A study of the immunomodulatory effects of coconut oil extract in broilers experimentally infected with velogenic Newcastle disease virus.

Objective: This study aims to evaluate the immunomodulatory effects of coconut oil extract (COE) in broilers experimentally infected with velogenic Newcastle disease virus (vNDV). Methods: A total of 150 broiler birds (day-old) were equally divided into five study groups i.e., negative control, positive control, COE-1, COE-2, and COE-3. On day 10, broilers of groups COE-1, COE-2, and COE-3 were supplemented with 1, 2, and 3 ml of COE respectively per liter of drinking water for 15 days. On day 13, 0.1ml/bird (10-5.25 ELD50) of vNDV was inoculated in broilers of positive control, COE-1, COE-2, and COE-3 groups intramuscularly. During this study, growth performance, morbidity, and mortality rates of each study group were recorded. The antibody titer against NDV was determined on days 7, 14, 21, 28, and 35. The levels of IgY and IgM were also determined on the 7th, 14th, and 21st days post-SRBC inoculation. On day 33, avian tuberculin was injected between the 1st and 2nd toes of the left side (intradermally) to measure lymphoproliferative responses. On day 35, the phagocytic activity in the blood was assessed through a carbon clearance assay by injecting carbon black ink into the right-wing vein. The visceral organs having gross lesions were also collected for histopathology. Results: The COE significantly improved the growth performance, and lowered the morbidity and mortality rates of broilers. There was a significant rise in antibody titers against NDV and levels of IgY and IgM antibodies against SRBC in COE-supplemented broilers. The lymphoproliferative response and phagocytic activity were also enhanced. Among COE-supplemented groups, the broilers of the COE-3 group showed a significant increase in growth performance and boosted immune defense. Conclusions: Coconut oil extract has the potential to boost the growth performance and immune status of broilers. It can be used effectively as a feed additive and alternative to antibiotics to prevent the spread of infectious poultry pathogens.

First Molecular-Based Confirmation of Dermacentor marginatus and Associated Rickettsia raoultii and Anaplasma marginale in the Hindu Kush Mountain Range.

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.

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

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

First report on tick-borne pathogens detected in ticks infesting stray dogs near butcher shops.

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.

Molecular detection of Coxiella spp. in ticks (Ixodidae and Argasidae) infesting domestic and wild animals: with notes on the epidemiology of tick-borne Coxiella burnetii in Asia.

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.

Facile one-step synthesis of gold nanoparticles using Viscum album and evaluation of their antibacterial potential.

Nanostructure gold nanoparticles (Au NPs) are well-known biological active materials, synthesised under different environment-friendly approaches that has gained significant interest in the field of biomedicine. This study investigated a novel, fast, easy, cost-effective and the eco-friendly method to synthesise Au NPs from mediated Viscum album Linn plant extract, where the plant metabolites act as stabilising and reducing agents. The synthesised Au NPs were analysed by UV/Vis spectroscopy that gave strong signals and a sharp absorption peak at 545nm due to the presence of surface plasmon resonance (SPR) bands. In addition, energy dispersive X-ray spectroscopy (EDX) showed that strong signals of Au NPs appeared at 9.7 and 2.3keV, as the rays of light passed. X-ray diffraction recognised the crystalline material and provided information on the cell unit that the synthesised Au NPs are face-centreed cubic in structure. The diffraction of X-ray spectra showed intense peaks at 38.44°, 44.7°, 44.9° and 77.8°. The mediated V. album plant extracts and synthesised Au NPs were screened against gram-positive and gram-negative (Enterobacter , Salmonella typhi , Escheria coli and Bacillus subtilis ) bacterial strains, confirming their antibacterial potential. Au NPs showed strong antibacterial activity due to its unique steric configuration. Au NPs damaged bacterial cell membrane leading to the leakage of the cytoplasm and death of the cell.

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.

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.

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.

Bioactive Compounds from Endophytic Bacteria Bacillus subtilis Strain EP1 with Their Antibacterial Activities.

Endophytic bacteria boost host plant defense and growth by producing vital compounds. In the current study, a bacterial strain was isolated from the Boswellia sacra plant and identified as Bacillus subtilis strain EP1 (accession number: MT256301) through 16S RNA gene sequencing. From the identified bacteria, four compounds-1 (4-(4-cinnamoyloxy)phenyl)butanoic acid), 2 (cyclo-(L-Pro-D-Tyr)), 3 (cyclo-(L-Val-L-Phe)), and 4 (cyclo-(L-Pro-L-Val))-were isolated and characterized by 1D and 2D NMR and mass spectroscopy. Moreover, antibacterial activity and beta-lactam-producing gene inhibition (δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine synthetase (ACVS) and aminoadipate aminotransferase (AADAT)) assays were performed. Significant antibacterial activity was observed against the human pathogenic bacterial strains (E. coli) by compound 4 with a 13 ± 0.7 mm zone of inhibition (ZOI), followed by compound 1 having an 11 ± 0.7 mm ZOI. In contrast, the least antibacterial activity among the tested samples was offered by compound 2 with a 10 ± 0.9 mm ZOI compared to the standard (26 ± 1.2 mm). Similarly, the molecular analysis of beta-lactam inhibition determined that compounds 3 and 4 inhibited the two genes (2- to 4-fold) in the beta-lactam biosynthesis (ACVS and AADAT) pathway. From these results, it can be concluded that future research on these compounds could lead to the inhibition of antibiotic-resistant pathogenic bacterial strains.

Comparison of Phytochemical Constituents and Pharmacological Activities of Various Solvent Extracts Obtained from Millettia speciosa Stem Powder.

Millettia speciosa is a plant extensively used as an important component in Chinese herbal medicine and food-based medicines. The present study was carried out to determine the total flavonoid content (TFC), volatile phytoconstituents, and pharmacological activities, i.e., antityrosinase, sunscreen, and anticancer activity, of different fractions of M. speciosa stem. Different organic solvents of increasing polarity, i.e., petroleum ether (PE), ethyl acetate (EtOAc), and methanol (MeOH), were used for extraction. The highest total flavonoid content, i.e., 48.30 ± 0.90%, was reported for PE extract. Various important phytocomponents were revealed by gas chromatography-mass spectroscopy (GC-MS) analysis. Based on abundance, the major compounds were n-hexadecanoic acid (16.654%), n-hexadecanoic acid (14.808%), and beta-sitosterol (6.298%) for PE, EtOAc, and MeOH extract, respectively. The significant antityrosinase activity, i.e., 70.97 ± 0.66%, with an IC50 value of 4.58 mg/mL was noted for PE extract followed by EtOAc extract, i.e., 59.84 ± 0.67%, with IC50 value of 6.10 mg/mL. The maximum sunscreen activity was reported for PE extract exhibiting the maximum absorbance value (0.633 ± 0.06) in the ultraviolet (UV) region, i.e., UVC, while EtOAc extract showed the second highest level of absorbance in the UVB range, i.e., 0.632 ± 0.07. The strongest anticancer activity (49.73 ± 0.49% cell viability) towards MCF-7 breast cancer cell line was reported for PE extract with IC50 197.51 µg/mL. Our results confirmed the presence of potential therapeutic components for each extract with significant biological functions, showing the importance of the M. speciosa stem as a source of biomedicine. To our knowledge, this is the first report on M. speciosa stem extending comprehensive research about its phytochemical profile and various significant pharmacological activities.

Genome-wide transcriptome analysis of the orphan crop tef (Eragrostis tef (Zucc.) Trotter) under long-term low calcium stress.

Calcium (Ca2+) is one of the essential mineral nutrients for plant growth and development. However, the effects of long-term Ca2+ deficiency in orphan crops such as Tef [(Eragrostis tef) (Zucc.) Trotter], which accumulate high levels of Ca in the grains, remained unknown. Tef is a staple crop for nearly 70 million people in East Africa, particularly in Ethiopia and Eritrea. It is one of the most nutrient-dense grains, and is also more resistant to marginal soils and climatic conditions than main cereals like corn, wheat, and rice. In this study, tef plants were grown in a hydroponic solution containing optimum (1 mM) or low (0.01 mM) Ca2+, and plant growth parameters and whole-genome transcriptome were analyzed. Ca+2-deficient plants exhibited leaf necrosis, leaf curling, and growth stunting symptoms. Ca2+ deficiency significantly decreased root and shoot Ca, potassium (K), and copper content in both root and shoots. At the same time, it greatly increased root iron (Fe) content, suggesting the role of Ca2+ in the uptake and/or translocation of these minerals. Transcriptomic analysis using RNA-seq revealed that members of Ca2+ channels, including the cyclic nucleotide-gated channels and glutamate receptor-like channels, Ca2+-transporters, Ca2+-binding proteins and Ca2+-dependent protein kinases were differentially regulated by Ca+2 treatment. Moreover, several Fe/metal transporters, including members of vacuolar Fe transporters, yellow stripe-like, natural resistance-associated macrophage protein, and oligo-peptide transporters, were differentially regulated between shoot and root in response to Ca2+ treatment. Taken together, our findings suggest that Ca2+ deficiency affects plant growth and mineral accumulation by regulating the transcriptomes of several transporters and signaling genes.

First genetic report of Ixodes kashmiricus and associated Rickettsia sp.

BACKGROUND: Hard ticks (Ixodidae) are hematophagous ectoparasites that transmit various pathogens to a variety of hosts including humans. Transhumant herds have been involved in the spread of ticks and associated Rickettsia spp., and studies on this neglected topic have been unexplored in many regions including Pakistan. This study aimed to investigate ticks infesting transhumant herds of sheep (Ovis aries) and goats (Capra hircus) in district Shangla, Khyber Pakhtunkhwa, Pakistan. METHODS: Of the 144 examined animals, 112 hosts (68 sheep and 44 goats) of transhumant herds were infested by 419 ticks of different life stages including nymphs (105; 25%), males (58; 14%) and females (256; 61%). For molecular analyses, DNA was extracted from 64 collected ticks and subjected to PCR for the amplification of tick 16S rDNA and ITS2 partial sequences and for the amplification of rickettsial gltA and ompA gene sequences. RESULTS: All tick specimens were identified as Ixodes kashmiricus based on morphological features. The obtained 16S rDNA and ITS2 sequences showed 95.7% and 95.3% identity, respectively, with Ixodes kazakstani reported from Kyrgyzstan. In the phylogenetic tree, the sequences clustered with members of the Ixodes ricinus species complex, including I. kazakstani and Ixodes apronophorus. Additionally, rickettsial gltA and ompA partial sequences were 99.7% identical to Rickettsia sp. endosymbiont of Ixodes spp. from Panama and Costa Rica and 99.2% with Rickettsia endosymbiont from the USA. Phylogenetically, the rickettsial gltA and ompA partial sequences from I. kashmiricus clustered with various haplotypes of Rickettsia endosymbiont, which were sister cladded to Rickettsia monacensis. CONCLUSIONS: This is the first genetic report of I. kashmiricus and associated Rickettsia sp. Large-scale tick surveillance studies across the country are needed to investigate Ixodes ticks and associated pathogens.

Generalized ß-Hyers-Ulam-Rassias Stability of Impulsive Difference Equations.

This paper describes the existence and uniqueness of the solution, ß-Hyers-Ulam-Rassias stability and generalized ß-Hyers-Ulam-Rassias stability of an impulsive difference system on bounded and unbounded discrete intervals. At the end, an example is given to illustrate the theoretical result.

Molecular Characterization and Assessment of Risk Factors Associated with Theileria annulata Infection.

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.

Spatio-Temporal Patterns of Ticks and Molecular Survey of Anaplasma marginale, with Notes on Their Phylogeny.

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.

Molecular Survey and Genetic Characterization of Anaplasma marginale in Ticks Collected from Livestock Hosts in Pakistan.

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.

Evaluation of Household Preparedness and Risk Factors for Cutaneous Leishmaniasis (CL) Using the Community Assessment for Public Health Emergency Response (CASPER) Method in Pakistan.

(1) Background: In endemic areas of Pakistan, local community knowledge and attitudes towards cutaneous leishmaniasis (CL) are critical elements in the effective control and management of the disease. A cross-sectional epidemiologic design was used to assess the disease concern, preparedness, practices, and preventive behavior of the households and to assist the personnel and health care professionals in strengthening their planning efforts and awareness of CL. (2) Methods: A two-stage cluster sampling process, i.e., Community Assessment for Public Health Emergency Response (CASPER) was conducted from September 2020 to March 2021 on present household-level information about community needs and health status regarding CL in a cost-effective, timely, and representative manner. (3) Results: In the current study, 67% of the respondents were aware of CL and its causative agent and showed a low level of pandemic preparedness. The majority (74%) of the respondents mentioned that they did not avoid sandfly exposure areas. The majority (84%) of respondents had unsatisfactory behavior towards using bed nets, sprays, or repellents. (4) Conclusion: In endemic areas of Pakistan, the inadequate concern and low preparedness of the local community toward CL are critical aspects in efficient control and management of the disease.

Analysis of miRNAs responsive to long-term calcium deficiency in tef (Eragrostis tef (Zucc.) Trotter).

MicroRNAs (miRNAs) play an important role in growth, development, stress resilience, and epigenetic modifications of plants. However, the effect of calcium (Ca2+) deficiency on miRNA expression in the orphan crop tef (Eragrostis tef) remains unknown. In this study, we analyzed expression of miRNAs in roots and shoots of tef in response to Ca2+ treatment. miRNA-seq followed by bioinformatic analysis allowed us to identify a large number of small RNAs (sRNAs) ranging from 17 to 35 nt in length. A total of 1380 miRNAs were identified in tef experiencing long-term Ca2+ deficiency while 1495 miRNAs were detected in control plants. Among the miRNAs identified in this study, 161 miRNAs were similar with those previously characterized in other plant species and 348 miRNAs were novel, while the remaining miRNAs were uncharacterized. Putative target genes and their functions were predicted for all the known and novel miRNAs that we identified. Based on gene ontology (GO) analysis, the predicted target genes are known to have various biological and molecular functions including calcium uptake and transport. Pairwise comparison of differentially expressed miRNAs revealed that some miRNAs were specifically enriched in roots or shoots of low Ca2+-treated plants. Further characterization of the miRNAs and their targets identified in this study may help in understanding Ca2+ deficiency responses in tef and related orphan crops.

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.