Circulation of foot-and-mouth disease serotypes, risk factors, and their effect on hematological and biochemical profiles among cattle and buffalo in Quetta, Balochistan, Pakistan.

Background and Aim: Foot-and-mouth disease (FMD) is an infectious disease of cloven-hoofed animals, including buffalo, cattle, sheep, goats, and pigs, causing major economic losses to the local farmers and, overall, to the national economy of the country. This study aimed to detect FMDV serotypes in year-round FMD outbreaks, hematological and biochemical changes, and oxidative stress in FMDV-infected cattle and buffaloes in the district of Quetta, Balochistan, Pakistan, and the socioeconomic impact of FMD outbreaks on farmers. Materials and Methods: We conducted a cross-sectional study in the district of Quetta, Balochistan, Pakistan, where FMD virus (FMDV) serotypes were detected by enzyme-linked immunosorbent assay (ELISA). Hematological, biochemical, and oxidative analyses were performed by analyzing the blood of FMDV-infected and non-infected animals. Information on the associated risk factors was obtained through a structured questionnaire by interviewing farmers in each FMD-affected farm. Results: Thirty-four out of 38 farms (89%, 95% confidence interval [CI]: 75%-97%) were positive for FMD by ELISA. Higher FMD infection was detected in farms with a herd size of <50 animals (50%, 17/34), followed by >100 animals (32%, 11/34) and 51-100 animals (18%, 6/34). Fifty-seven percent (114/200, 95% CI: 50%-64%) of animals were positive for FMD. Of these, 61% (69/114) were cattle and 39% (45/114) were buffalo. FMD positivity was higher in females (86%, 98/114) than in males (14%, 16/114) and higher in animals older than 2 years of age (52%, 59/114). On average, farmers lose U.S. dollars 3000 annually due to FMD outbreaks. Animals infected with FMDV had significantly (p ≤ 0.05) white blood cell counts and significantly (p ≤ 0.05) lower hemoglobin and total protein concentrations in buffalo and cattle, whereas infected cattle showed significantly (p ≤ 0.05) lower albumin levels. Globulin levels were lower in buffaloes infected. Alanine aminotransferase levels were lower in infected cattle (p ≤ 0.05). Creatinine levels were higher in infected buffalo (p ≤ 0.05). Urea and phosphorus concentrations were higher in FMDV-infected cattle and buffalo (p ≤ 0.05). Calcium levels were lower in infected cattle and buffalo (p ≤ 0.05). Catalase enzyme activity in infected cattle and buffaloes was significantly lower (p < 0.05). Lipid peroxidation was significantly higher in FMDV-infected cattle and buffalo (p ≤ 0.05). Conclusion: This study confirmed serotype O circulation among cattle and buffalo in year-long FMD outbreaks in the Quetta District of Balochistan. Blood analysis identified a parameter deviated from the normal level due to FMDV infection. In addition, the outbreak of FMD has a significant negative economic impact on livestock farmers.

Drug susceptibility, Prevalence and effect of Pseudomonas aeruginosa on wound healing of burn patients.

Objective: To determine the prevalence, antibiotic susceptibility and effect of Pseudomonas aeruginosa in relation to burn patients. METHODS: The cross-sectional study was conducted at the Centre for Advanced Studies in Vaccinology and Biotechnology, University of Balochistan, Quetta, Pakistan, from March 2018 to May 2021, and comprised pus swab cultures were isolated from inpatients with 2nd and 3rd degree burns aged up to 60 years at Bolan Medical Complex Hospital and Sandeman Provinical Hospital, the two main government tertiary care hospitals in Quetta. The samples were immediately cultured, and evaluated using biochemical tests, antibiotic susceptibility and molecular identification using polymerase chain reaction. Data was analysed using SPSS 20. RESULTS: Of the 720 burn wound samples, 424(58.9%) were positive for Pseudomonas aeruginosa; 304(42%) males and 120(16%) females (p<0.02). The overall mean age of the patients was 27.7±6.2 years (range: 1-60 years). The mean total burn surface area was not significantly different in positive 29.6±6.2% and negative 30.3±6.2% cases (p>0.05). The time leading to skin grafts in positive patients was 29.5±6.5 days compared to 22.3±6.3 days for negative patients (p< 0.007), and the time required for wound healing was 25.0±4.7 days and 16.7±5.2 days, respectively (p<0.001). Length of hospital stay of Pseudomonas aeruginosa positive patients was 38.0±7.8 days compared to 32.1±6.8 days for negative patients (p<0.001). Conclusion: Nosocomial infections and multidrug resistance species were observed frequently at the burn wound site. P. aeruginosa.

Contamination of ready-to-eat street food in Pakistan with Salmonella spp.: Implications for consumers and food safety.

OBJECTIVES: Ready-to-eat (RTE) food sold in Quetta, Pakistan was assessed for microbial contamination. METHODS: Equal numbers of samples were collected from four categories of RTE food - burgers, shawarma, pizza and sandwiches - from January 2018 to December 2018. Microbial contamination of individual food samples was assessed by quantifying the total aerobic count obtained from plating samples on bacterial growth medium. Salmonella spp. serovars were identified using polymerase chain reaction. RESULTS: Approximately 38% (121/320) of RTE food samples were not fit for human consumption. The most contaminated type of RTE food was shawarma (49%). Microbial contamination of food samples was higher in summer compared with the other seasons. Approximately 40% (49/121) of food samples that were not fit for human consumption were contamined with Salmonella spp. Salmonella enteritidis (69%) and Salmonella typhimurium (31%) were the only serovars among the samples testing positive for Salmonella spp. Of the 49 samples with high microbial counts, S. enteritidis was present in 34 samples and S. typhimurium was present in 15 samples. The antibiotic sensitivity results demonstrated that both S. enteritidis and S. typhimurium were resistant to amoxicillin. In addition, S. enteritidis was resistant to chloramphenicol and erythromycin, and S. typhimurium presented high resistance to erythromycin. Both S. typhimurium and S. enteritidis were highly sensitive to kanamycin. CONCLUSION: RTE food sold by street vendors in Quetta was found to be contaminated with Salmonella spp. and poses a great health risk to consumers. As such, consumption should be avoided, and the health authorities should take stringent action to ensure the quality of street food in order to reduce the healthcare burden.

Predominance of Central Asian strain (ST 26) in Mycobacterium tuberculosis isolates from Balochistan by spoligotyping.

INTRODUCTION: Tuberculosis is a chronic debilitating infectious disease causing a severe challenge to public health, especially in developing countries. The aim of this study was to examine genetic diversity in Mycobacterium tuberculosis strains circulating in the Balochistan region of Pakistan. METHODOLOGY: One hundred isolates collected from patients visiting the Fatima Jinnah TB Hospital in Quetta were subjected to genotype analysis by spoligotyping. RESULTS: Three main genotypes were identified: Central Asian Strain 1 (CAS1) (n = 89), East African Indian (EAI) strain (n = 7) and Latin American Mediterranean (LAM) strain (n = 3). The CAS1 clade (ST 26) had high genetic diversity represented by seven different spoligopatterns, of which one had major predominace (n = 75). CONCLUSIONS: This is the first insight into the genotype of M. tuberculosis strains in the Balochistan region that might serve as a base line study for control of tuberculosis in the community.

Effects of natural environment on reproductive histo-morphometric dynamics of female dromedary camel.

Camel shows a seasonal breeding pattern with enhanced reproductive activity during the period of low climatic temperature, high rainfall and better food conditions. Therefore, the study was conducted to explore the underlying seasonal effects on histological dimensions of reproductive tract in adult female one-humped camel (Camelus dromedarius) kept in the natural environment of Pakistan. A total 25 reproductive tracts were collected during spring, summer, autumn and winter seasons and were analysed for histo-morphometric parameters during different environmental conditions. A significant increase in number (p<0.05) and size (p<0.05) of surface with secondary and tertiary ovarian follicles was observed in winter season. The epithelial height (p<0.05) and luminal diameter (p<0.05) of infundibulum, ampulla and isthmus of uterine tubes were also significantly increased during winter season. Moreover, significantly increased length (p<0.05) and circumference (p<0.05) of uterine cornua, increased number (p<0.001) and diameter (p<0.001) of endometrial glands with enlarged surface and glandular epithelia (p<0.001) were found in winter compared to summer season. Therefore, we concluded that quiescent ovarian follicular and uterine glandular activities are the main reason of camel low breeding during summer season.

Comparative study of extrapolative factors linked with oxidative injury and anti-inflammatory status in chronic kidney disease patients experiencing cardiovascular distress.

BACKGROUND: Chronic kidney disease (CKD) is a group of heterogeneous abnormalities affecting the function and structure of the kidney and mostly further proceeds to cardiovascular damage prior to end stage renal disease (ESRD). The oxidative insult and inflammatory mediators have some undefined role in CKD and cardiovascular complications. It is therefore, aimed at to pin point the predictive factors in the development of cardiovascular disorder in patients with chronic kidney disease. METHODS: Fifty patients of CKD experiencing cardiovascular distress and twenty normal individuals having same age and sex acted as control during these observations. Blood samples (Each 5 ml) were drawn and subjected to centrifugation for 10-15 minutes to separate the serum at 4000-5000rpm. The levels of MDA, GSH, SOD, CAT, VIT C, VIT E, IL-1, TNF-alpha, nitric oxide (NO) and advanced oxidation protein products (AOPPs) were estimated and analyzed. RESULTS: The nitric oxide levels in the CKD patients decreased significantly (13.26±1.25 ng/ml) compared to controls (42.15±5.26 ng/ml). The serum vitamin E and C levels in these patients recorded 2.15±0.25 µg/ml and 0.97±0.09 µg/ml respectively as against their assigned controls which read 6.35±1.22 µg/ml and 3.29±0.25 µg/ml. Furthermore, a significantly higher level of Malondialdehyde (MDA) as1.25±0.07 nmol/ml was observed in CKD patients viz-a-viz relevant control. However, the serum SOD, catalase (CAT) and GSH levels in the same patients registered a significant decline as evident from respective figures 0.07±0.002 µg/dl, 1.22±0.012 µmol/mol, and 3.25±1.05 µg/dl. The control for these was observed as0.99±0.06 µg/dl, 3.19±0.05 µmol/mol, and 8.64±0.03 µg/dL. On the other hand, the IL-1 levels in the CKD patients found quite higher (402.5±18.26 pg/ml). This clearly points to substantial increase in oxidative insult and reduced NO levels leading to the renal and cardiovascular damage. CONCLUSION: Observations support the fact that the decrease in anti-oxidative capacity accompanied by higher inflammatory mediators in CKD is indicative of oxidative stress, consequently leading to CKD progression, in all probability to cardiovascular insult. The outcome reiterates that strategies be designed afresh to contain CKD progression to cardiovascular complications and ESRD. One way could be to focus on early detection of stress related to the disease. It requires analyzing the factors related to stress, such as the one reported here. Linking these factors with the symptoms could be a crucial step forward. And further, the disease could be monitored in a more disciplined manner.

DARPins Bioengineering and its Theranostic Approaches: Emerging Trends in Protein Engineering.

The therapeutic significance of bioengineering proteins has increased dramatically as a new generation of pharmacological drug with a great potential in medical treatment. Protein engineering has improved the use of new non immunoglobulin affinity proteins. The designed ankyrin repeat proteins abbreviated as DARPins is a classical example. These molecules have smaller structural size, unlike immunoglobulins, but similar target affinity is of great significance. Such molecules get more binding specificity and greater thermodynamic stability. These attributes made DARPins a favorite player in diagnostics and therapeutics of cancer. How DARPins are improved into vigorous and versatile scaffold for binding protein and selected by ribosome and phage display along with related aspects is discussed in this review.

Evaluation of Matrix Metalloproteinases, Cytokines and Their Potential Role in the Development of Ovarian Cancer.

BACKGROUND: Ovarian cancer is the 5th most common cause of deaths in the women among gynecological tumors. There are many growing evidences that stress and other behavioral factors may affect cancer progression and patient survival. The purpose of this study is to determine the key role of matrix metalloproteinases (MMPs), and cytokines in the aggregation and progression of ovarian cancer. METHODOLOGY: Stress variables (MDA, AGEs, AOPPs, NO), profile of antioxidants (SOD, Catalase, Vitamin E & A, GSH, GRx, GPx) and inflammatory biomarkers (MMP-9, MMP-2, MMP-11, IL-1α and TNF-α) were biochemically assessed from venous blood of fifty ovarian cancer patients and twenty healthy control subjects. The results of all parameters were analyzed statistically by independent sample t-test. RESULTS: The results of the study demonstrated that the levels of stress variables like MDA (3.38±1.12nmol/ml), AGEs (2.72±0.22 ng/ml), AOPPs (128.48±27.23 ng/ml) and NO (58.71±8.67 ng/ml) were increased in the patients of ovarian cancer as compared to control individuals whereas the profile of antioxidants like SOD, Catalase, Vitamin E, Vitamin A, GSH and GRx were decreased in ovarian cancer patients (0.11±0.08 µg/ml, 2.41±1.01µmol/mol of protein, 0.22±0.04 µg/ml, 45.84±9.07µg/ml, 4.88±1.18µg/ml, 5.33±1.26 µmol/ml respectively). But the level of GPx antioxidant was increased in ovarian cancer patients (6.58±0.21µmol/ml). Moreover the levels of MMP-9 (64.87±5.35 ng/ml), MMP-2 (75.87±18.82 ng/ml) and MMP-11 (63.58±8.48 ng/ml) were elevated in the patients. Similarly, the levels of various cytokines TNF-α and IL-1α were also increased in the patients of ovarian cancer (32.17±3.52 pg/ml and 7.04±0.85 pg/ml respectively). CONCLUSION: MMPs are commonly expressed in ovarian cancer which are potential extrapolative biomarkers and have a major role in metastasis. Due to oxidative stress, different cytokines are released by tumor associated macrophages (TAMs) that result in the cancer progression. Consequently, tissue inhibitors of matrix metalloproteinases (TIMPs) are the valuable therapeutic approaches to complement conservative anticancer strategies.

Autoantibody signatures defined by serological proteome analysis in sera from patients with cholangiocarcinoma.

BACKGROUND: The challenging diagnosis and poor prognosis of cholangiocarcinoma require the determination of biomarkers. Autoantibodies could be used in the clinic as diagnostic markers for the early detection of tumours. By proteomic approaches, several autoantibodies were proposed as potential markers. We tried in this study, to perform a serological proteome analysis, using various antigenic substrates, including tumours and human liver. METHODS: Sera from patients (n = 13) and healthy donors (n = 10) were probed on immunoblots performed using 2-dimensionally separated proteins from cholangiocarcinoma cell lines (CCLP1 and CCSW1), from the liver of healthy subject and interestingly, from tumour and adjacent non-tumour liver tissues from five patients with cholangiocarcinoma and tested with their corresponding serum. Spots of interest were identified using mass spectrometry and classified according gene ontology analysis. RESULTS: A comparison of the whole immunoblotting patterns given by cholangiocarcinoma sera against those obtained with normal control sera enabled the definition of 862 spots. Forty-five different proteins were further analysed, corresponding to (1) spots stained with more than four of 13 (30 %) sera tested with the CCLP1 or the CCSW1 cell line and with the normal liver, and (2) to spots immunoreactive with at least two of the five sera probed with their tumour and non-tumour counter-part of cholangiocarcinoma. Immunoreactive proteins with catalytic activity as molecular function were detected at rates of 93 and 64 % in liver from healthy subjects or cholangiocarcinoma non-tumour tissues respectively, compared to 43, 33, 33 % in tumour tissues, or CCSW1 and CCLP1 cell lines. A second pattern was represented by structural proteins with rates of 7 and 7 % in normal liver or non-tumour tissues compared to 14, 33 and 67 % in tumour tissue, CCSW1 or CCLP1 cell lines. Proteins with a binding function were detected at rates of 7 % in non-tumour tissue and 14 % in tumour tissue. Using the extracted tumour tissue, serotransferrin was targeted by all cholangiocarcinoma-related sera. CONCLUSIONS: Immunological patterns depended on the type of antigen substrate used; i.e. tumour versus non tumour specimens. Nevertheless, a combination of multiple autoantibodies tested with the most appropriate substrate might be more sensitive and specific for the diagnosis of cholangiocarcinoma.

SPRi-based strategy to identify specific biomarkers in systemic lupus erythematosus, rheumatoid arthritis and autoimmune hepatitis.

BACKGROUND: Heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 is a target for antinuclear autoantibodies in systemic Lupus erythematosus (SLE), rheumatoid arthritis (RA), and autoimmune hepatitis (AIH). AIM: To monitor molecular interactions between peptides spanning the entire sequence of hnRNP A2/B1 and sera from patients and healthy controls. METHODS: Sera from 8 patients from each pathology and controls were passed across a surface plasmon resonance Imagery (SPRi) surface containing 39 overlapping peptides of 17 mers covering the human hnRNP B1. Interactions involving the immobilised peptides were followed in real time and dissociation rate constants k(off) for each interaction were calculated. RESULTS: Several significant interactions were observed: i) high stability (lower k(off) values) between P55₋70 and the AIH sera compared to controls (p= 0.003); ii) lower stability (higher k(off) values) between P118₋133 and P262₋277 and SLE sera, P145₋160 and RA sera compared to controls (p=0.006, p=0.002, p=0.007). The binding curves and k(off) values observed after the formation of complexes with anti-IgM and anti-IgG antibodies and after nuclease treatment of the serum indicate that i) IgM isotypes are prevalent and ii) nucleic acids participate in the interaction between anti-hnRNAP B1 and P55₋70 and also between controls and the peptides studied. CONCLUSIONS: These results indicate that P55₋70 of hnRNP B1 is a potential biomarker for AIH in immunological tests and suggest the role of circulating nucleic acids, (eg miRNA), present or absent according to the autoimmune disorders and involved in antigen-antibody stability.

Immunoproteomic analysis of potentially severe non-graft-versus-host disease hepatitis after allogenic bone marrow transplantation.

UNLABELLED: The development of potentially severe non-graft-versus-host disease (GVHD) hepatitis resembling autoimmune hepatitis (AIH) has been reported after bone marrow transplantation (BMT). The aim of this study was to better characterize this form of hepatitis, particularly through the identification of autoantigens recognized by patient sera. Five patients who received an allogeneic BMT for the treatment of hematological diseases developed liver dysfunction with histological features suggestive of AIH. Before and during the onset of hepatic dysfunction, sera were tested on immunoblottings performed with cytosolic, microsomal, mitochondrial, and nuclear proteins from rat liver homogenate and resolved by two-dimensional electrophoresis. Antigenic targets were identified by mass spectrometry. During the year that followed BMT, all patients presented with GVHD. Acute hepatitis then occurred after the withdrawal, or during the tapering, of immunosuppressive therapy. At that time, no patients had a history of liver toxic drug absorption, patent viral infection, or any histopathological findings consistent with GVHD. Immunoreactive spots stained by sera collected at the time of hepatic dysfunction were more numerous and more intensely expressed than those stained by sera collected before. Considerable patient-dependent pattern heterogeneity was observed. Among the 259 spots stained exclusively by sera collected at the time of hepatitis, a total of 240 spots were identified, corresponding to 103 different proteins. Twelve of them were recognized by sera from 3 patients. CONCLUSIONS: This is the first immunological description of potentially severe non-GVHD hepatitis occurring after BMT, determined using a proteomic approach and enabling a discussion of the mechanisms that transform an alloimmune reaction into an autoimmune response. Any decision to withdraw immunosuppression after allogeneic BMT should be made with caution.