Construction of multi-epitope vaccine against the Rhipicephalus microplus tick: an immunoinformatics approach.

Rhipicephalus microplus, known as the hard tick, is a vector for the parasites Babesia spp. and Anaplasma marginale, both of which can cause significant financial losses to the livestock industry. There is currently no effective vaccine for R. microplus tick infestations, despite the identification of numerous prospective tick vaccine candidates. As a result, the current research set out to develop an immunoinformatics-based strategy using existing methods for designing a multi-epitope based vaccination that is not only effective but also safe and capable of eliciting cellular and humoral immune responses. First, R. microplus proteins Bm86, Subolesin, and Bm95 were used to anticipate and link B and T-cell epitopes (HTL and CTL) to one another. Antigenicity testing, allergenicity assessment, and toxicity screening were just a few of the many immunoinformatics techniques used to identify potent epitopes. Multi-epitope vaccine design was chosen based on the antigenic score 0.935 that is promising vaccine candidate. Molecular docking was used to determine the nature of the interaction between TLR2 and the vaccine construct. Finally, molecular dynamic simulation was used to assess the stability and compactness of the resulting vaccination based on docking scores. The developed vaccine was shown to be stable, have immunogenic qualities, be soluble, and to have high expression by in silico cloning. These findings suggest that experimental investigation of the multi-epitope based vaccine designed in the current study will produce achievable vaccine candidates against R. microplus ticks, enabling more effective control of infestations.

Immuno-bioinformatic approach for designing of multi-epitope merozoite surface antigen of Babesia bigemina and evaluation of its immunogenicity in inoculated calves.

Babesiosis is a parasitic disease caused by intraerythrocytic parasites of the genus Babesia, which infect both wild and domestic animals. Merozoite surface antigens (MSAs) have been identified as efficient immunogens in Babesia-infected animals. MSAs play a key role in the invasion process and have been proposed as potential targets for vaccine development. Epitope-based vaccines offer several advantages over whole protein vaccines as the immunogenic proteins are small and can induce both Th1 and Th2 immune responses, which are desirable for protection. However, the MSA, particularly gp45, is polymorphic in Babesia bigemina, posing a challenge to vaccine development. The purpose of this study was to develop a recombinant gpME (gp45-multi-epitope) for a vaccine against Babesia bigemina. B-cell, T-cell, and HLA epitope predictions were used to synthesize the gpME sequence from the consensus sequence of gp45. The gpME sequence was synthesized and cloned in the pET28α vector through the commercial biotechnology company to get pET28-gpME. The plasmid cloned with the gpME sequence comprising 1068 bp was expressed in a bacterial expression system. A band of 39 kDa of rec-gpME was obtained via SDS-PAGE and Western blotting. Rec-gpME @200ng was injected in calves 3 times at 2 weeks interval. The humoral response was evaluated through the indirect ELISA method. The ELISA with rec-gp45 protein showed a significant value of optical density. The recombinant protein containing multiple epitopes from the MSA gp45 may represent a promising candidate for a vaccine against Babesia bigemina.

Molecular prevalence of Anaplasma marginale in ruminants and Rhipicephalus ticks in northern Pakistan.

Anaplasma marginale is the most prevalent tick-borne haemoparasite of cattle and causes huge economic losses to the dairy industry worldwide. This study aimed to determine the occurrence of A. marginale infection in blood and tick samples collected from livestock animals in the districts located in Khyber Pakhtunkhwa (KPK), Pakistan. A total of 184 blood and 370 tick samples were included in this study. It has never been reported that sheep, goats, and cattle in Tank, Ghulam Khan, Birmil and Miran Shah areas were infected with A. marginale. All samples of blood and ticks were collected through random sampling from March 2021 to January 2022 from cattle, sheep and goats and screened through PCR for anaplasmosis by using primer pairs of Anaplasma spp. Three hundred and seventy ticks were collected from infested hosts (120/184, 64.21%). Among the four morphologically identified tick species, the highest occurrence was recorded for Rhipicephalus sanguineus (n=138, 37.29%), followed by Rhipicephalus microplus (n=131, 35.4%), Rhipicephalus annulatus (n=40, 10.81%), Hyalomma anatolicum (n=31, 8.37%), and Hyalomma marginatum (n=30, 8.1%). The occurrence of female tick was highest (n=160, 43.24%), followed by nymphs (n=140, 37.38%) and males ticks (n=70, 18.9%). Among these ticks, A. marginale was detected in female ticks of R. microplus, and R. sanguineus. Molecular identification of A. marginale was confirmed in 120 out of 184 blood samples and 6 out of 74 tick samples. Overall, occurrence of A. marginale in blood and tick samples was found to be 65.21% and 8.1% respectively. Species-wise occurrence in blood samples of goats were 71.11% followed by sheep 68.31% and cattle 50%. Specie-wise occurrence of A. marginale in tick samples of cattle were 12.5% followed by goats 6.89%. The obtained sequence showed similarity with A. marginale reported from Kenya and USA. We report the first PCR based detection of A. marginale infection in blood samples and in R. sanguineus ticks of goats simultaneously.

Occurrence of ticks and tick-borne mixed parasitic microbiota in cross-bred cattle in District Lahore, Pakistan.

The present study was focused on the incidence of ticks and tick-borne diseases (TTBD) in cross-bred cattle (Friesian x Sahiwal) of two farms (n = 2548) in district Lahore, Pakistan. We collected total of 572 ticks (adults and nymphs) and blood samples (10 ml) for microscopic i.e., blood smear test - Giemsa Stain (BST) and molecular analysis; Reverse Line Blot-General Primer-PCR (RLB-PCR) and Specie Specific Primer PCR (SP-PCR) from infested cattle (n = 100) from months of April to September. Results: The tick specie identified was Rhipicephalus microplus at both farms, with significant difference in infestations rate amongst both farms (p< 0.0001). The cross-bred cattle having higher ratio of Friesian blood and lower ratio of Sahiwal blood were mostly infested by ticks (p < 0.0458) and haemoparasites (p <0.474) and vice versa. The SP-PCR showed higher number of haemoparasites infection than BST, which revealed 16% T. annulata (p < 0.0001 and k value 0.485, 0.0001), 51% B. bigemina (p < 0.0001 and k value 0.485, 0.0001) and 15% A. marginale (p < 0.001 and k value 0.207, 0.001), respectively. The single infection with B. bigemina was 34% (n = 34/100) and A. marginale 6% (n = 6/100). The double infection with T. annulata/B. bigemina was 8% (n = 8/100) and B. bigemina/A. marginale 1% (n = 1/100). Whereas the triple infection with T. annulata/B. bigemina/A .marginale was 8% (n = 8/100). The phylogenetic study of isolated sequence of T. annulata revealed close homology to isolates from Iran (87%), B. bigemina to isolates from Cuba (94 to 100%) and A. marginale with isolates from Pakistan (99 to 98%).

Untapped potential of zeolites in optimization of food waste composting.

This study aims to examine the effect of zeolites in optimizing the process of food waste composting. A novel method of sequential hydrothermal was introduced to modify the natural zeolite and apply to in-vessel compost bioreactors. Raw and modified natural zeolites were applied at 10 and 15% (w/w) of the total waste and compared with un-amended control trial. Both raw and modified zeolites affected the composting process, but the notable results were observed for modified natural zeolite. The results for compost stability parameters were prominent at 15% modified natural zeolite concentration. The rapid and long-term thermophillic temperature and moisture content reduction to the optimum range was observed for modified natural zeolite. Furthermore, the total ammonium (NH4+) and nitrate (NO3-) concentration in modified natural zeolite were increased by 11.1 and 21.5% respectively as compared to raw zeolite. Compost stability against moisture contents (MC), electrical conductivity (EC), organic matters (OM), total carbon (TC), mineral nitrogen, nitrification index (NI) and germination index (GI) was achieved after 60 days of composting that was in accordance with the international compost quality standards. The findings of this study suggested the suitability of modified natural zeolite addition at 15% to the total waste as the optimum ratio for the composting of food waste in order to achieve a stable nutrient-rich compost.

Insecticide susceptibility status and major detoxifying enzymes activity in Anopheles subpictus from Kasur, Pakistan.

Anopheles subpictus s.l. Grassi (Diptera: Culicidae) is a malaria vector in South Asia, where insecticides are the mainstay for vector control interventions. Information on any variation in metabolic enzyme levels in mosquitoes is helpful with respect to adapting alternative strategies for vector control. The scarce data on the biochemical basis of insecticide resistance in malaria vectors of Pakistan limit the available information for vector control interventions within the country. The insecticide susceptibility status and its biochemical basis against dichlorodiphenyltrichloroethane (DDT) (4%), deltamethrin (0.05%) and permethrin (0.75%) in An. subpictus s.l. collected from all Tehsils of district Kasur were evaluated. For this purpose, a World Health Organization susceptibility bioassay was performed followed by the detection of altered metabolic enzyme activity using biochemical assays. Similarly, a significant difference in knock-down effect was observed among field collected and susceptible strain against all insecticides 24 h post exposure. The overall mean mortality rates of DDT, deltamethrin and permethrin were 27.86% [95% confidence interval (CI) = 29.65-26.06], 44.89% (95% CI = 46.23-43.54) and 78.82% (95% CI = 80.16-77.47), respectively. The biochemical assays revealed an elevated level of metabolic enzymes in the field population. The results provide evidence of resistance against organochlorine and pyrethroid groups in a field population of An. subpictus s.l. from district Kasur mediated by multiple metabolic mechanisms, including acetylcholinesterases, esterases, cytochrome P450 and glutathione S-transferases.

Histopathological Investigation of Skin and Hides Damage of Small and Large Ruminants due to Naturally Infested Ticks.

Ticks are important ectoparasites which transmit many disease pathogen to animals; these are labelled tick borne diseases (TBD). Tick induced damage to skin and hides has not received attention. Skin and hides are important for the leather product industry, particularly in Pakistan. Due to economic importance and financial loss by ticks in leather industry, the present study was designed to investigate skin and hides damage due to ticks at microscopic level. Naturally tick infested tissue samples of hides and skin were collected from slaughter houses. Primary lesions at tick feeding sites showed epidermal edema with adjacent dermal edema. Histopathological examination revealed degeneration of epidermal layer down to the basal layer. Epidermal and sub dermal layers often displayed focal necrosis infiltrated with neutrophils and mononuclear cells at tick bite sites. Hyperplasia of keratinocytes was also seen at sites of ruptured epidermis. Quality of leather depends upon the grain (Outer) surface skin/hides. Ticks infestation damages the outer surface, due to bites, inflammatory responses, and secondary bacterial infections that often become established at feeding sites. Control of ticks should be given consideration to reduce infestation induced losses in the leather industry in Pakistan.

Optimization of food waste compost with the use of biochar.

This paper aims to examine the influence of biochar produced from lawn waste in accelerating the degradation and mineralization rates of food waste compost. Biochar produced at two different temperatures (350 and 450 °C) was applied at the rates 10 and 15% (w/w) of the total waste to an in-vessel compost bioreactor for evaluating its effects on food waste compost. The quality of compost was assessed against stabilization indices such as moisture contents (MC), electrical conductivity (EC), organic matters (OM) degradation, change in total carbon (TC) and mineral nitrogen contents such as ammonium (NH4+) and nitrate (NO3-). The use of biochar significantly improved the composting process and physiochemical properties of the final compost. Results showed that in comparison to control trial, biochar amended compost mixtures rapidly achieved the thermophilic temperature, increased the OM degradation by 14.4-15.3%, concentration of NH4+ by 37.8-45.6% and NO3- by 50-62%. The most prominent effects in term of achieving rapid thermophilic temperature and a higher concentration of NH4+ and NO3- were observed at 15% (w/w) biochar. According to compost quality standard of United States (US), California, Germany, and Austria, the compost stability as a result of biochar addition was achieved in 50-60 days. Nonetheless, the biochar produced at 450 °C had similar effects as to biochar produced at 350 °C for most of the compost parameters. Therefore, it is recommended to produce biochar at 350 °C to reduce the energy requirements for resource recovery of biomass and should be added at a concentration of 15% (w/w) to the compost bioreactor for achieving a stable compost.

Serum sonic hedgehog (SHH) and interleukin-(IL-6) as dual prognostic biomarkers in progressive metastatic breast cancer.

Serum from one hundred and ten breast cancer patients and thirty healthy female volunteers, were prospectively collected and evaluated for serum levels of Shh and IL-6 using human Shh and IL-6 specific enzyme-linked immunoassays. All patients were regularly monitored for event free survival (EFS) and overall survival (OS). Overall outcome analysis was based on serum Shh and IL-6 levels. In patients with progressive metastatic BC, both serum Shh and IL-6 concentrations were elevated in 44% (29 of 65) and 63% (41 of 65) of patients, respectively, at a statistically significant level [Shh (p = 0.0001) and IL-6 (p = 0.0001)] compared to the low levels in healthy volunteers. Serum levels tended to increase with metastatic progression and lymph node positivity. High serum Shh and IL-6 levels were associated with poor EFS and OS opposite to the negative or lower levels in serum Shh and IL-6. The elevated levels of both serum Shh and IL-6 were mainly observed in BC patients who had a significantly higher risk of early recurrence and bone metastasis, and associated with a worse survival for patients with progressive metastatic BC. Further studies are warranted for validating these biomarkers as prognostic tools in a larger patient cohort and in a longer follow-up study.

Effect of aerobic exercise on patients with primary fibromyalgia syndrome.

Sixty eight adult patients of fibromyalgia were included in this prospective study from the Outpatient Department of Physical Medicine and Rehabilitation of Bangabandhu Sheikh Mujib Medical University (BSMMU), Dhaka during the period of January 2003 to June 2003. Study samples were assigned into two treatment groups: Group A (n = 38) with exercise by static bicycle and aerobic walking in addition to tricyclic antidepressant and analgesic and Group B (n = 30) was non exercise group, treated with tricyclic antidepressant and analgesic only. The total duration of treatment was 16 weeks. Pre-treatment (week 0) and post treatment (week 16) evaluation was performed in both groups. Evaluation parameters included pain grade, number of trigger points, occurrence of arousal at night, frequency of micturition and global evaluation by the physician. After 16 weeks, mean improvement of exercise group and non exercise group was 48% and 39% respectively but this difference was not statistically significant. Therefore, from this study it was observed that aerobic exercise showed no significant benefit to fibromyalgia patients.