Insights into the involvement of male Hyalomma anatolicum ticks in transmitting Anaplasma marginale, lumpy skin disease virus and Theileria annulata.

Ticks can transmit viruses, bacteria, and parasites to humans, livestock, and pet animals causing tick-borne diseases (TBDs) mechanically or biologically in the world. Lumpy skin disease virus, Anaplasma marginale, and Theileria annulata inflict severe infections in cattle, resulting in significant economic losses worldwide. The study investigated the potential transmissions of LSDV, A. marginale, and T. annulata through male Hyalomma anatolicum ticks in cattle calves. Two 6-month-old Holstein crossbred calves designated as A and B were used. On day 1, 15 uninfected female ticks (IIa) and infected batch of 40 male ticks (I) were attached on calf A for 11 days. Filial transmission of the infections was observed in female ticks (IIb) collected from calf A, where 8 female ticks had been co-fed with infected male ticks. The blood sample of calf B was found positive through PCR for the infections. The larvae and egg pools obtained from the infected ticks were also tested positive in PCR. The study confirmed the presence of these mixed pathogens and potential intra-stadial and transovarial transmissions of A. marginale, T. annulata, and LSDV in male and female ticks of H. anatolicum and experimental calves to establish the feasibility of infections through an in vivo approach.

Molecular detection of Toxoplasma gondii among commensal rodents from the Sahiwal division, Punjab, Pakistan.

Toxoplasmosis is a zoonotic disease in humans and animals caused by the protozoan parasite Toxoplasma gondii. Rodents not only act as intermediate hosts for T. gondii but are also a significant source of T. gondii infection for other hosts. There is limited information about the prevalence of this protozoan within rodents in Pakistan. The current study was conducted to determine the prevalence of T. gondii in commensal rodents as well as associated risk factors. A total of 236 rats including Rattus rattus and Rattus norvegicus were captured from three districts of the Sahiwal division in Punjab province, Pakistan. Brain samples were collected from each specimen. Data was gathered about rodent species, gender, age, location, districts, seasonality, and habitat type. PCR assays were used to screen for T. gondii from the brain samples. A Chi-square analysis was used to investigate the association between positive samples and risk factors. A total of 236 rodent spleen samples were examined for detection of the B1 and SAG3 genes of T. gondii DNA by conventional PCR, and 14 (5.9%) were positive. T. gondii prevalence was higher in R. norvegicus (8.2%) compared to R. rattus (4.6%). All risk factors were statistically non-significant. This study provides evidence of T. gondii in rodents in the Sahiwal division and indicates that other animals and humans are at risk of infection.

Comparative evaluation of cell-mediated immune response in calves immunized with live-attenuated and killed Theileria annulata vaccines.

Tropical theileriosis is a tick-borne disease caused by the protozoan Theileria annulata and transmitted by numerous species of Ixodid ticks of the genus Hyalomma. The main clinical signs are fever, lymphadenopathy, and anemia responsible for heavy economic losses, including mortality, morbidity, vaccination failure, and treatment cost. Development of poor cell-mediated immunity (CMI) has been observed in the case of many bovine pathogens (bacteria, viruses, and parasites). Quantification of CMI is a prerequisite for evaluating vaccine efficacy against theileriosis caused by T. annulata. The current study evaluated the CMI in calves administered with two types of T. annulata vaccine (live attenuated and killed). We prepared a live attenuated T. annulata vaccine by attenuation in a rabbit model and also prepared killed vaccine from non-attenuated T. annulata. For the evaluation of immune response in experimental groups including control, 20 calves were divided into four different groups (A, B, C, and D). They were either inoculated subcutaneously with live rabbit-propagated-Theileria-infected RBCs (5 × 106) (group A) or with killed T. annulata vaccine (2 × 109 schizonts) with Freund's adjuvant (group B), along with an infected group (group C) and a healthy control group (group D). The protection of vaccinated calves was estimated with challenge infection. Our results showed that with a single shot of live-attenuated and killed vaccine with a booster dose elicited cell-mediated immune responses in immunized calves. We observed a significant elevation in CD4 + and CD8 + T cells in immunized calves. A significant difference in the CD8 + T cell response between the post-challenge stage of killed and live vaccine (p < 0.0001) was observed, whereas no other difference was found at both pre- and post-immunization stages. A similar finding was recorded for the CD4 + T cells at a post-challenge stage, where a significant difference was seen between killed and live vaccine (p < 0.0001). Another significant difference was observed between the CD8 + T cells and CD4 + T cells at the post-challenge stage in the live vaccine group, where there was a significantly higher induction of CD4 + T cell response (p < 0.0001).

Seroprevalence of toxoplasmosis in humans and animals in Pakistan: a systematic review and metadata analysis.

OBJECTIVE: To review the seroprevalence of toxoplasmosis in Pakistan. METHODS: The systematic review comprised search on Science Direct, Google Scholar, PubMed and Scopus databases for studies related to the seroprevalence of toxoplasmosis in Pakistan published between 2006 and 2020 which used serological diagnostic tests to detect Toxoplasma gondii. Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines were used throughout the review and statistical analysis was done using forest plot and random effect model. RESULTS: Of the 7093 human studies initially found, 20(0.28%) were reviewed. Of the 16,432 animal studies, 16(0.09%) were selected for detailed review. The pooled seroprevalence of toxoplasmosis in humans, calculated in this review was found as (76%) (95% confidence interval: 69-83%). Seroprevalence of human toxoplasmosis was higher in Khyber Pakhtunkhwa (31.7%) than Punjab (20.4%). Pooled seroprevalence in animals calculated in this review was found as (69%) (95% confidence interval: 64-74%). Seroprevalence in animals was higher in Khyber Pakhtunkhwa (44.7%) than Punjab (29.4%). CONCLUSIONS: The seroprevalence of toxoplasmosis in both humans and animals should be studied it other parts of Pakistan as well.

Molecular epidemiology and characterization of Babesia bovis in cattle of Khyber Pakhtunkhwa province, Pakistan.

Babesiosis is a tick-borne disease found globally but most prominent in tropical and subtropical regions. It is responsible for huge mortality and morbidity, especially in developing countries like Pakistan. The current study was designed to determine the molecular epidemiology and characterization of Babesia bovis (B. bovis) infection in cattle populations of districts Mardan, Kohat and Swat of Khyber Pakhtunkhwa (KP) province of Pakistan. A total of 434 tick-infested animals were sampled. Blood samples were collected, processed and then examined initially by microscopy for the presence of Babesia and were later confirmed through PCR by targeting cytochrome b gene, and the PCR products were sequenced. Phylogenetic analysis of sequenced isolates of the current study showed close sequence similarity with the reported strain of China. A non-significant association (p > 0.05) was observed between the prevalence of infections and risk factors. The overall prevalence of infection in all three districts was 10.11%. In district Swat (12.61%), the prevalence was recorded as the highest for B. bovis infection followed by district Mardan (10.60%) and district Kohat (06.90%). The Friesian breed of cattle, females and adult animals were highly susceptible to B. bovis infection. The prevalence of infection was recorded highest during the summer season and lowest during the winter season. This study concludes that B. bovis infection is prevalent in three studied districts of KP province and the sequenced isolates of the current study showed close sequence similarity with the reported strain of China.

Challenges against CO2 abatement strategies in cement industry: A review.

Cement industry is an intensive source of fuel consumption and greenhouse gases (GHGs) emissions. This industry is responsible for 5% of GHGs emissions and is among the top industrial sources of carbon dioxide (CO2) emissions. Therefore, CO2 emissions reduction from cement production process has been always an appealing subject for researches in universities and industry. Various efforts have been carried out to mitigate the huge mass of CO2 emissions from the cement industry. Although, majority of these strategies are technically viable, due to various barriers, the level of CO2 mitigation in cement industry is still not satisfactory. Among numerous researches on this topic, only a few have tried to answer why CO2 abatement strategies are not globally practiced yet. This work aims to highlight the challenges and barriers against widespread and effective implementation of CO2 mitigation strategies in the cement industry and to propose practical solutions to overcome such barriers.

Recent advances in nucleic acid-based methods for detection of helminth infections and the perspective of biosensors for future development.

Pathogenic helminth infections are responsible for severe health problems and economic losses worldwide. Timely and accurate diagnosis of helminth infections is critical for adopting suitable strategies for pathogen control. Here, we review recent advances in nucleic acid-based diagnostic methods, including polymerase chain reaction, quantitative qPCR, loop-mediated isothermal amplification and recombinase polymerase amplification, and discuss their advantages and disadvantages for diagnosing helminth infections. In addition, we highlight recent advances in biosensors for the detection of nucleic acid biomarkers that can potentially be used for the diagnosis of helminth infection.

In vitro influence of Theileria annulata on the functions of bovine dendritic cells for stimulation of T lymphocyte proliferation.

The present study was performed on antigen-presenting cells (APCs) of Theileria annulata transformed dendritic cells (TaDCs) and monocyte-derived dendritic cells (MoDCs) to compare differences in antigen presentation and stimulation of T lymphocyte proliferation. Antigen presentation for T lymphocyte proliferation was analysed by flow cytometry. Additionally, the level of mRNA transcription of small GTPases of the Rab family expressed in the TaDC cell line was analysed by quantitative real-time polymerase chain reaction (Q-RT-PCR). The endocytosis rate of TaDCs was significantly (P < 0.01) lower than in MoDCs. In contrast, when T lymphocytes were co-cultured with TaDC-APCs T cell proliferation was similar, while co-culture with MoDC-APC stimulated proliferation of CD4+ cells to a greater degree than CD8+ cells. However, the efficacy of TaDC-APCs to stimulate T lymphocytes dropped as the number of passages of TaDC-APC increased. Likewise, the transcription level of Rab family genes also significantly (P > 0.001) declined with progressive passages (>50) of the TaDC cell line. We conclude that initially the TaDC cell line efficiently presents antigen to stimulate T lymphocyte proliferation to produce a cellular immune response against the presented antigen.

A systematic review on modelling approaches for economic losses studies caused by parasites and their associated diseases in cattle.

Parasites reside inside or outside their hosts and get host nutrition and blood. Here, we have emphasized economic losses in cattle caused by parasitic diseases due to ecto- and endo- parasites (flies, ticks, mites and helminths). We have outlined different methods/models including economic evaluation techniques and dynamic analysis as a major class, used for the calculation of economic losses caused by parasites in cattle. According to already conducted studies, a decrease in production is mentioned in quantity and percentage while financial losses are expressed in the form of account with respect to per head, herd or for the specific study area. The parasites cause the reduced production and financial losses due to control, treatment and mortality costs. We calculated the average decrease in milk production and organ condemnation as 1.16 L animal-1 day-1 and 12.95%, respectively, from overall cattle parasitic infections. Moreover, the average calculated financial and percentage losses were US$ 50.67 animal-1 year-1 and 17.94%, respectively. Economically important parasitic diseases mentioned here are caused by specific spp. of protozoans and helminths according to data collected from the literature. Protozoan diseases include tick-borne diseases, coccidiosis, neosporosis, trypanosomiasis and cryptosporidiosis. Losses due to tick-borne infections were encountered for decreased milk production, mortality, treatment and control. Losses from coccidiosis were due to decreased weight gain, treatment costs and mortality. While abortion losses were encountered in neosporosis. Trypanosomiasis caused losses due to a decrease in milk yield. Moreover, only diagnostic (conventional or molecular techniques) cost was taken into account for cryptosporidiosis. Economically important nematode parasites are Oesophagostomum spp., Cooperia spp., Trichostrongylus spp., Strongyloides spp., Ostertagia spp. and Haemonchus placei. Due to the zoonotic importance of echinococcosis, Echinococcus granulosus is the most economically important cestode parasite. Losses caused by echinococcosis were due to organ condemnation, carcass weight loss and decreases hide value, milk production and fecundity. While, fascioliasis is one of the most economically important trematodal disease, which causes cirrhosis of the liver due to parasite migration, and thus, the organ becomes inedible. So, it would be helpful for farmers and researchers to approach these methods/models for calculation of parasitic losses and should adopt suitable measures to avoid long-term economic losses.

Prevalence of Toxoplasma gondii oocysts through Copro-PCR in cats at Pet Center (UVAS), Lahore, Pakistan.

Toxoplasmosis is a major zoonotic disease of warm-blooded animals caused by Toxoplasma gondii. Cats are the only definitive host and they excrete environmentally resistant T. gondii oocysts in their faeces. Coproscopy was used to detect oocysts of enteric coccidians and then Copro-PCR was employed to test specifically for T. gondii in 470 cat samples. The prevalence of T. gondii oocysts was 2.3% (11/470) based on PCR. We observed 15 (3.2%) of 470 samples positive for coccidian oocysts by microscopy. The presence of Copro-DNA of T. gondii was found significantly higher (p<0.05) in males than females. We tested 11 samples of T. gondii oocysts in which 9 samples were from coccidian oocysts positive samples and 2 samples from negative faecal samples. Our results showed that PCR is the reliable method for the detection of faecal oocysts of T. gondii in cats as compared to microscopy. As per our knowledge, ours is first study for Copro-PCR prevalence of cats' T. gondii oocysts excretion in Pakistan.

Molecular epidemiology of bovine anaplasmosis in Khyber Pakhtunkhwa, Pakistan.

Bovine anaplasmosis is endemic in Pakistan where it reduces livestock productivity and leads to high mortality, especially in young animals. This study was aimed to identify the potential risk factors responsible for the occurrence and spread of anaplasmosis in cattle and buffaloes for the first time in Pakistan. A total of 900 (cattle = 479, buffalo = 421) blood samples were collected irrespective of age and sex from three distinct zones of Khyber Pakhtunkhhwa (KP) province of Pakistan. Polymerase chain reaction (PCR) technique was used for the molecular detection of anaplasmosis. Data collected on a piloted questionnaire including 11 predicting variables which were analyzed using R-statistical software, and association between the dependent and independent variables was assessed using univariable analysis. Automated and manual approaches were exercised, producing comparable models. Key risk factors identified in all the approaches included species of the animal, breed of animal, sex of animal, tick infestation status, previous tick history, tick control status, and acaricides used (odds ratio > 1). The 611 bp DNA fragment specific for 16S rRNA gene of Anaplasma spp. was produced from 165 samples. The samples were confirmed for anaplasmosis through sequencing and BLAST queries. The findings of the current study conclude that by enhancing the protective measures to control the identified risk factors can reduce the spread of anaplasmosis in Pakistan.

Risk management and loss prevention strategies for fertilizer industries.

Various toxic and flammable gases exist in the fertilizer industry whose release quantification is very important regarding emergency preparedness, planning and response, and well-being of the community. ALOHA threat zones and threat at a point coupled with MARPLOT are evaluated for ammonia, methane, carbon dioxide and hydrogen release, and outdoor and indoor concentrations of these gases in nearby residences and highways calculated. These footprints are calculated using ALOHA which requires inputs such as site data, site location, building type, gas name, atmospheric inputs, release source information and dispersion model to display the threat zone, which can then be shown on MARPLOT. Potential impact of these releases on the community is mitigated through releasing equipment isolations, water sprays for dilutions, dilutions through steam or air and emergency sirens for information. This article covers hazards in the fertilizer industry, and provides general guidelines for operational staff of any industry to mitigate hazards.

Carbon capture, utilization and storage opportunities to mitigate greenhouse gases.

Carbon capture, utilization and storage (CCUS) technologies are utmost need of the modern era. CCUS technologies adoption is compulsory to keep global warming below 1.5 °C. Mineral carbonation (MC) is considered one of the safest and most viable methods to sequester anthropogenic carbon dioxide (CO2). MC is an exothermic reaction and occur naturally in the subsurface because of fluid-rock interactions with serpentinite. In serpentine carbonation, CO2 reacts with magnesium to produce carbonates. This article covers CO2 mitigation technologies especially mineral carbonation, mineral carbonation by natural and industrial materials, mineral carbonation feedstock availability in Pakistan, detailed characterization of serpentine from Skardu serpentinite belt, geo sequestration, oceanic sequestration, CO2 to urea and CO2 to methanol and other chemicals. Advantages, disadvantages, and suitability of these technologies is discussed. These technologies are utmost necessary for Pakistan as recent climate change induced flooding devastated one third of Pakistan affecting millions of families. Hence, Pakistan must store CO2 through various CCUS technologies.

Behavior-based safety program for process industries.

Objectives. This article describes the reduction of unsafe behaviors observed at a fertilizer complex by implementation of a behavior-based safety (BBS) program via a behavior observation form developed by a multidisciplinary team. Methods. Six observation categories, i.e., position of people, reaction of people, personal protective equipment (PPE), tools used, operating procedures and housekeeping, are used to monitor safe and unsafe behaviors for a period of 18 months. Results. Safe behaviors increased from 57 to 70% and unsafe behaviors reduced from 40 to 26%. Behaviors of employees working in various sections of fertilizer complex such as ammonia, urea, utility, bagging/shipping and workshop were also observed. Non-compliance with PPE, housekeeping and standard operating procedures was also monitored in individual sections. Non-operational areas including the administration block, housing colony, maintenance workshop, warehouse, fire station and electrical substation were also observed. Among these, the maximum unsafe behaviors are for the housing colony and minimum for the electrical substation. Conclusion. It has been concluded that working on the housing colony, administration block and fire station areas will address 74% unsafe behaviors of non-operational areas. For practical applications, worldwide industries can implement this BBS program to enhance BBS, thus reducing unsafe behaviors and increasing employee morale.

Molecular epidemiology of Bartonella species from sympatric mammals collected in urban and rural areas of Punjab, Pakistan.

Bartonella can infect a variety of mammals including humans and has been detected in the Americas, Europe, Africa, and Asia. Roughly two-thirds of identified Bartonella species are found and maintained in rodent reservoirs, with some of these species linked to human infections. Rodents (N=236) were caught from the Sahiwal division of Punjab, Pakistan and tested for Bartonella using PCR targeting gltA and rpoB genes, followed by sequencing of rpoB-positive samples. Genetic relatedness to other published Bartonella spp. rpoB gene sequences were examined using BLAST and phylogenetic analysis. Overall, 7.62% (18/236) of rodents were positive for both gltA and rpoB fragments. Rattus rattus and R. norvegicus had 7.94% (12/151) and 7.05% (6/85) positivity rates for Bartonella DNA, respectively. Phylogenetic analysis revealed a close relatedness between Bartonella spp. from Pakistan to Bartonella spp. from China, Nepal, and Malaysia. This study is the first reported detection of Bartonella spp. in R. rattus and R. norvegicus from the Sahiwal area of Punjab, Pakistan.

Developments in mineral carbonation for Carbon sequestration.

Mineral technology has attracted significant attention in recent decades. Mineral carbonation technology is being used for permanent sequestration of CO2 (greenhouse gas). Temperature programmed desorption studies showed interaction of CO2 with Mg indicating possibility of using natural feedstocks for mineral carbonation. Soaking is effective to increase yields of heat-activated materials. This review covers the latest developments in mineral carbonation technology. In this review, development in carbonation of natural minerals, effect of soaking on raw and heat-activated dunite, increasing reactivity of minerals, thermal activation, carbonations of waste materials, increasing efficiency of carbonation process and pilot plants on mineral carbonation are discussed. Developments in carbonation processes (single-stage carbonation, two-stage carbonation, acid dissolution, ph swing process) and pre-process and concurrent grinding are elaborated. This review also highlights future research required in mineral carbonation technology.

Newly optimized ELISA kit and LAT reveal significantly higher seroprevalence in sheep raised in agro-ecological zone as against range-ecological zone, with a significant association of meteorological parameters.

BACKGROUND: Toxoplasma gondii is a zoonotic and foodborne intracellular parasite capable of inducing congenital infections, stillbirths and abortions in humans and animals, globally. The consumption of undercooked or raw mutton is "one of the vital risks" for acquiring toxoplasmosis: an asymptomatic condition in healthy persons, while life-threatening in immunodeficient individuals like "HIV/AIDS" patients. OBJECTIVES: The current study has multiple objectives: to optimize a newly ELISA kit for Sheep, to find out the seroprevalence of ovine toxoplasmosis of two ecological zones of the Punjab, Pakistan through LAT and newly Optimized Sheep ELISA kit, to do the comparison of efficacies of various tests (LAT with newly Optimized ELISA kit and newly Optimized ELISA kit with commercial ELISA kit) and to determine the different meteorological parameters as the risk factors for T. gondii infection in sheep. METHODS: A cross-sectional study was conducted on 400 sheep sera, 200 were collected from sheep raised on open grazing system by local farmers in the adjoining areas of Civil Veterinary Dispensaries (CVDs) of range-ecological zone i.e. tehsil Kot Chutta (Dera Ghazi khan). Similarly, the remaining 200 were collected from agro-ecological zone i.e. tehsil Sharaqpur (Sheikhupura), to evaluate the comparative efficacy of LAT with optimized ELISA kit and newly optimized ELISA kit with commercial ELISA kit. FINDINGS: The newly ELISA kit optimized against a commercial ELISA kit was found to have 100% sensitivity, 97.6% specificity with 98% Positive Predictive Value, 100% Negative Predictive Value, Cut off value = 0.505, 28.28 LR+, 0.0104 LR-, and 2719.23 DOR. Seroprevalence of toxoplasmosis was detected significantly (P < 0.01; χ2) higher in Sharaqpur (44.5% by LAT; 35.5% by ELISA) as compared to that in Kot Chutta (39.5% by LAT; 31% by ELISA). The highest seroprevalence was seen in the sheep of the 1-2 years age group (P < 0.01; χ2), whereas the lowest in the oldest animals (≥ 4 years). Investigation of meteorological data of both the regions reveals that the zone with higher seroprevalence has relatively higher rainfall, higher humidity, lower environmental temperatures, and higher altitude as the critical factors, potentially behind the significant difference seen in seroprevalence level. The partial correlation of both tests (newly optimized ELISA kit and LAT) was 0.991 at maximum temperature in Sharaqpur while it was 0.981 in Kot Chutta. INTERPRETATION: A novel significant correlation was found between the meteorological parameters (relative humidity, minimum, maximum, and average temperatures) divided into yearly units of both the ecological zones, and year-wise seroprevalence (birth years of age-wise groups) of the corresponding regions. We hypothesize that such environmental conditions increase the risk of toxoplasmosis in grazing sheep, owing to a more favorable environment for coccidian oocyst survival. The ELISA kit optimized in this study will be helpful for the detection of seroprevalence of ovine toxoplasmosis in other ecological zones of Pakistan as well as of any other country in the world. More studies are recommended involving regions from other ecological zones of Pakistan to further explore the seroprevalence of ovine toxoplasmosis and to ratify the novel correlation of meteorological parameters with seroprevalence.

IEg67 kDa Bovine Hydatid Cyst Antigen: A Candidate for Developing Sero-Diagnostic Assays for Cystic Echinococcosis, a Disease of One Health Importance.

Cystic echinococcosis (hydatidosis) is a world-wide zoonotic disease of mainly humans, livestock and dogs, caused by Echinococcus granulosus. The disease can negatively impact food production and animal welfare and causes socio-economic hardship. Here, we aimed to identify the local bovine hydatid cyst fluid (BHCF) antigen for developing a sero-diagnostic assay to be used for the pre-slaughter screening of food animals. In total, 264 bovines approved for slaughter in Pakistan were subjected to serum collection and post-mortem screening for hydatid cysts. These cysts were assessed microscopically to assess fertility and viability, and by PCR for molecular confirmation of species. A BHCF antigen was identified from positive sera via SDS-PAGE, confirmed by Western blot, and quantified via a bicinchoninic acid (BCA) assay. The quantified crude BHCF antigen (iEg67 kDa) was then used in ELISA screening to test all sera collected from known positive and negative animals based on hydatid cyst presence/absence. Of the 264 bovines examined, 38 (14.4%) showed hydatid cysts during post-mortem examination. All of these individuals, plus an additional 14 (total: 52; 19.6%) tested positive based on less time-consuming ELISA examination. Based on ELISA, occurrence in females (18.8%) was significantly higher than in males (9.2%) and was higher in cattle (19.5%) compared to buffalo (9.5%). The infection rate increased with age in both host species: cumulatively, 3.6% in animals aged 2-3 years, 14.6% in 4-5-year-olds and 25.6% in 6-7-year-olds. The occurrence of cysts in cattle was significantly higher in the lungs (14.1%) compared to their livers (5.5%), whereas the opposite was true in buffalo (6.6% livers, 2.9% lungs). For both host species, most cysts in the lungs were fertile (65%), while the majority in the liver were sterile (71.4%). We conclude that the identified iEg67 kDa antigen is a strong candidate for the development of a sero-diagnostic screening assay for the pre-slaughter diagnosis of hydatidosis.

Analysis of the antimicrobial potential of sericin-coated silver nanoparticles against human pathogens.

The antibacterial activity of synthetic antimicrobial agents is well known, but most of them have several side effects and are effective against selective microbes. Recently, major concern for the microbiologists is to investigate for some stable, non-toxic, cheap, and eco-friendly antimicrobial agents with a wide range of bactericidal potential. A cost-effective and environmentally friendly alternate has been proposed in the form of green synthesized nanoparticles. The Present study was designed to fabricate sericin-coated silver nanoparticles (S-AgNPs) using sericin as stabilizer and reductant of silver ions and their antibacterial potential was evaluated at various concentrations and temperatures (8, 40, and 50°C). Antimicrobial activities were assessed by the agar well diffusion method. Antibacterial activity of S-AgNPs was measured at different concentrations (1-6 mg/ml) whereas; antifungal activity was tested at 5-20 mg/ml of S-AgNPs. Nanoparticles were characterized by UV-visible spectrophotometer, Fourier transform infrared spectroscopy, and scanning electron microscopy. These nanoparticles significantly subdued the growth of Clostridium difficile (18.7 ± 0.9 mm), Proteus mirabilis (12.3 ± 0.3 mm) and Bacillus licheniformis (10.7 ± 0.9 mm) and Aspergillus flavus (18.7 ± 2.0 mm), Mucor mycetes (13 .0 ± 1.5 mm), Candida albicans (15.3 ± 0.3 mm) and Aspergillus niger (10.0 ± 0.6 mm). S-AgNPs were stable at all temperatures and the maximum growth inhibition was found at 8°C for all pathogenic strains. We concluded that the S-AgNPs could be a potential candidate to inhibit the growth of bacterial and fungal pathogens at a wide range of environmental conditions like temperature. In various biomedical applications including antimicrobial and wound dressings, S-AgNPs can be used in the future to treat various bacterial and fungal infections.

First Detection of Theileria sinensis-like and Anaplasma capra in Ixodes kashmiricus: With Notes on cox1-Based Phylogenetic Position and New Locality Records.

Ixodes ticks transmit Theileria and Anaplasma species to a wide range of animals. The spreading of ticks and tick-borne pathogens has been attributed to transhumant herds, and research on these uninvestigated issues has been neglected in many countries, including Pakistan. Recently, we used internal transcribed spacer (ITS) and 16S ribosomal DNA partial sequences to genetically characterize Ixodes kashmiricus ticks and their associated Rickettsia spp. However, the data on its cox1 sequence and associated Theileria spp. and Anaplasma spp. are missing. This study aimed to genetically characterize I. kashmiricus based on the cox1 sequence and their associated Theileria spp. and Anaplasma spp. The I. kashmiricus ticks were collected from small ruminants: sheep (Ovis aries) and goats (Capra hircus) of transhumant herds in district Shangla, Dir Upper and Chitral, Khyber Pakhtunkhwa (KP), Pakistan. Out of 129 examined hosts, 94 (72.87%) (56 sheep and 38 goats) were infested by 352 ticks, including adult females (175; 49.7%) followed by nymphs (115; 32.7%) and males (62; 17.6%). For molecular analyses, 121 ticks were subjected to DNA isolation and PCR for the amplification of the cox1 sequence for I. kashmiricus, 18S rDNA for Theileria spp. and 16S rDNA sequences for Anaplasma spp. The obtained cox1 sequence showed 89.29%, 88.78%, and 88.71% identity with Ixodes scapularis, Ixodes gibbosus, and Ixodes apronophorus, respectively. Phylogenetically, the present cox1 sequence clustered with the Ixodes ricinus complex. Additionally, the 18S rDNA sequence showed 98.11% maximum identity with Theileria cf. sinensis and 97.99% identity with Theileria sinensis. Phylogenetically, Theileria spp. clustered with the T. cf. sinensis and T. sinensis. In the case of Anaplasma spp., the 16S rDNA sequence showed 100% identity with Anaplasma capra and phylogenetically clustered with the A. capra. PCR-based DNA detection targeting the amplification of groEL and flaB sequences of Coxiella spp. and Borrelia spp., respectively, was unsuccessful. This is the first phylogenetic report based on cox1 and new locality records of I. kashmiricus, and the associated T. sinensis-like and A. capra. Significant tick surveillance studies are needed in order to determine the epidemiology of Ixodes ticks and their associated pathogens.