Anti-Inflammatory, Antipyretic, and Analgesic Potential of Chitin and Chitosan Derived from Cockroaches (Periplaneta americana) and Termites.

The chitin and chitosan biopolymers are extremely valuable because of their numerous industrial and pharmacological uses. Chitin and chitosan were extracted from the exoskeleton of Periplaneta americana (cockroaches) and termites using various acid and alkali techniques. The extraction process involves an initial demineralization step, during which integument dry powder was subjected to 500 mL (2.07 mol/L) of concentrated HCl at 100 degrees Celsius for 30 min, followed by meticulous rinsing with distilled water to restore the pH to its baseline. Deproteinization was conducted at 80 degrees Celsius using 500 mL (1 mol/L) of NaOH solution, which was repeated for 24 h. A total of 250 mL (0.06 mol/L) of NaOH was added at 100 degrees Celsius for 4 h to obtain chitosan, followed by extensive washing and subsequent drying. FTIR analysis was used to identify the functional groups in Periplaneta americana and termites. The crystallinity of these biopolymers, which have a face-centered cubic structure, was determined by X-ray diffraction analysis. This study assessed the analgesic properties of chitin and chitosan via an acetic-acid-induced writhing test in mice, revealing a significant reduction in writhing behavior following the chitin and chitosan extract. Notably, chitin exhibits the highest degree of analgesic activity compared to chitosan. Both chitin and chitosan show anti-inflammatory effects, with chitosan absorbing proton ions at sites of inflammation, while chitin effectively inhibits ear edema and elicits an analgesic response in mice. Furthermore, the present study revealed antipyretic activity, with termite chitin demonstrating the most significant effect at a concentration of 500 µL/mL, followed by chitosan and chitin at 100 µL/mL. These findings indicate the potential of using chitin and chitosan derived from termites and Periplaneta americana as natural anti-inflammatory compounds, implying prospective uses in anti-inflammatory, antipyretic, and analgesic capabilities.

Genotyping of ticks: first molecular report of Hyalomma asiaticum and molecular detection of tick-borne bacteria in ticks and blood from Khyber Pakhtunkhwa, Pakistan.

Multiple ticks (Acari: Ixodoidea) carrying Rickettsiales bacteria have significant importance for both human and animal health. Thus, the purpose of this work was to genetically analyze tick species and their associated Rickettsiales bacteria in animal hosts. In order to achieve these objectives, various animals (including camels, cattle, goats, sheep, dogs, and mice) were inspected in four districts (Mardan, Peshawar, Kohat, and Karak) of Khyber Pakhtunkhwa to collect ticks, while blood samples were collected from all the symptomatic and asymptomatic cattle in all four districts. A total of 234 ticks were obtained from 86 out of 143 (60.14%) host animals, which were morphologically identified as Rhipicephalus turanicus, Rhipicephalus microplus, Haemaphysalis cornupunctata, and Hyalomma asiaticum. Among these, their representative ticks (126/234, 53.85%) were processed for molecular confirmation using cytochrome c oxidase (cox1) gene. Obtained cox1 sequences of four different tick species showed 99.72%-100% maximum identity with their corresponding species reported from Pakistan, China, India, and Kazakhstan and clustered phylogenetically. This study presented the first genetic report of Hy. asiaticum ticks in Pakistan. Moreover, genetically confirmed tick species were molecularly analyzed by PCR for detection of Rickettsiales DNA using partial fragments of 16S rDNA, 190-kDa outer membrane protein A (ompA), and 120-kDa outer membrane protein B (ompB) genes. In addition, blood samples were analyzed to identify Rickettsiales bacteria using the aforementioned genes. Rickettsiales bacteria were found in 24/126 (19.05%) ticks and 4/16 (25.00%) in symptomatic cattle's blood. The obtained ompA and ompB sequences from Hy. asiaticum ticks showed 99.73%-99.87% with Candidatus Rickettsia shennongii and unidentified Rickettsia sp., whereas the obtained 16S rDNA sequences from cattle's blood and ticks (Hae. cornupunctata) showed 99.67% highest identity with Anaplasma phagocytophilum. The 16S rDNA sequence of Rickettsiales DNA from Rh. turanicus ticks showed 100% identity with Ehrlichia canis and unidentified Ehrlichia sp. Obtained sequences of Rickettsiales bacteria were grouped along with their respective species in phylogenetic trees, which were previously reported in Greece, Cuba, Iraq, Turkey, Pakistan, South Korea, and China (mainland and Taiwan). This extensive study explores the wide range of damaging ticks and their corresponding tick-borne bacteria in the area, suggesting a possible danger to both livestock and human communities.

Diversity, prevalence and risk factors associated to gastrointestinal tract parasites in wild and domestic animals from Pakistan.

The gastrointestinal tract (GIT) of animals contains various helminth parasites, responsible for subclinical and clinical parasitism. The health situation of animals is seriously affected by these parasites and leads massive financial losses for the livestock trade. The present study was conducted to determine the prevalence and associated risk factors of GIT parasites in small and large ruminants of Mardan district and zoo animals of Golden Life Safari Park Khyber Pakhtunkhwa. A total of 419 fecal samples were collected including 117 from goats, 110 from cattle, 80 from sheep, 66 from buffaloes, 40 from birds, four from deer, and two from leopards. The fecal samples were subjected to sedimentation and floatation to identify parasite eggs. Of a total of 419 fecal samples, 215 (51.31%) were found positive for GIT parasites eggs. The prevalence of GIT parasites was high in sheep (65%), followed by goats (62.39%), cattle (42.70%), and buffaloes (37.87%). In birds, GIT parasite eggs were identified in 11 (45.83%) and 7 (43.75%) fecal samples of Pheasant and Peacock, respectively. However, an absence of infection was noted in fecal samples from deer and leopards. Eggs of GIT Parasites belonging to one superfamily and four different genera were recognized in fecal samples of domestic animals and buffaloes, which were Trichostrongyloidea (19.81%), Strongyloides (9.31%), Trichuris (7.77%), Monezia (6.44%), and Fasciola (4.53%). For bird, the coccidia group comprises the highest representation of parasites (2.63%), followed by the Trichuris genus (1.67%). Settlement, management system, anthelminthic medications and body condition score were significantly associated with the occurrence of GIT parasites in feces (P < 0.05). Animals from rural settlements, rearing under poor management systems, with thin (poor) body condition and under warmed were more affected by GIT parasites. The diversity and the high prevalence of GIT parasites reported in this study highlights the urgent need for intervention through treatment and modification of management practices in the investigated areas of Pakistan.

Epidemiology and molecular characterization of Theileria annulata in cattle from central Khyber Pakhtunkhwa, Pakistan.

Theileria annulata is a tick-borne hemoprotozoan parasite responsible for tropical theileriosis in the bovine population, which causes substantial economic losses to the livestock sector. The present study has investigated, characterized, and shaped epidemiologic and phylogenetic profiles of T. annulata infection in the cattle population of central Khyber Pakhtunkhwa, Pakistan. A total of 600 blood samples were collected from cattle. Microscopy and PCR (18S rRNA taxonomic marker) assays were performed to detect T. annulata infection in cattle from the study area. The overall relative prevalence rates of T. annulata in the examined cattle population were 12.8% (microscopy) and 23.7% (PCR). District-wise analysis (microscopy/PCR) showed that cattle from district Mardan were found more infected (16.0%/28.0%), as compared to cattle from district Charsadda (13.5%/25.5%) and district Peshawar (9.0%/17.5%). Based on host demographic and ecological parameters analysis, theileriosis was found to be higher in young, female, crossbred, freely grazing, tick-infested, and irregular/no acaricides treated cattle. The univariate logistic analysis showed that host age, tick infestation, acaricides use, and feeding method were significant risk factors (P<0.05) whereas multivariate analysis indicated that host age, gender, tick infestation, acaricidal application, and feeding method were potential risk factors (P<0.05) for tropical theileriosis in the cattle population. Phylogenetic and sequence analysis showed that T. annulata 18S rRNA isolates shared homology and phylogeny with other isolates from Asia and Europe. This study has addressed the epidemiology and phylogeny of T. annulata circulating in bovid in the study area where gaps were still present. These findings will serve as a baseline and will facilitate future large-scale epidemiological investigations on tropical theileriosis in the cattle population at a national level.

Govaniadine Ameliorates Oxidative Stress, Inflammation, and Kupffer Cell Activation in Carbon Tetrachloride-Induced Hepatotoxicity in Rats.

Liver diseases such as hepatic carcinoma are one of the main health problems worldwide. Herbal drugs are largely used to treat liver injury in the indigenous system of medicine and may provide lead compounds for hepatoprotective drug discovery. The present study is investigated to test the Corydalis govaniana Wall. extract, fraction, and isolate therapeutically active constituents to explore their hepatoprotective, anti-inflammatory, and antioxidant activities. For this purpose, the antioxidant activity of govaniadine, caseadine, caseamine, and protopine was performed by assessing the scavenging events of the stable 2,2-diphenyl-1-picrylhydrazyl. Hepatoprotection of govaniadine was assessed in terms of reduction in serum enzymes (alanine aminotransferase, aspartate transaminase, and alkaline phosphatase) caused by CCl4-induced liver injury in rats and by histopathological techniques. All the compounds showed significant antioxidant activity with a percentage inhibition of 92.2, 86.7, 85.3, and 79.7, respectively, compared to propyl gallate 90.3%. Treatment with govaniadine reduced the serum enzyme level down to normal levels in the CCl4-treated group while inhibiting the increase of malondialdehyde, and the induction of superoxide dismutase and the glutathione level was upregulated. Histopathology showed ∼47% damage to the liver cells in the CCl4-treated group; reduction in this damaged area was found to be better upon using govaniadine. Immunohistochemistry results showed that govaniadine as compared to silymarin has exceedingly decreased the inflammation by halting the CCl4-induced activation of hepatic macrophages. In carrageenan-induced paw edema assay, govaniadine significantly alleviated the edema after 1-5 h at a dose of 20 mg/kg (26.00 and 28.5%), 50 mg/kg (22.05 and 27.0%), and 100 mg/kg (20.02 and 25.30%), respectively. The results of our experiments suggest that govaniadine showed antioxidant and hepatoprotective activity in liver injury. The hepatoprotective function of govaniadine may be associated to the scavenging of the free radical and attenuation of oxidative stress as well as inflammatory responses in the liver. Hence, govaniadine may be a lead compound for the hepatoprotective drug discovery process and further research is needed to find out their molecular mechanism of protection.

Molecular epidemiology and associated risk factors of Anaplasma marginale and Theileria annulata in cattle from North-western Pakistan.

Ticks and tick-borne pathogens are one of the major threats to livestock production worldwide. The aim of present study is to specify the molecular epidemiology and its associated risk factors of two well-distributed tick-borne pathogens Anaplasma marginale and Theileria annulata in cattle population from North-western Pakistan. Blood samples were Collected from 60 (32.6 %) local breed cattle, 101 (54.9 %) from crossbreed, and 24 (13.0 %) from exotic breed with total of 184 blood samples. Species-specific PCR assays were performed to detect the presence of A. marginale and T. annulata based on 16S rRNA and 18S rRNA genetic makers respectively. PCR results showed that A. marginale was 16.3 % prevalent and T. annulata was 29.9 % prevalent in the study area with a total prevalence rate of 46.2 % (85/184) of the tested blood samples. District wise analysis revealed that both pathogens were highly prevalent in district Dir Upper (13.6 %) and least prevalent in district Chitral (10.3 %). Univariable analysis of risk factors showed that only breed and acaricidal treatment were significant determinants (P < 0.05) for A. marginale infection, however, in case of T. annulata infection; breed, age, gender, grazing practice, and acaricidal treatment were potential determinants (P < 0.05). Multivariable analysis specified that breed and acaricidal treatment were considered as significant risk factors for A. marginale infection (P < 0.05) whereas acaricidal treatment was found to be a significant determinant for T. annulata infection (P < 0.05). Phylogenetic analysis indicated that A. marginale 16S rRNA and T. annulata 18S rRNA isolates showed similarities and shared phylogeny with same isolates reported from Asia. This is the first molecular report on the epidemiology and risk factors analysis of A. marginale and T. annulata infections in cattle population from NW Pakistan. Further large scale study is required to investigate molecular, epidemiological and genotypic aspects as well as potential risk factors analysis from the country to facilitate designing strategies to control tick-borne pathogen and reduce losses to cattle industry.

Genetic diversity, piroplasms and trypanosomes in Rhipicephalus microplus and Hyalomma anatolicum collected from cattle in northern Pakistan.

The two most important tick species in Pakistan are Rhipicephalus microplus and Hyalomma anatolicum. When associated with cattle, these have one or three host life cycles, respectively, with potential implications for their population genetics and for their vector role in the transmission of pathogens. To compare the two tick species in this context with molecular-phylogenetic methods, during the present study 123 ticks were collected from cattle in northern Pakistan. Two mitochondrial markers of 36 ticks were molecularly analyzed. All 11 R. microplus specimens had identical cox1 haplotypes, whereas the 25 H. anatolicum specimens had nine cox1 haplotypes. The latter belonged to two distinct phylogenetic lineages with high support. However, in the 16S rRNA gene these differences were less evident. Among the 113 ticks molecularly analyzed for tick-borne protozoa, the sequence of Babesia occultans was successfully amplified from two specimens of H. anatolicum. Theileria annulata was present in both R. microplus (10.4%) and H. anatolicum (27.3%), with significantly higher prevalence rate in the latter species. Only one tick, a H. anatolicum female, was positive in the PCR detecting Trypanosoma spp. Sequencing revealed the presence of a new genotype, with the closest phylogenetic relationship to stercorarian trypanosomes (in particular, to a tick-associated Trypanosoma sp. from Japan). In conclusion, the above differences between R. microplus and H. anatolicum may be partly related to their life cycles involving one host or three hosts, respectively. Among the others, host switching (reducing chances of inbreeding) and shorter periods spent on-host (reducing gene flow between cattle herds) are supposed to be important drivers of cox1 gene diversification in case of H. anatolicum as a three host tick species. These results highlight the importance of studying differences in intraspecific genetic diversity and piroplasm burdens between one host and three host ticks in the local scale. In addition, a Trypanosoma sp. molecularly identified in H. anatolicum is reported here for the first time from South Asia, deserving further evaluation concerning its host and vector species.

Facile and eco-benign synthesis of Au@Fe2O3 nanocomposite: Efficient photocatalytic, antibacterial and antioxidant agent.

The development of eco-benign experimental procedures for the synthesis of nanomaterials is a fundamental developing branch of green nanotechnology. In this paper, green synthetic route was followed to synthesize novel Au@Fe2O3nanocomposite using Citrus sinensis fruit extract as a reducing and stabilizing agent. The as synthesized Au@Fe2O3nanocomposite was successfully characterized by UV-visible spectroscopy, X-ray diffraction (XRD), Scanning electron microscope (SEM), Energy-dispersive X-ray (EDX), Fourier transform infrared (FT1R) spectrophotometry and Zeta potential. UV-vis spectroscopy showed two SPR peaks for Fe2O3 and coated Au at 290 and 520 nm respectively. XRD confirmed the crystallinity of Au@Fe2O3. Au@Fe2O3 nanocomposite showed better antioxidant activity to effectively scavenge DPPH. The Au@Fe2O3 has been also tested for antibacterial activity which showed an effective antibacterial activity against multi drug resistant E.coli and Bacillus subtilis. Furthermore, Au@Fe2O3 also demonstrated better photo catalytic activity for methylene blue (MB) degradation. We proposed that the existence of organic acids (citric acids) also played a significant role in the stabilization of Au@Fe2O3, and plant (Citrus sinensis Var Kozan yerly) containing such component may be more effective for the green synthesis of Au@Fe2O3 nanocomposite. The findings of this study prove the overwhelming therapeutic and photocatalytic potential of bio-inspired Au@Fe2O3nanocomposite which can be a novel candidate for the effective remediation of microbes and toxic organic pollutants.

Evaluation of prevalence and risk factors associated with Cryptosporidium infection in rural population of district Buner, Pakistan.

BACKGROUND: Cryptosporidium spp are important intestinal protozoan parasites that cause diarrhea in humans, domestic and wild animals. Its infection remains a main public health concern however, the epidemics in human being is still unclear, particularly in developing countries. There are several factors that may enhance the spreading of this parasite in human population especially in young children. METHODOLOGY: A questionnaire was designed to obtain the demographic and clinical data from the participants. A total of 425 stool samples were collected from suspected children (aged 3-10 years) in different hospitals and villages. The initial screening was performed with modified Ziehl Neelsen (mZN) staining technique followed by polymerase chain reaction (PCR). Several potential risk factors were also assessed through the obtained information from suspected individuals. RESULTS: Out of all 425 collected samples, 127 were observed positive by mZN with a prevalence of 29.88% (127/425). The 127 mZN positive samples together with 50 mZN negative samples were processed for molecular analysis through PCR assay. Among them, 71 out of 127 mZN positive samples and 4 out of 50 mZN negative samples were found positive by PCR. The molecular analysis showed that Cryptosporidium parvum was the main cause of infection in children. The results revealed that individuals exposed to diarrhea were more likely to be infected with Cryptosporidium infection while several environmental factors may also play a key role in spreading of this parasite. CONCLUSIONS/SIGNIFICANCE OF THE STUDY: The current high prevalence of Cryptosporidium infection may be due to the lack of awareness and routine based testing in identification of this parasite in District Buner. Further studies are required to determine the importance of Cryptosporidium infection in this area as well as across the country and to find out the possible risk factors that may be associated with the occurrence of this protozoan. There is, however, an urgent need for laboratory-based observational studies to develop a more dynamic estimate of the cryptosporidial disease burden in the region.

Molecular epidemiology of hcv among health care workers of khyber pakhtunkhwa.

BACKGROUND: Studies of the molecular epidemiology and risk factors for hepatitis C virus (HCV) in health care workers (HCWs) of Peshawar, Khyber Pakhtunkhwa region are scarce. Lack of awareness about the transmission of HCV and regular blood screening is contributing a great deal towards the spread of hepatitis C. This study is an attempt to investigate the prevalence of HCV and its possible association with both occupational and non-occupational risk factors among the HCWs of Peshawar. RESULTS: Blood samples of 824 HCWs, aged between 20-59 years were analysed for anti-HCV antibodies, HCV RNA and HCV genotypes by Immunochromatographic tests and PCR. All relevant information was obtained from the HCWs with the help of a questionnaire. The study revealed that 4.13% of the HCWs were positive for HCV antibodies, while HCV RNA was detected in 2.79% of the individuals. The most predominant HCV genotype was 3a and 2a. CONCLUSION: A program for education about occupational risk factors and regular blood screening must be implemented in all healthcare setups of Khyber Pakhtunkhwa province in order to help reduce the burden of HCV infection.