Comparative Molecular Virulence Typing and Antibiotic Resistance of Campylobacter Species at the Human-Animal-Environment Interface.

This study holds significant importance due to its focus on Campylobacter, the leading bacterial cause of gastroenteritis worldwide, responsible for ∼96 million cases each year. By investigating the prevalence of both Campylobacter jejuni and Campylobacter coli in humans, animals, and the environment, this research sheds light on the broader impact of these pathogens, which can harm both human and animal populations. Traditional microbiological methods were implemented followed by optimized multiplex polymerase chain reaction targeting 16S rDNA and virulence gene markers by using specific primers. The findings revealed that a total of 219 Campylobacter isolates were recovered from 528 collected specimens from human, animal, and environmental sources. Campylobacter species showed a prevalence of 41.5%, with C. jejuni accounting for 53% and C. coli for 47%. Antimicrobial resistance rates were high, with tetracycline at 89%, ceftriaxone at 75%, cefotaxime at 70%, erythromycin at 69%, nalidixic acid at 54%, ciprofloxacin at 39%, and gentamicin at 23%. Commonly prevalent virulence-associated genes observed in the Campylobacter were cadF at 93%, flaA at 91%, cdtB at 88%, cheY at 86%, sodB at 78%, and iamA at 32%. The study confirmed multidrug-resistant Campylobacter prevalence at the human-animal-environment interface, harboring virulence-associated genes with potential harm to humans. Data analysis showed a nonsignificant (p ≥ 0.05) correlation between virulence genes and antibiotic susceptibility. To effectively manage Campylobacter infections, a multifaceted strategy incorporating preventative interventions at different levels is required. This strategy should take into account practicability, effectiveness, and sustainability while strengthening surveillance systems and addressing the economics of disease prevention.

Guardians of the Herd: Molecular Surveillance of Tick Vectors Uncovers Theileriosis Perils in Large Ruminants.

Theileria sp. (Piroplasmida: Theileriidae) is one of the most widely known infections transmitted by hard ticks (Acari: Ixodidae) and has been linked to significant economic losses across the globe. The study's main emphasis was theileriosis, a disease that is common in Pakistan and has an incidence ranging from 0.6% to 33%. Through DNA screening of the vector ticks and host blood, this study sought to determine the risk of tick-borne theileriosis in populations of buffalos (Bubalus bubalis) and cattle (Bos indicus) in Toba Tek Singh district of Punjab, Pakistan. Identified tick species include Hyalomma anatolicum (35.4%), Rhipicephalus (Boophilus) microplus (30.2%), and R. sanguineus (25%). Tick specimens were collected from animals and their respective microenvironments. PCR assays targeting Theileria annulata were used to investigate the infection in the DNA extracted from the collected blood samples from large ruminants and salivary glands (SGs) of the Hyalomma ticks. The 18S rRNA of T. annulata was amplified using specific primers. Positive T. annulata amplicons were sequenced and verified using BLAST analysis. Overall, 50% of SGs contained T. annulate DNA. Female ticks, and those collected from cattle and from riverine environments had significantly higher (p < 0.05) rates of Theileria infection in their acini. Overall prevalence of Theileria infection was 35.9% in blood collected from large ruminants. Cattle had a substantially greater frequency of bovine theileriosis (43.2%) than buffalos (28.7%). Age and sex of large ruminants were significantly positively associated (p < 0.05) with Theileria infection. Furthermore, compared to non-riverine cattle (35%) and buffalo (19.5%), riverine cattle (52.2%) and buffalo (36.2%) showed a considerably higher prevalence. The results of this study, which is the first in Pakistan to examine the blood of large ruminants and vectorial function of Ixodid ticks in the transmission of T. annulata along with associated risk factors, offer an important insight for risk assessment of Theileria infection in livestock using vectorial infectivity.

Inflicting Significant Losses in Slaughtered Animals: Exposing the Hidden Effects of Parasitic Infections.

We started a campaign in the heart of Faisalabad, Punjab, Pakistan, to expose the hidden threats of parasitic illnesses in ruminants and the severe financial consequences associated with them. Our in-depth investigations focused on the prevalence, impact, and astounding financial losses brought on by organ contamination in slaughtered animals. Of the 384 slaughtered ruminants examined for gastrointestinal parasites, a prevalence of 44.79% was recorded. It is interesting to note that we found no conclusive association between parasitic infection and the various ruminant species under study (p > 0.05). However, goats (52.0%) had the highest numerical prevalence of parasitic infection, followed by cattle (46.1%), buffalo (46.0%), and sheep (34.7%) in that order. A significant finding (p < 0.05) showed that the majority of animals had light parasitism (46.5%), as opposed to those with moderate (30.2%) or severe loads (23.2%). Our research revealed substantial (p < 0.05) relationships between ruminant age, sex, and parasitic infection prevalence. In comparison to females (56.4%) and adults (48.1%), males (36.1%) and young (36.9%) ruminants showed considerably decreased infection rates (p < 0.05). On the other hand, we discovered a non-significant (p > 0.05) association between the months and the prevalence of parasitic infection. As a result of the condemnation of contaminated organs such as the rumen, lungs, and liver, an estimated financial loss of PKR 133,731,400 (USD = 466,939.2) was incurred. The yearly economic losses caused by liver condemnation were much greater than those caused by rumen and lung condemnation (p < 0.05). Our research not only reported a significantly higher abundance but also economic threats of the parasitic diseases among the slaughtered animals in Faisalabad, Punjab, Pakistan. Our findings highlighted the critical need for preventive and therapeutic interventions for parasitic infections in animals, in order to mitigate the economic losses through strengthened animal health.

Evaluation of trace elements in forages and their effect on gastrointestinal parasite burden in grazing sheep.

This study was designed to evaluate the trace elements (minerals) in forages fed to sheep and their effect on gastrointestinal parasite burdens. The ultimate objective was to determine the correlation between the burden of gastrointestinal (GI) parasites and the level of trace minerals in sheep serum as a result of the forages they grazed on. A total of 384 faecal samples were collected from sheep in each of the districts (Sialkot and Multan) and examined quantitatively using the McMaster technique. Serum collected from them and plants were pre-treated, and spectrophotometry was used to determine the concentration of trace minerals (Mn, Co, Cu, and Zn). The level of these trace elements differed significantly (P < 0.05) in forages from both districts. In the district of Sialkot, the highest concentrations (mg/Kg) of Zn (38.53 ± 0.16) were found in Cichorium intybus, Cu (41.57 ± 0.07) in Cynodon dactylon, Mn (39.61 ± 0.05) in Parthenium hysterophorus, and Co (1.42 ± 0.03) in Coronopus didymus. In the district of Multan, the highest concentrations (mg/Kg) of Zn (39.43 ± 0.46) were found in Cichorium intybus, Cu (25.76 ± 0.36) in Cynodon dactylon, Mn (34.29 ± 0.53) in Launaea nudicaulis, and Co (1.74 ± 0.08) in Brachiaria raptens. The prevalence of GI parasites in sheep populations in district Sialkot was 34%, while in district Multan, it was 32%. In tehsil Sialkot of district Sialkot, Zn and Cu were significantly (P < 0.05) correlated with eggs per gram (EPG) of faeces, while in tehsil Multan City of district Multan, only Cu was significantly (P < 0.05) correlated with EPG. The potential mechanism behind the role of trace minerals in lowering the burdens of GI parasites requires more investigation. It is recommended that plants with high content of trace minerals should be utilized as part of comprehensive preventive and control strategies against GI parasitism in ruminant animals like sheep.

Revealing novel cytb and nad5 genes-based population diversity and benzimidazole resistance in Echinococcus granulosus of bovine origin.

Cystic echinococcosis (CE) is a neglected zoonotic disease caused by Echinococcus granulosus (sensu stricto). The parasite affects a wide range of livestock and wild animals. In this study, the population diversity of the Echinococcus species was investigated based on mitochondrial cytochrome b (cytb) and NADH dehydrogenase subunit 5 (nad5) genes. In addition to this, ß-tubulin gene isoforms of Echinococcus granulosus were amplified to determine the resistance against benzimidazoles. For this purpose, 40 cyst samples from cattle (n = 20) and buffaloes (n = 20) were collected from the main abattoir of Sialkot. DNA extraction was performed using Qiagen Blood and Tissue Kits. Amplification was performed through PCR. Each amplicon was confirmed by GelRed™ stained agarose gel (2%). Samples were sequenced in a DNA analyzer and viewed for any misread nucleotide by using MEGA (v.11). Corrections in nucleotide sequence and multiple sequence alignment were made through the same software. NCBI-BLAST was used for sample specific sequences to identify them as belonging to a particular species. Diversity indices were estimated using DnaSP (v.6) while phylogenetic analysis was inferred using the Bayesian method using MrBayes (v.1.1). ß-tubulin gene isoforms sequence analysis was performed to find out the candidate gene causing benzimidazole resistance. All 40 isolates were found positive for E. granulosus. BLAST-based searches of sequences of each isolate for each gene (nad5 and cytb) confirmed their maximum similarity with the G1 genotype. Overall, high haplotype diversity (Hd nad5 = 1.00; Hd cytb = 0.833) and low nucleotide diversity (π nad5 = 0.00560; π = cytb = 0.00763) was identified based on diversity indices. For both the genes, non-significant values of Tajima's D (nad5 = -0.81734; cytb = -0.80861) and Fu's Fs (nad5 = -1.012; cytb = 0.731) indicate recent population expansion. Bayesian phylogeny-based results of nad5 and cytb sequences confirmed their genotypic status as distinct from other Echinococcus species. This study shed light on the status of benzimidazole resistance in Echinococcus granulosus for the very first time from Pakistan. The findings of this study will significantly add in the information available on genetic diversity of Echinoccous granulosus based on cytb and nad5 genes sequences.

A Participatory Approach in Assessing the Knowledge, Attitude, and Practices (KAP) of Stakeholders and Livestock Owners about Ticks and Tick-Borne Diseases from Sindh, Pakistan.

Ticks and tick-borne diseases (TTBDs) are responsible for significant losses in terms of treatment costs, decreased productivity (such as milk and meat), reduced reproductive ability, and financial crisis to livestock owners. In Pakistan, it is crucial to periodically assess the risk of TTBDs and ecological factors, potential causes of acaricidal resistance in tick fauna, and the intensive increase in the spread of TTBDs. Participatory epidemiological approaches are key to assessing the livestock owners' and stakeholders' knowledge, attitude, and practices (KAP) about TTBDs. The current study determined the KAP about ticks and tick-borne diseases of respondents from Sindh, Pakistan. A total of 240 respondents were interviewed from different ecological zones: among them, 42.5% (n = 102) of the respondents practiced the manual removal of ticks from animals, while acaricide usage was indicated by 137 respondents (57.0%) as occurring sometimes, 50 (20.8%) monthly, 41 (17.0%) fortnightly, and 12 (5%) weekly, during the peak infestation season. Ticks were 2.6 times [OR = 2.5 (95% Cl = 1.47-4.06)] and viruses were 1.89 times [OR = 188 (95% Cl = 1.09-2.9)] more likely to cause the development of disease in animals than any other pathogen. Despite the appropriate usage of acaricides, the knowledge of participants was inadequate. The findings of this study emphasize the need to take into account identified knowledge gaps and to take the initiative in carrying out appropriate education activities and extension programs to enhance the adoption of effective tick prevention and control strategies.

A new insight in immunomodulatory impact of botanicals in treating avian coccidiosis.

Avian coccidiosis is caused by genus Eimeria (E.) i.e. E. maxima, E. necatrix, E. tenella, E. acervulina, E. brunette and E. mitis and lead to three billion US dollar per year economic loss in poultry industry and reduces the growth performance of birds. To purge undesirable foreign agents, immune system produces a variety of molecules and cells that ultimately neutralize target particles in healthy organisms. However; when this particular system compromises, infection develops and the load of pathogens along with their virulence factors overcome both; innate and adaptive immune systems. Livestock and poultry sectors are important part of agriculture industry worldwide. Due to excessive use of chemotherapeutic agents, pathogens have developed resistance against these agents leading to the great economic losses. Numerous therapeutic approaches are in routine process for the treatment and prevention of various ailments but irrational use of antibiotics/chemicals has raised alarming concerns, like the development of drug resistant strains, residual effects in ultimate users and environmental pollution. These problems have led to the development of alternatives. In this regard, anticoccidial vaccine can be used as an alternative but due to high cost of production, plant derived biological response modifiers and antioxidants compounds are considered as a promising alternative. This review summarizes the immunotherapeutic effects of different compounds particularly with reference to avian coccidiosis.

Screening of Toxoplasmosis in Owned and Stray Dogs of District Faisalabad, Pakistan through Latex Agglutination and Indirect ELISA.

INTRODUCTION: Toxoplasma gondii (an intracellular protozoan) causes toxoplasmosis in warm-blooded animals, including humans and dogs. The present study was carried out to investigate the seroprevalence of canine toxoplasmosis in the owned and stray populations of dogs in Faisalabad District, Punjab, Pakistan. MATERIALS AND METHODS: Commercially available Latex Agglutination Test (LAT) kits were used for the screening of samples (139 stray and 150 owned), followed by confirmation through ELISA. For the statistical analyses, chi-square was used to correlate the prevalence of toxoplasmosis with various factors. RESULTS: The overall prevalence of toxoplasmosis, determined by the LAT, was 22.5% and, by ELISA, was 21.8%. A nonsignificant association of toxoplasmosis was determined among owned and stray dogs. Among owned dog breeds, Bulldogs showed 28.30% prevalence, and among stray dogs, the highest prevalence was determined in Bhakarwal dogs (39.29%). Young and female dogs showed a slightly higher prevalence of toxoplasmosis than adults and males, respectively. CONCLUSIONS: The present study determined by LAT and ELISA in owned dogs showed the same results, while a little variation was found in the stray dogs. It is concluded that both owned and stray dogs are infected with toxoplasmosis in Faisalabad District, and based on this, it is recommended that province-wide epidemiological studies be carried out to examine the prevalence of Toxoplasma and develop policies in order to control toxoplasmosis.

Seroprevalence and Molecular Evidence of Coxiella burnetii in Dromedary Camels of Pakistan.

Coxiellosis is a zoonosis in animals caused by Coxiella burnetii. A cross-sectional study was conducted on 920 (591 female and 329 male) randomly selected camels (Camelus dromedarius) of different age groups from 13 districts representative of the three different ecological zones in the Province Punjab, Pakistan to determine the prevalence and associated risk factors of coxiellosis. The blood samples were collected and tested for anti-C. burnetti antibodies using indirect multispecies ELISA. Real-time PCR was used for the detection of C. burnetii DNA to determine the prevalence in heparinized blood pools. Out of 920 investigated camels, anti-C. burnetii antibodies were detected in 288 samples (31.3%) (95% CI: 28.3-34.4%). The highest (78.6%) and lowest (1.8%) seroprevalence were detected in Rahimyar Khan (southern Punjab) and in Jhang (central Punjab), respectively. Potential risk factors associated with seropositivity of the Q fever in camels included desert area (42.5%; OR = 2.78, 95% CI 1.12-3.21) summer season (35.7%; OR = 2.3, 95% CI: 1.31-3.2), sex (female) (39.1; OR = 2.35, 95% CI: 1.34-2.98), tick infestation (51.3%;OR = 2.81, 95% CI: 1.34-3.02), age (>10 years; 46.4%; OR = 1.56, 95% CI: 0.33-2.05) and herd size (38.5%; OR = 1.21, 95% CI: 0.76-1.54). Coxiella burnetii DNA was amplified in 12 (20%) and 1 (10%) of 60 ELISA-negative and 10 suspected camels, respectively. DNA could not be detected in ELISA positive blood pools. This study emphasizes the seroprevalence and associated risk factors of coxiellosis as well as its potential to spill over to animals and humans in contact with these camel herds.

Molecular Epidemiology of Cryptosporidium parvum and Giardia lamblia in Different Water Bodies, Soil, and Vegetables in Pakistan.

There is a dearth of knowledge regarding transmission of Cryptosporidium (C) and Giardia (G) species through water and water-related sources in Pakistan. To this end, we conducted a study to evaluate the prevalence of these parasites in different water bodies, soil, and mixed raw vegetables in Pakistan. Researchers collected 200 samples from each reservoir including municipal water, sewage water, canal water, raw vegetables, soil of public parks, and soil of grazing areas. Researchers amplified the 18S ribosomal RNA gene of parasites using newly designed genus-specific primers through polymerase chain reaction testing. The sequencing analysis revealed that the obtained sequences belonged to C parvum and G lamblia. Phylogenetic clustering and sequence analysis of C parvum showed that the C parvum Pak1 (OM540369) and C parvum Pak2 (OM540370) as well as C parvum Pak3 (OM510450) and C parvum Pak6 (OM510445) were closely similar to each other. In the case of G lamblia, all the sequences appeared in the same clade. The epidemiological data showed lower prevalence of C parvum (11.5%) in all reservoirs, compared with G lamblia (20.5%). Among different reservoirs, prevalence of Cryptosporidium and Giardia was observed in sewage water (C = 13%, G = 26.5%), municipal water (C = 10%, G = 35%), canal water (C = 9.5%, G = 18.5%), raw vegetables (C = 5.5%, G = 8%), soil of public parks (C = 13%, G = 14%), and soil of grazing areas (C = 18.5%, G = 21.5%). Among targeted risk factors, poor hygienic conditions significantly affected the prevalence of parasites in sewage water (C = 20%, G = 41.2%), municipal water (C = 14.7%, G = 48.8.5%), raw vegetables (C = 11.3%, G = 15.1%), and soil of public parks (C = 19.5%, G = 21.9%). Similarly, contamination with sewage waste significantly affected (P < .05) the prevalence of these parasites in municipal water (C = 13.6%, G = 41.1%), canal water (C = 13.3%, G = 25%), raw vegetables (C = 12.1%, G = 15.1%), and soil of public parks (C = 23.3%, G = 15%). Results of this study illustrated a high risk of parasitic zoonosis through water bodies, soil, and vegetables.

Potential Mechanisms of Transmission of Tick-Borne Viruses at the Virus-Tick Interface.

Ticks (Acari; Ixodidae) are the second most important vector for transmission of pathogens to humans, livestock, and wildlife. Ticks as vectors for viruses have been reported many times over the last 100 years. Tick-borne viruses (TBVs) belong to two orders (Bunyavirales and Mononegavirales) containing nine families (Bunyaviridae, Rhabdoviridae, Asfarviridae, Orthomyxovirida, Reoviridae, Flaviviridae, Phenuviridae, Nyamiviridae, and Nairoviridae). Among these TBVs, some are very pathogenic, causing huge mortality, and hence, deserve to be covered under the umbrella of one health. About 38 viral species are being transmitted by <10% of the tick species of the families Ixodidae and Argasidae. All TBVs are RNA viruses except for the African swine fever virus from the family Asfarviridae. Tick-borne viral diseases have also been classified as an emerging threat to public health and animals, especially in resource-poor communities of the developing world. Tick-host interaction plays an important role in the successful transmission of pathogens. The ticks' salivary glands are the main cellular machinery involved in the uptake, settlement, and multiplication of viruses, which are required for successful transmission into the final host. Furthermore, tick saliva also participates as an augmenting tool during the physiological process of transmission. Tick saliva is an important key element in the successful transmission of pathogens and contains different antimicrobial proteins, e.g., defensin, serine, proteases, and cement protein, which are key players in tick-virus interaction. While tick-virus interaction is a crucial factor in the propagation of tick-borne viral diseases, other factors (physiological, immunological, and gut flora) are also involved. Some immunological factors, e.g., toll-like receptors, scavenger receptors, Janus-kinase (JAK-STAT) pathway, and immunodeficiency (IMD) pathway are involved in tick-virus interaction by helping in virus assembly and acting to increase transmission. Ticks also harbor some endogenous viruses as internal microbial faunas, which also play a significant role in tick-virus interaction. Studies focusing on tick saliva and its role in pathogen transmission, tick feeding, and control of ticks using functional genomics all point toward solutions to this emerging threat. Information regarding tick-virus interaction is somewhat lacking; however, this information is necessary for a complete understanding of transmission TBVs and their persistence in nature. This review encompasses insight into the ecology and vectorial capacity of tick vectors, as well as our current understanding of the predisposing, enabling, precipitating, and reinforcing factors that influence TBV epidemics. The review explores the cellular, biochemical, and immunological tools which ensure and augment successful evading of the ticks' defense systems and transmission of the viruses to the final hosts at the virus-vector interface. The role of functional genomics, proteomics, and metabolomics in profiling tick-virus interaction is also discussed. This review is an initial attempt to comprehensively elaborate on the epidemiological determinants of TBVs with a focus on intra-vector physiological processes involved in the successful execution of the docking, uptake, settlement, replication, and transmission processes of arboviruses. This adds valuable data to the existing bank of knowledge for global stakeholders, policymakers, and the scientific community working to devise appropriate strategies to control ticks and TBVs.

Concurrent Trypanosomosis, Babesiosis and Toxocarosis in a Dog: a Case Report.

Background: Among the blood-borne parasitic infections of dogs, trypanosomosis and babesiosis are highly prevalent in the Indian subcontinent and highly pathogenic. Methods: A six-month-old dog was presented with complaint of lethargy, anorexia and loss of body weight over the previous 15 days on October 11, 2021. A full clinical and targeted haematologic and parasitological examination was undertaken. Results: Wet smears were positive for the motile trypanosomes while stained thin blood smears showed large number of extracellular trypanosomes and intra-erythrocytic ring-like inclusion bodies of Babesia spp. A high burden of Toxocara canis was identified on coprological examination. Conclusion: This report describes the clinical manifestation of concurrent infection of haemoparasites and gastrointestinal nematodes in Pakistani dogs, and highlights the importance of prompt veterinary intervention.

Epidemiology, Diagnosis, and Control of Canine Infectious Cyclic Thrombocytopenia and Granulocytic Anaplasmosis: Emerging Diseases of Veterinary and Public Health Significance.

This review highlights the diagnostic methods used, the control strategies adopted, and the global epidemiological status of canine cyclic thrombocytopenia and granulocytic anaplasmosis at the animal-human interface. Canine anaplasmosis is an important worldwide disease, mainly caused by Anaplasma platys and A. phagocytophilum with zoonotic implications. A. platys chiefly infects platelets in canids, while A. phagocytophilum is the most common zoonotic pathogen infecting neutrophils of various vertebrate hosts. Diagnosis is based on the identification of clinical signs, the recognition of intracellular inclusions observed by microscopic observation of stained blood smear, and/or methods detecting antibodies or nucleic acids, although DNA sequencing is usually required to confirm the pathogenic strain. Serological cross-reactivity is the main problem in serodiagnosis. Prevalence varies from area to area depending on tick exposure. Tetracyclines are significant drugs for human and animal anaplasmosis. No universal vaccine is yet available that protects against diverse geographic strains. The control of canine anaplasmosis therefore relies on the detection of vectors/reservoirs, control of tick vectors, and prevention of iatrogenic/mechanical transmission. The control strategies for human anaplasmosis include reducing high-risk tick contact activities (such as gardening and hiking), careful blood transfusion, by passing immunosuppression, recognizing, and control of reservoirs/vectors.

Comparative anthelmintic efficacy of Arundo donax, Areca catechu, and Ferula assa-foetida against Haemonchus contortus.

In the present study, anthelmintic activities of Arundo (A.) donax L., Areca (Ar.) catechu L., and Ferula (F.) assa-foetida L. were determined. Leaves of A. donax L., latex of F. assa-foetida L. and seeds of Ar. catechu L. in different solvent fractions were subjected to in vitro (egg hatch assay; EHA, and adult motility assay; AMA) and in vivo (faecal egg count reduction test; FECRT) tests of anthelmintic activity using Haemonchus contortus model. In the AMA, crude aqueous methanol extracts (CAME) and ethyl acetate fractions of F. assa-foetida at 10 hr post-treatment showed maximum mortality of H. contortus at 12.5-50 mg mL-1. In the EHA, CAME of F. assa-foetida was identified as a potent ovicide based on its low LC50 (16.9 µg mL-1), followed in order by Ar. catechu and A. donax. Results from the FECRT also showed the extract of F. assa-foetida L. to be more effective than those of Ar. catechu L. and A. donax L., against the gastrointestinal parasitic nematodes. Chloroform and ethyl acetate fractions showed better anthelmintic activities against the adult worms in vitro, while CAME of these plants were better than their crude powders in vivo. It is recommended to document and investigate indigenous knowledge of possible medicinal plants to plan scientific trials that may justify their endorsement.

Hyalomma anatolicum resistance against ivermectin and fipronil is associated with indiscriminate use of acaricides in southwestern Balochistan, Pakistan.

Ivermectin and fipronil have been used regularly to control the hard tick Hyalomma anatolicum (Acari: Ixodidae) in domestic ruminants for more than a half-decade in Balochistan, Pakistan. Inappropriate and indiscriminate use of these acaricides has resulted in the development of resistances in tick species. In this work, acaricides (ivermectin and fipronil) resistance was evaluated in H. anatolicum through in vitro and in vivo bioassays in a horse farm of Quetta, Balochistan province, Pakistan. A participatory epidemiological survey was conducted to assess potential risk factors associated with the development of acaricide resistance in H. anatolicum. The results of the epidemiological survey revealed that the horse keepers did not follow the manufacturer's instructions for the use of acaricides and applied indiscriminate doses of acaricides. The results of in vitro bioassays (adult immersion test and larval immersion test) showed that fipronil and ivermectin have protective efficacy against H. anatolicum. The results of in vivo bioassay (adult-tick mortality assay) revealed that fipronil had a higher efficacy (78.16%) than ivermectin (49.94%). More than 80% of tick mortality was not achieved in any bioassays, even for the highest acaricide concentration (100 ppm), which suggests the development of acaricide resistance against fipronil and ivermectin. This study highlights the urgency to implement a country-wide awareness about resistance monitoring and effective tick control. Graphical abstract.

Comparative anthelmintic efficacy of Arundo donax, Areca catechu, and Ferula assa-foetida against Haemonchus contortus / Eficácia anthelmíntica comparativa de Arundo donax, Areca catechu e Ferula assa-foetida contra Haemonchus contortus

Abstract In the present study, anthelmintic activities of Arundo (A.) donax L., Areca (Ar.) catechu L., and Ferula (F.) assa-foetida L. were determined. Leaves of A. donax L., latex of F. assa-foetida L. and seeds of Ar. catechu L. in different solvent fractions were subjected to in vitro (egg hatch assay; EHA, and adult motility assay; AMA) and in vivo (faecal egg count reduction test; FECRT) tests of anthelmintic activity using Haemonchus contortus model. In the AMA, crude aqueous methanol extracts (CAME) and ethyl acetate fractions of F. assa-foetida at 10 hr post-treatment showed maximum mortality of H. contortus at 12.5-50 mg mL-1. In the EHA, CAME of F. assa-foetida was identified as a potent ovicide based on its low LC50 (16.9 µg mL-1), followed in order by Ar. catechu and A. donax. Results from the FECRT also showed the extract of F. assa-foetida L. to be more effective than those of Ar. catechu L. and A. donax L., against the gastrointestinal parasitic nematodes. Chloroform and ethyl acetate fractions showed better anthelmintic activities against the adult worms in vitro, while CAME of these plants were better than their crude powders in vivo. It is recommended to document and investigate indigenous knowledge of possible medicinal plants to plan scientific trials that may justify their endorsement.

Resumo No presente estudo, as atividades anti-helmínticas de Arundo (A.) donax L., Areca (Ar.) Catechu L. e Ferula (F.) assa-foetida L. foram determinadas. Folhas de A. donax L., látex de F. assa-foetida L. e sementes de Ar. catechu L. em diferentes frações de solvente foram submetidos a testes in vitro (teste de eclosão de ovos, EHA e ensaio de motilidade em adultos, AMA); e in vivo (teste de redução da contagem de ovos fecais, FECRT) de atividade anti-helmíntica, usando-se Haemonchus contortus. Na AMA, extratos aquosos brutos de metanol (CAME) e frações de acetato de etila de F. assa-foetida. Dez horas pós-tratamento, apresentaram mortalidade máxima de H. contortus em 12,5-50 mg mL-1. No EHA, CAME de F. assa-foetida foi identificado como um ovicida potente baseado em seu baixo LC50 (16,9 µg mL-1), seguido em ordem por Ar. catechu e A. donax. Os resultados do FECRT também mostraram que o extrato de F. assa-foetida L. é mais eficaz do que o de Ar. catechu L. e A. donax L., contra nematoides parasitas gastrointestinais. As frações clorofórmio e acetato de etila mostraram melhores atividades anti-helmínticas contra vermes adultos in vitro, enquanto o CAME dessas plantas foi melhor do que o pó bruto in vivo. Recomenda-se documentar e investigar o conhecimento indígena de possíveis plantas medicinais para planejar ensaios científicos que possam justificar seu endosso.

Tick-Borne Pathogens Shape the Native Microbiome Within Tick Vectors.

Ticks are blood-feeding arthropods and transmit a variety of medically important viral, bacterial, protozoan pathogens to animals and humans. Ticks also harbor a diverse community of microbes linked to their biological processes, such as hematophagy, and hence affect vector competence. The interactions between bacterial and/or protozoan pathogens and the tick microbiome is a black-box, and therefore we tested the hypothesis that the presence of a protozoan or bacterial pathogen will alter the microbial composition within a tick. Hence, this study was designed to define the microbial composition of two tick species, Hyalomma (H.) anatolicum and Rhipicephalus (R.) microplus. We used a combination of PCR based pathogen (Anaplasma marginale and Theileria species) and symbiont (Wolbachia species) identification followed by metagenomic sequencing and comparison of the microbial communities in PCR positive and negative ticks. A total of 1786 operational taxonomic units was identified representing 25 phyla, 50 classes, and 342 genera. The phylum Proteobacteria, Firmicutes, Actinobacteriota, and Bacteroidota were the most represented bacteria group. Alpha and beta diversity were not significantly affected in the presence or absence of Theileria sp. and A. marginale as see with H. anatolicum ticks. Interestingly, bacterial communities were significantly reduced in Theileria sp. infected R. microplus ticks, while also exhibiting a significant reduction in microbial richness and evenness. Putting these observations together, we referred to the effect the presence of Theileria sp. has on R. microplus a "pathogen-induced dysbiosis". We also identify the presence of Plasmodium falciparum, the causative agent of human malaria from the microbiome of both H. anatolicum and R. microplus ticks. These findings support the presence of a "pathogen-induced dysbiosis" within the tick and further validation experiments are required to investigate how they are important in the vector competence of ticks. Understanding the mechanism of "pathogen-induced dysbiosis" on tick microbial composition may aid the discovery of intervention strategies for the control of emerging tick-borne infections.

First Molecular Evidence of Anaplasma bovis and Anaplasma phagocytophilum in Bovine from Central Punjab, Pakistan.

Obligate intracellular bacteria belonging to the genus Anaplasma spp. are responsible for causing a hemolytic disease called anaplasmosis in animals, as well as in humans. This study was aimed at the molecular identification and genetic analysis of responsible causative agents of anaplasmosis beyond those already reported. A survey was performed during July and August 2018 in the Jhang District, Punjab, Pakistan. Four hundred and fifty blood samples from asymptomatic, tick-infested cattle were collected on FTA cards and tested for the Anaplasma spp. presence using nested-polymerase chain reaction (PCR) methods. The 16S ribosomal RNA gene sequences generated from the positive samples were used for genetic analysis of Anaplasma spp. The nested-PCR results showed the presence of two Anaplasma spp. with an overall prevalence rate of 10.44%, where the prevalence of A. bovis and A. phagocytophilum was 7.78% and 2.66%, respectively. The study portrayed new molecular data on the prevalence of Anaplasma spp. in the studied cattle population, indicating a potential threat to the human population as well.

Detection of West Nile virus lineage 1 sequences in blood donors, Punjab Province, Pakistan.

OBJECTIVES: This study was performed to determine the presence of West Nile virus (WNV) in mosquito specimens and human blood donors in Pakistan. METHODS: A total of 4150 mosquito specimens were collected using CO2-baited traps from five selected districts of Punjab Province, Pakistan. The mosquitoes were taxonomically identified using standard morphological keys, resulting in 166 pools. In addition, 1070 serum samples were collected from human blood donors. RNA was extracted from mosquito and human samples and screened for WNV using a reverse transcriptase PCR (RT-PCR). RESULTS: None of the mosquito pools tested positive for WNV, whereas three samples from asymptomatic humans tested positive. To determine the WNV strains, partial sequences were compared against a global representation of 23 WNV sequences. The study strains were determined to come from WNV lineage 1. CONCLUSIONS: This study is novel in reporting the circulation of lineage 1 WNV in Pakistan. Given its ability to transmit from human to human via blood transfusion, this highlights the urgent need for nationwide surveillance to assess the distribution and impact of WNV in Pakistan. Determining the source of human infection will require more extensive mosquito sampling.

Seroprevalence of Toxoplasma gondii in one-humped camels (Camelus dromedarius) of Thal and Cholistan deserts, Punjab, Pakistan.

Toxoplasma (T.) gondii is an important zoonotic protozoan infecting humans and a wide range of animals. In this study, we determine the seroprevalence and risk factors associated with the seroprevalence of T. gondii in one-humped camels (Camelus dromedarius) in Pakistan. Camels are still an important mean of transportation in some desert areas in Pakistan. In addition, they are the main source of meat and milk for people in those regions; therefore, they have the potential to transmit T. gondii to humans. In order to estimate the seroprevalence of T. gondii, a total of 897 sera samples were collected from camels in the Thal (n = 359) and Cholistan (n = 440) deserts, along with other districts of Chakwal (n = 44) and Faisalabad (n = 54) Punjab, Pakistan, through convenient and snowball sampling techniques. These samples were then analyzed by an indirect enzyme-linked immune-sorbent assay (ELISA) for the presence of T. gondii-specific antibodies, using purified recombinant micronemal protein 3 (MIC3) as an antibody-catching antigen. Our results showed an overall seroprevalence of T. gondii as 40.1% (Thal = 45%; Cholistan = 35.9%; other districts = 33.7%). Risk factor analysis suggested that infection rate was higher in older animals (70.6%). In addition, female camels carried frequent infection (48.8%) than males (22.4%). What's more, female animals having abortion history showed even higher infection rate (75%) compared to pregnant (68.4%) and non-pregnant (42.4%) animals. Our results reported high seroprevelance of T. gondii in camels in Pakistan which provided important information with respect to public health and disease controls.