Diversity of aquatic parasites in pristine spring waters in Tehsil Babuzai, Swat, Pakistan.

Global access to clean and safe drinking water remains a formidable challenge, contributing to a myriad of health issues. This research exposes the existence of waterborne parasites in seemingly pristine spring waters, indicating potential contamination. Daily extensive sampling of Seventeen water sources was conducted in the untarnished freshwater streams of Tehsil Babuzai, District Swat, Khyber Pakhtunkhwa, Pakistan, from February to September 2021. Employing a stringent filtration process, the collected samples were effectively concentrated to detect any waterborne parasites. Subsequent application of the wet mount technique, combined with the capabilities of a compound microscope, revealed a disconcerting reality: all examined samples tested positive for various parasites. Identified parasites included Schistosoma species, Ascaris lumbricoides, Trichiuria trichiuria, Taenia saginata, Entamoeba histolytica, Amoeba, Lacrymana olor, Tintinnids, Paramecium, Dileptus, Euglena, Loxodes striatus, Acanthocyclops lynceus, Spondylosium, Oscillatoria, Cyanobacteria, Cilindros, Cilindros cerro, Commensal amoeba mature cysts,, Filliform larva of Strongyloides, Cercaria larva, Larva of Taenia solium, Egg of Enterobius vermiculais, Egg of Isospora belli, Egg of Tapeworm, Egg of Schistosoma species, Egg of Toxocara, and Egg of Diphyllobothrium latum. These findings clearly demonstrate the presence of a diverse array of parasites in the freshwater springs of Tehsil Babuzai, Swat, Pakistan. Implementing robust water treatment protocols, conducting regular monitoring and testing, and raising awareness about the risks of waterborne parasites are crucial steps to safeguard public health in the region.

Spider diversity in different habitats at District Mardan, Khyber Pakhtunkhwa Pakistan.

Spider species are important for maintaining ecological balance, controlling insect populations, and providing potential sources of medicinal compounds. Highlighting the importance of spider species, the present study was aimed at exploring the diversity of spider fauna and species richness in various habitats in District Mardan, Khyber Pakhtunkhwa, Pakistan, from March 2021 to December 2021. Spiders were collected through different methods: pitfall traps, cryptic searching, ground hand collection, aerial hand collection, and vegetation beating. Then, the captured spiders were washed in alcohol and preserved in a combination of 80% alcohol and 20% glycerol for further observation. Identification was carried out with the help of available spider identification keys and literature. A total of 578 specimens representing 29 species from 17 families and 26 genera were collected and identified. Family Lycosidae was the most dominant family (13.49%), followed by Salticidae (10.2%), Araneidae (8.47%), Tetragnathidae (7.61%), Pholicidae (6.4%), Oxyopidae (5.88%), Thomisidae (5.7%), Clubionidae (5.53%), Sicariidae (5.19%), Scytodidae (5.01%), Corinnidae (4.67%), Agelenidae (4.32%), Cheiracanthiidae and Sparassidae (4.15%), Oecobiidae and Hersiliidae (3.97%) and Theridiidae was the rarest among all (1.21%). The most commonly identified species are Hippasa partita, Lycosa poonaesis, Lycosa terrestris, Draposa oakleyi, Plexippus paykulli, Menemerus nigli, Thyene imperialis, Thyene bivittate and Hasarius adansoni. Argiope versicolor, Neoscona theisi, Tetragnatha extensa, Crossopriza lyoni, and Oxyopes Javanus. During the present study, it was observed that spider species were abundantly found in warm areas from May to August, which demonstrates that the spider fauna of the study area is very rich but has yet to be explored.

Expansion of distribution ranges and current population status of Cheer pheasant (Catreus wallichii) in Northern Pakistan (AJ&K).

We studied the distribution and population status of the Cheer pheasant (Catreus wallichii) in Azad Jammu and Kashmir (AJ&K). Study was mainly conducted in four localities Jhelum Valley (JV), Machiara National Park (MNP), Haveli and Nar Sher Ali Khan (NSAK) from 2016-2020, for five breeding seasons. Population status of Cheer pheasant was determined by the call counts method. In total 105 breeding pairs (210 birds) were estimated highest (134) being recorded from Jhelum valley followed by MNP, Haveli and Nar Sher Ali Khan While applying correction factor 158 birds were recorded. Extensive surveys were carried out in an area of 19.23 km2. Our results indicate an expansion in distribution ranges of Cheer pheasant and population has established in several new sites. Highest Mean population density of 6.11pair/km2 was recorded in Qazi Nag area of Jhelum Valley. The mean population density of Cheer pheasant was slightly higher in the month of May. Furthermore, 58% of the calling birds were detected at elevations ranging from 2300-2600m, while 17% were found at elevations between 1900-2200m. Expansion in distribution ranges of Cheer might be due to reason that bird might have been underestimated in the past or conservation measures and community awareness programs taken by the Department of Wildlife and Fisheries, AJ&K.

Astragalus grahamianus extract: a novel source of bioactive compounds with antioxidant and neuroprotective activities.

The Astragalus grahamianus (AG) Royle ex. Benth is traditionally used for the treatment of various human disorders. The current research work is aimed to explore the neuroprotective anti-Parkinson effects of various fractions of Astragalus grahamianus (A. grahamianus). Fine powder of Astragalus grahamianus was extracted with 70% methanol and then fractionated with various solvents on the basis of polarity. Standard protocols were used to investigate the bioactive constituents present in the various plant fractions. In-vitro antioxidant potential of various fractions was checked using diverse free radicals. In-vivo rats model was used to determined the neuroprotective effects of methanol fraction of A. grahamianus. The results revealed that various fractions of A. grahamianus contain flavonoids, cardiac glycosides, steroids, gums, terpenes, proteins, and carbohydrates except chloroform fraction lake the presence of steroids, cardiac glycosides, gums and saponins, aqueous fraction of steroids, terpenoids, gums and saponins, n-Hexane fraction steroids, carbohydrates, alkaloids, gums and flavonoids. The highest amount of total phenolic contents was found in AGME (32.67 ± 2.3 mg GAE / g). The AGME also showed enhanced free radicals cations potential against DPPH, ABTS and H2O2, respectively. The correlation between AOA (antioxidant activity) and TPC (total phenolic contents) revealed to be substantial. Relative R2 values for ABTS, H2O2, and DPPH activity are 0.9974, 0.9845, and 0.9678, respectively. The in-vivo neuroprotective activities showed significant results. Our findings highlight significant antioxidant, and neuroprotective possessions of AGME attributed to powerful bioactive compounds.

Silicon nanoparticles vs trace elements toxicity: Modus operandi and its omics bases.

Phytotoxicity of trace elements (commonly misunderstood as 'heavy metals') includes impairment of functional groups of enzymes, photo-assembly, redox homeostasis, and nutrient status in higher plants. Silicon nanoparticles (SiNPs) can ameliorate trace element toxicity. We discuss SiNPs response against several essential (such as Cu, Ni, Mn, Mo, and Zn) and non-essential (including Cd, Pb, Hg, Al, Cr, Sb, Se, and As) trace elements. SiNPs hinder root uptake and transport of trace elements as the first line of defence. SiNPs charge plant antioxidant defence against trace elements-induced oxidative stress. The enrolment of SiNPs in gene expressions was also noticed on many occasions. These genes are associated with several anatomical and physiological phenomena, such as cell wall composition, photosynthesis, and metal uptake and transport. On this note, we dedicate the later sections of this review to support an enhanced understanding of SiNPs influence on the metabolomic, proteomic, and genomic profile of plants under trace elements toxicity.

Assessment of Babesia spp. prevalence in various domestic animals across Southern Punjab, Pakistan.

Parasitic diseases, notably babesiosis, exert a substantial impact on the global cattle industry, posing challenges to commerce, economies, and human health. This study, conducted in Southern Punjab, Pakistan, aimed to assess the prevalence of Babesia spp. across various livestock species using microscopic and PCR methods. A total of 180 blood samples (60 from each district) were systematically collected from apparently healthy animals, with 36 samples obtained from each domestic animal species, including camel, cattle, buffalo, goat, and sheep, noting that 12 samples were collected from each district for each animal species. Overall prevalence was determined to be 32.8% (59/180), with varying rates among species: 25.0% in cattle, 41.66% in buffalo, 30.55% in goats, 33.3% in sheep, and 33.3% in camels. Microscopic examination revealed slightly varied infection rates among large and small domestic animals (22.2%), while PCR results indicated a 32.8% overall infection rate in both large and small domestic animals, with no statistical significance. District-wise analysis showed regional variations, with Muzaffargarh recording a prevalence rate of 23.33% through microscopic examination, while Lodhran and Bahawalpur recorded 21.67%. PCR results revealed higher rates (38.33%, 26.67%, and 33.33%, respectively), underlining the importance of employing PCR for accurate detection. Examining ruminant types, large ruminants exhibited a 32.4% infection rate, while small domestic animals showed 33.3%, with no significant difference (p=0.897). District-wise prevalence showcased significant variation, with Muzaffargarh demonstrating a 25% prevalence, Lodhran 22%, and Bahawalpur 22%, through microscopic examination. PCR results displayed 38.33%, 27%, and 33.3%, respectively, with no statistical significance. Detailed analysis of individual districts highlighted variations in infection rates among camels, cattle, buffalo, goats, and sheep. The binomial test indicated significant differences through microscopic analysis (P=0.011) but non-significant variations through PCR (P=0.065), emphasizing the precision of PCR. Regional variations in prevalence, notably with Punjab exhibiting the highest frequency (33.87%) and KPK the lowest (13.24%), suggest potential influences from varying veterinary practices and environmental factors. This study underscores the pivotal role of PCR alongside microscopy for accurate babesiosis diagnosis. These findings contribute to the broader understanding of babesiosis prevalence, emphasizing the necessity of advanced molecular techniques for informed control measures.

Risk factors for surgical failure after primary scleral buckling for rhegmatogenous retinal detachment.

OBJECTIVE: To identify risk factors for surgical failure after scleral buckling (SB) for primary rhegmatogenous retinal detachment (RRD) repair. DESIGN: Single-centre retrospective consecutive case series. PARTICIPANTS: All patients who underwent SB for repair of primary RRD at Wills Eye Hospital between January 1, 2015, and December 31, 2018, were included. METHODS: Single-surgery anatomic success (SSAS) rate and risk factors associated with surgical failure were evaluated. A multivariable logistic regression model was completed to assess the effect of demographic, clinical, and operative variables on SSAS rate. RESULTS: A total of 499 eyes of 499 patients were included. Overall SSAS rate was 86% (n = 430 of 499). Using multivariate analysis, surgical failure was more likely in males (adjusted odds ratio [adjusted OR] = 2.98; 95% CI, 1.58-5.62; p = 0.0007) with a macula-off status on preoperative examination (adjusted OR = 2.15; 95% CI, 1.10-4.20; p = 0.03) and preoperative proliferative vitreoretinopathy (adjusted OR = 4.26; 95% CI, 1.10-16.5; p = 0.04). Time interval between initial examination and surgery (p = 0.26), distribution of buckle or band material used (p = 0.88), and distribution of tamponade used (p = 0.74) were not significantly different between eyes with and without surgical failure. CONCLUSION: Male sex, macula-off status, and preoperative proliferative vitreoretinopathy were factors with increased odds of surgical failure after SB for primary RRD repair. Operative characteristics, such as type of band or use of tamponade, were not associated with surgical failure.

Effects of microplastics in freshwater fishes health and the implications for human health / Efeitos dos microplásticos na saúde dos peixes de água doce e as implicações para a saúde humana

The presence of microplastics in aquatic environments has raised concerns about their abundance and potential hazards to aquatic organisms. This review provides insight into the problem that may be of alarm for freshwater fish. Plastic pollution is not confined to marine ecosystems; freshwater also comprises plastic bits, as the most of plastic fragments enter oceans via rivers. Microplastics (MPs) can be consumed by fish and accumulated due to their size and poor biodegradability. Furthermore, it has the potential to enter the food chain and cause health problems. Evidence of MPs s ingestion has been reported in >150 fish species from both freshwater and marine systems. However, microplastic quantification and toxicity in freshwater ecosystems have been underestimated, ignored, and not reported as much as compared to the marine ecosystem. However, their abundance, influence, and toxicity in freshwater biota are not less than in marine ecosystems. The interaction of MPs with freshwater fish, as well as the risk of human consumption, remains a mystery. Nevertheless, our knowledge of the impacts of MPs on freshwater fish is still very limited. This study detailed the status of the toxicity of MPs in freshwater fish. This review will add to our understanding of the ecotoxicology of microplastics on freshwater fish and give subsequent research directions.

A presença de microplásticos em ambientes aquáticos levantou preocupações sobre sua abundância e perigos potenciais para os organismos que vivem nesse meio. Esta revisão fornece informações sobre o problema que pode ser alarmante para os peixes de água doce. A poluição plástica não se limita aos ecossistemas marinhos; a água doce também contém pedaços de plástico, já que a maioria dos fragmentos de plástico entra nos oceanos por meio dos rios. Os microplásticos (MPs) podem ser consumidos pelos peixes e acumulados devido ao seu tamanho e baixa biodegradabilidade. Além disso, tem o potencial de entrar na cadeia alimentar e causar problemas de saúde. Evidências de ingestão de MPs foram relatadas em mais de 150 espécies de peixes de sistemas de água doce e marinhos. No entanto, a quantificação e a toxicidade de microplásticos em ecossistemas de água doce foram subestimadas, ignoradas e não relatadas tanto quanto em comparação com o ecossistema marinho. No entanto, sua abundância, influência e toxicidade na biota de água doce não são menores que nos ecossistemas marinhos. A interação de MPs com peixes de água doce, bem como o risco de consumo humano, permanece um mistério. Todavia, nosso conhecimento sobre os impactos das MPs em peixes de água doce ainda é muito limitado. Este estudo detalhou o status da toxicidade de MPs em peixes de água doce. Esta revisão aumentará nossa compreensão da ecotoxicologia de microplásticos em peixes de água doce e fornecerá direções de pesquisa subsequentes.

Microplastics assessment in Arabian Sea fishes: accumulation, characterization, and method development / Avaliação de microplásticos em peixes do mar arábico: acumulação, caracterização e desenvolvimento de métodos

Around the globe, plastic has been entering the aquatic system and is ingested by organisms. Identification, optimal digestion method, and characterization of the polymers to trace sources are of growing importance. Hence, the present work investigated microplastics accumulation, digestion protocol efficiency, and characterization of polymers with FTIR analysis in the guts of five fishes (Lethrinus nebulosus, Rastrelliger kanagurta, Acanthopagrus arabicus, Otolithes ruber, and Euryglossa orientalis) from the Karachi coastal area, Arabian Sea. A total of 1154 microplastics (MPs) were ingested by 29 out of 33 fish species (87%). The highest average MP/fish was recorded in Otolithes ruber (54) and the lowest in Rastrelliger kanagurta (19.42). Microfibers were the most abundant shape with the highest numbers (35.52%) as compared to the rest of the MPs identified. Transparent microfibers were recorded as the highest in numbers followed by red, black, blue, and green. In this study, KOH with different concentrations and exposure times along with oxidizing agent hydrogen peroxide was tested (Protocols 3 and 4). Results showed these bases were highly efficient in obtaining optimal digestion of the samples. FTIR analysis confirmed that the majority of the polymers found in the fish guts were polyethylene and polypropylene. This study validated for the first time the presence of these polymers of plastic in marine fish from Pakistan.

Em todo o mundo, o plástico tem entrado no sistema aquático e tem sido ingerido por organismos. A identificação, o método de digestão ideal e a caracterização dos polímeros para rastrear fontes são de crescente importância. Portanto, o presente trabalho investigou o acúmulo de microplásticos, a eficiência do protocolo de digestão e a caracterização de polímeros com análise de FTIR nos tratos digestivos de cinco peixes (Lethrinus nebulosus, Rastrelliger kanagurta, Acanthopagrus arabicus, Otolithes ruber e Euryglossa orientalis) do litoral de Karachi, no mar Arábico. Um total de 1154 microplásticos (MP) foram ingeridos por 29 das 33 espécies de peixes (87%) estudadas. A maior média MP/peixe foi registrada no Otolithes ruber (54), e a menor no Rastrelliger kanagurta (19,42). As microfibras foram a forma mais abundante e com os maiores números (35,52%), em comparação ao restante das MPs identificadas. As microfibras transparentes foram detectadas em maior número, seguidas por vermelho, preto, azul e verde. Neste estudo, o KOH foi testado em diferentes concentrações e tempos de exposição, juntamente com o agente oxidante peróxido de hidrogênio (Protocolos 3 e 4). Os resultados mostraram que essas bases foram altamente eficientes para a obtenção de digestão ideal das amostras. A análise de FTIR confirmou que a maioria dos polímeros encontrados nos tratos digestivos dos peixes eram polietileno e polipropileno. Este estudo validou pela primeira vez a presença desses polímeros de plástico em peixes marinhos do Paquistão.

Molecular based identification and phylogenetic relationship by using cytochrome b gene of Pangasius pangasius / Identificação de base molecular e relação filogenética usando o gene citocromo b de Pangasius pangasius

Molecular appraoch for identification of unknown species by using Cytochrome b gene is an effective and reliable as compared with morphological based identification. For DNA barcoding universal molecular genes were used to identify the species. Cytochrome b is a specific gene used for identification purpose. DNA barcoding is a reliable and effective method compared to the different traditional morphological methods of specie identification. So,in the present study which was conducted to identify the species, a total of 50 fish samples were collected from five different sites. DNA was extracted by using the Phenol Chloroform method from muscle tissue. Five sequences were sequenced (one from each site), analyzed, and identified specific species as Pangasius pangasius. Identified sequences were variable in length from 369 bp (Site 1), 364 bp (Site 2), 364 bp (Site 3), 352 bp (Site 4), and 334 bp (Site 5). Identity matches on the NCBI database confirmed the specific specie as P. pangasius. A distancing tree was drawn to show maximum likelihood among the same and different species. Yet, in many cases fishes on diverse development stages are difficult to identify by morphological characters. DNA-based identification methods offer an analytically powerful addition or even an alternative tool for species identification and phylogenetic study. This work intends to provide an updated and extensive overview on the DNA based methods for fish species identification by using Cytochrome b gene as targeted markers for identification purpose.

A abordagem molecular para identificação de espécies desconhecidas usando o gene citocromo b é eficaz e confiável em comparação com a identificação baseada na morfologia. Códigos de barras de DNA de genes moleculares universais foram usados ​​para identificar as espécies. O citocromo b é um gene específico usado para fins de identificação. O código de barras de DNA é um método confiável e eficaz em comparação com os diferentes métodos morfológicos tradicionais de identificação de espécies. Assim, no presente estudo, que foi realizado para identificar as espécies, um total de 50 amostras de peixes foram coletadas em cinco locais diferentes. O DNA foi extraído usando o método Fenol Clorofórmio do tecido muscular. Cinco sequências foram sequenciadas (uma de cada local), analisadas e identificadas espécies específicas, como Pangasius pangasius. As sequências identificadas tinham comprimento variável de 369 bp (Local 1), 364 bp (Local 2), 369 bp (Local 1), 364 bp (Local 3), 352 bp (Local 4) e 334 bp (Local 5). As correspondências de identidade no banco de dados do NCBI confirmaram a espécie específica como P. pangasius. Uma árvore de distanciamento foi desenhada para mostrar a máxima probabilidade entre elas e diferentes espécies. No entanto, em muitos casos, peixes em diversos estágios de desenvolvimento são difíceis de identificar por caracteres morfológicos. Os métodos de identificação baseados em DNA oferecem uma adição analiticamente poderosa ou mesmo uma ferramenta alternativa para identificação de espécies e estudo filogenético. Este trabalho pretende fornecer uma visão geral atualizada e abrangente sobre os métodos baseados em DNA para identificação de espécies de peixes usando o gene citocromo b como marcadores direcionados para fins de identificação.

Epidemiology and clinical features of cutaneous leishmaniasis in Khyber Pakhtunkhwa, Pakistan / Epidemiologia e características clínicas da leishmaniose cutânea em Khyber Pakhtunkhwa, Paquistão

Current cross-sectional study was carried out between September 2019 to January 2020 at the Department of Pathology, Mardan Medical Complex (MMC), Mardan, and District Headquarter Hospital North Waziristan, Khyber Pakhtunkhwa (KP), Pakistan. The objectives of the current study were to determine the prevalence of leishmaniasis and its associated risk factors in selected districts of KP province, Pakistan. Altogether, three hundred and seventy-four (n=374) leishmaniosis patients were included in the current study. Skin specimen from the ulcer border were collected. The slides were stained by Giemsa stain and examined for the presence of amastigote. The prevalence of leishmania infected patients in different region of KP were as follows: North Waziristan region 53.7 (n=201) District Mardan 34.7% (n=130); District Nowshera 6.7% (n=25), District Swabi 1.1% (n=4) and other Districts i.e. Dir, Malakand, Buner and Bajawarr were 3.7% (n=14). The frequency of leishmaniasis were more in male and majority of the infected patients were in the age group of <10 years. Among n=374 patients 95.7% (n= 358) had cutaneous leishmaniasis while 3% (n= 11) had mucocutaneous type of infection and 1.3% (n= 5) patients had both cutaneous and mucocutaneous infection. Upon treatment by Sodium stibogluconate (SSG) 97% (n=362) showed clinical signs of complete or partial recovery of their skin lesions. Conclusively, highest incidence of leishmania infection occurred during short study period and majority of the cases showed positive response to treatment.

O estudo transversal atual foi realizado entre setembro de 2019 e janeiro de 2020 no Departamento de Patologia Mardan Medical Complex (MMC), Mardan, e no District Headquarter Hospital North Waziristan, Khyber Pakhtunkhwa (KP), Paquistão. Os objetivos do presente estudo foram determinar a prevalência da leishmaniose e seus fatores de risco associados em distritos selecionados da província de KP, Paquistão. Ao todo, 374 (n = 374) pacientes com leishmaniose foram incluídos no presente estudo. Amostras de pele da borda da úlcera foram coletadas. As lâminas foram coradas pelo corante Giemsa e examinadas quanto à presença de amastigotas. As prevalências de pacientes infectados por leishmania em diferentes regiões de KP foram as seguintes: região do Waziristão Norte 53,7 (n = 201), distrito Mardan 34,7% (n = 130); distrito Nowshera 6,7% (n = 25), distrito Swabi 1,1% (n = 4) e outros distritos, ou seja, Dir, Malakand, Buner e Bajawarr foram 3,7% (n = 14). A frequência de leishmaniose foi maior no sexo masculino e a maioria dos pacientes infectados estava na faixa etária < 10 anos. Entre n = 374 pacientes, 95,7% (n = 358) tinham leishmaniose cutânea enquanto 3% (n = 11) tinham infecção do tipo mucocutâneo e 1,3% (n = 5) pacientes tinham infecção cutânea e mucocutânea. Após tratamento com estibogluconato de sódio (SSG), 97% (n = 362) apresentaram sinais clínicos de recuperação completa ou parcial de suas lesões cutâneas. Conclusivamente, a maior incidência de infecção por leishmania ocorreu durante o curto período de estudo e a maioria dos casos apresentou resposta positiva ao tratamento.

Human-black bear conflict: crop raiding by Asiatic black bear (Ursus thibetanus) in Azad Jammu and Kashmir, Pakistan / Conflito entre humanos e ursos-negros: invasão de plantações pelo urso-negro-asiático (Ursus thibetanus) em Azad Jammu e Caxemira, Paquistão

Asiatic black bear has long been in conflict with human beings crop raiding is a major cause of this conflict frequently noted in South Asia. Crops raided by black bears affected by temporal, spatial and anthropogenic attributes. Insight in this conflict and its mitigation is vital for the conservation of this threatened species. Present study aimed to evaluate crop raiding by black bears in the mountainous region of Azad Jammu and Kashmir. Field surveys were carried out to observe spatial and temporal crop raiding features between 2015-2020 and data gathered using designed questionnaires randomly tailored in villages nearby the forests. Results revealed that maize was the sole crop raided by black bears. A total of 28-acre area was raided by black bear in the fall season (Aug-November) resulting in a damage of 51 metric tons, whole raiding was carried out at night. Each respondent received crop damage on 0.09 acre with a loss of 0.17 metric ton yield. Crop quantity and area were significantly correlated to each other. District Neelum shared 49% of the total crop loss, while 47% of the maize was raided at the altitudinal range of 2100-2500 m. crop raiding was highly significantly () dependent upon distance to the forest. Linear regression revealed that maize quantity was determined by area, time and the total field area. Farmers faced 3.8 million PKRs loss due to crop damage by black bears. Despite the huge loss, the majority (23%) of the respondents did not respond to the query on mitigation measures indicating a poor adaptation of preventive measures. Preferred strategy to avoid crop damage was making noise (27.8%) when bears attacked their crops. A start of compensation scheme to the farmers is recommended that will have turned their negative attitude into a positive one toward the wildlife and black bear particularly. Study provides a new insight in human-bear conflict, particularly in spatial and temporal context of crop raiding in AJ&K.

O urso-negro-asiático está há muito tempo em conflito com os seres humanos. A invasão de plantações é uma das principais causas desse conflito frequentemente observado no sul da Ásia: cultivos invadidos por ursos-negros afetados por atributos temporais, espaciais e antropogênicos. A compreensão desse conflito, assim como sua mitigação, é vital para a conservação dessa espécie ameaçada. O presente estudo teve como objetivo avaliar a invasão de culturas por ursos-negros na região montanhosa de Azad Jammu e Caxemira. Pesquisas de campo foram realizadas para observar características espaciais e temporais de invasão de culturas, entre 2015 e 2020, e dados coletados usando questionários desenhados e adaptados aleatoriamente em aldeias próximas às florestas. Os resultados revelaram que o milho foi a única cultura atacada por ursos-negros. Uma área total de 28 acres foi invadida pelo urso-preto na temporada de outono (agosto-novembro), resultando em um dano de 51 toneladas ­ todo o ataque foi realizado à noite. Cada respondente recebeu danos na colheita em 0,09 acre, com uma perda de 0,17 tonelada de rendimento. A quantidade e a área da colheita foram significativamente correlacionadas entre si. O distrito de Neelum compartilhou 49% da perda total da colheita, enquanto 47% do milho foram invadidos na faixa altitudinal de 2100-2500 m. A invasão de culturas foi altamente significativa (χ2 = 1174,64;df = 308; p < 0,01), dependente da distância até a floresta. A regressão linear revelou que a quantidade de milho foi determinada por área, tempo e área total do campo. Os agricultores enfrentaram uma perda de 3,8 milhões de PKRs devido a danos nas colheitas causados ​​por ursos-negros. Apesar do grande prejuízo, a maioria (23%) dos respondentes não respondeu ao questionamento sobre as medidas de mitigação, indicando má adaptação das medidas preventivas. A estratégia preferida para evitar danos às plantações foi fazer barulho (27,8%) quando os ursos atacaram suas plantações. Recomenda-se o início de um esquema de compensação para os agricultores, que transformará sua atitude negativa em positiva em relação à vida selvagem e ao urso-negro em particular. O estudo fornece uma nova visão do conflito entre humanos e ursos, particularmente no contexto espacial e temporal de invasões de colheitas em AJ&K.

Inhibitory effect of thymoquinone from Nigella sativa against SARS-CoV-2 main protease. An in-silico study / Efeito inibitório da timoquinona de Nigella sativa contra a principal protease do SARS-CoV-2. Um estudo in silico

Nigella sativa is known for the safety profile, containing a wealth of useful antiviral compounds. The main protease (Mpro, 3CLpro) of severe acute respiratory syndrome 2 (SARS-CoV-2) is being considered as one of the most attractive viral target, processing the polyproteins during viral pathogenesis and replication. In the current investigation we analyzed the potency of active component, thymoquinone (TQ) of Nigella sativa against SARS-CoV-2 Mpro. The structures of TQ and Mpro was retrieved from PubChem (CID10281) and Protein Data Bank (PDB ID 6MO3) respectively. The Mpro and TQ were docked and the complex was subjected to molecular dynamic (MD) simulations for a period 50ns. Protein folding effect was analyzed using radius of gyration (Rg) while stability and flexibility was measured, using root means square deviations (RMSD) and root means square fluctuation (RMSF) respectively. The simulation results shows that TQ is exhibiting good binding activity against SARS-CoV-2 Mpro, interacting many residues, present in the active site (His41, Cys145) and also the Glu166, facilitating the pocket shape. Further, experimental approaches are needed to validate the role of TQ against virus infection. The TQ is interfering with pocket maintaining residues as well as active site of virus Mpro which may be used as a potential inhibitor against SARS-CoV-2 for better management of COVID-19.

Nigella sativa é conhecida pelo perfil de segurança, contendo uma grande variedade de compostos antivirais úteis. A principal protease (Mpro, 3CLpro) da síndrome respiratória aguda grave 2 (SARS-CoV-2) está sendo considerada como um dos alvos virais mais atraentes, processando as poliproteínas durante a patogênese e replicação viral. Na presente investigação analisamos a potência do componente ativo, timoquinona (TQ) de Nigella sativa contra SARS-CoV-2 Mpro. As estruturas de TQ e Mpro foram recuperadas de PubChem (CID10281) e Protein Data Bank (PDB ID 6MO3), respectivamente. O Mpro e o TQ foram acoplados e o complexo foi submetido a simulações de dinâmica molecular (MD) por um período de 50ns. O efeito de dobramento de proteínas foi analisado usando o raio de giração (Rg) enquanto a estabilidade e a flexibilidade foram medidas usando a raiz quadrada média dos desvios (RMSD) e a raiz média quadrada da flutuação (RMSF), respectivamente. Os resultados da simulação mostram que o TQ está exibindo boa atividade de ligação contra o SARS-CoV-2 Mpro, interagindo em muitos resíduos presentes no sítio ativo (His41, Cys145) e também o Glu166, facilitando o formato da bolsa. Além disso, são necessárias abordagens experimentais para validar o papel do TQ contra a infecção pelo vírus. O TQ está interferindo nos resíduos de manutenção do bolso, bem como no sítio ativo do vírus Mpro, que pode ser usado como um potencial inibidor contra o SARS-CoV-2 para um melhor gerenciamento da Covid-19.

Studying biodiversity of spiders species in seven different localities of Charsadda District, Khyber Pakhtunkhwa, Pakistan / Estudando a biodiversidade de espécies de aranhas em sete localidades diferentes do distrito de Charsadda, Khyber Pakhtunkhwa, Paquistão

The current research work aims to provide knowledge about the diversity of spiders' fauna and their occurrence throughout the year from District Charsadda Khyber Pakhtunkhwa, Pakistan. Research data were collected from March-2015 to January-2017 from seven different localities of Charsadda District by using the camera, bottle, plastic bags, paraffin films, field book and 70% of ethylene alcohol and 20% of glycerine were used as chemicals. By using special identification keys, spiders were differentiated into families, genera and species. During the study time, a total of 2734 specimens of spiders were collected belonging from 35 genera, 15 families and 44 species were identified. Salticidae was the dominant family according to genera studied plus spiders samples numbers collected with 10 genera and 616 species specimens count. The high occurrence of spiders was studied during July. The result of the current study also shows a reduction of spider's species in December due to lowering the temperature. The current study shows that Salticidae were the dominant family as capered to other species. The occurrence of spiders species greatly depends on changing the weather condition. The present study also shows great fluctuation in spider's occurrence with changing of hot climate to colder during the study duration. Moreover, the wet season plays a great role in spiders' population increase and growth.

O objetivo do presente trabalho de pesquisa é fornecer conhecimento sobre a diversidade da fauna de aranhas e sua ocorrência ao longo do ano no distrito Charsadda Khyber Pakhtunkhwa, Paquistão. Os dados da pesquisa foram coletados no período de março de 2015 a janeiro de 2017 em sete diferentes localidades do distrito de Charsadda por meio de câmera, garrafa, sacos plásticos, filmes de parafina, livro de campo, e 70% de álcool etílico e 20% de glicerina foram utilizados como produtos químicos. Usando chaves de identificação especiais, as aranhas foram diferenciadas em família, gênero e espécie. Durante o período de estudo foram coletados 2.734 espécimes de aranhas pertencentes a 35 gêneros, 15 famílias e 44 espécies. Salticidae foi a família dominante de acordo com os gêneros estudados mais o número de amostras de aranhas coletadas com 10 gêneros e contagem de 616 espécies. Altas ocorrências de aranhas foram estudadas durante o mês de julho. O resultado do estudo atual também mostra redução das espécies de aranhas no mês de dezembro devido à diminuição da temperatura. O estudo atual mostra que os Salticidae foram a família dominante como alcaparras para outras espécies. A ocorrência de espécies de aranhas depende muito da mudança das condições climáticas. O presente estudo também mostra grande flutuação na ocorrência de aranhas com a mudança do clima quente para mais frio durante a duração do estudo. Além disso, a estação chuvosa desempenha um grande papel no aumento e crescimento da população de aranhas.

Population and risk assessment of sympatric pheasant species in Palas Valley, Pakistan / População e avaliação de risco de espécies simpátricas de Faisão no Vale Palas, Paquistão

Pheasants are declining everywhere in the world and therefore updated information about their population and habitats are important for conservation and management. The present study was conducted in the Palas Valley, District Kohistan, Pakistan in late spring (May and June) 2020 and early spring (March and April) 2021 to assess the population and anthropogenic stress. The major focus was on three sympatric pheasant species, including Western Horned Tragopan (Tragopan melanocephalus), Himalayan Monal (Lophophorus impejanus), and Koklass Pheasant (Pucrasia macrolopha). We used the "Call Count Method" for the population assessment in the field, and a questionnaire survey was conducted to document the risk assessment of local residents of the valley. The population assessments revealed that the Koklass Pheasant is more adapted to increasing anthropogenic activities and its population appeared more or less similar as 22 years ago. In the past 22 years, Western Tragopan and Himalayan Monal have lost about 40-50% of their populations. Human interference in the form of illegal hunting, deforestation, and overgrazing was found to be common in the valley. The study concludes that the Palas Valley habitat is ideal for pheasant species; however, human interference in the form of urbanization, habitat fragmentation, illegal hunting, and deforestation is occurring at a rapid pace, causing havoc in the pheasant population.

Os faisões estão diminuindo em todo o mundo, por isso informações atualizadas sobre sua população e seus habitats são importantes para conservação e manejo. O presente estudo foi realizado no vale Palas, distrito de Kohistan, Paquistão, no final da primavera (maio e junho) de 2020 e início da primavera (março e abril) de 2021 para avaliar a população e o estresse antropogênico. O foco principal foram três espécies simpátricas de faisão, incluindo Tragopan de Chifres Ocidental (Tragopan melanocephalus), Monal-do-Himalaia (Lophophorus impejanus) e Faisão Koklass (Pucrasia macrolopha). Foi utilizado o "Método de Contagem de Chamadas" para avaliar a população em campo, e foi realizada uma pesquisa por questionário para documentar a avaliação de risco dos moradores locais do vale. As avaliações populacionais revelaram que o Faisão Koklass está mais adaptado às crescentes atividades antrópicas e que sua população se mantém mais ou menos semelhante à população de 22 anos atrás. Nos últimos 22 anos, Tragopan de Chifres Ocidental e Monal-do-Himalaia perderam cerca de 40 a 50% de suas populações. A interferência humana, sob a forma de caça ilegal, desmatamento e sobrepastoreio, era comum no vale. O estudo conclui que o habitat do vale Palas é ideal para espécies de faisão; no entanto, a interferência humana, por meio de urbanização, fragmentação de habitat, caça ilegal e desmatamento, está ocorrendo em ritmo acelerado, causando estragos na população de faisões.

Prevalence of insect pests on maize crop in District Mansehra, Khyber Pakhtunkhwa, Pakistan / Prevalência de insetos-praga na cultura de milho no distrito de Mansehra, Khyber Pakhtunkhwa, Paquistão

The maize crop is used as food for humans, livestock and poultries forms, it is also used in bread making, corn flakes, corn syrup, corn starch and corn oils. The field study consisted of one experimental trial, about the incidence of that insect pest complex on maize cultivar Azam during the Kharif season 2020 at the Agricultural, Research Station, Baffa, Mansehra. The trial was laid out in the Randomized Complete Block Design (RCBD), and then it was divided into three replications. The result obtained from the trial showed that a number of the pest species were recorded during the experimental period; however, the population was noticed at a low level. The obtained insect species were corn leaf aphid (6.90 ± 5.5) per square inch, corn leafhopper (1.32 ± 0.63), maize stem borer (0.63 ± 0.29), corn flea beetle (0.43 ± 0.28), Thrips (0.38 ± 0.22), Hairy caterpillar (0.21 ± 0.22), Grasshopper (0.17 ± 0.11) and shoot fly (0.11 ± 0.08) throughout the season

A cultura do milho é utilizada na alimentação humana, pecuária e avícola, bem como na panificação, flocos de milho, xarope de milho, amido de milho e óleos de milho. O estudo de campo consistiu em um ensaio experimental sobre a incidência desse complexo de insetos-praga na cultivar de milho Azam durante a temporada de Kharif 2020 na Estação de Pesquisa Agrícola, Baffa, Mansehra. O ensaio foi estabelecido no Randomized Complete Block Design (RCBD) e, em seguida, dividido em três repetições. O resultado obtido no ensaio mostrou que várias espécies de pragas foram registradas durante o período experimental; no entanto, foi observado um baixo nível da população. As espécies de insetos obtidas foram pulgão-da-folha-do-milho (6,90 ± 5,5) - por polegada quadrada -, cigarrinha-do-milho (1,32 ± 0,63), broca-do-caule-do-milho (0,63 ± 0,29), besouro-da-pulga-do-milho (0,43 ± 0,28), tripses (0,38 ± 0,22), lagarta-cabeluda (0,21 ± 0,22), gafanhoto (0,17 ± 0,11) e mosca (0,11 ± 0,08) ao longo da temporada

Phytochemical screening, antimicrobial activity, in vitro and in vivo antioxidant activity of Berberis lycium Royle root bark extract / Triagem fitoquímica, atividade antimicrobiana, atividade antioxidante in vitro e in vivo do extrato de casca de raiz de Berberis lycium Royle

Antioxidants are materials that scavenge or remove free radicals from living systems. The oxidation process ends in the production of free radicals. These free radicals are the chief birthplace of cancerous cells. Antioxidizing agents remove free radical intermediates by terminating oxidation processes by being oxidized themselves. On the other hand, infectious diseases affect the world on a large scale. To fight these diseases several synthetic compounds have been used. Plant based medications play important role in this regard. So, the current research aimed to investigate the antibacterial and antioxidant effect of Berberis lycium Royle root bark (BLR) extract. Berberis lycium Royle was used for phytochemical analysis and also as antimicrobial and antioxidant agents. The antimicrobial activity was evaluated by the agar well diffusion method. Current study revealed that BLR was rich in phytochemicals and toxic against tested pathogenic bacteria. BLR showed the highest activity against S. pyogenes (13.3±0.8 mm). The lowest antibacterial activity was reported against E. coli (0±0 mm). In case of minimum inhibitory concentration, it was observed that BLR with 10 µg/mL concentration showed the highest activity while 2.5 µg/mL of BLR showed the least inhibitory activity. The highest In vitro antioxidant activity was recorded as 65% at 100 µg/mL. In case of in vivo antioxidant activity level of CAT, GSH and SOD were decreased while that of MDA was enhanced in groups treated with CCl4 as compared to the control group. BLR extract treatment reversed all these changes significantly. Current results indicate that BLR is effective against bacterial pathogens and also has antioxidant potential.

Os antioxidantes são materiais que eliminam ou removem os radicais livres dos sistemas vivos. O processo de oxidação termina na produção de radicais livres. Esses radicais livres são o principal local de nascimento das células cancerosas. Os agentes antioxidantes removem os intermediários dos radicais livres ao encerrar os processos de oxidação ao serem eles próprios oxidados. Por outro lado, as doenças infecciosas afetam o mundo em grande escala. Para combater essas doenças, diversos compostos sintéticos têm sido utilizados. Os medicamentos à base de plantas desempenham um papel importante a este respeito. Assim, o objetivo da pesquisa atual é investigar o efeito antibacteriano e antioxidante do extrato da casca da raiz de Berberis lycium Royle (BLR). Berberis lycium Royle foi utilizado para análises fitoquímicas e também como agentes antimicrobianos e antioxidantes. A atividade antimicrobiana foi avaliada pelo método de difusão em ágar em poço. A partir do estudo atual, observou-se que o BLR era rico em fitoquímicos e tóxico contra bactérias patogênicas testadas. BLR apresentou maior atividade contra S. pyogenes (13,3 ± 0,8 mm). A menor atividade antibacteriana foi relatada contra E. coli (0 ± 0 mm). No caso de concentração inibitória mínima, observou-se que BLR com concentração de 10 µg / mL apresentou maior atividade, enquanto BLR 2,5 µg / mL apresentou menor atividade inibitória. A maior atividade antioxidante in vitro foi registrada como 65% a 100 µg / mL. No caso do nível de atividade antioxidante in vivo de CAT, GSH e SOD diminuiu, enquanto o de MDA aumentou nos grupos tratados com CCl4 em comparação com o grupo controle. O tratamento com extrato de BLR reverteu todas essas mudanças significativamente. Os resultados atuais indicam que o BLR é eficaz contra patógenos bacterianos e também tem atividade antioxidante.

GCMS characterization and biological potential of the seeds and aerial part of Galium tricorne Stokes / Caracterização GCMS e potencial biológico das sementes e parte aérea de Galium tricorne Stokes

Natural products have long been proven very effective against various challenging diseases including cancer and bacterial infections. Galium tricorne is one of the important source of natural products, which has not been explored till date in spite of its profound ethnomedicinal prominence. The current study has been designed to explore the biological potential of G. tricorne and to extract and isolate chemical constituents from its aerial part and seeds respectively along with identification of their chemical constituents. Phytochemical screening was performed to figure out the presence of secondary metabolite in G. tricorne. Crude Methanolic extract (Gt.Crd), which was obtained from the aerial part while the fatty acids were extracted from the seeds, which were later on analyzed by GCMS. Similarly, Well Diffusion and MTT method were used for antibacterial activity and cancer cell line assay respectively. To evaluate the cytotoxic potential, brine shrimps were used. Likewise, in Gas Chromatography-Mass Spectroscopy (GC-MS) analysis a total number of 23 compounds were identified in Gt.Crd extract out of which 7 compounds were sorted out to have some sort of toxicity profile. In the same fashion, 5 fatty acids were identified in the seeds of G. tricorne. Moreover, among the fractions, chloroform fraction (Gt.Chf) exhibited greater zone of inhibition (ZOI) 20.37 mm followed by Gt.Crd 18.40 mm against S. aureus and S. pyogenes respectively. In cytotoxicity Gt.Chf was more active followed by ethyl acetate fraction (Gt.Eta) by exhibiting 88.32±0.62% (LC50=60 µg/mL) and 73.95±2.25% (LC50=80 µg/mL) respectively at 1000 µg/mL concentration of the tested sample. Gt.Chf exhibited greater cell line inhibitory activity (IC50=61 µg/mL) against HeLa cell line. Similarly, Gt.Crd displayed IC50 values of 167.84 µg/mL and 175.46 µg/mL against HeLa and NIH/3T3 cell line respectively. Based on the literature review and screenings, it may be concluded that the aerial part and seeds of G. tricorne are the rich sources of bioactive compounds. The results of the current study also authenticate the scientific background for the ethnomedicinal uses of G. tricorne.

Os produtos naturais têm se mostrado muito eficazes contra várias doenças desafiadoras, incluindo câncer e infecções bacterianas. O gálio tricorne é uma importante fonte de produtos naturais, ainda pouco explorada, apesar de sua profunda proeminência etnomedicinal. O presente estudo foi desenvolvido para explorar o potencial biológico de G. tricorne e extrair e isolar constituintes químicos de sua parte aérea e sementes, respectivamente, juntamente com a identificação de seus constituintes químicos. A triagem fitoquímica foi realizada para descobrir a presença de metabólito secundário em G. tricorne, extrato metanólico bruto (Gt.Crd) que foi obtido da parte aérea enquanto os ácidos graxos foram extraídos das sementes, que posteriormente foram analisadas por GCMS. Da mesma forma, os métodos Well Diffusion e MTT foram usados ​​para atividade antibacteriana e ensaio de linha de células cancerígenas, respectivamente. Para avaliar o potencial citotóxico, foram utilizadas artémias. Da mesma forma, na análise de cromatografia gasosa-espectroscopia de massa (GC-MS) um número total de 23 compostos foi identificado no extrato de Gt.Crd, dos quais 7 compostos foram selecionados para ter algum tipo de perfil de toxicidade. Da mesma forma, 5 ácidos graxos foram identificados nas sementes de G. tricorne. Além disso, entre as frações, a fração clorofórmio (Gt.Chf) apresentou maior zona de inibição (ZOI), 20,37 mm, seguida de Gt.Crd 18,40 mm contra S. aureus e S. pyogenes, respectivamente. Na citotoxicidade Gt.Chf foi mais ativo seguido pela fração acetato de etila (Gt.Eta), exibindo 88,32±0,62% (LC50=60 µg/mL) e 73,95±2,25% (LC50=80 µg/mL) respectivamente a 1000 µg/ concentração de mL da amostra testada. Gt.Chf exibiu maior atividade inibitória da linha celular (IC50 = 61 µg/mL) contra a linha celular HeLa. Da mesma forma, Gt.Crd apresentou valores de IC50 de 167,84 µg/mL e 175,46 µg/mL contra linha celular HeLa e NIH/3T3, respectivamente. Com base na revisão de literatura e triagens, pode-se concluir que a parte aérea e as sementes de G. tricorne são as ricas fontes de compostos bioativos. Os resultados do presente estudo também autenticam a base científica para os usos etnomedicinais de G. tricorne.

Transformation of Rhodococcus Pigment Production Hydroxylase (PPH) gene into Camelina sativa: an alternative marker for the detection of transgenic plants / Transformação do gene Rhodococcus Pigment Production Hydroxylase (PPH) em Camelina sativa: um marcador alternativo para a detecção de plantas transgênicas

Production of transgenic plants with desired agronomic and horticultural traits has gained great importance to fulfill demands of the growing population. Genetic transformation is also a fundamental step to study basics of plant sciences. Different transformation protocols have been developed and used which are reliable and efficient. These protocols used antibiotic or herbicide resistance genes incorporated along with gene of interest to identify transformed plants from non-transformed ones. These marker genes may pose a threat to human and environment. Use of visual markers enables direct and easier observation of transformed plants with more precision. In current study a gene cassette with 'pigment production hydroxylase (PPH) gene under fiber specific promoter (GhSCFP) and downstream Nos-terminator was designed. After checking the structural and functional efficiency of codon optimized gene using bioinformatics tools, the cassette was sent for chemical synthesis from commercial source. The pigment gene cassette (PPH_CEMB), cloned in pCAMBIA-1301, was transformed into Agrobacterium through electroporation. Agrobacterium-mediated floral dip method was used to transform Camelina sativa inflorescence. After seed setting a total of 600 seed were observed for change in color and out of these, 19 seeds developed a reddish-brown coloration, while the remaining 581 seeds remained yellow. The transformation efficiency calculated on basis of color change was 1.0%. PCR analysis of leaves obtained after sowing reddish seeds confirmed the transformation of pigment production gene, while no PCR amplification was observed in leaves of plants from wild type seeds. From the results it is evident that Agrobacterium-mediated transformation of C. sativa inflorescence is very efficient and environment friendly technique not only for detection of transformed plants but also to study basic cellular processes.

A produção de plantas transgênicas com características agronômicas e hortícolas desejadas vem ganhando grande importância por atender às demandas da crescente população. A transformação genética também é um passo fundamental para estudar os fundamentos das ciências das plantas. Foram desenvolvidos e utilizados diferentes protocolos de transformação, ​​que são confiáveis ​​e eficientes. Esses protocolos usaram genes de resistência a antibióticos ou herbicidas incorporados ao gene de interesse para identificar plantas transformadas e não transformadas. Esses genes marcadores podem representar uma ameaça ao ser humano e ao meio ambiente. O uso de marcadores visuais permite a observação direta e fácil de plantas transformadas com mais precisão. No estudo atual, foi projetado um cassete de genes com o gene da hidroxilase de produção de pigmentos (PPH) sob promotor específico de fibra (GhSCFP) e terminador NOS a jusante. Após verificar a eficiência estrutural e funcional do gene otimizado por códons usando ferramentas de bioinformática, o cassete foi enviado para síntese química a partir de fonte comercial. O cassete do gene do pigmento (PPH_CEMB), clonado em pCAMBIA-1301, foi transformado em Agrobacterium por eletroporação. O método de imersão floral mediado por Agrobacterium foi usado para transformar a inflorescência de Camelina sativa. Após a formação de sementes, foi observado um total de 600 sementes com mudança de cor, das quais 19 desenvolveram uma coloração marrom-avermelhada, enquanto as 581 restantes permaneceram amarelas. A eficiência de transformação calculada com base na mudança de cor foi de 1%. A análise de PCR das folhas obtidas após a semeadura de sementes avermelhadas confirmou a transformação do gene produtor de pigmentos, enquanto não foi observada amplificação por PCR em folhas de plantas de sementes do tipo selvagem. A partir dos resultados, é evidente que a transformação da inflorescência de C. sativa mediada por Agrobacterium é uma técnica muito eficiente e favorável ao ambiente não só para a detecção de plantas transformadas, mas também para estudar processos celulares básicos.

A perspective of the prevalent H9N2 virus with a special focus on molecular and pathological aspects in commercial broiler chicken in Punjab, Pakistan / Perspectiva do vírus H9N2 prevalente com foco especial em aspectos moleculares e patológicos em frangos de corte comerciais em Punjab, Paquistão

Frequent outbreaks of avian influenza H9N2 virus in Pakistan revealed that this subtype has become endemic in the poultry industry and, besides economic losses, poses a threat to public health. The present study describes the molecular characterization and pathological alterations in naturally infected broiler chickens with the current H9N2 field strain and their phylogenomic dynamics. In this study, tissue samples (trachea, lung, kidney and intestine) from 100 commercial chicken flocks were collected from July 2018 to August 2019. Samples were subjected to molecular detection, phylogeny and subsequent pathological examination. The complete length of the HA gene was successfully amplified in five samples. Nucleotide sequencing revealed positive samples placed in a clade belonging to the B2 sub-lineage of the G1 genotype and categorized as LPAIV based on the amino acid sequence of the HA gene at the cleavage site (PAKSSR/G). Genetic analysis of the haemagglutinin (HA) gene revealed nt: 80.5%-99.5%; aa: 83.8%-98.9% homology to H9N2 strains reported previously from Pakistan, neighbouring countries, and (A/Quail/Hong Kong/G1/97). Gross lesions include a slight airsacculitis, mild hemorrhages, diffuse congestion and purulent exudate in tracheal mucosa, fibrinonecrotic cast in the trachea lumen and mild pulmonary congestion. Histopathological alterations include sloughing of epithelial cells and infiltration of inflammatory cells in the trachea, mononuclear cells (MNCs) infiltration, pulmonary congestion and exudate in the lumen of parabronchi, peritubular congestion in the kidneys with degeneration of tubular epithelial cells and degenerative changes in the intestinal villi epithelial cells and goblet cell hyperplasia. Immunohistochemistry analysis confirmed the presence of AIVH9N2 antigen in the trachea, lungs, kidney and intestine. Electron microscopy revealed ultrastructural changes in the trachea, including degenerated cilia, mitochondrial swelling and enlarged endoplasmic reticulum. Based on all essential analysis, the present study revealed the distribution of the H9N2 virus of G1 genotype in Punjab, Pakistan, with mild to moderate pathogenicity.

Surtos frequentes do vírus da gripe aviária H9N2 no Paquistão revelaram que esse subtipo se tornou endêmico na avicultura e, além das perdas econômicas, representa uma ameaça à saúde pública. O presente estudo descreve a caracterização molecular e as alterações patológicas em frangos de corte naturalmente infectados com a atual cepa H9N2 e sua dinâmica filogenômica. Neste estudo, amostras de tecidos (traqueia, pulmões, rim e intestino) de 100 lotes comerciais de frangos foram coletadas de julho de 2018 a agosto de 2019. As amostras foram submetidas à detecção molecular, filogenia e posterior exame patológico. O comprimento completo do gene HA foi amplificado com sucesso em cinco amostras. O sequenciamento de nucleotídeos revelou amostras positivas colocadas em um clado pertencente à sublinhagem B2 do genótipo G1 e categorizado como LPAIV com base na sequência de aminoácidos do gene da hemaglutinina (HA) no local de clivagem (PAKSSR/G). A análise genética do gene da HA revelou: nt = 80,5%-99,5%; aa = 83,8%-98,9% de homologia com cepas de H9N2 relatadas anteriormente no Paquistão e em países vizinhos (A/Quail/Hong Kong/G1/97). As lesões macroscópicas incluíram aerossaculite leve, hemorragias leves, congestão difusa e exsudato purulento na mucosa traqueal, cilindro fibrinonecrótico no lúmen da traqueia e congestão pulmonar leve. As alterações histopatológicas incluíram descamação de células epiteliais, infiltração de células inflamatórias na traqueia, infiltração de células mononucleares (MNCs), congestão pulmonar e exsudato no lúmen dos parabrônquios, congestão peritubular nos rins com degeneração das células epiteliais tubulares, alterações degenerativas nas células epiteliais das vilosidades intestinais e hiperplasia de células caliciformes. A análise imunoistoquímica confirmou a presença do antígeno AIVH9N2 na traqueia, nos pulmões, no rim e no intestino. A microscopia eletrônica revelou alterações ultraestruturais na traqueia, incluindo cílios degenerados, inchaço mitocondrial e retículo endoplasmático aumentado. Com base em todas as análises, o presente estudo revelou a distribuição do vírus H9N2 do genótipo G1 em Punjab, Paquistão, com patogenicidade de leve a moderada.