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Abstract Anaplasma spp. and Rickettsia spp. are intracellular vector-borne pathogens and harbored by a wide range of ticks and vertebrate hosts. Aim of this study was to molecularly characterize Anaplasma spp. and Rickettsia spp. in different ticks collected from livestock hosts in nine districts of Khyber Pakhtunkhwa (KP), Pakistan. In total, 862 ticks were collected from cattle, goats and sheep. Highest tick’s infestation was observed on cattle 56.14% (32/57), followed by goats 45.45% (40/88), and sheep 42.05% (45/107). Rhipicephalus microplus (305/862, 35.38%) was predominant species, followed by Haemaphysalis sulcata (243/862, 28.19%), Hyalomma anatolicum (133/862, 15.42%), Haemaphysalis bispinosa (120/862, 13.92%), and Hyalomma kumari (61/862, 7.07%). A subset of 135 ticks were screened for Anaplasma spp. and Rickettsia spp. based on the amplification of partial 16 S rDNA and outer-membrane protein A (ompA) fragments, respectively. In total, 16 ticks (11.85%) were positive for Anaplasma spp. and Rickettsia spp. Obtained 16 S rDNA sequences for Anaplasma spp. detected in Ha. bispinosa and Ha. sulcata showed 99.98% identity with Anaplasma bovis, while other detected in Rh. microplus showed 99.84% identity with Candidatus Anaplasma boleense. Similarly, detected ompA sequence in Ha. sulcata showed 100% identity with Rickettsia sp. and 97.93% with Rickettsia slovaca, and another sequence detected in Rh. microplus showed 100% identity with Candidatus Rickettsia shennongii. In phylogenetic trees, these sequences clustered with corresponding species from Pakistan, China, Turkey, South Korea, South Africa, and Herzegovina. This is the first study reporting detection of A. bovis in Ha. bispinosa and Ha. sulcata, Ca. A. boleense in Rh. microplus collected from goats, and R. slovaca-like in Ha. sulcata. Our results enforce the need for regular surveillance of Rickettsiales in hard ticks infesting livestock in the region.

Abstract Tick-borne bacteria of the genera Rickettsia and Coxiella cause several emerging veterinary and human infectious diseases. Ticks of the genus Hyalomma are medically important vectors due to their potential role in the transmission of pathogens to vertebrate hosts. There is an inadequate knowledge on tick-borne Rickettsia spp. and Coxiella spp. in ticks infesting transhumant camels in Pakistan. In this study, we conducted a molecular survey for screening of Rickettsia spp. and Coxiella spp. in ticks infesting camels. Seven hard tick species including Hyalomma dromedarii, Hyalomma anatolicum, Hyalomma scupense, Hyalomma isaaci, Hyalomma turanicum, Hyalomma asiaticum, and Rhipicephalus sanguineus s.l were confirmed on camels in three distinct physiographic regions of Khyber Pakhtunkhwa, Pakistan. A subset of morphologically identified ticks were subjected to molecular assays for the genetic characterization of ticks and the detection and genetic characterization of Rickettsia and Coxiella species using standard genetic markers. Ticks screened for pathogens resulted in the detection of Rickettsia aeschlimannii and Candidatus Rickettsia shennongii and Coxiella burnetii. The molecular analysis further reveals the presences of an undetermined Rickettsia aeschlimannii-like species, that is making a distinct phylogenetic clade with R. aeschlimannii. The detection of pathogens in camel ticks poses potential health hazards as these ticks frequently bites humans. Molecular screening of Rickettsia spp. and Coxiella spp. associated with camel ticks is a preliminary step toward the surveillance of evaluating their zoonotic threats in the region.

Rickettsia felis, responsible for flea-borne spotted fever, is a rising zoonotic pathogen posing an increasing global threat due to its expanding geographical distribution. The rise in antibiotic-resistant strains of this pathogen underscores the urgent need for new therapeutic interventions. This study employed a comprehensive subtractive proteomics analysis of the R. felis proteome, aiming to identify essential, non-host homologous, and pathogen-specific proteins, which were subsequently evaluated as potential new drug targets. These findings offer valuable insights into the development of therapeutic strategies against rickettsiosis. The analysis revealed 343 proteins that are non-homologous to the host, including 108 essential proteins, 25 unique metabolic pathways, and 11 distinct proteins. Out of these, 10 proteins were druggable in which two associated with virulence, and one related to resistance (succinate dehydrogenase). Through a rigorous screening process and extensive literature review, succinate dehydrogenase emerged as a promising drug target. Protein interaction partners for succinate dehydrogenase were identified using the STRING database. To further assess the functionality of succinate dehydrogenase, structure-based studies were conducted. Approximately 18,000 ZINC compounds were screened, leading to the finding of six potential inhibitors: ZINC67847806, ZINC67982856, ZINC67974679, ZINC67895371, ZINC05668040, and ZINC05670149. Absorption, distribution, metabolism, excretion, and toxicity (ADMET) profiling confirmed that most compounds met the preferred pharmacokinetic properties, except for ZINC67895371 and ZINC67847806, which exhibited positive ames test results, and ZINC05670149, ZINC67895371, and ZINC67847806, showed hepatotoxicity. All compounds were found to be non-sensitizing to the skin. Based on these findings, further experimental validation of ZINC67974679, ZINC67982856, and ZINC05668040 is recommended.

There is limited information on the occurrence of Hyalomma turanicum and Hyalomma asiaticum ticks, as well as associated Rickettsia, Anaplasma, and Ehrlichia species in Pakistan. Addressing this knowledge gap, the current study aimed at morphomolecular confirmation of these ticks and molecular assessment of associated Rickettsiales bacteria (Rickettsia, Anaplasma, and Ehrlichia spp.) in Balochistan, Pakistan. A total of 314 ticks were collected from 74 of 117 (63.2%) hosts, including 41 of 74 (55.4%) goats and 33 of 74 (44.5%) sheep. Subsequently, a subset of microscopically identified ticks was subjected to DNA extraction and PCR to amplify 16S rDNA and cox1 fragments. Additionally, gltA, ompA, and ompB fragments were targeted for Rickettsia spp. and 16S rDNA fragments for both Anaplasma and Ehrlichia spp. The 16S rDNA and cox1 sequences of Hy. turanicum demonstrated 100% identity with those of the same species previously reported from Pakistan. The 16S rDNA and cox1 sequences of Hy. asiaticum exhibited 99.52 and 100% identities, respectively, with corresponding species reported from China, Kazakhstan, and Turkey. The gltA, ompA, and ompB fragments associated with Hy. turanicum showed 100% identities with Rickettsia aeschlimannii reported from Egypt, Italy, Kazakhstan, Kenya, Pakistan, and Senegal. The 16S rDNA sequences of Anaplasma sp. and Ehrlichia sp. associated with both Hy. asiaticum and Hy. turanicum exhibited 99.67 and 100% identities with unknown Anaplasma sp. and Ehrlichia sp. reported from Morocco and Pakistan, respectively. In the 16S rDNA and cox1 phylogenetic trees of ticks, Hy. turanicum and Hy. asiaticum from the current study clustered with their respective species. Similarly, in gltA, ompA, and ompB phylogenetic trees of Rickettsia, R. aeschlimannii of the present study clustered with the same species, whereas Anaplasma sp. and Ehrlichia sp. of this study clustered with undetermined Anaplasma spp. and Ehrlichia spp. in the 16S rDNA phylogenetic tree of Anaplasmataceae. Among the DNA samples from the screened ticks, a coinfection rate of R. aeschlimannii, Anaplasma sp., and Ehrlichia sp. (2 of 80, 2.5%) was observed in Hy. turanicum, whereas individual infection rates were noted as follows: R. aeschlimannii (8 of 80, 10%), Anaplasma sp. (5 of 80, 6.3%), and Ehrlichia sp. (5 of 80, 6.3%). This study marks the first record of molecular characterization of Hy. turanicum and Hy. asiaticum as well as the detection of associated R. aeschlimannii, Anaplasma sp., and Ehrlichia sp. in Balochistan, Pakistan.

Klebsiella pneumoniae is a Gram-negative bacterium associated with a wide range of community- and hospital-acquired infections. The emergence of clonal hypervirulent strains resistant to last-resort antimicrobial agents has become a global concern. The Kingdom of Saudi Arabia (KSA), with its diverse population and high tourism traffic, serves as a platform where the spread of multidrug-resistant (MDR) strains are facilitated. However, the knowledge of epidemiology and population diversity of MDR K. pneumoniae in KSA is scarce. We conducted a comprehensive genomic survey on 352 MDR K. pneumoniae isolates systematically collected from bloodstream and urinary tract infections in 34 hospitals across 15 major cities in KSA during 2022 and 2023. Whole-genome sequencing on the isolates was performed, followed by genomic epidemiology and phylodynamic analysis. Our study revealed a dynamic population characterized by the rapid expansion of several dominant clones, including, ST2096, ST147, and ST231, which were estimated to have emerged within the past decade. These clones exhibited widespread dissemination across hospitals and were genetically linked to global strains, particularly from the Middle East and South Asia. All major clones harboured plasmid-borne ESBLs and carbapenemase genes, with plasmidome analysis identifying multiple IncH, IncA/C and IncL plasmids underlying the MDR-hypervirulent phenotype. These plasmids were shared between major clones and became acquired on the same time scales as the expansion of the dominant clones. Our results report ST2096 as an emerging MDR-hypervirulent clone, emphasizing the need for monitoring of the circulating clones and their plasmid content in the KSA and broader West Asia.

The genus Amblyomma contains the highest percentage of reptile-associated ticks, and comprises approximately nine subgenera. One of these subgenera is Adenopleura, which also encompasses Amblyomma javanense, and its type species Amblyomma compressum. This study describes a new Amblyomma species associated with Bengal monitor lizards (Varanus bengalensis) based on morphology and its mitogenome in Khyber Pakhtunkhwa, Pakistan. Reptiles belonging to different genera were examined for Amblyomma ticks and only the monitor lizard was infested with ticks in the District Bajaur. Collected Amblyomma cf. javanense ticks were analyzed and formally described as a new species. Overall, 57 A. cf. javanense ticks were collected on monitor lizards (4/27) with a 15% prevalence of infestation, 2.1 mean abundance, and 14.3 mean intensity. Ticks comprised males (n = 23, 40%), females (n = 14, 25%) and nymphs (n = 20, 35%), while no larvae were found. BLAST analysis of A. cf. javanense sequences showed the following maximum identities; 98.25% with undetermined Amblyomma species based on 12S rRNA, 96.07% with A. javanense based on 16S rRNA, 99.56% and 90.95% with an Amblyomma sp. and A. javanense, respectively, based on ITS2. Moreover, the mitochondrial genome of A. cf. javanense showed maximum identities of 80.75%, 80.48% and 79.42% with Amblyomma testudinarium, A. javanense, and Amblyomma sp., respectively. The phylogenetic analysis of A. cf. javanense revealed that its 12S rRNA and 16S rRNA are closely related to an Amblyomma sp. and A. javanense, respectively, from Sri Lanka, its ITS2 is closely related to A. javanense from China and an Amblyomma sp. from Sri Lanka, and its mitogenome is closely related to A. javanense and Amblyomma sp. from China. The pairwise distance analysis resulted in divergence of 0–1.71% (12S rRNA), 0–17.5% (16S rRNA), 0–9.1% (ITS2) and 0–20.5% (mitochondrial genome). We also contributed the full-length mitochondrial genome sequence of A. compressum and showed that this species does not share a most recent common ancestor with A. javanense. As the subgenus Adenopleura is paraphyletic, this study could help to understand the systematics and phylogeny of this taxon.

Ticks and tick-borne diseases (TBDs) pose potential health threats to small-scale farmers of grazing animals in the upper highlands of Balochistan, Pakistan. This study was conducted based on a questionnaire survey involving 153 farmers of grazing animals in seven districts to access their knowledge, attitudes, and practices regarding ticks and TBDs. Odds ratios and 95% confidence intervals, based on Fisher’s test, were used to assess risk factors for determining preventive measures. The findings revealed a low level of knowledge among the participants. For instance, there was a lack of awareness of the effects of climate change and the economic impact of ticks on animal health. The essential precautions, such as the non-indiscriminate use of acaricides, wearing dark-colored clothing, and limiting children’s interaction with grazing animals, were often overlooked. However, the farmers had a positive attitude towards tick control, but they mostly relied on the knowledge of local communities. The neglect of such measures places these farmers and their children at risk of contracting TBDs. This study also indicates minimal involvement from the government in educating farmers and controlling ticks. The role of stakeholders, including the government, non-governmental organizations, veterinary doctors, and local farmer communities, is crucial to address these issues and to implement effective training programs that address misconceptions about ticks and TBDs. Overall, this study highlights the importance of implementing awareness and education programs to address the misconceptions about ticks and TBDs among farmers.

A novel tick-borne orthonairovirus called the Yezo virus (YEZV), primarily transmitted by the Ixodes persulcatus tick, has been recently discovered and poses significant threats to human health. The YEZV is considered endemic in Japan and China. Clinical symptoms associated with this virus include thrombocytopenia, fatigue, headache, leukopenia, fever, depression, and neurological complications ranging from mild febrile illness to severe outcomes like meningitis and encephalitis. At present, there is no treatment or vaccine readily accessible for this pathogenic virus. Therefore, this research employed an immunoinformatics approach to pinpoint potential vaccine targets within the YEZV through an extensive examination of its structural proteins. Three structural proteins were chosen using specific criteria to pinpoint T-cell and B-cell epitopes, which were subsequently validated through interferon-gamma induction. Six overlapping epitopes for cytotoxic T-lymphocytes (CTL), helper T-lymphocytes (HTL), and linear B-lymphocytes (LBL) were selected to construct a multi-epitope vaccine, achieving a 92.29% coverage of the global population. These epitopes were then fused with the 50S ribosomal protein L7/L12 adjuvant to improve protection against international strains. The three-dimensional structure of the designed vaccine construct underwent an extensive evaluation through structural analysis. Following molecular docking studies, the YEZV vaccine construct emerged as a candidate for further investigation, showing the lowest binding energy (−78.7 kcal/mol) along with favorable physiochemical and immunological properties. Immune simulation and molecular dynamics studies demonstrated its stability and potential to induce a strong immune response within the host cells. This comprehensive analysis indicates that the designed vaccine construct could offer protection against the YEZV. It is crucial to conduct additional in vitro and in vivo experiments to verify its safety and effectiveness.

Bartonella henselae is a Gram-negative bacterium causing a variety of clinical symptoms, ranging from cat-scratch disease to severe systemic infections, and it is primarily transmitted by infected fleas. Its status as an emerging zoonotic pathogen and its capacity to persist within host erythrocytes and endothelial cells emphasize its clinical significance. Despite progress in understanding its pathogenesis, limited knowledge exists about the virulence factors and regulatory mechanisms specific to the B. henselae strain Houston-1. Exploring these aspects is crucial for targeted therapeutic strategies against this versatile pathogen. Using reverse-vaccinology-based subtractive proteomics, this research aimed to identify the most antigenic proteins for formulating a multi-epitope vaccine against the B. henselae strain Houston-1. One crucial virulent and antigenic protein, the PAS domain-containing sensor histidine kinase protein, was identified. Subsequently, the identification of B-cell and T-cell epitopes for the specified protein was carried out and the evaluated epitopes were checked for their antigenicity, allergenicity, solubility, MHC binding capability, and toxicity. The filtered epitopes were merged using linkers and an adjuvant to create a multi-epitope vaccine construct. The structure was then refined, with 92.3% of amino acids falling within the allowed regions. Docking of the human receptor (TLR4) with the vaccine construct was performed and demonstrated a binding energy of −1047.2 Kcal/mol with more interactions. Molecular dynamic simulations confirmed the stability of this docked complex, emphasizing the conformation and interactions between the molecules. Further experimental validation is necessary to evaluate its effectiveness against B. henselae.

Pioglitazone, an anti-diabetic drug, has been previously shown to ameliorate kidney damage through anti-inflammatory and antioxidant effects. In this study, we employed an integrative bioinformatics approach to study the possible mechanisms involved in the mitigative effect of pioglitazone against colistin-induced nephrotoxicity. Next, we validated the results obtained from the bioinformatics study by pre-treating human kidney-2 (HK-2) cell line with pioglitazone 100 μM for 30 minutes and then treating the cells with colistin sulfate 1200 μM for 24 hours. Inflammatory signaling by cytokines and the nuclear factor erythroid 2 related factor 2 (Nrf2) signaling pathways were predicted to be involved in the ameliorative effect of pioglitazone against colistin-induced nephrotoxicity. The nuclear factor kappa B subunit p65 (NF-κB p65) and Nrf2 were among the predicted transcription factors regulating the hub genes. Moreover, miR-24, miR-16, and miR-21 were identified as potential pathogenic miRNAs regulating the hub genes. In contrast, miR-17, miR-27a, and miR-146a were identified as potential protective miRNAs. The in vitro study indicated that pioglitazone pre-treatment increased cell viability in HK-2 cells exposed to colistin. Pioglitazone pre-treatment reduced the expression of pro-inflammatory cytokine genes (IL6 and TNF). Moreover, pioglitazone reduced the protein expression of NF-κB p65 and increased the protein expression of Nrf2. The protective effect of pioglitazone against colistin-induced toxicity in HK-2 cells is related to its anti-inflammatory and antioxidant activity through modulating NF-κB-mediated inflammatory signaling and Nrf2-mediated antioxidative stress signaling.

Alectorobius species are soft ticks primarily infesting birds, such as swallows, while Dermacentor species are hard ticks mainly infesting mammals, such as small ruminants. This study for the first time reported on the morphological and molecular bases of two tick species, namely A. coniceps and a Dermacentor sp. in Pakistan. The former species was examined in swallows’ nests in Khyber Pakhtunkhwa province, while the latter species was examined in small ruminants in Balochistan province. In total, 25 ticks were collected, with 14 ticks morphologically identified as A. coniceps (males = 9 and females = 5) and 11 ticks identified as Dermacentor sp. (males = 7 and females = 4). Following morphological identification, molecular identification was gained by obtaining 16S rDNA and cox1 sequences for these ticks. The BLAST results for the 16S rDNA and cox1 sequences from A. coniceps shared a maximum identity of 97.46% and 96.49% with the same species from Malta. The BLAST analysis of the 16S rDNA and cox1 sequences from Dermacentor sp. showed maximum identities of 98.42% and 97.45% with Dermacentor pavlovskyi from China. The phylogenetic analysis based on 16S rDNA and cox1 of A. coniceps showed a close evolutionary relationship with the same species. The case of Dermacentor sp., based on 16S DNA and cox1, indicated a close evolutionary relationship with Dermacentor pavlovskyi from China.

Ticks are important ectoparasites that transmit various pathogens causing morbidity and mortality in humans and animals. Saudi Arabia faces several challenges that can contribute to the emergence and spread of antimicrobial resistance (AMR) bacteria. These challenges require collaborative efforts to successfully achieve significant control of AMR in the country. The present study aims to isolate bacteria from camels' tick Hyalomma dromedarii in Al-Jouf province to identify and determine these isolates' antimicrobial susceptibilities. Forty-nine ticks were collected from dromedary camels and morphologically classified as H. dromedarii. Ticks were then homogenized and plated individually, which resulted in the isolation of 55 bacteria. The results showed that the bacterial isolates belong to 20 different species. About 71% (n = 39) of the total isolates were identified as Gram-positive bacteria comprised of 11 different species, while 29% (n = 16) of the total isolates were Gram-negative bacteria comprised of 9 different species. The most prevalent isolate within the total samples was Staphylococcus lentus (22.45%, 11/49), followed by Staphylococcus pseudintermedius (18.37%, 9/49) and Sphingomonas paucimobilis (16.33% 8/49). The antimicrobial susceptibility profile of Gram-positive bacteria showed that 100% (n = 31) were resistant to benzylpenicillin; 90.3% (n = 28) were resistant to oxacillin; 58.1% (n = 18) were resistant to clindamycin; 48.4% (n = 15) were resistant to vancomycin. In addition, 32.3% (n = 10) were resistant to trimethoprim/sulfamethoxazole and rifampicin; 25.8% (n = 8) were resistant to erythromycin; 16.1% (n = 5) were resistant to teicoplanin; 6.5% (n = 2) were resistant to tetracycline. All Gram-positive bacteria were 100% susceptible to linezolid, gentamicin, tobramycin, levofloxacin, moxifloxacin, tigecycline, and nitrofurantoin. In antimicrobial susceptibility tests for the Gram-negative bacteria, 57.14% (n = 8) of the identified bacteria were resistant to ampicillin, whereas 50% (n = 7) were resistant to cefoxitin and ceftazidime. About 28.57% (n = 4) of the Gram-negative bacteria were resistant to ceftriaxone, trimethoprim/sulfamethoxazole. In addition, 21.43% (n = 3) were resistant to amoxicillin/clavulanic acid and cephalothin; 14.29% (n = 2) were resistant to cefepime and nitrofurantoin; 7.14% (n = 1) were resistant to piperacillin/tazobactam and tigecycline. However, all Gram-negative bacteria were susceptible to other examined antimicrobials. This is the first study that investigates the role of the hard tick as a potential reservoir for AMR pathogens within our region.

Tick-borne Rickettsia spp. have long been known as causative agents for zoonotic diseases. We have previously characterized Rickettsia spp. in different ticks infesting a broad range of hosts in Pakistan; however, knowledge regarding Rickettsia aeschlimannii in Haemaphysalis and Hyalomma ticks is missing. This study aimed to obtain a better understanding about R. aeschlimannii in Pakistan and update the knowledge about its worldwide epidemiology. Among 369 examined domestic animals, 247 (66%) were infested by 872 ticks. Collected ticks were morphologically delineated into three genera, namely, Rhipicephalus, Hyalomma, and Haemaphysalis. Adult females were the most prevalent (number ₌ 376, 43.1%), followed by nymphs (303, 34.74%) and males (193, 22.13%). Overall, genomic DNA samples of 223 tick were isolated and screened for Rickettsia spp. by the amplification of rickettsial gltA, ompA, and ompB partial genes using conventional PCR. Rickettsial DNA was detected in 8 of 223 (3.58%) ticks including nymphs (5 of 122, 4.0%) and adult females (3 of 86, 3.48%). The rickettsial gltA, ompA, and ompB sequences were detected in Hyalomma turanicum (2 nymphs and 1 adult female), Haemaphysalis bispinosa (1 nymph and 1 adult female), and Haemaphysalis montgomeryi (2 nymphs and 1 adult female). These rickettsial sequences showed 99.71–100% identity with R. aeschlimannii and phylogenetically clustered with the same species. None of the tested Rhipicephalus microplus, Hyalomma isaaci, Hyalomma scupense, Rhipicephalus turanicus, Hyalomma anatolicum, Rhipicephalus haemaphysaloides, Rhipicephalus sanguineus, Haemaphysalis cornupunctata, and Haemaphysalis sulcata ticks were found positive for rickettsial DNA. Comprehensive surveillance studies should be adopted to update the knowledge regarding tick-borne zoonotic Rickettsia species, evaluate their risks to humans and livestock, and investigate the unexamined cases of illness after tick bite among livestock holders in the country.

Ticks pose significant threats to hosts by transmitting Borrelia spp., which are grouped into Lyme borreliae, relapsing fever borreliae (RF), and reptiles- and monotremes-associated borreliae. The RF borreliae encompass a group of Borrelia species predominantly transmitted by soft ticks, but some of its members can also be transmitted by hard ticks. Information on the detection and genetic characterization of tick-borne RF borreliae, including Borrelia theileri, is notably rare in Asia, particularly in Pakistan. Herein, we employed molecular techniques to detect borreliae in hard ticks collected from domestic animals in Khyber Pakhtunkhwa, Pakistan. Ticks were subjected to morphological analysis, followed by DNA extraction and PCR amplification of partial fragments of borrelial 16S rRNA and flaB genes. A total of 729 ticks were collected from 264 hosts, with Haemaphysalis cornupunctata (12.9%; 94/729) being the most prevalent, followed by Hyalomma anatolicum (11.7%; 85/729), Rhipicephalus microplus (10.0%; 73/729), Haemaphysalis kashmirensis (9.1%; 66/729), Haemaphysalis bispinosa (8.5%; 62/729), Rhipicephalus sanguineus (8%; 58/729), Haemaphysalis montgomeryi (6.2%; 45/729), Rhipicephalus turanicus (5.5%; 40/729), Hyalomma dromedarii and Ixodes kashmirensis (4.4%; 32/729 each), Rhipicephalus haemaphysaloides (4.1%; 30/729), Haemaphysalis sulcata and Hyalomma scupense (3.8%; 28/729 each), Haemaphysalis danieli (2.9%; 21/729), Hyalomma kumari (2.6%; 19/729), and Hyalomma isaaci (2.2%; 16/729). Based on 16S rRNA detection of Borrelia spp., only R. turanicus yielded positive results, resulting in an overall infection rate of 0.3% (2/160), while using flaB-based detection, four tick species including R. microplus, R. turanicus, Ha. sulcata, and Ha. cornupunctata showed positive results, yielding an overall infection rate of 6.9% (11/160). The amplified DNA fragments of borrelial 16S rRNA and flaB in R. turanicus from goats shared maximum identities of 100 and 99.40% with Borrelia theileri, respectively. Amplified borrelial flaB fragments in R. microplus from cows and sheep displayed 100% identity with B. theileri, while flaB fragments in Ha. cornupunctata and Ha. sulcata from goats revealed identities of 99.32 and 99.75% with undetermined RF Borrelia spp., respectively. Phylogenetic analysis revealed clustering of B. theileri from R. microplus and R. turanicus with the same species, while Borrelia spp. from Ha. cornupunctata and Ha. sulcata with undetermined RF Borrelia spp. Notably, this research marks the first documentation of B. theileri in R. turanicus and the identification of RF Borrelia spp. in Ha. cornupunctata and Ha. sulcata.

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

The nucleotide-binding site-leucine-rich repeat (NBS–LRR) gene family is the largest group of disease resistance (R) genes in plants and is active in response to viruses, bacteria, and fungi usually involved in effector-triggered immunity (ETI). Pangenome-wide studies allow researchers to analyze the genetic diversity of multiple species or their members simultaneously, providing a comprehensive understanding of the evolutionary relationships and diversity present among them. The draft pan-genome of three Mangifera indica cultivars (Alphonso, Hong Xiang Ya, and Tommy atkins) was constructed and Presence/absence variants (PAVs) were filtered through the ppsPCP pipeline. As a result, 2823 genes and 5907 PAVs from H. Xiang Ya, and 1266 genes and 2098 PAVs from T. atkins were added to the reference genome. For the identification of CC-NBS-LRR (CNL) genes in these mango cultivars, this draft pan-genome study has successfully identified 47, 27, and 36 members in Alphonso, H. Xiang Ya, and T. atkins respectively. The phylogenetic analysis divided MiCNL proteins into four distinct subgroups. All MiCNL genes are unevenly distributed on chromosomes. Both tandem and segmental duplication events played a significant role in the expansion of the CNL gene family. These genes contain cis-elements related to light, stress, hormone, and development. The analysis of protein-protein interactions (PPI) revealed that MiCNL proteins interacted with other defense-responsive proteins. Gene Ontology (GO) analysis indicated that MiCNL genes play a role in defense mechanisms within the organism. The expression level of the identified genes in fruit peel was observed under disease and cold stress which showed that Mi_A_CNL13 and 14 were up-regulated while Mi_A_CNL15, 25, 30, 31, and 40 were down-regulated in disease stress. On the other hand, Mi_A_CNL2, 14, 41, and 45 were up-regulated and Mi_A_CNL47 is down-regulated in cold stress. Subsequently, the Random Forest (RF) classifier was used to assess the multi-stress response of MiCNLs. It was found that Mi_A_CNL14 is a gene that responds to multiple stress conditions. The CNLs have similar protein structures which show that they are involved in the same function. The above findings provide a foundation for a deeper understanding of the functional characteristics of the mango CNL gene family.

This study aimed to detect Hepatozoon spp. in ticks infesting asymptomatic domestic animals and to provide insight into their potential spillover from wild to domestic animals. In total, 537 tick specimens were collected in Khyber Pakhtunkhwa, Pakistan, and morphologically identified. The most prevalent tick species was Haemaphysalis cornupunctata (69; 12.8%), followed by Haemaphysalis kashmirensis (62; 11.5%), Rhipicephalus microplus (58; 10.8%), Haemaphysalis montgomeryi (51; 9.5%), Rhipicephalus sanguineus (49; 9.1%), each Haemaphysalis bispinosa and Haemaphysalis sulcata (43; 8.0%), each Hyalomma anatolicum and Rhipicephalus turanicus (37; 6.9%), Rhipicephalus haemaphysaloides (33; 6.1%) Hyalomma scupense (30; 5.6%), and Hyalomma isaaci (25; 4.7%). The extracted DNA from a subset of each tick species was subjected to PCR to amplify 18S rRNA fragments of Hepatozoon spp. By BLAST analysis, the Hepatozoon sp. detected in Hy. anatolicum infesting cows and in Ha. sulcata infesting sheep showed 99.7% maximum identity with Hepatozoon colubri. Similarly, the Hepatozoon sp. detected in R. haemaphysaloides infesting goats shared 99.49% maximum identity with Hepatozoon ayorgbor, and the Hepatozoon sp. detected in R. sanguineus infesting dogs exhibited 99.7% identity with Hepatozoon canis. Having an overall infection rate (9.3%; 16/172), the highest infection rate was recorded for each H. canis, and H. colubri (3.5%; 6/172), followed by H. ayorgbor (2.3%; 4/172). In the phylogenetic tree, H. colubri clustered with corresponding species from Iran, H. ayorgbor clustered with the same species from Croatia, Ghana, and Portugal, and H. canis clustered with the conspecifics from Iran, Israel, Romania, and Zambia. Regarding the potential spillover of Hepatozoon spp. from wildlife through ticks, free ranging animals was at higher risk compared to confined animals (RR = 3.05), animals consuming food from wildlife habitats were at higher risk compared to those consuming domestic food (RR = 3.06), and animals residing in farm buildings located in wildlife habitats were at higher risk compared to those residing in farm buildings located in villages (RR = 3.28). In addition to the first report on H. canis in R. sanguineus in Pakistan, this is the earliest data showing H. ayorgbor in R. haemaphysaloides and H. colubri in Ha. sulcata and Hy. anatolicum. These preliminary findings suggest a potential spillover of Hepatozoon spp. from wild to domestic animals via ticks under certain risk factors.

Public health is a major concern for several developing countries due to infectious agents transmitted by hematophagous arthropods such as ticks. Health risks due to infectious agents transmitted by ticks infesting butcher-associated stray dogs (BASDs) in urban and peri-urban regions have been neglected in several developing countries. To the best of the authors’ knowledge, this is the first study assessing public health risks due to ticks infesting BASDs in Pakistan’s urban and peri-urban areas. A total of 575 ticks (390 from symptomatic and 183 from asymptomatic BASDs) were collected from 117 BASDs (63 symptomatic and 54 asymptomatic); the ticks belonged to 4 hard tick species. A subset of each tick species’ extracted DNA was subjected to polymerase chain reaction (PCR) to amplify the 16S rDNA and cox1 sequences of the reported tick species, as well as bacterial and protozoal agents. The ticks’ 16S rDNA and cox1 sequences showed 99–100% identities, and they were clustered with the sequence of corresponding species from Pakistan and other countries in phylogenetic trees. Among the screened 271 ticks’ DNA samples, Anaplasma spp. was detected in 54/271 (19.92%) samples, followed by Ehrlichia spp. (n = 40/271, 14.76%), Rickettsia spp. (n = 33/271, 12.17%), Coxiella spp. (n = 23/271, 4.48%), and Hepatozoon canis (n = 9/271, 3.32%). The obtained sequences and phylogenetic analyzes revealed that the pathogens detected in ticks were Ehrlichia minasensis, Ehrlichia sp., Hepatozoon canis, Coxiella burnetii, Coxiella sp., Anaplasma capra, Anaplasma platys, Anaplasma sp., Rickettsia massiliae, “Candidatus Rickettsia shennongii” and Rickettsia aeschlimannii. Tick-borne pathogens such as E. minasensis, H. canis, A. capra, A. platys, and R. aeschlimannii, were detected based on the DNA for the first time in Pakistan. This is the first report on public health risks due to ticks infesting BASDs. These results not only provided insights into the occurrence of novel tick-borne pathogens in the region but also revealed initial evidence of zoonotic threats to both public health and domestic life.

Tick-borne Coxiella spp. are emerging in novel regions infecting different hosts, but information regarding their occurrence is limited. The purpose of this study was the molecular screening of Coxiella spp. in various ticks infesting goats, sheep, camels, cattle, wild mice, and domestic fowls (Gallus gallus domesticus) in various districts of Khyber Pakhtunkhwa, Pakistan. Morphologically identified tick species were confirmed by obtaining their cox1 sequences and were molecularly screened for Coxiella spp. by sequencing GroEL fragments. Almost 345 out of 678 (50.9%) hosts were infested by nine tick species. Regarding the age groups, the hosts having an age >3 years were highly infested (192/345, 55.6%), while gender-wise infestation was higher in female hosts (237/345, 68.7%). In collected ticks, the nymphs were outnumbered (613/1,119, 54.8%), followed by adult females (293/1,119, 26.2%) and males (213/1,119, 19.7%). A total of 227 ticks were processed for molecular identification and detection of Coxiella spp. The obtained cox1 sequences of nine tick species such as Hyalomma dromedarii, Hyalomma anatolicum, Haemaphysalis cornupunctata, Haemaphysalis bispinosa, Haemaphysalis danieli, Haemaphysalis montgomeryi, Rhipicephalus haemaphysaloides, Rhipicephalus microplus, and Argas persicus showed maximum identities between 99.6% and 100% with the same species and in the phylogenetic tree, clustered to the corresponding species. All the tick species except Ha. danieli and R. microplus were found positive for Coxiella spp. (40/227, 17.6%), including Coxiella burnetii (15/40, 6.7%), Coxiella endosymbionts (14/40, 6.3%), and different Coxiella spp. (11/40, 4.9%). By the BLAST results, the GroEL fragments of Coxiella spp. showed maximum identity to C. burnetii, Coxiella endosymbionts, and Coxiella sp., and phylogenetically clustered to the corresponding species. This is the first comprehensive report regarding the genetic characterization of Coxiella spp. in Pakistan's ticks infesting domestic and wild hosts. Proper surveillance and management measures should be undertaken to avoid health risks.

Control of ticks and tick-borne pathogens is a priority for human and animal health. Livestock-holders extensively rely on acaricide applications for tick control. Different groups of acaricides including cypermethrin and amitraz have been consistently used in Pakistan. There has been a gap in understanding the susceptibility or resistance of Rhipicephalus microplus, the most prevalent tick in Pakistan, to acaricides. The present study aimed to molecularly characterize cypermethrin and amitraz targeted genes such as voltage-gated sodium channel (VGSC) and octopamine tyramine (OCT/Tyr) of R. microplus ticks in Khyber Pakhtunkhwa (KP), Pakistan to monitor the acaricides resistance. Tick specimens were collected from cattle and buffaloes in northern (Chitral, Shangla, Swat, Dir, and Buner), central (Peshawar, Mardan, Charsadda, Swabi, and Nowshera), and southern districts (Kohat, Karak, Lakki Marwat, Tank, and Dera Ismail Khan) of KP, Pakistan. Different concentrations of commercially available cypermethrin (10%) and amitraz (12.5%) were prepared for in vitro larval immersion tests (LIT). In LIT, the average mortality rate of immersed larvae was recorded that was increased gradually with an increase in the concentration of specific acaricide. The larvae’s highest mortality rates (94.5% and 79.5%) were observed at 100-ppm of cypermethrin and amitraz, respectively. A subset of 82 R. microplus ticks was subjected to extract genomic DNA, followed by PCR to amplify partial fragments of VGSC (domain-II) and OCT/Tyr genes. The BLAST results of the consensus sequence of VGSC gene (domain-II) showed 100% identity with the acaricides susceptible tick sequence from the United States (reference sequence). Obtained identical sequences of OCT/Tyr genes showed maximum identity (94-100%) with the identical sequences reported from Australia (reference sequence), India, Brazil, Philippines, USA, South Africa, and China. Thirteen single nucleotide polymorphisms (10 synonymous and three non-synonymous) were observed at various positions of partial OCT/Tyr gene fragments. The SNP at position A-22-C (T-8-P) in OCT/Tyr gene has been linked to amitraz resistance in R. microplus ticks. Molecular analysis and LIT bioassay’s findings indicate the availability of resistant R. microplus ticks in the KP region. To our understanding, this is the first preliminary study to monitor cypermethrin and amitraz resistance via molecular profiling of cypermethrin and amitraz targeted genes (VGSC and OCT/Tyr) in combination with in vitro bioassays (LIT) in R. microplus ticks from Pakistan.

Despite significant advances in the treatment of triple-negative breast cancer, this disease continues to pose a clinical challenge, with many patients ultimately suffering from relapse. Tumor cells that recover after entering into a state of senescence after chemotherapy or radiation have been shown to develop a more aggressive phenotype, and to contribute to disease recurrence. By combining the PARP inhibitor (PARPi), talazoparib, with radiation, senescence was enhanced in 4T1 and MDA-MB-231 triple-negative breast cancer cell lines (based on SA-β-gal upregulation, increased expression of CDKN1A and the senescence-associated secretory phenotype (SASP) marker, IL6). Subsequent treatment of the radiation- and talazoparib-induced senescent 4T1 and MDA-MB231 cells with navitoclax (ABT-263) resulted in significant apoptotic cell death. In immunocompetent tumor-bearing mice, navitoclax exerted a modest growth inhibitory effect when used alone, but dramatically interfered with the recovery of 4T1-derived tumors induced into senescence with ionizing radiation and talazoparib. These findings support the potential utility of a senolytic strategy in combination with the radiotherapy/PARPi combination to mitigate the risk of disease recurrence in triple-negative breast cancer.

Ticks of the genus Dermacentor Koch, 1844 (Acari: Ixodidae) are poorly known systematically due to their habitation in harsh topographic environments and high mountains. Dermacentor ticks are diversely distributed in the Palearctic, Nearctic, and Oriental regions. There is no available information on the occurrence of Dermacentor marginatus in Pakistan; thus, the current investigation aimed the first morphological and molecular confirmation of this species and associated Anaplasma marginale and Rickettsia raoultii. Ticks were collected from goats (Capra hircus) and morphologically identified. Genomic DNA was extracted from 18/26 (69.23%) tick specimens, including 11 males and 7 females (1 unfed and 6 fed females). Extracted DNA was subjected to PCR for the amplification of genetic markers like 16S rDNA and cox1 for ticks, 16S rDNA for Anaplasma spp., and gltA and ompB for Rickettsia spp. A total of 26 D. marginatus ticks composed of 19 males (73.07%) and 7 females (26.9%) [1 (3.84%) unfed and 6 (23.07%) fed females] were collected from goats. According to amplicons via BLAST analysis, the 16S rDNA sequence showed 97.28–98.85% identity and the cox1 sequence showed 95.82–98.03% identity with D. marginatus. Additionally, the 16S rDNA sequence for Anaplasma sp. was detected in D. marginatus that showed 100% identity with Anaplasma marginale. Rickettsial gltA and ompB sequences for Rickettsia sp. showed 100% identity with Rickettsia raoultii. In phylogenetic analysis, ticks’ 16S rDNA and cox1 sequences clustered with the same species. In phylogenetic analysis, A. marginale based on 16 rDNA clustered with A. marginale, while gltA and ompB sequences clustered with R. raoultii. This is the first study on the genetic characterization of D. marginatus and associated A. marginale and R. raoultii in Pakistan. The northern areas of Pakistan, which need to be explored in terms of ticks and associated pathogens due to their zoonotic threats, have been neglected due to the inaccessible climatic conditions.

Emerging evidence has shown that the therapy-induced senescent growth arrest in cancer cells is of durable nature whereby a subset of cells can reinstate proliferative capacity. Promising new drugs named senolytics selectively target senescent cells and commit them into apoptosis. Accordingly, senolytics have been proposed as adjuvant cancer treatment to cull senescent tumor cells, and thus, screening for agents that exhibit senolytic properties is highly warranted. Our study aimed to investigate three agents, sorafenib, rapamycin, and venetoclax for their senolytic potential in doxorubicin-induced senescence in HCT116 cells. HCT116 cells were treated with one of the three agents, sorafenib (5 µM), rapamycin (100 nM), or venetoclax (10 µM), in the absence or presence of doxorubicin (1 µM). Senescence was evaluated using microscopy-based and flow cytometry-based Senescence-associated-β-galactosidase staining (SA-β-gal), while apoptosis was assessed using annexin V-FITC/PI, and Muse caspase-3/-7 activity assays. We screened for potential genes through which the three drugs exerted senolytic-like action using the Human Cancer Pathway Finder PCR array. The three agents reduced doxorubicin-induced senescent cell subpopulations and significantly enhanced the apoptotic effect of doxorubicin compared with those treated only with doxorubicin. The senescence genes IGFBP5 and BMI1 and the apoptosis genes CASP7 and CASP9 emerged as candidate genes through which the three drugs exhibited senolytic-like properties. These results suggest that the attenuation of doxorubicin-induced senescence might have shifted HCT116 cells to apoptosis by exposure to the tested pharmacological agents. Our work argues for the use of senolytics to reduce senescence-mediated resistance in tumor cells and to enhance chemotherapy efficacy.

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.

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.

Ticks are hematophagous ectoparasites that transmit pathogens to animals and humans. Updated knowledge regarding the global epidemiology of tick-borne Rickettsia hoogstraalii is dispersed, and its molecular detection and genetic characterization are missing in Pakistan. The current study objectives were to molecularly detect and genetically characterize Rickettsia species, especially R. hoogstraalii, in hard ticks infesting livestock in Pakistan, and to provide updated knowledge regarding their global epidemiology. Ticks were collected from livestock, including goats, sheep, and cattle, in six districts of Khyber Pakhtunkhwa (KP) Pakistan. Overall, 183 hosts were examined, of which 134 (73.2%), including goats (number = 39/54, 72.2%), sheep (23/40, 57.5%), and cattle (71/89, 80%) were infested by 823 ticks. The most prevalent tick species was Rhipicephalus microplus (number = 283, 34.3%), followed by Hyalomma anatolicum (223, 27.0%), Rhipicephalus turanicus (122, 14.8%), Haemaphysalis sulcata (104, 12.6%), Haemaphysalis montgomeryi (66, 8.0%), and Haemaphysalis bispinosa (25, 3.03%). A subset of 210 ticks was selected and screened for Rickettsia spp. using PCR-based amplification and subsequent sequencing of rickettsial gltA and ompB fragments. The overall occurrence rate of R. hoogstraalii was 4.3% (number = 9/210). The DNA of Rickettsia was detected in Hy. anatolicum (3/35, 8.5%) and Ha. sulcata (6/49, 12.2%). However, no rickettsial DNA was detected in Rh. microplus (35), Rh. turanicus (35), Ha. montgomeryi (42), and Ha. bispinosa (14). The gltA and ompB fragments showed 99–100% identity with R. hoogstraalii and clustered phylogenetically with the corresponding species from Pakistan, Italy, Georgia, and China. R. hoogstraalii was genetically characterized for the first time in Pakistan and Hy. anatolicum globally. Further studies should be encouraged to determine the role of ticks in the maintenance and transmission of R. hoogstraalii in different hosts.

Argasid ticks have the vectorial potential for transmitting disease-causing pathogens to avian hosts, resulting in economic losses that may not be fully estimated. Borrelia species are the responsible agents of borreliosis in poultry, animals and humans. Our previous studies have reported a high prevalence of Argas persicus infesting domestic fowls in Khyber Pakhtunkhwa (KP), Pakistan. However, molecular screening and genetic characterization of Borrelia spp. in A. persicus have been neglected in Pakistan. In this study, we focused on the molecular epidemiology and genetic characterization of Borrelia spp. associated with A. persicus ticks infesting domestic fowls and ducks, and Carios vespertilionis infesting bats in selected districts of KP. Overall, 1818 ticks, including females (415; 23%), males (345; 19%), nymphs (475; 26%) and larvae (583; 32%), were collected from 27 locations in nine districts (Peshawar, Mardan, Swabi, Charsadda, Chitral, Lakki Marwat, Bannu, Bajaur and Hangu) from domestic fowls, ducks and their shelters, and bats. A subset of 197 ticks was selected for DNA extraction and PCR to amplify fragments of the cytochrome c oxidase (cox) gene for ticks and flagellin B (flaB) for the detection and genetic characterization of associated Borrelia spp. Among these, only Borrelia anserina DNA was detected in 40 ticks (27.2%) of different life stages, where highest prevalence was found in female ticks (18; 45%), followed by nymphs (12; 30%), larvae (7; 17.5%) and males (3; 7.5%). Tick infestation in shelters (1081; 77%) was higher than on hosts (323; 23%). The resultant cox amplicons of A. persicus showed 100% identity with the same species reported from Pakistan, China, Iran, Kenya, Kazakhstan, Algeria and Egypt and C. vespertilionis show 100% identity with the species reported from Pakistan, China, Japan, Kenya, Vietnam, Spain, Netherlands, the United Kingdom and Hungry, and clustered with the aforementioned species in the phylogenetic tree. The obtained Borrelia sequences showed 100% identity with B. anserina and revealed a close resemblance to the relapsing fever group and clustered in a monophyletic clade with B. anserina from India, Iran and Brazil in a phylogenetic tree. These results establish the first molecular characterization of B. anserina in A. persicus infesting domestic fowls and ducks in the region, as well as their shelters. To effectively control zoonotic consequences, country-wide surveillance research should be encouraged to screen soft ticks infesting various birds for associated pathogens.

Ticks are hematophagous ectoparasites that transmit different pathogens such as Rickettsia spp. to domestic and wild animals as well as humans. Genetic characterizations of Rickettsia spp. from different regions of Pakistan are mostly based on one or two genetic markers and are confined to small sampling areas and limited host ranges. Therefore, this study aimed to molecularly screen and genetically characterize Rickettsia spp. in various tick species infesting camels, sheep, and goats. All the collected tick specimens were morphologically identified, and randomly selected tick species (148) were screened molecularly for the detection of Rickettsia spp. by amplifying three rickettsial DNA fragments, namely, the citrate-synthase gene (gltA), outer-membrane protein A (ompA), and outer-membrane protein B (ompB). After examining 261 hosts, 161 (61.7%) hosts were found infested by 564 ticks, including 287 (50.9%) nymphs, 171 (30.3%) females, and 106 (18.8%) males in five districts (Kohat, Dera Ismail Khan, Lower Dir, Bajaur, and Mansehra). The highest occurrence was noted for Hyalomma dromedarii (number = 72, 12.8%), followed by Haemaphysalis sulcata (n = 70, 12.4%), Rhipicephalus turanicus (n = 64, 11.3%), Rhipicephalus microplus (n = 55, 9.7%), Haemaphysalis cornupunctata (n = 49, 8.7%), Hyalomma turanicum (n = 48, 8.5%), Hyalomma isaaci (n = 45, 8.0%), Haemaphysalis montgomeryi (n = 44, 7.8%), Hyalomma anatolicum (n = 42, 7.5%), Haemaphysalis bispinosa (n = 38, 6.7%), and Rhipicephalus haemaphysaloides (n = 37, 6.6%). A subset of 148 ticks were tested, in which eight (5.4%) ticks, including four Hy. turanicum, two Ha. cornupunctata, one Ha. montgomeryi, and one Ha. bispinosa, were found positive for Rickettsia sp. The gltA, ompA, and ompB sequences revealed 100% identity and were phylogenetically clustered with Rickettsia raoultii reported in China, Russia, USA, Turkey, Denmark, Austria, Italy, and France. Additionally, various reports on R. raoultii from Palearctic and Oriental regions were summarized in this study. To the best of our knowledge, this is the first report regarding genetic characterization and phylogenetic analysis of R. raoultii from Pakistan. Further studies to investigate the association between Rickettsia spp. and ticks should be encouraged to apprise effective management of zoonotic consequences.

The market for nanoparticles has grown significantly over the past few decades due to a number of unique qualities, including antibacterial capabilities. It is still unclear how nanoparticle toxicity works. In order to ascertain the toxicity of synthetic cobalt iron oxide (CoFe2O4) nanoparticles (CIONPs) in rabbits, this study was carried out. Sixteen rabbits in total were purchased from the neighborhood market and divided into two groups (A and B), each of which contained eight rabbits. The CIONPs were synthesized by the co-precipitation method. Crystallinity and phase identification were confirmed by X-ray diffraction (XRD). The average size of the nanoparticles (13.2 nm) was calculated by Scherrer formula (Dhkl = 0.9 λ/β cos θ) and confirmed by TEM images. The saturation magnetization, 50.1 emug−1, was measured by vibrating sample magnetometer (VSM). CIONPs were investigated as contrast agents (CA) for magnetic resonance images (MRI). The relaxivity (r = 1/T) of the MRI was also investigated at a field strength of 0.35 T (Tesla), and the ratio r2/r1 for the CIONPs contrast agent was 6.63. The CIONPs were administrated intravenously into the rabbits through the ear vein. Blood was collected at days 5 and 10 post-exposure for hematological and serum biochemistry analyses. The intensities of the signal experienced by CA with CIONPs were 1427 for the liver and 1702 for the spleen. The treated group showed significantly lower hematological parameters, but significantly higher total white blood cell counts and neutrophils. The results of the serum biochemistry analyses showed significantly higher and lower quantities of different serum biochemical parameters in the treated rabbits at day 10 of the trial. At the microscopic level, different histological ailments were observed in the visceral organs of treated rabbits, including the liver, kidneys, spleen, heart, and brain. In conclusion, the results revealed that cobalt iron oxide (CoFe2O4) nanoparticles induced toxicity via alterations in multiple tissues of rabbits.

Ixodid ticks are responsible for the transmission of various intracellular bacteria, such as the Rickettsia species. Little Information is available about the genetic characterization and epidemiology of Rickettsia spp. The current study was designed to assess the tick species infesting various livestock hosts and the associated Rickettsia spp. in Pakistan. Ticks were collected from different livestock hosts (equids, cattle, buffaloes, sheep, goats, and camels); morphologically identified; and screened for the genetic characterization of Rickettsia spp. by the amplification of partial fragments of the gltA, ompA and ompB genes. Altogether, 707 ticks were collected from 373 infested hosts out of 575 observed hosts. The infested hosts comprised 105 cattle, 71 buffaloes, 70 sheep, 60 goats, 34 camels, and 33 equids. The overall occurrence of Rickettsia spp. was 7.6% (25/330) in the tested ticks. Rickettsia DNA was detected in Rhipicephalus haemaphysaloides (9/50, 18.0%), followed by Rhipicephalus turanicus (13/99, 13.1%), Haemaphysalis cornupunctata (1/18, 5.5%), and Rhipicephalus microplus (2/49, 4.1%); however, no rickettsial DNA was detected in Hyalomma anatolicum (71), Hyalomma dromedarii (35), and Haemaphysalis sulcata (8). Two Rickettsia agents were identified based on partial gltA, ompA, and ompB DNA sequences. The Rickettsia species detected in Rh. haemaphysaloides, Rh. turanicus, and Rh. microplus showed 99–100% identity with Rickettsia sp. and Candidatus Rickettsia shennongii, and in the phylogenetic trees clustered with the corresponding Rickettsia spp. The Rickettsia species detected in Rh. haemaphysaloides, Rh. turanicus, Rh. microplus, and Ha. cornupunctata showed 100% identity with R. massiliae, and in the phylogenetic trees it was clustered with the same species. Candidatus R. shennongii was characterized for the first time in Rh. haemaphysaloides, Rh. turanicus, and Rh. microplus. The presence of SFG Rickettsia spp., including the human pathogen R. massiliae, indicates a zoonotic risk in the study region, thus stressing the need for regular surveillance.

Abstract Determination of the seroprevalence and risk factors that are associated with West Nile virus (WNV) in horses is essential for adoption of effective prevention strategies. Our objective in this study, therefore, was to determine the seroprevalence and to identify the risk factors associated with WNV infection in the most densely horse-populated governorates in Egypt. A cross-sectional study was conducted in 2018 on 930 horses, which were distributed over five governorates in the Nile delta of Egypt. The horses, which were randomly selected, were serologically tested through use of an ID screen West Nile competition enzyme-linked immunosorbent assay (ELISA) to detect anti-WNV immunoglobulin G (IgG) and plaque reduction neutralization tests (PRNT; gold standard) to confirm the seropositive status of animals and to avoid cross reaction with other flavi-viruses. Four variables (geographical location, breed, sex and age) were considered in the risk analysis. Univariable and stepwise forward multivariable logistic regression methods were used for risk-factor analysis. The odds ratio (OR) was used as an approximate measure of relative risk. A total of 156 (16.8%; 95% confidence interval (CI) 14.4–19.2; P

Tick sialome is comprised of a rich cocktail of bioactive molecules that function as a tool to disarm host immunity, assist blood-feeding, and play a vibrant role in pathogen transmission. The adaptation of the tick’s blood-feeding behavior has lead to the evolution of bioactive molecules in its saliva to assist them to overwhelm hosts’ defense mechanisms. During a blood meal, a tick secretes different salivary molecules including vasodilators, platelet aggregation inhibitors, anticoagulants, anti-inflammatory proteins, and inhibitors of complement activation; the salivary repertoire changes to meet various needs such as tick attachment, feeding, and modulation or impairment of the local dynamic and vigorous host responses. For instance, the tick’s salivary immunomodulatory and cement proteins facilitate the tick’s attachment to the host to enhance prolonged blood-feeding and to modulate the host’s innate and adaptive immune responses. Recent advances implemented in the field of “omics” have substantially assisted our understanding of host immune modulation and immune inhibition against the molecular dynamics of tick salivary molecules in a crosstalk between the tick–host interface. A deep understanding of the tick salivary molecules, their substantial roles in multifactorial immunological cascades, variations in secretion, and host immune responses against these molecules is necessary to control these parasites. In this article, we reviewed updated knowledge about the molecular mechanisms underlying host responses to diverse elements in tick saliva throughout tick invasion, as well as host defense strategies. In conclusion, understanding the mechanisms involved in the complex interactions between the tick salivary components and host responses is essential to decipher the host defense mechanisms against the tick evasion strategies at tick-host interface which is promising in the development of effective anti-tick vaccines and drug therapeutics.

As a vector of wide range of pathogenic agents, ticks pose health threats to wild and domestic animals, and humans. Information is unavailable about the prevalence and spatial survey of Hyalomma kumari ticks and associated Rickettsia spp. in Pakistan. Concerning this knowledge gap, the present study aimed to molecularly detect Rickettsia species associated with H. kumari infesting small ruminants in Khyber Pakhtunkhwa (KP), Pakistan. A total of 409 H. kumari ticks were collected from 163/295 infested hosts with an infestation rate of 55.25%. A total of 204 females, 158 males, and 47 nymphs were collected. Goats were heavily infested by 224 ticks having an infestation rate of 58.33% (98/168), whereas sheep were infested by 185 ticks having a lesser infestation rate of 51.18% (65/127). Genomic DNA extracted from ticks was used for the amplification of tick (cox I, 16S rRNA, ITS-2) species and Rickettsia (gltA, ompA, and ompB) partial genes. Eighty-three ticks were subjected to PCR, and 8/83 (9.6%) were found positive for rickettsial agents. The cox I and 16S rRNA sequences of H. kumari showed 98.90–99.74% identity with H. kumari sequences reported from Pakistan, and phylogenetically clustered to the corresponding species reported from Pakistan and India. The obtained rickettsial gltA, ompA, and ompB sequences showed 100% identity with Rickettsia sp. of the Rickettsia conorii reported from Pakistan. In the phylogenetic trees, rickettsial sequences clustered with uncharacterized Rickettsia sp. from Pakistan and R. conorii from Israel, Russia, South Africa, and India. The present molecular based detection of H. kumari-associated R. conorii will facilitate effective surveillance in the region.

Emerging resistance to colistin in Acinetobacter baumannii clinical strains is concerning because of the limited therapeutic choices for these important clinical pathogens. We studied the in vitro activities of different colistin-based antimicrobial agent combinations against colistin-resistant Acinetobacter baumannii. Fourteen clinical isolates of colistin-resistant Acinetobacter baumannii were obtained between 2015 and 2016. To identify colistin-based combinations with synergistic activities, multiple two antimicrobial combinations based on 8 commercially available drugs were evaluated by the checkerboard method. The most effective colistin-based combinations were vancomycin, aztreonam, ceftazidime and imipenem which showed synergistic activities against all examined strains. Colistin-rifampin showed synergy against four strains. Colistin-tigecycline and colistin-amikacin mostly showed indifferent results. By using the checkerboard tests, we were able to find the most promising colistin-based combinations that may provide more therapeutic options against colistin-resistant Acinetobacter baumannii.

Haemaphysalis ticks are globally distributed with the greatest diversity in the Oriental region. This study aimed to primarily provide information on the morphology, host record, and preliminary phylogenetic position of a poorly known tick Haemaphysalis danieli. Herds comprised of goats and sheep were examined for this tick species in Upper Dir, Khyber Pakhtunkhwa, Pakistan. A total of 127 ticks, including males (n = 15, 11.8%) and females (n = 112, 88.2%), were collected, and morphologically identified as H. danieli. The morphological identification was confirmed through the 16S rDNA and cytochrome c oxidase (cox1) sequences. Phylogenetic analysis inferred based on 16S rDNA and cox1 showed a close evolutionary relationship of H. danieli with a conspecific from China and an undetermined Haemaphysalis sp. from China and Anatolia. A total of 32/223 (14.3%) goats in two different herds were the only host infested by H. danieli. The earliest study provided the morphological description of H. danieli male, host record, and phylogenetic position. The information provided herein could assist in minimizing the knowledge gap regarding the systematic and taxonomy of Haemaphysalis species.

Rhipicephalus microplus tick highly affects the veterinary sector throughout the world. Different tick control methods have been adopted, and the identification of tick-derived highly immunogenic sequences for the development of an anti-tick vaccine has emerged as a successful alternate. This study aimed to characterize immunogenic sequences from R. microplus ticks prevalent in Pakistan. Ticks collected in the field were morphologically identified and subjected to DNA and RNA extraction. Ticks were molecularly identified based on the partial mitochondrial cytochrome C oxidase subunit (cox) sequence and screened for piroplasms (Theileria/Babesia spp.), Rickettsia spp., and Anaplasma spp. PCR-based pathogens-free R. microplus-derived cDNA was used for the amplification of full-length cysteine protease inhibitor (cystatin 2b), cathepsin L-like cysteine proteinase (cathepsin-L), glutathione S-transferase (GST), ferritin 1, 60S acidic ribosomal protein (P0), aquaporin 2, ATAQ, and R. microplus 05 antigen (Rm05Uy) coding sequences. The cox sequence revealed 100% identity with the nucleotide sequences of Pakistan’s formerly reported R. microplus, and full-length immunogenic sequences revealed maximum identities to the most similar sequences reported from India, China, Cuba, USA, Brazil, Egypt, Mexico, Israel, and Uruguay. Low nonsynonymous polymorphisms were observed in ATAQ (1.5%), cathepsin-L (0.6%), and aquaporin 2 (0.4%) sequences compared to the homologous sequences from Mexico, India, and the USA, respectively. Based on the cox sequence, R. microplus was phylogenetically assembled in clade C, which includes R. microplus from Pakistan, Myanmar, Malaysia, Thailand, Bangladesh, and India. In the phylogenetic trees, the cystatin 2b, cathepsin-L, ferritin 1, and aquaporin 2 sequences were clustered with the most similar available sequences of R. microplus, P0 with R. microplus, R. sanguineus and R. haemaphysaloides, and GST, ATAQ, and Rm05Uy with R. microplus and R. annulatus. This is the first report on the molecular characterization of clade C R. microplus-derived immunogenic sequences.

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

The increasing prevalence of antimicrobial-resistant (AMR) bacteria along with the limited development of antimicrobials warrant investigating novel antimicrobial modalities. Emerging inorganic engineered nanomaterials (ENMs), most notably silver nanoparticles (AgNPs), have demonstrated superior antimicrobial properties. However, AgNPs, particularly those of small size, could exert overt toxicity to mammalian cells. This study investigated whether combining AgNPs and conventional antimicrobials would produce a synergistic response and determined the optimal and safe minimum inhibitory concentration (MIC) range against several wild-type Gram-positive and -negative strains and three different clinical isolates of AMR Klebsiella pneumoniae. Furthermore, the cytotoxicity of the synergistic combinations was assessed in a human hepatocyte model. The results showed that the AgNPs (15–25 nm) were effective against Gram-negative bacteria (MIC of 16–128 µg/mL) but not Gram-positive strains (MIC of 256 µg/mL). Both wild-type and AMR K. pneumoniae had similar MIC values following exposure to AgNPs. Importantly, co-exposure to combinations of AgNPs and antimicrobial agents, including kanamycin, colistin, rifampicin, and vancomycin, displayed synergy against both wild-type and AMR K. pneumoniae isolates (except for vancomycin against AMR strain I). Notably, the tested combinations demonstrated no to minimal toxicity against hepatocytes. Altogether, this study indicates the potential of combining AgNPs with conventional antimicrobials to overcome AMR bacteria.

Theileria annulata is a tick-associated parasite that causes tropical theileriosis in livestock and is responsible for huge economic losses. Studies have been neglected on the effect of Theileria spp. on cattle in Khyber Pakhtunkhwa (KP), Pakistan. The present study was designed to determine the genetic diversity and assess the risk factors associated with Theileria infection in selected districts of KP. Information on the risk factors related to the Theileria infection was collected through a questionnaire. Blood samples were collected from symptomatic cattle from January 2019 to February 2020, identified morphologically through microscopic examination, and processed for molecular characterization using the 18S rRNA gene as a genetic marker. Of the 555 cattle examined (136, 24.5%) and (294, 53%) were found positive for Theileria spp. by microscopic examination and a PCR test, respectively. Based on the PCR test, the highest prevalence of infection was found in district Upper Dir (46/75, 61.3%), followed by Lower Dir (54/90, 60%); Malakand (51/88, 57.9%); Peshawar (40/80, 50%); and Charsadda (52/112, 46.4%), with the lowest in Bajaur (51/110, 46.34%). A BLAST analysis of the 18S rDNA sequences showed 99.5% identity with T. annulata. In a phylogenetic tree, the 18S rDNA sequence of T. annulata clustered with sequences from Pakistan, China, and Italy. A significant association was observed between the prevalence of infection and different host characteristics. The highest infection was found in adult cattle (216/360, 60%); females (218/377, 57.8%); and Holstein Friesian (120/180, 66.6%). Theileria infection was significantly associated with management practices. Higher infection rates were observed in free-grazing cattle (190/412, 42.2%); those kept in unhygienic conditions (246/405, 60.7%); cattle in combined farming systems (165/255, 64.8%); and those in congested stall systems (150/218, 68.8%). Seasonal patterns were found to be significantly associated with infection, and a higher infection rate was observed in summer (215/350, 61.4%) than in winter (79/205, 38.5%). Identified risk factors should be considered in designing practical control approaches to reduce the burden of Theileria infection. Large scale studies are required to explore the diversity of Theileria species in KP, Pakistan.

Hard ticks (Ixodida: Ixodidae) are medically important ectoparasites that feed on all classes of terrestrial vertebrates. Recently, we molecularly characterized hard ticks and associated Anaplasma spp. in the northern and central regions of Khyber Pakhtunkhwa (KP), Pakistan; however, this knowledge was missing in the southern regions. This study aimed to investigate tick prevalence, host range, genetic diversity, and molecular survey of Anaplasma spp. in a wide range of tick species in two distinct physiographic regions of southern KP. A total of 1873 hard ticks were randomly collected from 443/837 hosts (cattle, Asian water buffaloes, horses, goats, sheep, dogs, and camels) in Lakki Marwat, Bannu, and Orakzai districts of KP. Overall, 12 tick species were morphologically identified, among which Hyalomma dromedarii was the most prevalent species (390/1873, 20.9%), followed by Hy. anatolicum (294, 15.7%), Rhipicephalus microplus (262, 14%), Hy. scupense (207, 11.1%), R. sanguineus (136, 7.3%), R. turanicus (121, 6.5%), Haemaphysalis cornupunctata (107, 5.7%), R. haemaphysaloides (110, 5.9%), Ha. montgomeryi (87, 4.6%), Hy. isaaci (58, 3.1%), Ha. bispinosa (54, 2.9%), and Ha. sulcata (47, 2.5%). The extracted DNA from a subset of each tick species was subjected to PCR to amplify cox1 or 16S rRNA sequences of ticks and 16S rRNA sequences of Anaplasma spp. The tick cox1 sequences showed 99–100% identities with the sequences of the same species, whereas 16S rRNA sequences of R. turanicus, Ha. montgomeryi and Ha. sulcata showed 97–100% identities with the corresponding species. The 16S rRNA sequence of Ha. cornupunctata showed 92% identity with the species from the same subgenus, such as Ha. punctata. The 16S rRNA sequence of Anaplasma spp. showed 100% identity with Anaplasma marginale. Moreover, 54 ticks were found positive for A. marginale with a total infection rate of 17.2%. The highest infection rate was recorded in Hy. dromedarii (31.1%) and the lowest in each R. haemaphysaloides and R. sanguineus (20%). All the cox1 or 16S rRNA sequences in phylogenetic trees clustered with the same species, except Ha. cornupunctata, which clustered with the Ha. (Aboimisalis) punctata. In this study, Ha. cornupunctata was reported for the first time at the molecular level. The genetic characterization of ixodid ticks and molecular detection of associated A. marginale will assist in the epidemiological surveillance of these parasites in the region.

Introduction: Tuberculosis (TB) remains a global public health threat affecting people in many developing countries, including the Kingdom of Saudi Arabia. Maintaining a long-term treatment regimen has always been the cornerstone of successful treatment outcomes among tuberculosis patients. In the Jeddah region, the National Tuberculosis Control and Prevention Program is now treating TB patients by means of a community mobile outreach team approach.The objective of this study was to compare the effectiveness of the community mobile outreach approach in improving treatment outcomes (success rate) among local tuberculosis patients with those being treated with a facility-based directly observed treatment, short-course (DOTS). Study design: Our study consisted of a two-sample, parallel design [1:1], statistician -blind randomized control trial with 200 newly diagnosed, TB patients as subjects. Setting/participants: The patients had all presented at the Madain Alfahd Primary Health Care Center, Jeddah. Between Nov 2017 and Nov 2018, a total of 221 TB patients were screened of whom 200 were randomly selected using randomly generated sequences. Intervention: Patients in the intervention sample group were treated by means of mobile outreach teams with oral anti-TB treatment under the DOTS, and control group patients were given the traditional facility-based DOTS treatment according to the WHO recommendations and national guidelines. Main outcome: The primary outcome was the level of overall treatment success rate. It was finally determined and compared in the two sample groups using chi-square analysis and relative risk assessment. Results: In the analysis stage, 97 patients were in the intervention group, while the control group consisted of 76. The overall response rate was 86.5% (173/200). We found that the percentage of overall treatment success rate among the patients served by the mobile outreach team was 97%, compared to 76% in the non-mobile team treated patients. The relative risk of treatment success rate among the intervention group was 1.27 (95% CI = 1.13–1.43) times greater than that amongst the control group. Log-rank test (log-rank statistics = 18.91; p

Ticks transmit pathogens to animals and humans more often than any other arthropod vector. The rural economy of Pakistan mainly depends on livestock farming, and tick infestations cause severe problems in this sector. The present study aimed to molecularly characterize the Anaplasma spp. in hard ticks collected from six districts of Khyber Pakhtunkhwa, Pakistan. Ticks were collected from various livestock hosts, including cattle breeds (Holstein-Friesian, Jersey, Sahiwal, and Achai), Asian water buffaloes, sheep, and goats from March 2018 to February 2019. Collected ticks were morphologically identified and subjected to molecular screening of Anaplasma spp. by amplifying 16S rDNA sequences. Six hundred seventy-six ticks were collected from infested hosts (224/350, 64%). Among the nine morphologically identified tick species, the highest occurrence was noted for Rhipicephalus microplus (254, 37.6%), followed by Hyalomma anatolicum (136, 20.1%), Rhipicephalus haemaphysaloides (119, 17.6%), Rhipicephalus turanicus (116, 17.1%), Haemaphysalis montgomeryi (14, 2.1%), Hyalomma dromedarii (11, 1.6%), Haemaphysalis bispinosa (10, 1.5%), Hyalomma scupense (8, 1.2%), and Haemaphysalis kashmirensis (8, 1.2%). The occurrence of tick females was highest (260, 38.5%), followed by nymphs (246, 36.4%) and males (170, 25.1%). Overall, the highest occurrence of ticks was recorded in the Peshawar district (239, 35.3%), followed by Mardan (183, 27.1%), Charsadda (110, 16.3%), Swat (52, 7.7%), Shangla (48, 7.1%), and Chitral (44, 6.5%). Among these ticks, Anaplasma marginale was detected in R. microplus, R. turanicus, and R. haemaphysaloides. The 16S rDNA sequences showed high identity (98–100%) with A. marginale reported from Australia, China, Japan, Pakistan, Thailand, Uganda, and the USA. In phylogenetic analysis, the sequence of A. marginale clustered with the same species reported from Australia, China, Pakistan, Thailand, Uruguay, and the USA. Further molecular work regarding the diversity of tick species and associated pathogens is essential across the country.

Background and Objectives: The development of radioresistance is a fundamental barrier to successful glioblastoma therapy. Autophagy is thought to play a role in facilitating the DNA repair of DNA damage foci in radiation-exposed tumor cells, thus, potentially contributing to their restoration of proliferative capacity and development of resistance in vitro. However, the effect of autophagy inhibitors on DNA damage repair is not fully clear and requires further investigation. Materials and Methods: In this work, we utilized M059K (DNA-PKcs proficient) and M059J (DNA-PKcs deficient) glioma cell lines to investigate the role of autophagy inhibitors in the DNA repair of radiation-induced DNA damage. Cell viability following radiation was determined by trypan blue exclusion in both cell lines. Cell death and autophagy assays were performed to evaluate radiation-induced cell stress responses. DNA damage was measured as based on the intensity of phosphorylated γ-H2AX, a DNA double-stranded breaks (DSBs) marker, in the presence or absence of autophagy inhibitors. Results: The cell viability assay showed that M059J cells were more sensitive to the same dose of radiation (4 Gy) than M059K cells. This observation was accompanied by an elevation in γ-H2AX formation in M059J but not in M059K cells. In addition, the DAPI/TUNEL and Senescence-associated β-galactosidase (SA-β-gal) staining assays did not reveal significant differences in apoptosis and/or senescence induction in response to radiation, respectively, in either cell line. However, acridine orange staining demonstrated clear promotion of acidic vesicular organelles (AVOs) in both cell lines in response to 4 Gy radiation. Moreover, DNA damage marker levels were found to be elevated 72 h post-radiation when autophagy was inhibited by the lysosomotropic agent bafilomycin A1 (BafA1) or the PI3K inhibitor 3-methyl adenine (3-MA) in M059K cells. Conclusions: The extent of the DNA damage response remained high in the DNA-PKcs deficient cells following exposure to radiation, indicating their inability to repair the newly formed DNA-DSBs. On the other hand, radioresistant M059K cells showed more DNA damage response only when autophagy inhibitors were used with radiation, suggesting that the combination of autophagy inhibitors with radiation may interfere with DNA repair efficiency.

Protozoa, helminths and ectoparasites are the major groups of parasites distributed worldwide. Currently, these parasites are treated with chemotherapeutic antiprotozoal drugs, anti-helminthic and anti-ectoparasitic agents, but, with the passage of time, resistance to these drugs has developed due to overuse. In this scenario, nanoparticles are proving to be a major breakthrough in the treatment and control of parasitic diseases. In the last decade, there has been enormous development in the field of nanomedicine for parasitic control. Gold and silver nanoparticles have shown promising results in the treatments of various types of parasitic infections. These nanoparticles are synthesized through the use of various conventional and molecular technologies and have shown great efficacy. They work in different ways, that include damaging the parasite membrane, DNA (Deoxyribonucleic acid) disruption, protein synthesis inhibition and free-radical formation. These agents are effective against intracellular parasites as well. Other nanoparticles, such as iron, nickel, zinc and platinum, have also shown good results in the treatment and control of parasitic infections. It is hoped that this research subject will become the future of modern drug development. This review summarizes the methods that are used to synthesize nanoparticles and their possible mechanisms of action against parasites.

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

Noha Attia,1–3,* Mohamed Mashal,1,* Cristina Soto-Sánchez,4,5 Gema Martínez-Navarrete,4,5 Eduardo Fernández,4,5 Santiago Grijalvo,4,6 Ramón Eritja,4,6 Gustavo Puras,1,4 Jose Luis Pedraz1,4,* 1NanoBioCel Group, Laboratory of Pharmaceutics, School of Pharmacy, University of the Basque Country (UPV/EHU), Vitoria-Gasteiz, Spain; 2Medical Histology and Cell Biology Department, Faculty of Medicine, University of Alexandria, Alexandria, Egypt; 3Department of Basic Sciences, The American University of Antigua-College of Medicine, Coolidge, Antigua and Barbuda; 4Networking Research Centre of Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Vitoria-Gasteiz, Spain; 5Neuroprothesis and Neuroengineering Research Group, Miguel Hernández University, Elche, Spain; 6Institute of Advanced Chemistry of Catalonia (IQAC-CSIC), Barcelona, Spain *These authors contributed equally to this work Background: Gene therapy can be an intriguing therapeutic option in wide-ranging neurological disorders. Though nonviral gene carriers represent a safer delivery system to their viral counterparts, a thorough design of such vehicles is crucial to enhance their transfection properties.Purpose: This study evaluated the effects of combined use of two nonionic surfactants, poloxamer 188 (P) and polysorbate 80 (P80) into nanovesicles – based on 2,3-di(tetradecyloxy)propan-1-amine cationic lipid (D) – destined for gene delivery to central nervous system cells.Methods: Niosome formulations without and with poloxamer 188 (DP80 and DPP80, respectively) were prepared by the reverse-phase evaporation technique and characterized in terms of size, surface charge, and morphology. After the addition of pCMS-EGFP plasmid, the binding efficiency to the niosomes was evaluated in agarose gel electrophoresis assays. Additionally, transfection efficiency of complexes was also evaluated in in vitro and in vivo conditions.Results: In vitro experiments on NT2 cells revealed that the complexes based on a surfactant combination (DPP80) enhanced cellular uptake and viability when compared with the DP80 counterparts. Interestingly, DPP80 complexes showed protein expression in glial cells after administration into the cerebral cortices of rats.Conclusion: These data provide new insights for glia-centered approach for gene therapy of nervous system disorders using cationic nanovesicles, where nonionic surfactants play a pivotal role. Keywords: gene therapy, nonviral gene vectors, poloxamer 188, niosomes, cationic lipids

Bovine leukaemia virus (BLV) is a deltaretrovirus that is closely related to human T-cell leukaemia virus types 1 and 2 (HTLV-1 and -2). It causes enzootic bovine leukosis (EBL), which is the most important neoplastic disease in cattle. Most BLV-infected cattle are asymptomatic, which potentiates extremely high shedding rates of the virus in many cattle populations. Approximately 30% of them show persistent lymphocytosis that has various clinical outcomes; only a small proportion of animals (less than 5%) exhibit signs of EBL. BLV causes major economic losses in the cattle industry, especially in dairy farms. Direct costs are due to a decrease in animal productivity and in cow longevity; indirect costs are caused by restrictions that are placed on the import of animals and animal products from infected areas. Most European regions have implemented an efficient eradication programme, yet BLV prevalence remains high worldwide. Control of the disease is not feasible because there is no effective vaccine against it. Therefore, detection and early diagnosis of the disease are essential in order to diminish its spreading and the economic losses it causes. This review comprises an overview of bovine leukosis, which highlights the epidemiology of the disease, diagnostic tests that are used and effective control strategies.

Abstract Background Cisplatin is widely used chemotherapeutic agent for cancer treatment with limited uses due to its neurotoxic side effect. The aim of this study was to determine the potential preventive effects of rutin on the brain of cisplatin- neurotoxic rat model. Methods Forty rats were divided into four groups. Group-1 (control group) was intra-peritoneal (IP) injected with 2.5 ml/kg saline. Group-2 (rutin group) was orally administrated 30 mg/kg rutin dissolved in water for 14 days. Group-3 (cisplatin group) was IP received 5 mg/kg cisplatin single dose. Group-4 (rutin and cisplatin group) was orally administrated 30 mg/kg rutin dissolved in water for 14 days with a single dose of 5 mg/kg cisplatin IP on day ten. Brain tissues from frontal cortex was used to extract RNA, the gene expression levels of paraoxonase-1 (PON-1), PON-2, PON-3, peroxisome proliferator-activated receptor delta (PPAR-δ), and glutathione peroxidase (GPx) was investigated by Real-time PCR. Results Cisplatin significantly decreased the expression levels of PON-1, PON-3, PPAR-δ and GPX whereas significantly increased PON-2 expression levels. Co-administration of Rutin prevented the cisplatin-induced toxicity by restoring the alteration in the studied genes to normal values as in the control group. Conclusion This study showed that Rutin has neuroprotective effect and reduces cisplatin- neurotoxicity with possible mechanism via the antioxidant pathway.

Abstract Background Cisplatin (CP) is commonly used in the treatment of different types of cancer but nephrotoxicity has been a major limiting factor. Therefore, the present study aimed to study the possible protective effect of rutin against nephrotoxicity induced by cisplatin in rats. Methods Forty male Wistar albino rats were randomly divided into 4 groups. Rats of group 1 control group intraperitoneal (i.p.) received 2.5 ml/kg, group 2 CP group received single dose 5 mg/kg cisplatin i.p. group 3 rutin group orally received 30 mg/kg rutin group 4 (CP plus rutin) received CP and rutin as in group 2 and 3. Kidneys were harvested for histopathology and for the study the gene expression of c-Jun N-terminal kinases (JNK), Mitogen-activated protein kinase 4 (MKK4), MKK7, P38 mitogen-activated protein kinases (P38), tumor necrosis factors alpha (TNF-α), TNF Receptor-Associated Factor 2 (TRAF2), and interleukin-1 alpha (IL-1-α). Results The cisplatin single dose administration to rats induced nephrotoxicity associated with a significant increase in blood urea nitrogen (BUN) and serum creatinine and significantly increase Malondialdehyde (MDA) in kidney tissues by 230 ± 5.5 nmol/g compared to control group. The animal treated with cisplatin showed a significant increase in the expression levels of the IL-1α (260%), TRFA2 (491%), P38 (410%), MKK4 (263%), MKK7 (412%), JNK (680%) and TNF-α (300%) genes compared to control group. Additionally, histopathological examination showed that cisplatin-induced interstitial congestion, focal mononuclear cell inflammatory, cell infiltrate, acute tubular injury with reactive atypia and apoptotic cells. Rutin administration attenuated cisplatin-induced alteration in gene expression and structural and functional changes in the kidney. Additionally, histopathological examination of kidney tissues confirmed gene expression data. Conclusion The present study suggested that the anti-oxidant and anti-inflammatory effect of rutin may prevent CP-induced nephrotoxicity via decreasing the oxidative stress, inhibiting the interconnected ROS/JNK/TNF/P38 MAPK signaling pathways, and repairing the histopathological changes against cisplatin administration.