Consanguineous Marriages and the Perception of Wife-Beating Justification in Pakistan: An Application of Fairlie Decomposition Analysis.

Pakistan has a significant occurrence of both consanguineous marriages and intimate partner violence (IPV), which may be interlinked. The practice of consanguineous marriages could potentially influence women to rationalize and accept instances of IPV. Such attitudes perpetuate a culture of violence against women, creating difficulties for victims to reject or escape from it. Pakistan has high prevalence rate of consanguineous marriages and IPV. However, no research has been done to explain the difference in acceptance of IPV between women in consanguineous and non-consanguineous marriages. This study used Pakistan Demographic and Health Survey data and applied association tests, logistic regression, and the Fairlie decomposition analysis. The Fairlie decomposition helps us identify the relative contribution of different socioeconomic factors in the inequalities in IPV between the two types of marriages. Five dimensions of wife-beating justification are used as outcome variables. Age, education, and empowerment of women, husband education, woman witness parental violence, region, place of residence, and household wealth status are used as independent variables. The logistic regression results indicated that women in consanguineous marriages of younger age and who witnessed parental violence are more likely to justify wife-beating than those who belong to wealthy households and enjoy more empowerment. Compared to the Punjab province, women residing in Sindh and Baluchistan are less likely and in the Khyber Pakhtunkhwa province are more likely to justify wife-beating. The Fairlie decomposition analysis shows that household wealth status, woman education, and empowerment are the main contributors in explaining the gap between the wife-beating justification of the two groups. The IPV gap can be reduced up to 77% if the distribution of women in different wealth quantiles of the consanguineous marriage group is identical to the non-consanguineous marriage group. Furthermore, woman education level is the second highest contributor. Consanguineous marriages are a prevalent cultural practice in Pakistan and are associated with several negative health and social outcomes. Addressing this issue requires a sustained and comprehensive effort by the government, civil society, and international partners, with a particular focus on women from poor households with less education.

Dynamics of inflammation-associated plasma proteins following faecal microbiota transplantation in patients with psoriatic arthritis and healthy controls: exploratory findings from the FLORA trial.

OBJECTIVES: The gut microbiota can mediate both pro and anti-inflammatory responses. In patients with psoriatic arthritis (PsA), we investigated the impact of faecal microbiota transplantation (FMT), relative to sham transplantation, on 92 inflammation-associated plasma proteins. METHODS: This study relates to the FLORA trial cohort, where 31 patients with moderate-to-high peripheral PsA disease activity, despite at least 3 months of methotrexate treatment, were included in a 26-week, double-blind, randomised, sham-controlled trial. Participants were allocated to receive either one gastroscopic-guided healthy donor FMT (n=15) or sham (n=16). Patient plasma samples were collected at baseline, week 4, 12 and 26 while samples from 31 age-matched and sex-matched healthy controls (HC) were collected at baseline. Samples were analysed using proximity extension assay technology (Olink Target-96 Inflammation panel). RESULTS: Levels of 26 proteins differed significantly between PsA and HC pre-FMT (adjusted p<0.05), of which 10 proteins were elevated in PsA: IL-6, CCL20, CCL19, CDCP1, FGF-21, HGF, interferon-γ (IFN-γ), IL-18R1, monocyte chemotactic protein 3, and IL-2. In the FMT group, levels of 12 proteins changed significantly across all timepoints (tumour necrosis factor (TNF), CDCP1, IFN-γ, TWEAK, signalling lymphocytic activation molecule (SLAMF1), CD8A, CD5, Flt3L, CCL25, FGF-23, CD6, caspase-8). Significant differences in protein levels between FMT and sham-treated patients were observed for TNF (p=0.002), IFN-γ (p=0.011), stem cell factor (p=0.024), matrix metalloproteinase-1 (p=0.038), and SLAMF1 (p=0.042). FMT had the largest positive effect on IFN-γ, Axin-1 and CCL25 and the largest negative effect on CCL19 and IL-6. CONCLUSIONS: Patients with active PsA have a distinct immunological plasma protein signature compared with HC pre-FMT. FMT affects several of these disease markers, including sustained elevation of IFN-γ. TRIAL REGISTRATION NUMBER: NCT03058900.

Immune modulation of goat monocytes by Fasciola gigantica Legumain-1 protein (Fg-LGMN-1).

Legumains belonging to C_13 peptidase family of proteins, and are ubiquitously disseminated among all vertebrate and invertebrate organisms, and have been implicated in innumerable biological and cellular functionality. Herein, we characterized and evaluated immunoregulatory characteristics of Legumain-1 from Fasciola gigantica (Fg-LGMN-1) during its interaction with host immune cells. The isopropyl-ß-d-thiogalactopyranoside (IPTG) stimulated RFg-LGMN-1 protein was positively detected by rat serum containing anti-RFg-LGMN-1 polyclonal antibodies. Furthermore, the uptake of RFg-LGMN-1 by goat monocytes was successfully confirmed using Immunofluorescence Assay (IFA). The immunohistochemical analysis revealed the native localization of LGMN-1 protein on the periphery and internal structures such as suckers, pharynx, and genital pore of the adult parasite, thereby validating its presence in excretory-secretory (ES) products of F. gigantica. The RFg-LGMN-1 co-incubated with concanavalin-A (Con-A) stimulated the increase of interleukin 2 (IL-2), IL-10, and IL-17 in monocytes derived from peripheral blood mononuclear cells (PBMCs) in the concentration-dependent manner. However, the IL-4 cytokine in response to the RFg-LGMN-1 protein declined. These results illuminated the role of LGMN-1 during the parasite-host interface. Our findings elaborated additional evidence that Legumain protein play a role in the manipulating host immune responses during parasite infections. However, further evaluation of RFg-LGMN-1 protein in context of its immunomodulatory roles should be conducted to enhance our understandings of the mechanisms employed by F. gigantica to evade host immune responses.

Cost-benefit ratio of anthelmintic treatment and its comparative efficacy in commercial dairy farms.

Intestinal parasitic infection is one of the major challenges in obtaining optimal production and maintaining the health and welfare of all animals including cattle and buffaloes. Anti-parasitic treatments appear to be a reliable countermeasure. However, the effectiveness and selection of suitable anthelmintics require situational assessments in a given locality. In the current study, the efficacy and impact of benzimidazole (albendazole) were assessed in a total of 400 (100 each) on the performance of buffaloes, buffalo-heifer, cattle, and cattle-heifers at two commercial dairy farms in the Province of Punjab, Pakistan. Additionally, the cost-benefit ratio was calculated by assessing the inputs (medication, feed, and labor cost) and outputs (milk and weight gain). The qualitative and quantitative examination of helminth eggs in each type of animal indicated a prevalence of 73.3, 78.3, 76.6, and 85.0% in cattle, cattle-heifers, buffaloes, and buffaloes-heifers, respectively. Specifically, a highest rate (10.0-13.3%) of Haemonchus sp. infection was only observed in cattle and heifers, while Fasciola sp. infections (10.0-11.6%) were the most often found species in buffaloes and heifers. The highest anthelmintic impacts (egg per gram of feces, p < 0.001) were observed on day 14 post-medication. Until 60 days of post-anthelmintic treatment, an average increase of 0.8 and 0.7 L in milk production per day in cattle and buffaloes, respectively while a total of 11.45 and 9.45 kg body weight were noticed in cattle-heifer and buffaloes-heifer, respectively. Cumulative cost-benefit analysis indicated a positive correlation between treated and non-treated animals. These findings reiterate the importance of anthelmintic drugs in reducing the impacts of parasites on the productivity, health, and well-being of an animal under high infection challenges.

Comparison of sleep quality among COVID-19 patients and non-COVID-19 population in Pakistan: A cross sectional study during the COVID-19 pandemic.

Background: Adverse effects on the health and well-being changes may also express as a decreased sleep quality in COVID-19 patients. This study aimed to assess sleep quality among confirmed COVID-19 patients and the non-COVID-19 Pakistani population. Methods: An online cross-sectional survey was conducted between April and September 2020 in Punjab province, Pakistan. Information about demographic characteristics, COVID-19 diseased status, prior knowledge about COVID-19, and sleep quality using the Pittsburgh Sleep Quality Index (PSQI) was collected. Results: A total of 597 participants were included in this study, 296 (49.6%) COVID-19 patients and 301(50.4%) non-COVID-19 population. The PQSI was used to measure seven distinct sleep components; subjective quality, latency, duration, efficiency, disturbances, medication, and daytime dysfunction. Where mean ± standard deviation (SD) were 0.96 ± 0.743, 1.47 ± 1.032, 0.97 ± 1.006, 0.61 ± 0.995, 1.13 ± 0.649, 0.23 ± 0.651, 1.02 ± 0.861 respectively in total population (N = 597). Sleep latency, sleep duration, and sleep efficiency did not show a significant difference in the T-Test. While sleep quality, sleep disturbances, sleep medication, and daytime dysfunction showed a significant difference between both populations. Conclusion: In conclusion, we highlighted the poor sleep quality in COVID-19 patients as compared to the non-COVID-19 population.

The utility of remdesivir in SARS-CoV-2: A single tertiary care center experience from a developing country.

BACKGROUND: Remdesivir is a monophosphoramidate prodrug of an adenosine analog, and it has a broad-spectrum antiviral activity against paramyxoviruses, flaviviruses, and coronaviruses. Remdesivir is associated with decreased hospital stay and improved outcomes in coronavirus- disease 2019 (COVID-19). METHODOLOGY: Of 846 suspected COVID-19 patients admitted to the hospital, 612 SARS-CoV-2 nasopharyngeal RT-PCR positive patients were evaluated for enrollment in this prospective cohort study. 159 RT-PCR positive patients were given remdesivir. Their clinical, biochemical parameters, hospital stay, and outcomes related to morbidity and mortality were followed. RESULTS: Out of the 159 patients, 141 recovered after remdesivir use. The Chi-square test for independence examined the relation between the day of the first dose, dose of remdesivir, and clinical outcome. The standardized case fatality ratio (CFR) in the 453 hospitalized patients who did not receive remdesivir was 32.89% (N = 149) as compared to 11.32% (N = 18) in the patients who received remdesivir. These findings are in keeping with the therapeutic value of remdesivir in symptomatic SARS-CoV-2 infection of varying severity. CONCLUSION: The use of remdesivir is associated with a decrease in the severity of the SARS-CoV-2 infection. Its use is also associated with a decreased length of hospital stay and lower mortality than the patients who did not receive remdesivir.

The interactions of flaviviruses with cellular receptors: Implications for virus entry.

Flaviviruses are a group of enveloped viruses that enter the host cells through receptor-mediated endocytosis. The entry of flaviviruses into the cells is a multi-step process which involves several host factors that trigger the uptake of the virus. The initial step in the virus life cycle is the interactions between viral envelope proteins and the specific receptors on the surface of host cell. To date, several receptors have been identified such as glycosaminoglycans, tight junction proteins, laminin receptor and phosphatidylserine receptors. Moreover, the viruses may utilize integrins and C-type lectin receptors on the surface of host cells as the initial attachment factors. This mini-review will focus on recent progresses in the understanding of virus attachment, internalization, and membrane fusion with specific emphasis on the cellular receptors.

Tocilizumab in severe COVID-19 - A randomized, double-blind, placebo-controlled trial.

Background: The therapeutic effectiveness of interleukin-6 receptor inhibitor in critically ill hospitalized patients with coronavirus disease 2019 (COVID-19) is uncertain. Methods: To evaluate the efficacy and safety of the outcome as recovery or death of tocilizumab for severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection, we conducted a randomized, double-blinded, placebo-controlled phase 2 trial in critically ill COVID-19 adult patients. The patients were randomly assigned in a 4:1 ratio to receive standard medical treatment plus the recommended dose of either tocilizumab or the placebo drug. Randomization was stratified. The primary outcome was the recovery or death after administration of tocilizumab or a placebo drug. The secondary outcomes were clinical recovery or worsening of the patients' symptoms and inflammatory markers and discharge from the hospital. Results: Of 190 patients included in this study, 152 received tocilizumab, and 38 received a placebo. The duration of hospital stay of the interventional group was 12.9 ± 9.2, while the placebo group had a more extended hospital stay (15.6 ± 8.8). The mortality ratio for the primary outcome, ie, mortality or recovery in the tocilizumab group was 17.8%; p = 0.58 by log-rank test). The mortality ratio in the placebo group was 76.3%; p = 0.32 by log-rank test). The inflammatory markers in the tocilizumab group significantly declined by day 16 compared to the placebo group. Conclusions: The use of tocilizumab was associated with decreased mortality, earlier improvement of inflammatory markers, and reduced hospital stay in patients with severe COVID-19.

Effects of sodium bicarbonate and chromium propionate supplementation on growth performance, blood indices of Beetal bucks under heat stress.

The objective of the current experiment was to determine the effects of sodium bicarbonate (SBC) and chromium propionate (Cr) supplementation on dry matter intake (DMI), growth performance, blood indices, feed sorting behavior, and digestibility of nutrients during the hot and humid condition in Beetal bucks. Twenty-eight Beetal bucks were randomly assigned to four concentrates treatments (n = 7 bucks/treatment) under 2 × 2 factorial arrangements. The factors were (1) chromium supplementation, basal diet without any supplementation (C) and basal diet with Cr @ 1.5 mg Cr/head/day (Cr), and (2) sodium bircbonate supplementation, basal diet supplemented with SBC @15 g/kg of DM (SBC) and diet containing SBC @ 15 g/kg of DM) and Cr @ 1.5 mg/day/animal (SBC + Cr). Chromium was drenched to each animal during the morning feeding. The average daily noon temperature-humidity index (THI) was 86.37 ± 4.01. The daily DMI and ADG was higher (P < .0001) in the SBC, and trend for daily DMI was observed (P < 0.01) for the Cr. The feed to gain ratio was tended to improve (P = 0.056) by the supplementation of Cr. Plasma glucose, cholesterol, and catalase concentration were lower (P < 0.05) in the Cr supplemented, whereas plasma BUN and TPP were not influenced (P > 0.05) by the Cr or the SBC supplementation. The feed selection index indicated that supplementation of Cr and SBC had no effects on selection or rejection of feed particles. In Cr-supplemented bucks, there was a trend for higher ADF digestibility. Digestibility of dry matter, organic matter, NDF, and ADF were not affected by Cr or SBC supplementation. In conclusion, co supplementation of Cr @ 1.5 mg/d and SBC @ 15 g/kg resulted in highest DMI, ADG and improved the feed efficiency in heat-stressed fattening bucks by alleviating negative impacts of HS.

Overcoming open defecation for healthier environment in case of Pakistan.

With the growing awareness of the linkage among open defecation (OD), environment, and health, it is important to understand the factors responsible for OD. It is a necessary step toward developing a strategy to end open defecation for ensuring a better environment and human health. There is no such study available for Pakistan. The study, therefore, aims to bridge this gap. Using household data of Pakistan Demographic and Health Survey (PDHS) 2017-2018, an association of OD with potential predictors, analysis of variance, and a logistic regression model are employed to develop the evidence. The results suggest that place of residence, education, poverty status, social norms, geopolitical regions, and living space significantly predict the OD behavior in Pakistan. This study recommends two things: first is to facilitate the households and communities to own latrines, second is to change the behavior through intervention. However, political commitment and effective administration will be key to ascertain ending OD.

The Role of Social Norm in Acceptability Attitude of Women Toward Intimate Partner Violence in Punjab, Pakistan.

Intimate partner violence (IPV) is a public health and human rights issue with the social and economic impacts felt by women. Punjab is a male-dominated society, where partner violence is generally accepted as a cultural norm and viewed as normal behavior within a marriage. It stems from attitudes that partner violence is a private matter and usually a justifiable response to misbehavior on the part of the wife. The focus of the current study is to investigate the impact of this cultural norm in acceptability attitude of women toward IPV in Punjab province. A large-scale sample size of more than 46,000 from Multi-Indicator Cluster Survey (MICS) 2018 has been used. The impact of the cultural norm on the wife-beating attitude has been investigated in five dimensions: wife going out without telling him, neglecting the children, arguing with him, refusing to have sex with him, and burning the food. The logistic regression results indicate that wife-beating is accepted as a community norm. It has been found that if wife-beating is accepted by community, then the individual female is more than 3 times more likely to consider it justifiable in all five dimensions. The other important factors are education level, household wealth, ethnicity, media exposure, and wife-husband age difference. The level of education of women plays the most important role in rejecting the wife-beating norm, as an increase in level of education is associated with an increase in the probability of rejecting the wife-beating. So, the current study suggests that there is a dire need to increase the female education level to overcome this issue.

Proteomic Analysis of Exosome-Like Vesicles Isolated From Saliva of the Tick Haemaphysalis longicornis.

Extracellular vesicles (EVs), are considered as vehicles of cellular communication. Parasites usually release EVs in their excretory-secretory products to modulate host environment. However, little is known about the secretion of EVs by ticks. In this study, we show for the first time that the tick Haemaphysalis longicornis secretes EVs in saliva that resembles exosomes. EVs were purified from pilocarpine induced saliva of partially engorged H. longicornis ticks. Electron microscopy analysis revealed the presence of exosome-like vesicles with a size of 100 nm. Proteomic analysis by LC-MS/MS identified a total of 356 proteins in tick-derived EVs. Proteome data of tick-derived EVs was validated by Western blot analysis. Immunodetection of Hsp70 and GAPDH proteins indicated that the proteomics data of tick-derived EVs were highly reliable. Bioinformatics analysis (Gene Ontology) indicated association of certain biological and molecular functions with proteins which may be helpful during tick development. Likewise, KEGG database revealed involvement of vesicular proteins in proton transport, detoxification, ECM-receptor interaction, ribosome, RNA transport, ABC transporters, and oxidative phosphorylation. The results of this study provide evidence that EVs are being secreted in tick saliva and suggest that tick saliva-derived EVs could play important roles in host-parasite relationships. Moreover, EVs could be a useful tool in development of vaccines or therapeutics against ticks.

Inhaler Use Technique in Chronic Obstructive Pulmonary Disease Patients: Errors, Practices and Barriers.

Background Inhaled medications are the main therapeutic treatment of chronic obstructive pulmonary disease (COPD) and inhaler technique remained important that can increase medication efficacy, reducing dose and side effects. Poor inhaler technique is multi-factorial and the quality of inhaler technique has not previously assessed in Pakistan. We conducted a study to examine a range of competing factors that impact COPD patient willingness, practices, and preference in using their inhalers. Methods A cross-sectional of 765 patients with COPD were interviewed and assessed by qualitative questionnaires. Objective inhalation technique and steps assessment was performed; satisfaction, preferences, perception, and practice of different types of inhaler devices were evaluated at a single cross-sectional visit at the study enrolment. Results The study included 765 participants of mean age 58.7 years (SD ±7.8); 32% males and 68% females. Almost all of the females were exposed to biomass fuel smoke exposure (99%) and pipe (Huka) smokers 53%, while most male participants were cigarette smokers (92%). Only 6.3% of participants were able to perform correct steps of inhaler use, and few educated patients completed 7-steps. 66% of patients were using dry powder inhalers (DPI) inhaler devices and mostly performed the steps 1, 2, and 4 (98%) correctly, while 44% who were using metered-dose inhalers (MDI) completed only steps 2 and 4 correctly (88%). The majority of participants reported the particular inhaler devices was prescribed by the visiting consultants (54%). Interestingly, they were using two inhalers together (47%) relieving symptoms of dyspnea (83%) and cough (73%). The inhaler use technique was demonstrated to most of the patients by the pharmacy salesman (38.4%), while 15.8% reported that their doctors taught them the inhaler technique. 54.2% reported reason for poor adherence to inhaler use as they understand it might not work lately and 75.2% were not aware of any side effects associated with the regular use of an inhaler. Conclusions Poor inhaler technique is highly prevalent and the associated errors did not appear to be dependent on device type. Most of the participants had not receive proper training about the correct use and were not involved in decision making about the choice of inhaler device.

miRNA profile of extracellular vesicles isolated from saliva of Haemaphysalis longicornis tick.

Extracellular vesicles (EVs) play a key role in host-parasite interactions. Previous studies have shown that parasites can release microRNA (miRNA) containing EVs, which can transfer their contents to host cells and regulate gene expression in recipient cells. However, a little is known about the secretion of EVs by the ticks. This study was therefore, carried out to examine the saliva of ticks for the presence of miRNA containing EVs. Vesicles were purified from saliva of partially engorged Haemaphysalis longicornis ticks. Transmission electron microscopy (TEM) was carried out to confirm that vesicles within saliva were EVs based on size and morphology. Total RNA was extracted from EVs and was analyzed by deep sequencing to determine miRNA profile. TEM analysis confirmed the presence of extracellular vesicle-like structures within tick saliva. RNA-seq analysis showed that tick-derived EVs contained small non-coding RNA populations including miRNAs. The analysis of tick-derived EVs identified 36 known miRNAs, 34 novel miRNAs and 842 novel Piwi-interacting RNAs (piRNA). The results of this study provide evidence that EVs containing miRNAs can be secreted by the ticks and suggest that vesicles could transfer these miRNAs to modulate host cell functions.

Newcastle disease virus induces testicular damage and disrupts steroidogenesis in specific pathogen free roosters.

Newcastle disease (ND), which is caused by Newcastle disease virus (NDV), can cause heavy economic losses to the poultry industry worldwide. It is characterised by extensive pathologies of the digestive, respiratory, and nervous systems and can cause severe damage to the reproductive system of egg-laying hens. However, it is unknown whether NDV replicates in the male reproductive system of chickens and induces any pathologies. In this study, we selected a representative strain (i.e. ZJ1) of the most common genotype (i.e. VII) of NDV to investigate whether NDV can induce histological, hormonal, and inflammatory responses in the testes of specific pathogen free (SPF) roosters. NDV infection increased the expression of toll like receptor TLR3, TLR7, MDA5, IFN-α, IFN-ß, IFN-γ, IL-8, and CXCLi1 in the testes of NDV-infected roosters at 5 days post-infection (dpi). Severe histological changes, including decrease in the number of Sertoli cells and individualized, shrunken spermatogonia with pyknotic nuclei, were observed at 3 dpi. At 5 dpi, the spermatogenic columns were disorganized, and there were fewer cells, which were replaced by necrotic cells, lipid vacuoles, and proteinaceous homogenous material. A significant decrease in the plasma concentrations of testosterone and luteinizing hormone (LH) and the mRNA expression of their receptors in the testes, steroidogenic acute regulatory protein, cytochrome P450 side-chain cleavage enzyme, and 3ß-hydroxysteroid dehydrogenase in the NDV-infected group was observed relative to those in the control group (P < 0.05). Collectively, these results indicate that NDV infection induces a severe inflammatory response and histological changes, which decrease the steroidogenesis.

Phenotypic and Genotypic Comparison of a Live-Attenuated Genotype I Japanese Encephalitis Virus SD12-F120 Strain with Its Virulent Parental SD12 Strain.

The phenotypic and genotypic characteristics of a live-attenuated genotype I (GI) strain (SD12-F120) of Japanese encephalitis virus (JEV) were compared with its virulent parental SD12 strain to gain an insight into the genetic changes acquired during the attenuation process. SD12-F120 formed smaller plaque on BHK-21 cells and showed reduced replication in mouse brains compared with SD12. Mice inoculated with SD12-F120 via either intraperitoneal or intracerebral route showed no clinical symptoms, indicating a highly attenuated phenotype in terms of both neuroinvasiveness and neurovirulence. SD12-F120 harbored 29 nucleotide variations compared with SD12, of which 20 were considered silent nucleotide mutations, while nine resulted in eight amino acid substitutions. Comparison of the amino acid variations of SD12-F120 vs SD12 pair with those from other four isogenic pairs of the attenuated and their virulent parental strains revealed that the variations at E138 and E176 positions of E protein were identified in four and three pairs, respectively, while the remaining amino acid variations were almost unique to their respective strain pairs. These observations suggest that the genetic changes acquired during the attenuation process were likely to be strain-specific and that the mechanisms associated with JEV attenuation/virulence are complicated.

Localized expression and inhibition effect of miR-184 on blood digestion and oviposition in Haemaphysalis longicornis (Acari: Ixodidae).

BACKGROUND: The hard tick Haemaphysalis longicornis (Ixodidae) is widely distributed in East Asia, China, Australia and New Zealand. It can transmit many infectious pathogens, including the causative agents of human rickettsiosis, bovine theileriosis, bovine babesiosis and canine babesiosis. Therefore, a greater understanding of H. longicornis biology might aid in the development of more effective control measures against the tick and tick-borne pathogens. METHODS: We analyzed the expression of miR-184 in different developmental stages and various tissues of H. longicornis using real-time PCR (qRT-PCR). Antagomir (Ant-184) was used to knock-down miR-184, whilst Ms-Ant and non-injected ticks were used as the negative and blank controls, respectively. We used online software tools (RNAhybrid and TargetScan) to predict the putative target genes of miR-184. RESULTS: The expression of miR-184 was highest in unfed nymphs and lowest in unfed larvae. The tissue distribution of miR-184 showed abundant expression in the midgut. To investigate the probable roles of miR-184, antagomir (Ant-184) was used to knock-down miR-184 (t(4) = 12.32, P = 0.0002). After inhibiting miR-184, other biological factors were examined in each group. The engorged body weight was significantly reduced in the treated group (Ant-184) in contrast to control groups (t(22) = 2.19, P = 0.0388). The mean duration of the egg-laying days was significantly increased (33.5 ± 1.91) and the number of eggs (t(10) = 3.147, P = 0.0137), and egg mass (t(10) = 3.4472, P = 0.0063) were significantly reduced in the treated group. During oviposition, eggs were monitored and in half of the ticks of the Ant-184 group the eggs were completely desiccated, lacked embryo development and did not hatch. We analyzed the expression of Vg proteins (Vg1, Vg2, Vg3) in semi-engorged ticks, engorged ticks, ticks at day 2 after engorgement and egg stage in Ant-184, non-injected and Ms-Ant groups, and found significant variation. CONCLUSIONS: This study provides information on the role of miR-184 in H. longicornis ticks. The data suggest that miR-184 targets Vg proteins and affects blood digestion and oviposition.

Research progress on the composition and function of parasite-derived exosomes.

Parasites use excretory-secretory pathways to communicate with the host. Characterization of exosomes within the excretory-secretory products reveal by which parasites manipulate their hosts. Parasite derived exosomes provide a mechanistic framework for protein and miRNAs transfer. Transcriptomics and proteomics of parasite exosomes identified a large number of miRNAs and proteins being utilized by parasites in their survival, reproduction and development. Characterization of proteins and miRNAs in parasite secreted exosomes provide important information on host-parasite communication and forms the basis for future studies. In this review, we summarize recent advances in isolation and molecular characterization (protein and miRNAs) of parasite derived exosomes.

A microRNA profile of saliva and role of miR-375 in Haemaphysalis longicornis (Ixodida: Ixodidae).

BACKGROUND: Tick saliva contains many bioactive molecules that are involved in attachment to the host, blood-feeding and transmission of pathogens. MicroRNAs (miRNAs) are a class of short non-coding RNAs with a length of 19-24 nucleotides. They act as regulators of gene expression by binding to their target mRNA at the post-transcriptional level and control a variety of cellular functions, including regulation of growth, metabolism and development. The detection and characterizations of miRNAs from tick saliva may help explain the molecular mechanisms involved in the interaction between ticks, pathogens and hosts. They may also contribute to the discovery of vaccines, which can control ticks and the pathogens they transmit. RESULTS: An RNA library was generated from the saliva of fed adult Haemaphysalis longicornis ticks, containing 17.4 million clean reads of 18-30 nucleotides. Overall, 319 known miRNAs and 1 novel miRNA were found. The 10 most abundantly expressed miRNAs present in tick saliva were miR-100_2, miR-315, miR-184_1, miR-100-5p_2, miR-5307, miR-184-3p_3, Let-7-5p_6, miR-71_5, miR-1-3p_6 and miR-10-5p_2. miR-375, one of the abundantly expressed, was subjected to quantitative real-time PCR analysis (qRT-PCR) in various tick developmental stages, as well as in different tissues isolated from adult ticks. The expression of miR-375 in different tick development stages was highest in unfed nymphs and lowest in the egg stage. In the tissues of adult ticks, miR-375 was most highly expressed in the salivary gland. To investigate the possible role of miR-375, Ant-375 was used to inhibit the miR-375. The treated group (Ant-375) had a reduced number of eggs (t(10) = 2.652, P = 0.0242), eggs that were partially desiccated, and reduced egg hatchability (t(10) = 2.272, P = 0.044) compared to Ms-Ant and the non-injected control. CONCLUSIONS: This is the first study to investigate the miRNA profile in tick saliva and the role of miR-375 in H. longicornis. The identification and characterization of miRNA in tick saliva may help to reveal the molecular mechanisms of interactions among ticks, pathogens and hosts, and suggest new vaccine strategies to control tick-borne diseases.

Molecular Detection and Prevalence of Hepatozoon canis in Dogs from Punjab (Pakistan) and Hematological Profile of Infected Dogs.

The intraleukocytic parasite, Hepatozoon canis, causes the sometimes fatal tick borne disease canine hepatozoonosis. In this study, dogs from Islamabad, Lahore, and Multan Districts of the Punjab region of Pakistan were surveyed to investigate the presence and prevalence of H. canis infection and to determine the effects of the parasite on hematological parameters. Blood samples were collected from 151 domestic dogs (149 pet, 2 stray) of both sexes and varying ages. Data on sex, age, tick infestation, and clinical factors (body temperature, mucous membrane status, and presence of hematuria and vomiting) were collected. Using PCR, 18 dogs (11.9%) were found positive for the presence of H. canis DNA. Partial sequences of the 18S rRNA gene shared 99-100% similarity with the corresponding H. canis isolates. This epidemiological survey revealed higher prevalence of H. canis in Islamabad (11/49, 22.4%) compared to Lahore (3/52, 5.8%) and Multan (4/50, 8%) in Pakistan. No investigated epidemiological or clinical factors were found to be associated with the presence of H. canis (p > 0.05) in dogs. H. canis positive dogs exhibited higher minimum inhibitory dilution (p = 0.04), mixed inclusion (p = 0.008) and relative distribution width of red blood cells (p = 0.02), and lower hematocrit (p = 0.03) and mean hemoglobin content (p = 0.03) than did dogs in which H. canis was not detected. We are recommending this PCR-based protocol to the veterinary practitioners for the detection and/or confirmation of H. canis in dogs suspected for hepatozoonosis to improve their health status.