In vitro assessment of the anthelmintic activity of copper oxide and zinc oxide nanoparticles on egg and adult stages of Fasciola hepatica: evidence on oxidative stress biomarkers, and DNA damage.

OBJECTIVES: Fasciolosis is of significant economic and public health importance worldwide. The lack of a successful vaccine and emerging resistance in flukes to the drug of choice, triclabendazole, has initiated the search for alternative approaches. In recent years, metallic nanoparticles have been extensively investigated for their anthelmintic effects. This study investigates the in vitro anthelmintic activity of copper oxide and zinc oxide nanoparticles against Fasciola hepatica. METHODS: The in vitro study was based on egg hatchability test (EHA), adult motility inhibition tests, DNA damage, ROS levels, as well as several biomarkers of oxidative stress, including glutathione peroxidase (GSH) and glutathione S-transferase (GST), superoxide dismutase (SOD) and malondialdehyde (MDA). For this purpose, different concentrations of copper oxide nanoparticles (CuO-NPs) and Zinc oxide nanoparticles (ZnO-NPs) (1, 4, 8, 12, and 16 ppm) were used to evaluate the anthelmintic effect on different life stages, including egg and adults of Fasciola hepatica, over 24 h. RESULTS: In vitro treatment of F. hepatica worms with both CuO-NPs and ZnO-NPs could significantly increase ROS production and oxidative stress induction (decreased SOD, GST and GSH and increased MDA) compared to control group. CONCLUSIONS: Based on the results, it seems that CuO-NPs and ZnO-NPs may be effective in the control and treatment of F. hepatica infection. Further research is needed to investigate their potential for in vivo use in the treatment of parasitic infections.

Molecular Identification, Multigenic Characterization and Phylogenetic Analysis of Ctenocephalides Canis Fleas from Humans and Domestic Animals in Iran.

BACKGROUND: Fleas belonging to the Pulicidae are prevalent ectoparasites infesting mammals and birds in Iran. This study focused on genetically identifying and characterizing Ctenocephalides canis collected both off-host and infesting humans and various domestic animals in the country. METHODS: A total of 918 adult flea samples were collected from 10 sites in western and northwestern Iran between April 2018 and May 2019. Out of these, 71 specimens were found off-host, while the remaining fleas were collected from humans (121), sheep (126), goats (184), and dogs (416). Morphological identification at the genus level was performed on all fleas, and ten selected specimens selected based on the sampling sites and hosts were subjected to molecular detection at the species level by using partial amplification and sequencing of the internal transcribed spacer 1 and 2, as well as the cytochrome oxidase I (COXI) markers. RESULTS: The morphological identification confirmed all fleas as Ctenocephalides spp. Alignment and phylogenetic analysis of nuclear and mitochondrial partial sequences confirmed the presence of C. canis. However, molecular divergence was observed among the ten isolates based on the ITS1 and ITS2 with diversity rates estimated at 0.15% and 3.36%, respectively. Notably, the analysis of the COXI marker revealed no molecular divergence among the partial sequences representing the ten studied isolates from C. canis. CONCLUSIONS: This study explores the diversity of C. canis in the western and northwestern regions of Iran, providing insights into their molecular taxonomy and potential role as disease vectors in these areas.

In vitro anti-Trichomonas gallinae effects of Ziziphus vulgaris L. and Camellia sinensis (L.) Kuntze extracts.

BACKGROUND: Trichomonas gallinae is a parasite that causes canker and severe loss and death, especially in young pigeons. Metronidazole (MTZ) is the recommended drug for treating avian trichomoniasis. Due to drug resistance, non-chemical alternatives, such as medicinal plant extracts, are also considered possible therapies for this disease. OBJECTIVES: This study compares the antitrichomonal effects of MTZ with extracts of Camellia sinensis and Ziziphus vulgaris on T. gallinae in vitro. METHODS: Samples of T. gallinae were taken from infected pigeons. Multi-well plates with different concentrations (5, 10, 25, 50 and 100 µg/mL) of plant extracts were used for the in vitro study. RESULTS: The minimum inhibitory concentration (MIC) of C. sinensis extract was 25 µg/mL over 24 h, compared to 50 µg/mL for MTZ. The MIC value of the Z. vulgaris extracts was 50 µg/mL. CONCLUSIONS: The results suggest that the extracts of Z. vulgaris and C. sinensis, as potential natural agents, could have anti-avian trichomoniasis properties. This study also shows that MTZ, C. sinensis and Z. vulgaris are equally effective in preventing the growth of T. gallinae trophozoites in the culture.

Study on species distribution and seasonal dynamics of equine tick infestation in Ardabil province, northwest of Iran.

Ticks are important ectoparasites in equids, causing economic losses in animal husbandry in Iran and worldwide. This study was aimed to determine frequency and species diversity of hard ticks in equids in Ardabil province, during the four seasons in 2021. A total of 240 equids (187 horses, 53 donkeys) were randomly selected and examined. Ixodid ticks were collected from body surface of examined animals and identified. Of all examined equids, 32.5% horses, and 4.58% donkeys were infested with a total number of 412 ixodid ticks. Tick indices (tick number per animal) were 4.62. There was significant difference between prevalence and different age groups of infested animals. The highest prevalence of hard ticks was found in ≥3 years-old equids in the region. Of all examined ticks (412), three genera including Hyalomma spp., Rhipicephalus spp. .and Dermacentor spp. with four species. i.e. Hyalomma anatolicum (73.05%), H. marginatum (17.23%), Rhipicephalus bursa (7.76%) and Dermacentor marginatus (1.94%) were identified. Ixodid ticks' infestations occurred throughout the year with the highest prevalence in spring. The findings indicated that the equids had a high species diversity and ixodid tick infestation, necessitating additional research to identify any possible tick vectors for the hemoparasite transmission in equids of this region.

In Vitro anthelmintic efficacy of medicinal plant essential oils against Marshallagia marshalli: Evidence on oxidative/nitrosative stress biomarkers, DNA damage, and egg hatchability.

One of the major public health problems is drug resistance in parasitic diseases. It is therefore important to find new active ingredients to combat parasites. Herbal products such as essential oils (EOs) may show promise in treating infections caused by gastrointestinal nematodes (GINs). This study investigated the in vitro anthelmintic activity of the EOs of Lavandula angustifolia and Quercus infectoria against Marshallagia marshalli. The in vitro study was based on an egg hatch test (EHT), adult and larval motility inhibition tests, DNA damage, and several biomarkers of oxidative/nitrosative stress, including superoxide dismutase [SOD], catalase [CAT], and glutathione peroxidase [GSH -Px], protein carbonylation [PCO], malondialdehyde [MDA], total antioxidant status [TAS], and nitric oxide levels [NO]. Different concentrations of Lavandula angustifolia and Quercus infectoria EOs (1, 5, 10, 25 and 50 mg/ml) were used to determine the anthelmintic effect on three stages of the life cycle of M. marshalli, i.e. eggs, larvae and adult parasites, for 24 hr. The results showed that EOs of L. angustifolia and Q. infectoria play an important role as anthelmintics. These essential oils significantly reduced the egg hatching and motility of larval and adult worms. This anthelmintic effect is dependent on concentration and time. Furthermore, the EOs of L. angustifolia and Q. infectoria caused oxidative/nitrosative stress (reduced SOD, GSH-Px and CAT and increased MDA, PCO and NO) and DNA damage, thereby providing significant antihelminthic effects. Based on the results, it seems that the EOs extracted from L. angustifolia and Q. infectoria may be effective in the control and treatment of M. marshalli infections. Further research is needed to investigate their potential for in vivo use in the treatment of parasitic infections.

Frequency and genotyping of Giardia duodenalis in dogs of Urmia, northwest of Iran.

Giardia duodenalis is a zoonotic protozoan infecting various vertebrates such as humans and domestic animals. The aim of this study was to determine the frequency and genotypes of G. duodenalis using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in dogs of Urmia, Iran. Overall, 246 stool specimens were collected from 100 pet, 49 stray, and 97 shelter dogs in the Urmia, Iran. Totally, seven samples (2.48%) were microscopically positive in terms of Giardia cyst. The PCR-RFLP analysis revealed that three (1.21%) and two (0.83%) samples have the C and D genotypes, respectively. In addition, two samples (0.83%) were belonged to the AI sub-group. A significant association was determined between the frequency of Giardia infection and life style, age, and stool form of dogs. The findings of the study showed the high frequency of Giardia infection in stray dogs and the dogs under one-year-old. Furthermore, the C and D genotypes of G. duodenalis were predominant in dogs of Urmia, Iran.

Pyrethroids resistance in Pulex irritans and Ctenocephalides canis in west and northwest Iran.

Resistance to the knockdown effect of pyrethroid insecticides occurs due to mutations at target sites of pyrethroids, meaning the voltage-gated sodium channels gene (VGSC) in the membrane of the neurons. In fleas, this mutation occurs at two sites in the sodium channel in neurons: one is the replacement of leucine with phenylalanine (L1014F) and the other is the replacement of threonine with valine (T929V). In this study, 81 Pulex irritans and 47 Ctenocephalides canis fleas were collected from five provinces in the west and northwest of Iran. Adult fleas were exposed to cypermethrin 0.75%, and the mortality rate was calculated after 1 and 8 hr, and the mutation sites in the VGSC gene were investigated. The lethality of cypermethrin 0.75% for P. irritans was 40.00 - 57.14% after 1 hr and 60.00 - 73.91% after 8 hr. The lethality of this dose for C. canis after 1 and 8 hr of exposure was 33.33 - 41.17% and 66.66 - 80.33%, respectively. The VGSC sequence analysis indicated two mutation sites in the resistant and one mutation site in the susceptible fleas. The VGSC sequence analysis of susceptible P. irritans showed that 5.50% of them were homozygous susceptible and 94.45% were hetero-zygous susceptible. Susceptible C. canis were 5.26% homozygous and 94.73% heterozygous susceptible. All the resistant fleas were homozygous. The development of pyrethroid resistance and high-frequency L1014F mutation in fleas suggest that pyrethroids are likely to be ineffective in controlling fleas. Therefore, monitoring pyrethroid resistance and its underlying mechanisms are necessary for controlling fleas and finding new alternative control methods.

Neospora caninum, a cause of abortion in donkeys (Equus asinus) in Iran.

Neospora caninum is an Apicomplexan parasite that can cause enormous economic losses due to abortions in cattle. The present study investigated the role of Neospora spp. infection in equine abortion in Iranian donkeys using molecular and phylogenetic analyses. Twenty-nine-aborted fetuses and 29 blood samples from their dams were collected from six different regions in the West and Northwest of Iran. They were tested for N. caninum by PCR at the Nc5 locus, followed by sequencing of five of the PCR products. The overall molecular prevalence was 34.5% in blood samples and the prevalence by DNA detection in the aborted fetuses was 13.8%. Evidence of transplacental transmission from positive jennies to their fetuses was detected in 40% of aborting jennies. Comparison of the five partial Nc5 sequences (227 bp length) exhibited 98-100% similarity with N. caninum GenBank sequences. This is the first molecular study and genetic characterization of N. caninum in Iranian donkeys suggests that N. caninum may be a significant cause of abortion in donkeys.

In vitro assessment of anti-Trichomonas effects of Zingiber officinale and Lavandula angustifolia alcoholic extracts on Trichomonas gallinae.

Trichomonas gallinae is a parasite that acts as a canker-causing agent and leads to significant loss and mortality, especially in young birds. Metronidazole is the approved drug used for the treatment of trichomoniasis. A non-chemical alternativess such as medical plant extracts are also used to treat this disease due to drug resistance. This study aimed to assess in vitro antitrichomonal effects of Lavandula angustifolia and Zingiber officinale extracts on T. gallinae compared with metronidazole. The T. gallinae samples were obtained from infected pigeons. Multi-well plates filled with different concentrations (5.00, 10.00, 25.00, 50.00, and 100 µg mL-1) were used to perform in vitro analysis. The Z. officinale extract's minimum inhibitory concentration (MIC) in the 24-hr period was 25.00 µg mL-1, while it was 50.00 µg mL-1 for metronidazole. The MIC value obtained for L. angustifolia extract in 24-hr was 50.00 µg mL-1. The results indicated that the extracts of Z. officinale and L. angustifolia could act as potential natural agents against trichomoniasis. Furthermore, this study delineated the equal efficiency of L. angustifolia and Z. officinale with that of metronidazole in inhibiting the growth of Trichomonas gallinae trophozoites in culture media.

In Vitro Assessment Antiparasitic Effect of Selenium and Copper Nanoparticles on Giardia deodenalis Cyst.

BACKGROUND: Application of chemotherapy to treatment of parasitic disease of man and animals can be problematic due to different adverse effects. As a result, there is an increasing interest in nanoparticles as new therapeutic tools against these diseases. This study was designed to evaluate the effect of selenium and copper oxide nanoparticles on Giardia deudenalis cysts in vitro, as well as comparing it to that of metronidazole. METHODS: The cysts were taken from the stools of patients in Urmia, Iran, during 2017-2018. The cysts were taken from stool and were concentrated and isolated on 0.85 M-sucrose. Then, selenium and copper oxide nanoparticles were prepared at concentrations of 0.15, 0.3, and 0.6 mg/ml. The effect of nanoparticle's various concentrations at 10, 15, 30, 60, and 180 min were evaluated and compared to control groups. Obtained data was recorded and statistically analyzed. RESULTS: Copper oxide nanoparticles at a concentration of 0.6 mg/ml and selenium nanoparticles at a concentration of 0.3 mg/ml had the same effect as of metronidazole in killing of Giardia cysts. The cytotoxic effects of selenium and copper oxide nanoparticles, compared with metronidazole, on Giardia cysts, showed an increase of fatality rate due to extend exposure time and nanoparticle's concentration (P<0.05). CONCLUSION: Selenium and copper oxide nanoparticles are as efficient as metronidazole, for killing Giardia cysts in vitro.

Hematological and Biochemical Changes in Naturally Occurring Equine Piroplasmosis in Donkeys (Equus asinus) of Northwest of Iran.

PURPOSE: Equine piroplasmosis is a tick-borne protozoal disease affecting horses, mules, donkeys and zebras and can lead to severe economic losses in the equine industry. There is limited information on clinical pathologic changes of equine piroplasmosis in donkeys. This study aims to investigate the impacts of babesiosis on various hematological and biochemical properties in naturally infected donkeys with Theileria equi and Babesia caballi. METHODS: A total of 200 apparently healthy donkeys were randomly examined for the presence of equine piroplasmosis in blood samples. Of 200 samples, 11(5.5%) animals were positive for T. equi and 3(1.5%) of the samples were infected with B. caballi. 14 uninfected donkeys, were also selected as a control group. RESULTS: The results of the comparison showed that infected donkeys presented a significant elevation (P < 0.05) of total leukocyte count, number of lymphocyte, monocyte, neutrophil and eosinophil, while showed a significant decreased in (P < 0.05) hemoglobin concentration (Hb), packed cell volume (PCV), red blood cells (RBCs), mean corpuscular volume (MCV) and mean corpuscular hemoglobin concentration (MCHC). The infected donkeys had significantly higher levels of total protein and total bilirubin (P < 0.05), whereas the amount of albumin was significantly lower in comparison to healthy animals (P < 0.05). It is interesting to note that the amounts of urea, cholesterol, creatinine, triglyceride, LDL and HDL, CK, AST, LDH, GGT, and Malondialdehyde were significantly higher (P < 0.05) in infected donkeys. CONCLUSIONS: The current study describes laboratory findings in the affected donkeys which were good criteria for diagnosis and control of equine piroplasmosis in donkeys.

Molecular detection and identification of Giardia duodenalis in cattle of Urmia, northwest of Iran.

Giardia duodenalis is one of the most prevalent intestinal protozoa infecting humans and domestic animals. The aim of this study was to identify subspecies of G. duodenalis by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) method from fecal samples of naturally infected cattle in the Urmia, West Azerbaijan province, Iran. Overall, 246 fecal specimens were collected from the cattle (diarrheic and healthy) and microscopically examined for G. duodenalis. The PCR-RFLP analysis of glutamate dehydrogenase (gdh) locus was used to identify the genotypes found in cattle. In this method, 432 bp expected size was amplified and then specific restriction NlaIV enzyme was used for subspecies detection. Totally, 23 (9.34%) specimens were microscopically positive for giardia cyst out of 246 examined samples. The PCR-RFLP analysis revealed that 19 samples (82.60%) have the genotype E and 4 samples (17.39%) belong to the subgroup AI. Our findings indicated that G. duodenalis infection is prevalent in cattle of Urmia and the non-zoonotic genotype E predominates in cattle in this region.

Detection of Theileria equi and Babesia caballi using microscopic and molecular methods in horses in suburb of Urmia, Iran.

Equine piroplasmosis is a severe disease of horses caused by the intra-erythrocyte protozoan, Theileria equi and Babesia caballi. The aim of this study was to identify equine piroplasmosis based on molecular and morphometrical features in horses in suburb of Urmia, West Azerbaijan province, Iran. From April to September 2011, a total number of 240 blood samples were collected randomly from horses of 25 villages. The specimens were transferred to the laboratory and the blood smears stained with Geimsa, and the morphological and biometrical data of parasite in any infected erythrocyte were considered. Extracted DNA from each blood sample was used in multiplex PCR in order to confirm the presence of B. caballi and T. equi. Microscopic observation on 240 blood smears determined that 15 (6.25%) and 5 (2.80%) samples were infected by T. equi and B. caballi, respectively. The mixed infections occurred in 2 (0.83%) samples. The results of the PCR assays showed 26 (10.83%), 14 (5.83%) and 4 (1.66%) were distinguished as T. equi, B. caballi and mixed infection, respectively. Differences in infection rates were statistically nonsignificant between male and female horses and among different age groups. Our findings indicated that T. equi and B. caballi were prevalent in horse population.

Susceptibility of different life stages of Ornithodoros lahorensis to entomopathogenic fungi Metarhizium anisopliae and Beauveria bassiana.

The use of chemicals for the control of arthropod pests can be problematic due to the potential for both environmental contamination and resistance development. As a result, there is an increasing interest in nonchemical alternatives, such as biocontrol by entomopathogenic fungi. In the present study, three strains of Metarhizium anisopliae (V245, 3247, and 4456) and one strain of Beauveria bassiana (LM 3.2) were evaluated under laboratory conditions for their virulence towards three life stages of Ornithodoros lahorensis. Groups of eggs, larvae, and adult ticks were treated by immersion in two different suspensions (10(5) and 10(7) conidia/ml) of each fungal strain. All treatment and control groups were observed during a 3-week period, and the hatchability of eggs and mortality percentage of larvae and adult ticks were assessed. The effect of fungal strains on egg hatchability and larva and adult mortality was significant and dose dependent compared to the control groups (P < 0.05). The results also showed that the greatest biopesticidal effect was due to strain 4456 of M. anisopliae and LM 3.2 strain of B. bassiana at all tested concentrations, making these fungi potential biological control agents of O. lahorensis reducing the use of chemical acaricides.