi-organ, advanced and prolonged critical care resources are needed. Interpretation of our data is limited by a higher mortality of the earlier members of the cohort, a change in therapeutic practice over time and institution of social distancing.

Cryptococcal infection (CI) is an uncommon fungal disease that poses a particular fatal risk to liver transplant (LT) recipients because of the potential rapid development and dissemination of the disease. Depending on the pathophysiology, CI may manifest with a wide range of clinical presentations that may delay early diagnosis and timely treatment. Additionally, most anticryptococcal therapies may threaten LT recipients owing to the associated hepatotoxicity of these medications. We report a case of a 25-year-old woman who received an LT for cryptogenic cirrhosis and developed rapidly progressive CI with pulmonary, myocardial, and cerebral involvement within a month of transplantation. She presented with severe pulmonary hypertension refractory to medical management and subsequently died despite our efforts. Herein, we review the etiology of cryptococcosis, the natural history of cryptococcal disease, and standard treatments for CI, and we highlight peculiarities of Cryptococcus neoformans infection in solid organ transplant recipients.

Pulmonary infections are frequent complications in abdominal solid-organ transplantation (aSOT) which may threaten patient and allograft survival. Accurate diagnosis and treatment of pulmonary infections in this population can be challenging. Immunosuppressive therapy not only increases the risk of acquiring opportunistic and non-opportunistic infections, but it also impairs the inflammatory responses associated with microbial invasion which in an otherwise normal host produce clinical and radiologic responses that allow for early identification of the offending pathogen. Serologic testing is not a reliable diagnostic modality. Direct microbiological sampling is often necessary to make a definitive diagnosis early in the clinical course to optimize timely, targeted therapy while reducing the risk of developing antimicrobial resistance, and minimize adverse effects of therapy, if any. Fiber-optic bronchoscopy (FOB) with bronchoalveolar lavage (BAL) or transbronchial lung biopsy (TBB) offers such diagnostic advantage and possesses a potential therapeutic value too. This comprehensive review discusses the potential benefits of FOB alongside its risks and complications, indications and contraindications, and techniques. Additionally, the essay highlights FOB's utility and yield specifically with regard to type and timing of infections in aSOT patients.

Bluetongue (BT), caused by bluetongue virus (BTV), is a vector-borne disease of small ruminants that has the potential to spread across international borders. Despite large populations of susceptible animals and borders with BTV endemic countries, little is known of the disease burden and prevalent serotypes in the province of Balochistan in Pakistan. We conducted a cross-sectional study to determine seroconversion and prevalent serotypes in selected districts of the province using a competitive enzyme-linked immunosorbent assay (cELISA) and real-time polymerase chain reaction (RT-PCR). Sera (n = 876) were collected from clinically healthy sheep and goats originating from the districts of Quetta (n = 300), Mastung (n = 201), Killa Saifullah (n = 75) and Kech (n = 300). None of the study herds (n = 97) were seronegative for BTV, and at the individual level, the overall prevalence of BTV seroconversion was 47.26% (n = 414/876, 95% CI = 43.92%-50.63%). A higher percentage of goats (50.87%, 95% CI = 45.99%-55.73%) were seropositive for anti-VP7 immunoglobulins (IgG) than sheep (44.21%, 95% CI = 39.81%-48.70%). Odds ratios of seroconversion for goats were associated with breed type (χ2 = 16.84, p = .01), parity (χ2 = 23.66, p = .00) and presence of vector (χ2 = 2.63, p = .10), whereas for sheep, it was associated with breed type (χ2 = 13.80, p = .01) and parity (χ2 = 53.40, p = .00). Serotype 8 was the most prevalent (26.82%, 95% CI = 14.75%-43.21%) followed by an equal prevalence of serotypes 2 and 9 (7.31%, 95% CI = 1.91%-21.01%). To the best of our knowledge, this is the first study conducted in Balochistan province and the results indicate that there is a necessity to initiate intervention strategies to control BT disease burden not only in this region of Pakistan but also in adjacent areas of the neighbouring countries, Iran and Afghanistan.

Newcastle disease virus (NDV) live vaccines are supplied in lyophilized form and usually administered through conventional routes (drinking water, spray, or eye drop) following reconstitution in a diluent. Virus inactivation due to physico-chemical properties of the diluent at the time of administration may lead to vaccine failure. The present study aimed to evaluate the survival of NDV live vaccine strain immersed in 5 pH-amended water samples (pH 5.00, pH 6.00, pH 7.00, pH 8.00, and pH 9.00) by sequential determination of virus infectivity on Vero cells for 3 hours. Minimum reduction in virus infectivity was recorded in the water with neutral or slightly alkaline pH, while the virus was relatively less stable at extreme pH conditions. Maximum reduction of infectivity was observed in the water with pH 9.00 in which the virus was completely inactivated within 3 hours. Addition of stabilizers (Cevamune® or skimmed milk) slightly altered the pH and total dissolved solids (TDS) values of the virus-charged water samples. In the stabilizer-added water samples, minimum reduction in infectivity was observed in the water with neutral pH, followed by the ones with a pH of 8.00, 6.00, 5.00, and 9.00. In all types of water samples, T-90 values (time required for 90% reduction in virus infectivity) were highest (485 minutes) at neutral pH (pH 7.00) and lowest (102 to 134 min) at an extreme alkaline condition (pH 9.00). Results of the present study indicate that water with a pH range of 7.00 to 8.00 is suitable for administration of NDV live vaccines. However, the addition of Cevamune® or skimmed milk may have beneficial effects on preserving the infectivity of the virus, even at extreme pH conditions.

Histoplasma capsulatum is typically an indolent disease among immunocompetent patients. However, immunocompromised patients, such as solid organ transplant recipients, are at risk of developing severe histoplasmosis. Yet post-transplant histoplasmosis is a rare pathology, representing less than five percent of invasive fungal infections among transplant recipients. Furthermore, patients tend to present with nonspecific clinical symptoms, complicating timely diagnosis and delaying treatment. Disease features that may be more representative of H. capsulatum infection, such as anemia, leukopenia and pulmonary involvement are often not present until late in the disease course, when the patient is at greater risk of decompensation. Unlike H. capsulatum infections among immunocompetent hosts, extrapulmonary infection among immunocompromised hosts is more the rule than the exception. Treatment with liposomal amphotericin B followed by oral itraconazole is the standard therapy, but special considerations must be made for patients with hepatic and/or renal insufficiency, underlying cardiac abnormalities or malabsorptive pathologies and doses of immunosuppressants will need to be adjusted for drug interactions. Herein we present a case of H. capsulatum infection presenting with generalized lymphadenopathy post-renal transplant. Keywords: Infectious disease, Histoplasma, Transplant, Fungal infection

Purpose The stages of development of a health system–wide antimicrobial stewardship program (ASP) using existing personnel and technology are described. Summary Small hospitals with limited resources may struggle to meet ASP requirements, particularly facilities without onsite infectious disease physicians and/or experienced infectious disease pharmacists. Strategies for ASP development employed by Avera Health, a 33-hospital health system in the Midwest, included identifying relevant drug utilization and resistance patterns, education and pathway development, and implementation of Web-based conferencing to provide pharmacists throughout the system with access to infectious disease expertise on a daily basis. These efforts resulted in an evolving single-system ASP that has leveraged existing resources to overcome some system barriers. Program outcomes to date include a reduction in the use of a targeted agent, improved pathogen susceptibility trends, and rates of hospital-associated Clostridium difficile infection below national benchmarks. Conclusion The Avera Health ASP grew from a collaborative project targeting levofloxacin overuse and resistance among key bacteria to a formal, health system–wide ASP in a rural setting. This program used existing personnel to provide standardized processes, educational campaigns, and antimicrobial expertise through the use of technology. This ASP program may provide helpful examples of ASP strategies for other rural health systems with similar resources.

Silver nanoparticles in the range of 10–40 nm were synthesized chemically and by laser ablation, employed for in vitro antibacterial action against human pathogenic bacterium. Their formation was evidenced by UV-visible spectrophotometer; particle size confirmed by atomic force microscopy, crystal structure determined by X-ray diffraction and chemical composition investigated by Fourier transform infrared spectroscopy. The calculated MIC (minimum inhibitory concentration) of chemically synthesized nanoparticles with 30–40 nm in size are 2.8 µg/mL, 4.37 µg/mL, 13.5 µg/mL and 2.81 µg/mL for E. coli, S. aureus, B. subtillis and Salmonella, respectively. Whereas laser ablated nanoparticles exhibit MIC of 2.10 µg/mL, 2.36 µg/mL and 2.68 µg/mL for E. coli, S. aureus and Salmonella, respectively.

Copper nanoparticles (Cu NPs) have been synthesized by using laser ablation method, using deionized water as main solvent. The formation of Cu NPs is confirmed by UV–visible spectrophotometer (UV–Vis), atomic force microscopy (AFM) and X-ray diffraction (XRD). Cu NPs fabricated by laser ablation have diameter in the range from 14 to 55 nm. Structural analysis revealed the face-centered cubic (fcc) crystal structure of Cu NPs. The antibacterial activity of Cu NPs has been evaluated in vitro against strains of Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). The fabricated Cu NPs show considerable antibacterial activity against both bacterial strains. The bacterial activity of Cu NPs was found to depend on the microbial species.

Background: Organosphophorous compounds are the commonly available insecticides in households. Therefore, children are vulnerable to accidental poisoning. It is associated with significant morbidity and mortality. The aim of this study was to study the clinical profile of organophosphorous poisoning in children.Methods: This was prospective study conducted over a period of two years from January 2018 to January 2020 in department of Pediatrics, Government Medical College Srinagar, Jammu and Kashmir, India. All the patients in the age group of 1-18 years with history and examination suggestive of organophosphorous poisoning were included in this study.Results: In this study there were total of 54 patients. In majority of cases poisoning was accidental. Oral consumption was most common route of poisoning. The most common symptoms were excessive salivation (100%), vomiting (72%), abdominal pain (26%), diarrohea (13%), agitation (11%) and convulsions (3.7%). The most common signs were miosis (78%), bradycardia (59%), fasciculation (57%) and altered sensorium (13%). Respiratory failure and circulatory collapse were two main complications which contributed to mortality in this study.Conclusions: Organophosphorous poisoning is one of the most common poisoning in children. Early diagnosis and treatment is of pivotal importance to prevent mortality.

Background: Neonatal sepsis is leading cause of mortality in children. The clinical presentation of neonatal sepsis is non-specific and variable. This study was undertaken to study clinical and etiological profile of neonatal sepsis.Methods: This was a prospective study conducted over a period of one year from March 2018 to March 2019. The patients with clinically suspected sepsis were included in this study. Detailed history and examination was done in all patients. In addition to baseline investigations, C-reactive protein and blood culture was done in all patients. Blood culture was done prior to administration of antibiotics.Results: In this study there were total of 102 patients out of which 54 were male and 48 were female. Among 102 patients, 69 patients were premature born before 37 weeks of gestation. Prematurity emerged to be the most common risk factor. In this study 62% patients had EOS (72 hours of life). The most common presenting feature were refusal of feeds, lethargy, respiratory distress and hypothermia. Other features were seizures, abdominal distension, apnea and sclerema. Blood culture was positive in 41% patients. The most common organism isolated on culture was Kleibsella followed by E. coli.Conclusions: Neonatal sepsis is leading cause of mortality in children. Early diagnosis and treatment is of paramount importance to prevent mortality. The clinical presentation of neonatal sepsis is non-specific and variable. So, high index of suspicion is required to detect sepsis at earliest. Gram negative organism like Kleibsella and E. coli are the common causative organism in neonatal sepsis.

Background: Enteric fever is a common public health problem with variable clinical presentation. The aim of study was to study the clinical spectrum of enteric fever in children.Methods: This was a prospective study conducted over period of one year from January 2019 to January 2020 in the Department of Paediatrics at Govt Medical College Srinagar. It included all patients in the age group of 1-18 years who were clinically suspected to have enteric fever and had either a positive blood culture for Salmonella or a positive Widal test.Results: This study included total of 76 patients out of which 36 were males and 40 were females. The most common presenting symptoms were fever anorexia, vomiting, diarrohea, abdominal pain, headache and constipation. The most common signs were coated tongue, toxic look, hepatomegaly, splenomeagly, pallor, jaundice and abdominal distension. Complications were seen in in 8 (10.5%) patients. Myocarditis was seen in 3 patients. Encephalopathy and hepatitis was seen in 2 patients each. Pneumonia was seen in 1 patient. Majority of patients had normal white blood cell count (4000-11000/cumm). Leukopenia (11000/cumm) was seen in 15% patients. Thrombocytopenia was seen in 9% patients. Blood culture was positive in 36 (47.36%) patients. Salmonella typhi was seen in 33 patients whereas Salmonella paratyphi A was seen in 3 patients. All culture positive cases were sensitive to ceftriaxone, cefixime and azithromycin. Ciprofloxacin resistance was seen in 11 (14.4%) patients.Conclusions: Enteric fever is a common public health problem with fever as most common presenting symptom. Culture yield can be increased in enteric fever by drawing blood culture prior to administration of antibiotics. Ceftriaxone is highly efficacious as monotherapy in enteric fever.

Given the global evolutionary dynamics of Newcastle disease viruses (NDVs), it is imperative to continue extensive surveillance, routine monitoring and characterization of isolates originating from natural reservoirs (waterfowls). In this report, we isolated and characterized two virulent NDV strains from clinically healthy mallard (Anas platyrhynchos). Both isolates had a genome of 15,192 nucleotides encoding six genes in an order of 3´-NP-P-M-F-HN-L-5´. The biological characteristics (mean death time: 49.5-50 hr, EID50108.5 ml-1) and presence of a typical cleavage site in the fusion (F) protein (112R-R-Q-K-R↓F117) confirmed the velogenic nature of these isolates. Phylogenetic analysis classified both isolates as members of genotype VII within class-II. Furthermore, based upon the hypervariable region of the F gene (375 nt), isolates showed clustering within sub-genotype VIIi. Similarity index and parallel comparison revealed a higher nucleotide divergence from commonly used vaccine strains; LaSota (21%) and Mukteswar (17%). A comparative residues analysis with representative strains of different genotypes, including vaccine strains, revealed a number of substitutions at important structural and functional domains within the F and hemagglutinin-neuraminidase (HN) proteins. Together, the results highlight consistent evolution among circulating NDVs supporting extensive surveillance of the virus in waterfowl to better elucidate epidemiology, evolutionary relationships and their impacts on commercial and backyard poultry.

Bluetongue (BT) is a vector-borne disease of immense economic importance for small and large ruminants. Despite frequent disease reports from neighboring countries, a little is known about current disease status and prevalent serotypes in Pakistan. We screened a total of 1312 healthy animals for group-specific antibodies and serotype-specific genome for BT virus through competitive ELISA and real-time PCR, respectively. An overall prevalence of group-specific VP7 antibodies [28.81% (n = 378/1312, 95% CI = 26.4–31.4)] was observed. The prevalence was higher in goats [40.75% (n = 194/476, 95% CI = 36.4–45.3)] followed by buffalo [29.34% (n = 81/276, 95% CI = 24.3–34.9)], sheep [18.40% (n = 60/326, 95% CI = 14.5–22.9)] and cattle [17.94% (n = 42/234, 95% CI = 13.56–23.4)]. The odds of seropositivity were more in buffalo of Nili breed (OR = 2.06, 95% CI = 1.19–3.58) as well as those found with a presence of vector (OR = 2.04, 95% CI = 1.16–3.59). Buffalo and cattle with history of abortion [(OR = 3.95, 95% CI = 1.33–11.69) and (OR = 5.89, 95% CI = 1.80–19.27) respectively] were much likely to be infected with the disease. Serotype 8 was detected in all animal species while, serotypes 4 and 6 were detected in sheep, 2, 6 and 11 in goat, and 2 and 16 in buffalo. The study concludes a much frequent exposure of different serotypes of Bluetongue virus (BTV) in small and large ruminants and indicates its expansion to enzootic range worldwide.

A novel contaminant was isolated from Madin Darby Bovine Kidney (MDBK) cells. The organism was unable to grow on standard microbiological media by conventional techniques, but grew well in Dulbecco's Modified Eagle's Medium (DMEM) containing high glucose concentration. The organism formed a white biofilm on the bottom without any signs of turbidity. Upon genome sequence analysis of 16 S rDNA, the contaminant was identified as Sphingomonas sp. Shah, a member of the group α-Proteobacteria. Neutral red dye uptake method confirmed clear cytotoxic potential of the bacterium on A-549 cells. The organism was capable of invading and infecting different mammalian cell lines: MDBK, ZZ-R, 293-T, A549, and HeLa cells. Infected cells showed a variety of cytopathic effects including vacuolation at perinuclear area, cytoplasmic granulation and membrane blebbing. Microscopic analysis of the infected cells revealed the presence of cytoplasmic vacuoles harboring motile organisms. Apparently local serum preparations seem to be the source of this contamination, which is imperceptibly passed on from one culture passage to the other and ultimately leading to serious cytopathic manifestations. J. Cell. Biochem. 116: 934–942, 2015. © 2015 Wiley Periodicals, Inc.

The aim of the present study was to evaluate the anticoccidial efficacy of three different forms of Aloe vera including powder form, aqueous and methanol extracts. Although powdered and aqueous forms of Aloe vera also exhibited anticoccidial activity but its methanol extracts at higher concentration (4g) showed better weight gain and less oocyst per gram count in comparison of Amprolium (anticoccidial drug). We concluded that due to its anticoccidial activity Aloe vera can contribute to minimize the use of growth promoter antibiotics in poultry feed.

The following Protocols were used to diagnose serologically and sequence the isolated equine influenza virus, in Pakista.

Background A widespread epidemic of equine influenza (EI) occurred in nonvaccinated equine population across multiple districts in Khyber Pakhtunkhwa Province of Pakistan during 2015–2016. Objectives and methods An epidemiological surveillance study was conducted from Oct 2015 to April 2016 to investigate the outbreak. EI virus strains were isolated in embryonated eggs from suspected equines swab samples and were subjected to genome sequencing using M13 tagged segment specific primers. Phylogenetic analyses of the nucleotide sequences were concluded using Geneious. Haemagglutinin (HA), Neuraminidase (NA), Matrix (M) and nucleoprotein (NP) genes nucleotide and amino acid sequences of the isolated viruses were aligned with those of OIE recommended, FC-1, FC-2, and contemporary isolates of influenza A viruses from other species. Results HA and NA genes amino acid sequences were very similar to Tennessee/14 and Malaysia/15 of FC-1 and clustered with the contemporary isolates recently reported in the USA. Phylogenetic analysis showed that these viruses were mostly identical (with 99.6% and 97.4% nucleotide homology) to, and were reassortants containing chicken/Pakistan/14 (H7N3) and Canine/Beijing/10 (H3N2) like M and NP genes. Genetic analysis indicated that A/equine/Pakistan/16 viruses were most probably the result of several re-assortments between the co-circulating avian and equine viruses, and were genetically unlike the other equine viruses due to the presence of H7N3 or H3N2 like M and NP genes. Conclusion Epidemiological data analysis indicated the potential chance of mixed, and management such as mixed farming system by keeping equine, canine and backyard poultry together in confined premises as the greater risk factors responsible for the re-assortments. Other factors might have contributed to the spread of the epidemic, including low awareness level, poor control of equine movements, and absence of border control disease strategies.

EI in non-vaccinated population causes disruption and economic losses. To identify the risk factors associated with the EI epidemics in equids in Pakistan, a 1:1 matched case control study was conducted during 2015-2016. Including a total of 197 laboratory confirmed cases and negative controls, matched on the basis of geography, time of sampling, specie and age. A piloted questionnaire was used to collect data regarding risk factors associated with the occurrence of EI in face to face interviews. Conditional logistic regression was performed to analyze the data. A total of 16 out of 23 variables were found associated as risk factors in Univariable conditional logistic regression analysis. Multivariable conditional logistic-regression analysis was also performed. Monthly removal of manure doubles the risk of EI (EI) compared to its daily removal. Due to lack of vaccination; the spread of disease was favored by high equine density. Investigating the index-case it was recorded that infected cases were imported from Afghanistan. Most of these risk factors related to biosecurity and management were due to low awareness level regarding EI amongst the respondents. These findings are in line with the results of many other studies identifying similar risk factors for EI infection in various countries. Adopting protective practices, vaccination and controlling the risk factors identified in the present study could reduce the spread and future outbreaks of EI in Pakistan.

A total of 300 meat samples comprising mutton, beef, and chicken meat (n = 100) collected from either local butcher shops or large meat outlets situated at various areas of Lahore City located in Punjab province of Pakistan were tested for the isolation of Clostridium perfringens. Prevalence of the organism was highest in the chicken (6 %) followed by mutton (5 %) and beef (1 %). Contamination level was high (10/150) in the samples collected from local butcher shops in comparison to the samples collected from large meat outlets (2/150). All of the raw meat samples were negative for the presence of alpha, beta and epsilon toxins of C. perfringens as detected through ELISA. Out of a total number of 12 isolates only half were capable of producing enterotoxins when cultured in trypticase glucose yeast (TGY) broth. Toxinotyping of the isolates showed that 3 were of type A while one each of the remaining three belonged to type B, C, and D. Antibiotic susceptibility testing of the toxin producing isolates revealed that C. perfringens were susceptible to chloramphenicol, ciprofloxacin, metronidazole, and ceftriaxone. All of the other drugs were relatively less effective with a least activity of amoxicillin against the isolates.

Background: Group A bovine Rotaviruses (BRV) are one of the main factors of neonatal calf diarrhea and mortality around the world. The current study was carried out to assess the genetics of BRV circulating in the Pakistani livestock and characterize the antigenic genes of Rotavirus contributing towards calf diarrhea. Methods: A total of 200 fecal samples were collected from diarrheic buffalo and cattle calves from ten districts of Punjab, Pakistan. Samples were selected on the basis of agro-ecological zones of the province. Fecal samples were analyzed for the presence of BRV using Ag-capture ELISA. Positive samples were subjected to PCR for the amplification of VP4 and VP6 genes and subsequent sequencing. Phylodynamics was performed to infer genetic clustering and evolutionary distances of characterized strains of BRV in accordance to the BRV available publically. Results: In ELISA, a total of 12 calf samples (5 cattle and 7 buffalo), 6%, were found positive. The sequencing and phylogenetic analysis of both genes showed that Pakistani BRV (BRV/QOL/13) depicted maximum identity (98%) of the VP4 gene with Indian BRV VP4 gene. Pakistani BRV VP6 gene (Pakistan/MM85/QOL) showed maximum identity of 98% with Indian BRV A VP6 gene (accession No. JF720873, EF200565). Conclusions: Bovine rotavirus presents 6% of calf diarrhea in Pakistani cattle and buffalo samples. The VP4 and VP6 genes show closed relationships with bovine rotavirus reported from Indian isolates.

Enteric bacterial human pathogens, i.e., Escherichia coli, Staphylococcus aureus, Bacillus subtilis and Klebsiella pneumoniae, are the major cause of diarrheal infections in children and adults. Their structure badly affects the human immune system. It is important to explore new antibacterial agents instead of antibiotics for treatment. This project is an attempt to explain how gold nanoparticles affect these bacteria. We investigated the important role of the mean particle size, and the inhibition of a bacterium is dose-dependent. Ultra Violet (UV)-visible spectroscopy revealed the size of chemically synthesized gold nanoparticle as 6–40 nm. Atomic force microscopy (AFM) analysis confirmed the size and X-ray diffractometry (XRD) analysis determined the polycrystalline nature of gold nanoparticles. The present findings explained how gold nanoparticles lyse Gram-negative and Gram-positive bacteria.

Ranaviruses infect fish, amphibians, and reptiles. The present study was conducted to compare the persistence of amphibian and reptilian ranaviruses in a pond habitat. The 4 viruses used in this study included 2 amphibian ranaviruses, Frog virus 3 (FV3, the type species of the genus Ranavirus) and an isolate from a frog, and 2 ranaviruses of reptilian origin (from a tortoise and from a gecko). A sandwich germ-carrier technique was used to study the persistence of these viruses in sterile and unsterile pond water (PW) and soil obtained from the bank of a pond. For each virus, virus-loaded carriers were placed in each of the 3 substrates, incubated at 4 and 20°C, and titrated at regular intervals. Serial data were analyzed using a linear regression model to cal- culate T-90 values (time required for 90% reduction in the virus titer). Resistance of the viruses to drying was also studied. All 4 viruses were resistant to drying. At 20°C, T-90 values of the viruses were 22 to 31 d in sterile PW and 22 to 34 d in unsterile PW. Inactivation of all 4 viruses in soil at this temperature appeared to be non-linear. T-90 values at 4°C were 102 to 182 d in sterile PW, 58 to 72 d in unsterile PW, and 30 to 48 d in soil. Viral persistence was highest in the sterile PW, followed by the unsterile PW, and was lowest in soil. There were no significant differences in the survival times between the amphibian and reptilian viruses. The results of the present study sug- gest that ranaviruses can survive for long periods of time in pond habitats at low temperatures.

Objective: To determine the role of water fleas in accumulating avian influenza viruses (AIV) from the surrounding water and to estimate their role as a vector of AIV. Methods: Water fleas were exposed to H4N6 and H5N1 AIV-contaminated water in a closed system. The potential of water fleas to take up and retain the viruses was estimated by quantitative real-time RT-PCR (qRRT-PCR) and titration on cell culture. Results: Contamination trials showed that significantly higher amounts of viral RNA were detectable per gram of water fleas as compared to per milliliter of the surrounding water at 1, 4, and 6 days of incubation. Viral infectivity was only detectable in the water samples collected immediately after mixing the virus in water containing the water fleas, while no virus was detectable in any of the water fleas or water samples collected afterwards. Conclusions: Water fleas are able to accumulate AIV from surrounding water based upon the qRRT-PCR detection of viral RNA. Additional studies are necessary to investigate the inactivation potential of water fleas on viral infectivity.