Mitochondrial genome and nuclear ribosomal RNA analysis place Alveonasus lahorensis within the Argasinae and suggest that the genus Alveonasus is paraphyletic.

Two major families exist in ticks, the Argasidae and Ixodidae. The Argasidae comprise 2 sub-families, Argasinae and Ornithodorinae. The placement into subfamilies illuminate differences in morphological and molecular systematics and is important since it provides insight into evolutionary divergence within this family. It also identifies fundamental gaps in our understanding of argasid evolution that provide directions for future research. Molecular systematics based on mitochondrial genomics and 18S/28S ribosomal RNA confirmed the placement of various genera and subgenera into the Argasinae: Argas (including Argas and Persicargas), Navis, Ogadenus, Otobius lagophilus, Proknekalia, Secretargas and the Ornithodorinae: Alectorobius, Antricola (including Antricola and Parantricola), Carios, Chiropterargas, Nothoaspis, Ornithodoros (including Microargas, Ornamentum, Ornithodoros sensu strictu, Pavlovskyella), Otobius sensu strictu, Reticulinasus and Subparmatus. The position of Alveonasus remains controversial since traditional taxonomy placed it in the Ornithodorinae, while cladistic and limited molecular analysis placed it in the Argasinae. The current study aimed to resolve the systematic position of Alveonasus using mitochondrial genomic and 18S/28S ribosomal RNA systematics by sequencing the type species Alveonasus lahorensis from Pakistan. In addition, the mitochondrial genomes for Argas reflexus and Alectorobius kelleyi are reported from Germany and the USA, respectively. The systematic data unambiguously place Alveonasus in the Argasinae and also suggest that Alveonasus may be another paraphyletic genus.

Exploring the Potent Combination of Quercetin-Boronic Acid, Epalrestat, and Urea Containing Nanoethosomal Keratolytic Gel for the Treatment of Diabetic Neuropathic Pain: In Vitro and In Vivo Studies.

Transdermal penetration of therapeutic moieties from topical dosage forms always remains a challenge due to the presence of permeation impeding keratin which should be addressed. The purpose of the study was to formulate quercetin and 4-formyl phenyl boronic acid (QB complex) used for the preparation of nanoethosomal keratolytic gel (EF3-G). The QB complex was confirmed by Fourier transform infrared spectroscopy while skin permeation, viscosity, and epalrestat entrapment efficiency were used for the optimization of nanoethosomal gel. The keratolytic effect of the proposed nanoethosomal gel with urea (QB + EPL + U) was calculated in rat and snake skin. The spherical shape of nanoethosomes was confirmed by scanning electron microscopy. According to the findings of stability studies, viscosity decreases as temperature increases, proving their thermal stability. The negative charge of optimized EF3 with 0.7 PDI proved narrow particle size distribution with homogeneity. Optimized EF3 showed two folds increase of epalrestat permeation in highly keratinized snake skin as compared to rats' skin after 24 h. Antioxidant behaviors of EF3 (QB) > QB complex > quercetin > ascorbic acid proved reduction of oxidative stress in DPPH reduction analysis. Interestingly, the hot plate and cold allodynia test in the diabetic neuropathic rat model reduced 3-fold pain as compared to the diabetic control group which was further confirmed by in vivo biochemical studies even after the eight week. Conclusively, ureal keratolysis, primary dermal irritation index reduction, and improved loading of epalrestat render the nanoethosomal gel (EF3-G) ideal for the treatment of diabetic neuropathic pain.

Assessing the impacts of climate change on streamflow dynamics: A machine learning perspective.

This study investigates changes in river flow patterns, in the Hunza Basin, Pakistan, attributed to climate change. Given the anticipated rise in extreme weather events, accurate streamflow predictions are increasingly vital. We assess three machine learning (ML) models - artificial neural network (ANN), recurrent neural network (RNN), and adaptive fuzzy neural inference system (ANFIS) - for streamflow prediction under the Coupled Model Intercomparison Project 6 (CMIP6) Shared Socioeconomic Pathways (SSPs), specifically SSP245 and SSP585. Four key performance indicators, mean square error (MSE), root mean square error (RMSE), mean absolute error (MAE), and coefficient of determination (R2), guide the evaluation. These models employ monthly precipitation, maximum and minimum temperatures as inputs, and discharge as the output, spanning 1985-2014. The ANN model with a 3-10-1 architecture outperforms RNN and ANFIS, displaying lower MSE, RMSE, MAE, and higher R2 values for both training (MSE = 20417, RMSE = 142, MAE = 71, R2 = 0.94) and testing (MSE = 9348, RMSE = 96, MAE = 108, R2 = 0.92) datasets. Subsequently, the superior ANN model predicts streamflow up to 2100 using SSP245 and SSP585 scenarios. These results underscore the potential of ANN models for robust futuristic streamflow estimation, offering valuable insights for water resource management and planning.

Intercomparison of SWAT and ANN techniques in simulating streamflows in the Astore Basin of the Upper Indus.

The current research work was carried out to simulate monthly streamflow historical record using Soil and Water Assessment Tool (SWAT) and Artificial Neural Network (ANN) at the Astore Basin, Gilgit-Baltistan, Pakistan. The performance of SWAT and ANN models was assessed during calibration (1985-2005) and validation (2006-2010) periods via statistical indicators such as coefficient of determination (R2), Nash-Sutcliffe efficiency (NSE), percent bias (PBIAS), and root-mean-square error (RMSE). R2, NSE, PBIAS, and RMSE values for SWAT (ANN with Architecture (2,27,1)) models during calibration are 0.80 (0.88), 0.73 (0.82), 15.7 (0.008), and 79.81 (70.34), respectively, while during validation, the corresponding values are 0.71 (0.86), 0.66 (0.95), 17.3 (0.10), and 106.26 (75.92). The results implied that the ANN model is superior to the SWAT model based on the statistical performance indicators. The SWAT results demonstrated an underestimation of the high flow and overestimation of the low flow. Comparatively, the ANN model performed very well in estimating the general and extreme flow conditions. The findings of this research highlighted its potential as a valuable tool for accurate streamflow forecasting and decision-making. The current study recommends that additional machine learning models may be compared with the SWAT model output to improve monthly streamflow predictions in the Astore Basin.

The impact of the COVID-19 on China's fisheries sector and its countermeasures.

The COVID-19 pandemic has affected China's economic development, international exchanges and other aspects to varying degrees, among which the impact on China's fishery development should not be underestimated. The fishery is a traditional and vast industry in China. The ongoing COVID-19 outbreak has also exposed potential problems in China's fishery development, such as the lack of specialised aquatic product quality and safety supervision and testing institutions. New problems have also arisen, such as the decline in the market demand for fish and aquatic products, affecting fishers' income, livelihood and so on. These deficiencies require China to establish a matching legal system to correspondingly solve the current legal and political problems. This paper will systematically discuss the impact of the COVID-19 on China's fisheries sector from the perspectives of 'fishers' and 'aquatic products' after the outbreak of the COVID-19 pandemic and analyse how to deal with various problems by referring to relevant experience in light of China's national conditions and characteristics.

Molecular characterization and RSV Co-infection of Nicotiana benthamiana with three distinct begomoviruses.

Geminiviruses constitute a family of plant viruses with characteristic twinned quasi-icosahedral virions and a small circular DNA genome. Geminiviruses, especially begomoviruses, cause substantial economic losses in tropical and subtropical regions globally. Geminiviruses use the host's transcriptional mechanisms to synthesize their mRNAs. They are considered as an attractive model to understand the transcription mechanism of their host plants. Experiments were conducted to identify transcriptional start sites (TSSs) of the three begomoviruses, i.e., Cotton leaf curl Multan virus (CLCuMuV), Corchorus yellow vein virus (CoYVV), and Ramie mosaic virus (RamV). We first rub-inoculated Rice stripe tenuivirus (RSV), a segmented negative-sense RNA virus that uses cap-snatching to produce capped viral mRNAs, into N. benthamiana. After the inoculation, RSV-infected N. benthamiana were super-infected by CoYVV, CLCuMuV, or RamV, respectively. The capped-RNA leaders snatched by RSV were obtained by determining the 5'-ends of RSV mRNA with high throughput sequencing. Afterwards, snatched capped-RNA leaders of RSV were mapped onto the genome of each begomovirus and those matching the begomoviral genome were considered to come from the 5' ends of assumed begomoviral mRNAs. In this way, TSSs of begomoviruses were obtained. After mapping these TSSs onto the genome of the respective begomovirus, it was found very commonly that a begomovirus can use many different TSSs to transcribe the same gene, producing many different mRNA isoforms containing the corresponding open reading frames (ORFs).

Probabilistic risk assessment of water distribution system in Hyderabad, Pakistan reveals unacceptable health hazards and areas for rehabilitation.

Poor water quality exacerbates multidimensional poverty in developing nations. Often centralized treatment facilities generate acceptable water quality, but the water is contaminated during distribution. Methods to assess sources of contamination in water distribution systems are lacking. A case study of two methods, human risk assessment linked to water distribution system sampling was conducted in Hyderabad, Pakistan to determine areas requiring infrastructure rehabilitation. Water samples from source water (i.e., the Indus River), treatment plant effluent and from taps in the water distribution system were analyzed by atomic adsorption spectroscopy for metals and metalloids (As, Cd, Cr, Hg, and Pb) and water quality parameters (dissolved and suspended solids, pH, conductivity, and total organic carbon). Source water exceeded acceptable drinking water levels for As, Cd, total Cr, and Pb, while the treatment plant effluent concentrations were acceptable. Concentrations of all metals and metalloids, except Hg, increased in the water distribution system post-treatment, exceeding safe drinking limits in at least one location, suggesting contamination of the water during distribution. A deterministic and a probabilistic risk assessment were conducted to evaluate two scenarios: (1) unrestricted use of piped water for all household purposes, including as drinking water and (2) restricted use of the water for purposes other than drinking in the household, including only dermal and inhalation exposure pathways. The water was deemed unsafe for unrestricted use as the sole source of drinking water by both risk assessment methods. Yet when an alternative source of drinking water was assumed and the piped water was used only for bathing and dish washing, the probabilistic risk assessment revealed acceptable health risks to the population, while the overly conservative deterministic risk assessment suggested unacceptable risks. The combined methods of water sampling, risk assessment and correlation analysis suggested areas for rehabilitation of the water distribution system in Hyderabad, Pakistan and these methods can be adopted in other developing nations to target limited funds for infrastructure rehabilitation.

A Rare Case of Chromoblastomycosis in a 12-year-old boy.

Chromoblastomycosis is a chronic fungal infection of the subcutaneous tissue. The infection usually results from a traumatic injury and inoculation of the microorganism by a specific group of dematiaceous fungi, resulting in the formation of verrucous plaques. The fungi produce sclerotic or medlar bodies (also called muriform bodies or sclerotic cells) seen on direct microscopic examination of skin smears. The disease is often found in adults due to trauma. We report a case of chromoblastomycosis in a 12-year-old child in whom the infection started when he was only 4 years old with secondary involvement of bones, cartilage, tongue and palatine tonsils. The child was not immunosuppressed.

Description of a new tick species, closely related to Amblyomma javanense (Supino, 1897), associated with Varanus bengalensis (Squamata: Varanidae) in Pakistan.

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.

Reflections on the environmental damage compensation regime in Chinese civil legislations.

The chapter on Tort Liability of the new Chinese Civil Code has broadened the types of environmental torts and expanded the scope of environmental damages. After such changes, however, deficiencies still exist. Most significantly, environmental torts are not determined by illegality, which means whether national emission standards have been violated is irrelevant. As long as damages are caused, the principle of liability without fault shall be applied. Conflicts in Chinese environmental law have led to discrepancies and inconsistencies in related judicial decisions. In this regard, this paper argues that the "theory of tolerance limit" should be adopted to redefine the meaning of illegality and further identify the conception of liability without fault for environmental damages. In addition, the punitive damages system created by the Civil Code also lacks clarity in its judgment criteria. Since the purpose of private law is reparation rather than punishment, this paper recommends that the scope of punitive damages should be clarified through compensation for loss to maintain consistency in civil legislation.

The impact of the monetary gold principle on the determination of jurisdiction in the mixed maritime disputes.

Mixed maritime disputes are maritime disputes that involve the concurrent consideration of any dispute concerning sovereignty or other rights over continental or insular land territory, which according to Article 298(1)(a)(i) of United Nations Convention on the Law of the Sea, should be excluded from the jurisdiction of a court or tribunal referred to in Article 287. However, by means of treaty interpretation or consensus of two parties, International Tribunal for the Law of the Sea and other Annex VII tribunals have increasingly broken through the limits to actively exercise jurisdiction. Judgements under the influence of this tendency will be firmly resisted by states whose sovereign interests are damaged. Moreover, these judgements will not only fail to resolve disputes, but will create more serious international law and international political disputes, such as the judgement on preliminary objections of Mauritius v. Maldives delimitation case. In this judgement, the Special Chamber of International Tribunal for the Law of the Sea circumvented the Monetary Gold principle by citing the International Court of Justice advisory opinion and United Nations General Assembly resolution, which had no legally binding force. It is proposed that this judgement could open Pandora's box of challenges to general principles of international law and jurisdiction over sovereignty disputes. For States with mixed maritime disputes to resolve the issues mentioned above, it is necessary to respond to lawsuits actively, avoid adverse consequences caused by default judgements and reach regional consensus, and avoid unfavourable International Court of Justice advisory opinions and United Nations General Assembly resolutions.

Genetic characterization of Haemaphysalis (Rhipistoma) indica and Haemaphysalis (Segalia) montgomeryi ticks (Ixodoidea: Ixodidae).

Haemaphysalis species are medically important hard ticks (Ixodidae) that parasitize nearly all domestic as well as wild hosts, mostly in the Oriental region. Genetic diversity of Haemaphysalis ticks in South Asia has gained little attention, despite its economic, medical, and veterinary importance. The purpose of this study was to genetically characterize Haemaphysalis indica and Haemaphysalis montgomeryi ticks to obtain their preliminary phylogenetic relationship. A total of 1171 ticks were collected from Indian mongooses (Herpestes javanicus and Herpestes edwardsii), sheep and goats in Pakistan. The collected ticks were morphologically identified as H. indica (n = 121, 10.3%) from mongooses and H. montgomeryi (n = 1050, 89.7%) from sheep and goats. Phylogenetic trees inferred from 12S rDNA, 16S rDNA, cox1, and ITS2 sequences revealed an evolutionary relationship of H. indica with the tick species belonging to the subgenus Rhipistoma, and H. montgomeryi with the tick species belonging to the subgenus Segalia, validating the described taxonomy based on the morphological descriptions of these ticks. Further genetic studies are essential to overcome morphology-based systematic issues associated with Haemaphysalinae ticks.

Molecular evidence of Borrelia theileri and closely related Borrelia spp. in hard ticks infesting domestic animals.

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.

First report on detection of Hepatozoon ayorgbor in Rhipicephalus haemaphysaloides and Hepatozoon colubri in Haemaphysalis sulcata and Hyalomma anatolicum: risks of spillover of Hepatozoon spp. from wildlife to domestic animals.

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.

First record of Alectorobius coniceps (Ixodoidea: Argasidae) and Dermacentor sp. (Ixodoidea: Ixodidae) in Pakistan.

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.

Phylogenetic Position of Haemaphysalis kashmirensis and Haemaphysalis cornupunctata, with Notes on Rickettsia spp.

Despite high diversity in the Oriental region, ticks of the genus Haemaphysalis have been neglected regarding their genetic data and vector potential. This study aimed to genetically characterize three species of the genus Haemaphysalis: Haemaphysalis cornupunctata, Haemaphysalis kashmirensis, and Haemaphysalis montgomeryi infesting goats and sheep, and Rickettsia spp. associated with these tick species in the Hindu Kush Himalayan range of Pakistan. Altogether, 834 ticks were collected by examining 120 hosts including goats (64/120, 53.3%) and sheep (56/120, 46.6%), in which 86 (71.6%) hosts were found to be tick-infested. The morphologically identified ticks were subjected to DNA extraction and PCR for the amplification of partial 16S rDNA and cox fragments. Rickettsia spp. associated with the collected ticks were detected through the amplification of gltA, ompA and ompB partial fragments. The 16S rDNA of H. cornupunctata and H. montgomeryi showed a maximum identity of 100% with the sequences of the same species, whereas the 16S rDNA of H. kashmirensis showed the highest identity of 93-95% with Haemaphysalis sulcata. The cox sequence of H. montgomeryi displayed 100% identity with the same species. In comparison, the cox sequences of H. cornupunctata and H. kashmirensis showed maximum identities of 87.65-89.22% with Haemaphysalis punctata and 89.34% with H. sulcata, respectively. The gltA sequence of Rickettsia sp. from H. kashmirensis showed the highest identity of 97.89% with Rickettsia conorii subsp. raoultii, while the ompA and ompB fragments from the same DNA samples revealed 100% and 98.16% identity with Rickettsia sp. and "Candidatus Rickettsia longicornii", respectively. Another gltA sequence amplified from H. montgomeryi ticks showed 100% identity with Rickettsia hoogstraalii, while the attempts to amplify ompA and ompB for R. hoogstraalii were unsuccessful. In the phylogenetic tree, the 16S rDNA of H. cornupunctata clustered with the corresponding species while its cox clustered with H. punctata. Both 16S rDNA and cox sequences of H. kashmirensis clustered with H. sulcata. The gltA sequence of Rickettsia sp. was clustered individually in the spotted fever (SF) group of Rickettsia, while the gltA sequence of R. hoogstraalii was clustered with the same species in the transition group of Rickettsia. In the SF group, the rickettsial ompA and ompB sequence clustered with undetermined Rickettsia sp. and "Candidatus Rickettsia longicornii", respectively. This is the earliest study regarding the genetic characterization of H. kashmirensis. This study indicated that ticks belong to the genus Haemaphysalis have the potential of harboring and/or transmitting Rickettsia spp. in the region.

Ornithodoros (Pavlovskyella) ticks associated with a Rickettsia sp. in Pakistan.

BACKGROUND: Soft ticks (Ixodida: Argasidae) are medically important ectoparasites that mainly feed on birds and mammals, which play a key role in their geographic distribution and dispersion. Despite their importance, studies on soft ticks are scarce for many regions and countries of the world, including Pakistan. METHODS: In this study, 2330 soft ticks-179 larvae (7.7%), 850 nymphs (36.4%), 711 males (30.5%) and 590 females (25.3%)-were collected from animal shelters in 18 locations within five districts of Khyber Pakhtunkhwa, Pakistan. A subset of the collected ticks was processed for DNA extraction and polymerase chain reaction (PCR) for the amplification of tick 12S ribosomal DNA (rDNA), 16S rDNA and cytochrome c oxidase subunit I (cox1), and rickettsial 16S rDNA gene fragments. The obtained sequences were used for the construction of a phylogenetic tree. RESULTS: All the specimens were morphologically identified as Ornithodoros, and were morphologically similar to Ornithodoros tholozani. The genus was confirmed by sequencing partial 12S rDNA, 16S rDNA and cox1 gene fragments. Additionally, a Rickettsia sp. was detected in some of the collected ticks by PCR targeting 16S rDNA. The morphological relatedness of the tick specimens with O. tholozani was confirmed by phylogenetic analysis, in which the Ornithodoros sp. clustered with Ornithodoros tholozani and Ornithodoros verrucosus, both of which belong to the subgenus Pavlovskyella and have been previously reported from Israel, Ukraine and Iran. The phylogenetic tree also indicated that the Ornithodoros sp. from Pakistan corresponds to an undetermined species. Furthermore, the associated Rickettsia sp. grouped with the limoniae group of Rickettsia species previously reported from Argas japonicus ticks from China. CONCLUSIONS: This is the first molecular study of an Ornithodoros species from Pakistan. Further studies are essential to confirm its identity and possible pathogenicity with regard to its associated microorganisms in the studied region.

Development of ß-cyclodextrin/polyvinypyrrolidone-co-poly (2-acrylamide-2-methylpropane sulphonic acid) hybrid nanogels as nano-drug delivery carriers to enhance the solubility of Rosuvastatin: An in vitro and in vivo evaluation.

The present study is aimed at enhancing the solubility of rosuvastatin (RST) by designing betacyclodextrin/polyvinypyrrolidone-co-poly (2-acrylamide-2-methylpropane sulphonic acid) crosslinked hydrophilic nanogels in the presence of crosslinker methylene bisacrylamide through free-radical polymerization method. Various formulations were fabricated by blending different amounts of betacyclodextrin, polyvinylpyrrolidone, 2-acrylamide-2-methylpropane sulphonic acid, and methylene bisacrylamide. The developed chemically crosslinked nanogels were characterized by FTIR, SEM, PXRD, TGA, DSC, sol-gel analysis, zeta size, micromeritics properties, drug loading percentage, swelling, solubility, and release studies. The FTIR spectrum depicts the leading peaks of resultant functional groups of blended constituents while a fluffy and porous structure was observed through SEM images. Remarkable reduction in crystallinity of RST in developed nanogels revealed by PXRD. TGA and DSC demonstrate the good thermal stability of nanogels. The size analysis depicts the particle size of the developed nanogels in the range of 178.5 ±3.14 nm. Drug loading percentage, swelling, solubility, and release studies revealed high drug loading, solubilization, swelling, and drug release patterns at 6.8 pH paralleled to 1.2 pH. In vivo experiments on developed nanogels in comparison to marketed brands were examined and better results regarding pharmacokinetic parameters were observed. The compatibility and non-toxicity of fabricated nanogels to biological systems was supported by a toxicity study that was conducted on rabbits. Efficient fabrication, excellent physicochemical properties, improved dissolution, high solubilization, and nontoxic nanogels might be a capable approach for the oral administration of poorly water-soluble drugs.

New records of Amblyomma gervaisi from Pakistan, with detection of a reptile-associated Borrelia sp.

Ticks are hematophagous ectoparasites of terrestrial and semi-aquatic vertebrates that may transmit microorganisms to their hosts. Spirochetes of the genus Borrelia are common in ticks and an incipient group has been identified in association with reptiles and their tick parasites. To overcome the knowledge deficit, this study aimed to morphologically and molecularly identify ticks infesting wild lizards and to molecularly assess Borrelia spp. associated with these ticks in Pakistan. For this purpose, free-ranging monitor lizards (Varanus bengalensis) from Khyber Pakhtunkhwa province, Pakistan, were examined for tick infestations. A total of 776 ticks were collected from 36/63 lizards, resulting in a prevalence of 57% (95% CI 44.7-69.3%), overall mean intensity of 21.5 (95% CI 18.9-24.1) ticks per infested lizard, and overall mean abundance of 12.3 (95% CI 9.25-15.4) ticks per examined lizard. All ticks were morphologically identified as Amblyomma gervaisi. The morphological identification of the ticks was molecularly confirmed through sequencing fragments of the mitochondrial 16S rRNA gene. In addition, a fragment of nuclear second internal transcribed spacer (ITS2) was generated for the first time for A. gervaisi. Phylogenetic analysis inferred from tick 16S rRNA gene partial sequences predicted a close evolutionary relationship of the collected A. gervaisi ticks with conspecific sequences from India, which shared 94.5% identity. Through two PCR assays targeting fragments of the borrelial genes, 16S rRNA and flaB, 19 (18%) out of 108 ticks yielded borrelial DNA. Phylogenetic analyses inferred from DNA sequences of the two borrelial genes revealed that the Borrelia sp. from A. gervaisi detected in this study belonged to the reptile-associated Borrelia group (REP). This is the first molecular report of ticks infesting monitor lizards and associated Borrelia sp. in Pakistan. The preliminary phylogenetic analyses of A. gervaisi may assist in understanding the molecular epidemiology of Amblyomma spp.

Description of Male, Redescription of Female, Host Record, and Phylogenetic Position of Haemaphysalis danieli.

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.