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Hyalomma anatolicum and Rhipicephalus microplus are tick species that are important vectors of numerous pathogens affecting both humans and livestock. Endosymbionts, such as Coxiella-like endosymbionts (CLE), Francisella-like endosymbionts (FLE), and Candidatus Midichloria, play a crucial role in the physiology and vector competence of these ticks. In this study, we investigated the microbial composition of H. anatolicum and R. microplus from four geographically distinct regions of Pakistan to assess whether environmental differences influence their microbiomes. We analyzed the ticks’ gut microbiome targeting the V3-V4 hypervariable region of 16S rRNA for Illumina 16S metagenome NGS sequencing and processed overall 144 ticks. Analysis of gut bacterial composition resulted in observation of 1200 R. microplus and 968 H. anatolicum unique amplicon sequencing variants (ASVs). Relative abundance, Alpha diversity (Shannon, Faith’s phylogenetic distance) and beta diversity metrics (Bray–Curtis, Jaccard and UniFrac) were analyzed and revealed that H. anatolicum ticks have significantly unique and diverse microbial communities with Acinetobacter indicus and Francisella-like endosymbionts dominating as opposed to Candidatus Midichloria. Rhipicephalus microplus exhibited results consistent with the previous studies with no major changes in microbiome including Coxiella-like endosymbionts as the major contributor. These findings suggest that geographical and environmental factors play a significant role in shaping the tick microbiome, with potential consequences for disease transmission and tick survivability. Further research is needed to elucidate the functional roles of these microbial shifts and their impact on public health and livestock in affected regions.

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 FROM AUTHOR]

Complex fracture reconstruction, malunion, delayed and nonunion are challenging problems often require special attention. Autogenous bone graft for osseous healing has been considered gold standard for decades. It contains viable osteoprogenitor cells, cytokines and growth factors with appropriate tissue matrix resulting in osteogenesis, osteoinduction and osteoconduction. Orthobiologics are biological materials used to augment bone and soft tissue cure, with multifactorial effects. In our study, three orthobiologics i.e., Bone Marrow Aspirate (BMA), Platelet Rich Plasma (PRP) and Decellularized Fish Scale (DFS) were used in conjunction to autogenous bone graft. Objective was to transplant as many cells as possible with osteogenic potential along with the graft. These three regimens were evaluated in terms of clinical vital parameters, hematology, serum analysis, bone biomarkers, mechanical assessment, radiographic evaluation and histomorphometry. We concluded that Bone-BMA combination showed best results in fracture healing and bone remodeling while Bone-PRP combination was below par than Bone-BMA but was superior to Bone-DFS combination. More clinical research may be required before widespread use of these regimens. Furthermore, it is paramount that veterinary orthopedic surgeons must have contemporary knowledge regarding autogenous grafts and orthobiologics, so they can utilize them correctly. [ABSTRACT FROM AUTHOR]

The rise in greenhouse gases, particularly carbon dioxide (CO2) emissions, in the atmosphere is one of the major causes of global warming and climate change. The production of ordinary Portland cement (OPC) emits harmful CO2 gases, which contribute to sporadic heatwaves, rapid melting of glaciers, flash flooding, and food shortages. To address global warming and climate change challenges, this research study explores the use of a cement-less recycled aggregate concrete, a sustainable approach for future constructions. This study uses fly ash, an industrial waste of coal power plants, as a 100% substitute for OPC. Moreover, this research study also uses recycled coarse aggregates (RCAs) as a partial to complete replacement for natural coarse aggregates (NCAs) to preserve natural resources for future generations. In this research investigation, a total of 60 pull-out specimens were prepared to investigate the influence of steel bar diameter (9.5 mm, 12.7 mm, and 19.1 mm), bar embedment length, d b (4 d b and 6 d b ), and percentage replacements of NCA with RCA (25%, 50%, 75%, and 100%) on the bond stress behavior of cement-less RA concrete. The test results exhibited that the bond stress of cement-less RCA concrete decreased by 6% with increasing steel bar diameter. Moreover, the bond stress decreased by 5.5% with increasing bar embedment length. Furthermore, the bond stress decreased by 7.6%, 7%, 8.8%, and 20.4%, respectively, with increasing percentage replacements (25%, 50%, 75%, and 100%) of NCA with RCA. An empirical model was developed correlating the bond strength to the mean compressive strength of cement-less RCA concrete, which matched well with the experimental test results and predictions of the CEB-FIP model for OPC. The CRAC mixes exhibited higher costs but significantly lower embodied CO2 emissions than OPC concrete. [ABSTRACT FROM AUTHOR]

Theileriosis is a protozoan parasite that is transmitted by ticks and infects a wide range of animals worldwide. This study aims to assess the molecular prevalence and related risk factors of theileriosis in Balochistan, Pakistan. Standard microscopy methods, polymerase chain reaction (PCR), 18S small subunit ribosomal RNA gene sequencing, and phylogenetic analysis were used. For this purpose, a total of 408 blood samples were collected from tick-infested cattle in Zhob, Loralai, and Quetta districts of Balochistan, Pakistan. Microscopy and subsequent PCR analysis confirmed the highest prevalence of Theileria annulata in Loralai district (11.76% and 12.75%), followed by Zhob district (11.27% and 12.25%), and Quetta district (8.34% and 9.56%), respectively. Moreover, the prevalence of T. annulata was higher in young cattle (85.82%), followed by female cattle (58.87%), and exotic crossbred cattle (33.33%) in the study area. However, various variable such as sex, area, and breeds of the cattle were not significantly correlated (P>0.05) with the presence of T. annulata, except for the age of animals (P<0.05). In addition, sequencing and phylogenetic analyses revealed that the isolated T. annulata was closely related to the isolates from Türkiye, Italy, Egypt, Iran, and Pakistan. Hence, these findings will contribute to the development of more effective control strategies for theileriosis in the cattle population of Balochistan, as well as in Pakistan on a broader scale. [ABSTRACT FROM AUTHOR]

Visceral leishmaniasis is a neglected zoonotic protozoal disease caused by Leishmania infantum and Leishmania donovani that is transmitted by sandflies (Phlebotamine flies). In November 2020, a case of leishmaniasis was diagnosed in a captive tiger through microscopy and L. infantum was confirmed by PCR and sequencing analysis. DNA sequencing of the amplicon revealed close homology with Leishmania sequences available in GenBank. Alignments and phylogenetic analyses of the Leishmania infantum from a tiger in Pakistan indicated 94-100 % identity with Leishmania from animals and 98.8-100% with Leishmania from humans origins suggesting the need for screening of animals before transporting, and of humans before taking care of captive animals, in order to prevent transboundary spread of Leishmania. [ABSTRACT FROM AUTHOR]

Toxoplasmosis is a zoonotic parasitic disease which is caused by Toxoplasma gondii that infects all warm-blooded mammals including human beings. In the current study, it was explored to Toxoplasma-specific IgG titer while comparing quantitative ELISA and WB. Ten human sera samples labelled as 53, 217, 28, 277, 229, 07, 204, 275, 241 and 04, (known positive as a result of earlier testing in a commercial ELISA kit. were analysed through quantitative ELISA and WB. The WB analysis revealed antibody titers ranging from 800 to12800 with one sample (28) possessing a titer of 1:800, two samples (07, 241) at 1:1600, five samples (53, 277, 204, 275, 04) at 1:3200, one sample (217) at 1:6400 and one sample (229) at 1:12800. The same dilutions of these samples were subjected to our indigenous quantitative ELISA (coated with recombinant SAG1) to determine and antibody (IgG) titer that ranged from 200 to 1600 with two samples (28, 277) at a titer of 1:200, with four samples (53, 07, 204, 241) at a titer of 1:400, with three samples (217, 275, 04) at a titer of 1:800 and with one sample (229) at a titer of 1:1600. In our study, the titers of T. gondii-specific-IgG antibodies revealed in WB were found much higher as compared to those in ELISA but there is a high positive correlation (rs = 0.714) analysed by spearman's rho test. [ABSTRACT FROM AUTHOR]

Trypanosoma evansi is an important hemoparasite of a variety of animal species worldwide. This parasite is a threat to the health of domestic animals as well as wild animals, particularly those managed in captivity. The current study investigated the presence of T. evansi in captive tigers (Panthera tigris tigris) and lions (Panthera leo) in Pakistan. In total, 24 blood samples from 11 tigers and 3 lions (n = 14) were collected during the course of roughly 3 yr (2016–2018). Eighteen samples were subjected to both microscopic and molecular evaluation for the presence of T. evansi; the remaining 6 samples were processed for PCR only. Of the 18 samples tested by both methods, 3 (16%) and 8 (44%) were positive by microscopy and PCR, respectively. This highlights the higher sensitivity of PCR over microscopy for detection of trypanosomes. Of the 24 total samples evaluated by PCR, 12 (50%) were positive. The three sequences obtained showed 99% identity with variant surface glycoprotein genes of the different isolates of T. evansi. The sensitivity, specificity, positive predictive value, and negative predictive value of microscopy in identifying T. evansi was 37.5, 100, 100, and 66.7%, respectively, considering PCR as the gold standard. We recommend rigorous monitoring of captive tigers and lions for hemoparasites, particularly in winter and early spring in areas with high infection rate of this parasite, preferably via PCR. [ABSTRACT FROM AUTHOR]

Simple Summary: Toxoplasmosis is a zoonotic disease caused by T. gondii infection. The main aims of this study were to assess the seropositivity to toxoplasmosis of an exotic breed of cattle (n = 400) from different farms using the Latex Agglutination Test, validate the Cattle Toxo IgG ELISA kit with the help of the commercially available ELISA kit and compare the efficacy of the LAT and Cattle Toxo IgG ELISA kit for assessing seropositivity of cattle to toxoplasmosis. Four hundred cattle sera were collected from an exotic breed of cattle in Pakistan. These sera were evaluated through an LAT and Cattle Toxo IgG ELISA kit. Of 400 samples, 90 were elected and screened through a commercially available ELISA kit. The sensitivity and specificity of the Cattle Toxo IgG ELISA kit came out to be 100% and 96.15%, and in LAT, it was found as 26.31% and 61.53%, respectively. The Cattle Toxo IgG ELISA kit revealed 29.75% (119/400) seropositivity, 6% less than that found through LAT. The results of this study show that Cattle Toxo IgG ELISA kit is a highly accurate and reliable serodiagnostic tool to diagnose bovine toxoplasmosis. Toxoplasma gondii is a protozoan parasite that causes toxoplasmosis in warm-blooded vertebrates, globally. The main aims of this study were to assess the seropositivity to toxoplasmosis of an exotic breed of cattle (n = 400) from different farms using the Latex Agglutination Test and validate Cattle Toxo IgG ELISA kit. Of a total of 400 cattle sera that were evaluated by LAT, 143 (35.75%) were found positive. Based on these data, 90 samples (n = 60 seronegative by LAT; n = 30 seropositive by LAT) were elected for screening through a commercially available ELISA kit. The same 90 samples were screened through a Cattle Toxo IgG ELISA kit for validation purposes. Of 90 samples, 40 were seropositive in the Cattle Toxo IgG ELISA kit (100% sensitivity), and 38 were seropositive in a commercially available ELISA kit. All 50 samples in the Cattle Toxo IgG ELISA kit (96.15% specificity) were also seronegative in the commercially available ELISA kit. Hence, the sensitivity and specificity of the Cattle Toxo IgG ELISA kit came out to be 100% and 96.15%, and in LAT, it was found as 26.31% and 61.53%, respectively. Therefore, the Cattle Toxo IgG ELISA kit is a highly reliable serodiagnostic tool to diagnose bovine toxoplasmosis. [ABSTRACT FROM AUTHOR]

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