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Tropical theileriosis is a lymphoproliferative disease caused by Theileria annulata and is transmitted by Ixodid ticks of the genus Hyalomma. It causes significant losses in livestock, especially in exotic cattle. The existing methods for controlling it, chemotherapeutic agents and a vaccine based on an attenuated schizont stage parasite, have several limitations. A promising solution to control this disease is the use of molecular vaccines based on potential immunogenic proteins of T. annulata. For this purpose, we selected five antigenic sequences of T. annulata, i.e. SPAG-1, Tams, TaSP, spm2, and Ta9. These were subjected to epitope prediction for cytotoxic T lymphocytes, B-cells, and helper T lymphocytes. CTL and B-cell epitopes with a higher score whereas those of HTL with a lower score, were selected for the construct. A single protein was constructed using specific linkers and evaluated for high antigenicity and low allergenicity. The construct was acidic, hydrophobic, and thermostable in nature. Secondary and tertiary structures of this construct were drawn using the PSIPRED and RaptorX servers, respectively. A Ramachandran plot showed a high percentage of residues in this construct in favorable, allowed, and general regions. Molecular docking studies suggested that the complex was stable and our construct could potentially be a good candidate for immunization trials. Furthermore, we successfully cloned it into the pET-28a plasmid and transformed it into the BL21 strain. A restriction analysis was performed to confirm the transformation of our plasmid. After expression and purification, recombinant protein of 49 kDa was confirmed by western blotting. An ELISA detected increased specific antibody levels in the sera of the immunized animals compared with the control group, and flow cytometric analysis showed a stronger cell-mediated immune response. We believe our multi-epitope recombinant protein has the potential for the large-scale application for disease prevention globally in the bovine population. This study will act as a model for similar parasitic challenges.

Abstract Coccidiosis causes huge economic losses worldwide. Current study evaluated the effect of biosynthesized Zinc oxide nanoparticles (ZnONPs) using Nigella sativa, on Eimeria tenella infected broilers. Scanning electron microscopy showed spherical ZnONPs with 50–100 nm diameter, Fourier transforms infrared spectroscopy revealed the functional groups involved in the reduction of zinc acetate dihydrate to ZnONPs, UV–vis spectroscopy showed a peak at 354 nm, and Zeta potential exhibited stability at − 30 mV. A total of 150, a day-old broiler chicks were divided into 5 equal groups. Control negative: uninfected and untreated; Control positive: Infected and untreated; 3rd, 4th and 5th group were infected orally with 5 × 104 sporulated oocysts of Eimeria tenella and treated with 60 mg/kg ZnONPs, 1% Nigella sativa seeds and amprolium 125 ppm, respectively. ZnONPs significantly (p

Background. Isoflurane (ISO) has been extensively uses in general anesthesia and reported to cause deoxyribonucleic acid (DNA) damage in prolonged surgical procedures. Dexmedetomidine (DEX) is an adrenergic agonist and having antioxidant activity that may reduce the genotoxic potential (DNA damage) and oxidative stress induced by ISO in patients undergoing major neurosurgical procedures. Methods and Findings. Twenty-four patients of ASA (American Society of Anesthesiologists) classes I and II were randomly divided into two groups (n = 12). Group A patients received ISO, while group B patients received DEX infusion for maintenance of anesthesia. Venous blood samples were collected at different time intervals and used to evaluate the oxidative stress marker malondialdehyde (MDA) and endogenous antioxidants superoxide dismutases (SOD) and catalases (CAT). A single-cell gel electrophoresis (SCGE)-comet assay was used to investigate the genotoxic potential of ISO. Conclusion. Increased level of antioxidants and decreased value of MDA and genetic damage index were seen in group B (P

Ticks transmit a number of bacterial, protozoal and viral pathogens that cause many diseases like ehrlichiosis, hemorrhagic fever, theileriosis, babesiosis and anaplasmosis in livestock. This study was designed for molecular characterization of BM86 gene of Rhipcephalus (Boophilus) microplus tick. In this study, the BM86 gene was amplified, using primers fl anked by restriction enzyme sites. The molecular detection of R. microplus was studied in three districts belonging to diff erent ecological zones in the province of the Punjab, Pakistan. Tick samples were collected and initially screened through microscopy and further analyzed by PCR and sequencing, the phylogenetic tree generated by using the MEGA 7 through Neighbor Joining method using best model through the phylogenetic analysis of R. microplus. Pairwise comparisons of nucleotide sequences showed nucleotide diff erences ranging between 0.007 and 0.01%. Haplotype and nucleotide diversity in Bm86 gene was found among diff erent districts. Six single nucleotide polymorphism were seen sequences of BM 86 from indigenous tick population collected from the Punjab province. More interestingly out of these 6 polymorphism we got 2 from district Okara, 3 from district Sahiwal, and 1 in Mandi Bahauddin. Conserved regions were observed among the local strains for BM86 gene. A common convergence in similar clade was with the local Pakistan R. microplus. Local mean diversity was 0.005 and overall mean diversity was 0.038. Field strain has been isolated as candidate specie for local tick vaccine, which in turn will increase the efficacy of future tick vaccine including reduction of economic burden on the farmer.

Coccidiosis is a protozoan disease that is characterized by diffuse diarrhea, dehydration, emaciation accompanied by moderate morbidity and mild mortality in animals and birds. The current study targeted the molecular characterization of Eimeria isolates in captive deer from different localities in Lahore. The host species was the Cervidae family, such as Hog deer (Axis porcinus) and Punjab urial (Ovis aries vignei). The Eimeria crandallis was isolated from zoo animals. The DNA was extracted from oocysts and amplified by using reported oligonucleotide primers that exhibited the 809 bp product. These were analyzed by using the small subunit 18S rRNA gene-based evolutionary relationship with 36 other Eimeria species reported in caprine, cervinae, bovines, avians, and rodents. Light microscopic examination exhibited 3.29% (7/213) Eimeria-positive fecal samples with morphological features, including sub-spherical forms, the presence of micropyle with polar cap, and oocysts diameters (μm) ranging from 24.32 ± 1.61 to 18.94 ± 1.51. The phylogenetic tree constitutes four distinct clusters with relatively higher values. The evolutionary network showed that sequences were clustered in the monophyletic group of Eimeria species reported in caprine and cervinae. The nucleotide and amino acid sequence similarity matrix analysis exhibited 99.5–99.9% identity of the study isolates with Eimeria crandallis (AF336339). This study provides relevant baseline data to develop strategic control measures for coccidiosis in zoo animals. However, further investigations are required to place the hog deer and Punjab urial-derived E. crandallis into the caprine-originated cluster.

The emergence of a pathogen responsible for a mysterious respiratory disease was identified in China and later called a novel coronavirus. This disease was named COVID-19. The present study seeks to determine the epidemiological and clinical characteristics of COVID-19 in Pakistan. This report will exhibit a linkage between epidemiology and clinical aspects which in turn can be helpful to prevent the transmission of the virus in Pakistan. A retrospective, multiple center study was performed by collecting the data from patients' with their demographics, epidemiological status, history of co-morbid conditions, and clinical manifestations of the disease. The data was collected from 31 public-sector and 2 private hospitals across Pakistan by on-field healthcare workers. A Chi-square test was applied to assess the relationship between categorical data entries. A total of 194 medical records were examined. The median age of these patients was found to be 34 years. A total of 53.6% active cases were present including 41.2% males and 12.4% females till the end of the study. Adults accounted for most of the cases (94.3%) of COVID-19. Fever (86.60%), cough (85.05%), fatigue (36.60%), dyspnea (24.74%), and gastrointestinal discomfort (10.31%) were among the most frequently reported signs and symptoms by the patients. However, 4.12% of the total patient population remained asymptomatic. The median duration of hospital stay was found to be 14 (0–19) days. The earliest source of the spread of the virus may be linked to the foreigners traveling to Pakistan. Spread among men was more as compared to women. A few cases were found to be positive, due to the direct contact with pets or livestock. Hypertension (7.73%), diabetes (4.64%), cardiovascular conditions (2.58%) were the most common co-morbidities. The percentage mortality was 2.50% with the highest mortality among elders.

Toxoplasmosis is a globally distributed disease of warm-blooded animals. It is caused by the opportunistic parasite Toxoplasma gondii (T. gondii). One-third of the global human population is believed to be infected with T. gondii. Cats serve as final host of T. gondii and are the main source of contamination of soil and water. This study aimed to detect genotypes of T. gondii in cats. Fecal samples (n = 400) were collected from districts of South Punjab (Khanewal and Sahiwal), and were processed by polymerase chain reaction (PCR) followed by sequencing and phylogenetic analysis. The obtained oligonucleotide sequences (T. gondii) were submitted to the GenBank database, and the evolutionary tree was constructed using MEGA-X software. Seven fecal samples (3.5%) from cats were positive. Five out of thirteen fecal samples (38.46%) found to be positive for T. gondii with microscopy were confirmed by PCR. After phylogenetic analysis with 3 clonal types and atypical strains, isolates of T. gondii in current study were more closely linked to a typical strain (AF249696). Besides genotyping from cats, seroprevalence from humans and ruminants is still considered to be the best and easiest way to identify the Toxoplasma. Blood samples were collected from sheep and goats (n = 2000 each), and human blood samples (n = 400) were collected from the same vicinity. Seroprevalence was determined using a commercial enzyme-linked immunosorbent assay (ELISA) kit. In Khanewal, the blood samples of 292 goats (29.2%) and 265 sheep (26.5%), and 6 fecal samples from cats (3%) were positive. Out of 200 human blood samples, 52 were positive, with a seroprevalence of 26%. In the Sahiwal district, the blood samples from 49 humans, 235 sheep and 348 goats were positive, with seroprevalence of 24.5%, 23.5% and 34.8%, respectively. The present study revealed the current circulating genotype of T. gondii from cats in the districts Khanewal and Sahiwal and the seroprevalence of the organism in small ruminants and humans living in the same vicinity. Further genotype analyses of the organism from ruminants and humans are needed.

Background: Theileria equi is a tick borne protozoan parasite which causes piroplasmosis among equines worldwide. The present study was aimed to determine seroprevalence of T. equi in donkeys, horses, and mules from two equine populated districts (Peshawar and Charsadda) of Khyber Pakhtunkhwa (KPK), Pakistan. Methods: A total of 393 equine (195 horses, 194 donkeys and 4 mules) serum samples were collected from five and four randomly selected localities in Charsadda (n = 193) and Peshawar (n = 200), respectively. The presence of antibodies to T. equi was determined using a commercially available competitive enzyme-linked immunosorbent assay. Results: An overall seroprevalence of 38.2% (n=150) was observed among all the tested animals suggesting a higher seropositivity among equids belonging to Charsada (50.3%) as compared to Peshawar (27.5%). Binary logistic regression analysis revealed that being a donkey (OR 2.94), having tick infestation (OR 4.32), history of voiding red (i.e., blood containing) urine (OR 3.97) and anemia (OR 2.1) were the factors significantly associated with the seroprevalence of T. equi. For animals with higher anti-T. equi antibody titers, a strong association of seroprevalence for T. equi was recorded with species, age, sex, tick infestation, anemia and history of hematuria. Conclusion: The present study indicates a high level of exposure of working equids to T. equi in KPK region, Pakistan. Future studies should focus on tick vector identification and other factors responsible for spread of the disease. [ABSTRACT FROM AUTHOR]