Gastrointestinal parasites of dogs and foxes in the Zanjan province of Iran: With an emphasis on Echinococcus species.

Canids harbor many zoonotic parasites and play an important role in the spread of parasites in the human environment. Estimation of parasitic infection among canids as definitive hosts may help competent authorities design efficient control programs. This study was conducted to determine the prevalence of intestinal parasites in dogs and foxes with an emphasis on Echinococcus spp. A total of 500 fecal samples of dogs and 30 fecal samples of foxes were studied in the summer, autumn, and winter of 2021 in the Zanjan province using the formalin-ethyl acetate concentration technique, followed by multiplex PCR. The overall prevalence of gastrointestinal parasite infection was estimated to be 19.05%. The prevalence was 24.8%, 10.2%, and 26.7% in stray, shelter dogs and foxes, respectively. No parasites were found among pet and guard dog samples. PCR results on Taenidae eggs showed that 2.4% of samples were positive for Echinococcus granulosus and none contained E. multilocularis. Noteworthy is that E. granulosus was identified only in stray dog samples. The higher prevalence of E. granulosus infection in stray dogs in this province emphasizes the importance of monitoring the food sources consumed by these animals.

Phylogenetic analysis of Acanthamoeba isolated from soil samples and nasal cavities of patients with malignancy: a public health concern in the northwest of Iran.

BACKGROUND: The genus Acanthamoeba is reported from various environmental sources and can cause multiple complications, including chronic amoebic aeratitis and amoebic granulomatous encephalitis. This study investigated the presence and genotyping of Acanthamoeba in the soil of parks and patients with malignancies referred to health centers in Zanjan city, Iran. METHODS: In this cross-sectional study, 200 soil samples were collected from amusement parks in Zanjan city from September 2017 to May 2018. Samples were cultured on 1.5% non-nutrient agar, and the Acanthamoeba genus was identified using the morphological method. PCR was performed on all positive environmental samples, and six microscopically positive clinical samples belonged to our previous study. DNA sequencing of 18S rRNA was performed to analyze the genetic pattern of some PCR-positive isolates. RESULTS: Microscopic results showed that 96 (48%) soil samples were positive. PCR confirmed all positive cases of clinical samples and 84 soil samples. Out of the PCR-positive samples, 20 soil samples and five clinical samples were sequenced successfully. All soil isolates belonged to the T4 genotype, and three and two clinical samples belonged to T4 and T5 genotypes, respectively. CONCLUSION: : The presence of Acanthamoeba in both the environment and clinical samples of Zanjan city suggests paying greater attention to the infections caused by it.

The effects of glucose and ascorbic acid on in vitro development of Echinococcus granulosus metacestodes.

Echinococcus granulosus-developed metacestodes in the cultured medium are used for the assessment of its susceptibility to different compounds; however, this procedure is time-consuming and risky. In the present study, aspirated protoscoleces from the infected sheep were used to evaluate the effects of glucose, as an energy source, as well as ascorbic acid, as an antioxidant vitamin, on larval development. Protoscoleces were maintained in RPMI1640 culture media containing 10% fetal calf serum, as well as different concentrations of glucose (4, 6, and 8 mg/ml) and ascorbic acid (25, 50, and 100 µg/ml). A culture medium containing 4 mg/ml of glucose was served as the control. Larger cysts were achieved in a shorter time from the medium enriched with 6 mg/ml of glucose (740 ± 20 µm) compared to the control group (420 ± 40 µm). However, in the groups treated with ascorbic acid, the number of cysts was higher in 100 µg/ml (32.5 ± 0.7) compared to the control group (12.5 ± 0.7). Additionally, the mature cysts were achieved on the 7th day of cultivation with 100 µg/ml of ascorbic acid compared to 18 days in the control group.

Sequence analysis, intra-genotyping variation, and phylogenetic study of nad1 gene in Echinococcus granulosus sensu lato genotypes from intermediate hosts in southwestern Iran.

Cystic echinococcosis (CE) is caused by the larval stage of Echinococcus granulosus sensu lato (s. l.). The disease is cosmopolitan, and Iran is a highly endemic area for CE. This parasite exhibits high genetic diversity, which can be related to its life cycle, transmission, and pathogenesis. This study was aimed at determining the phylogenetic relationship and intra-genotyping variation of E. granulosus s.l. in a vast area in the southwest of Iran (SWI). Eighty hydatid cyst isolates of intermediate hosts (i.e., cattle, sheep, goat, buffalo, camel, and human) were collected. The sequence analysis of the nad1 gene exhibited the three genotypes of G1 (n = 70, 87.5%), G3 (n = 8, 10%), and G6/G7 (n = 2, 2.5%). Also, 16, 2, and 1 unique haplotypes were identified for the G1, G3, and G6/G7 genotypes, respectively. According to the phylogenetic tree topology, the nad1 gene similarities were found for some G1 isolates in some vast areas, and the G1 genotype showed a heterogeneous population worldwide. The only SWI G6/G7 haplotype was at a distant position in E. canadensis clade, indicating the notable difference of this haplotype from other isolates from Iran and other countries. The presence of the G6/G7 genotype in the SWI may be due to the transmission of the genotype from other regions or the role of camel/wild boar or other possible hosts in the expansion of this genotype in SWI. The results of the present study can be used in CE control programs, molecular epidemiology, and phylogenetic studies in Iran and other countries for future goals.

In vitro evaluation of albendazole-loaded nanostructured lipid carriers on Echinococcus granulosus microcysts and their prophylactic efficacy on experimental secondary hydatidosis.

To enhance the therapeutic effects of albendazole (ABZ) on Echinococcus granulosus protoscoleces and metacestodes, ABZ-loaded nanostructured lipid carriers (ABZ-NLCs) are prepared by the hot high-speed homogenization method. Protoscoleces and microcysts were treated in vitro with free ABZ and ABZ-NLCs (concentrations of 1, 5, and 10 µg/ml), and the corresponding effects were monitored by methylene blue exclusion test and scanning and transmission electron microscopy. Chemoprophylactic treatment was performed on Balb/C mice 1 day before intraperitoneal injection of viable protoscoleces. The drugs were administered daily by intragastric inoculation for a period of 30 days. The prophylactic efficacy was assessed based on the number and weight of cysts developed in treated mice. The ultrastructural alterations in cysts were examined by transmission electron microscopy. After 18 days, all the protoscoleces incubated with 10 µg/ml ABZ-NLCs were killed, while 51.25 ± 4.03% of the protoscoleces incubated with 10 µg/ml free ABZ were still viable. Microcysts treated with ABZ-NLCs underwent degenerative alterations in a shorter time than when free ABZ was applied. The mean weight of the cysts recovered from mice of ABZ-NLCs group was significantly lower than that of the free ABZ group (P < 0.05), yielding prophylactic efficacy of 92.45% and 38.53%, respectively. The cysts treated with ABZ-NLCs showed marked ultrastructural changes in the germinal layer. This study demonstrated that both in vitro and in vivo treatments with ABZ-NLCs are significantly more efficient than treatment with free ABZ against E. granulosus protoscoleces, metacestodes, and prevention of cyst development in mice.

Induction of Immunogenic Response in BALB/c Mice by Live and Killed Form of Recombinant Lactococcus lactis Displaying EG95 of Echinococcus granulosus.

Background: Cystic echinococcosis is a zoonotic parasitic infection caused by Echinococcus granulosus worldwide and is associated with economic losses among livestock animals. EG95 is an immunogenic antigen from the E. granulosus. Lactococcus lactis has been prested as a safe vehicle for antigen delivery. The goal of this study was to design a novel L. lactis strain displaying EG95 as a vaccine delivery system. Methods: The eg95 encoding gene fragment fused to the M6 anchoring protein was cloned into the pNZ7021 vector, and L. lactis NZ9000 displaying recombinant EG95 was constructed. The expression of an approximately 32-kDa EG95 protein was confirmed by Western blotting and immunofluorescence analysis. The immune responses were evaluated in BALB/c mice immunized orally and subcutaneously with the live and killed recombinant L. lactis, respectively. Results: Total IgG level in mice immunized with heat-killed recombinant L. lactis (pNZ7021-eg95) significantly increased compared to the control group. Mucosal IgA was significantly higher in mice received live recombinant L. lactis (pNZ7021-eg95) compared to the control mice. Splenic lymphocytes from immunized mice represented the high levels of IFN-γ and the low-levels of IL-4 and IL-10. Conclusion: Our results indicate that immunization with EG95-expressing L. lactis can induce both specific humoral and cellular immune responses in mice.

Prevalence and molecular analysis of Sarcocystis infections in cattle in Northwest Iran and the first global report of S. gigantea in cattle.

Cattle are intermediate host for several species of Sarcocystis, including S. cruzi, S. hirsuta, and S. hominis with high prevalence worldwide. The present study aimed to determine the prevalence of Sarcocystis infection, species identification, and phylogenetic analysis of the parasite in cattle in Northwest Iran. The samples of diaphragm and esophagus from 290 cattle were collected from slaughterhouses in Northwest Iran and subjected to macroscopic, microscopic, and histopathology examinations, PCR-RFLP, sequencing and phylogenetic analyses. Tissue cysts of Sarcocystis spp. were detected in 92% of cattle by digestion and microscopic tests. Based on the PCR-RFLP and specific PCR, 87.9% and 1.03% of isolates were identified as S. cruzi, and S. hominis, respectively. Macrocyst was seen in a single sample that was identified as S. gigantea. The haplotype network exhibited the extension of the various haplotypes of S. cruzi between neighboring provinces in Northwest Iran. Heterogeneity analysis of S. cruzi 18S-rRNA sequences indicated genetic diversity among S. cruzi isolates (Haplotype diversity: 0.733-0.854) consisting 16 haplotypes; however, the nucleotide differences showed low diversity (0.01481 to 0.03351). Pair wise sequence distance matrix amongst S. cruzi sequences indicated an intra-species divergence of 0%-7.8% and identity of 92.6%-100%. Sarcocystis infection is highly prevalent in cattle in Northwest Iran, with the predominance of S. cruzi, and genetic variants of this species are unequivocally distributing in Northwest provinces. First global detection of S. gigantea in cattle reflects new insights of transmission dynamic and biology of this parasite in Iran.

Antiparasitic effects of Zataria multiflora essential oil nano-emulsion on larval stages of Echinococcus granulosus.

Various protoscolicidal agents are indicated for the prevention of recurrence of cystic echinococcosis caused by the larval stage of Echinococcus granulosus; however, most of these chemicals have adverse side effects. This study evaluated the effects of Zataria multiflora essential oil (ZEO) nano-emulsion and emulsion at the concentrations of 1, 2, 5, 10, 15, and 20 µl/ml on E. granulosus protoscoleces. Albendazole (5 mg/ml), normal saline, and nano-emulsion without ZEO served as control groups. Optimal concentrations of ZEO nano-emulsion and emulsion on the microcysts were also investigated. ZEO emulsion at a concentration of 20 µl/ml for the duration of 15 min and nano-emulsion for the duration of 10 min resulted in the death of 100% of the protoscoleces. Additionally, densely packed aggregates were formed inside the microcysts treated with ZEO nano-emulsion and emulsion at a concentration of 20 µl/ml after 40 min, but the complete destruction of laminated layers did not occur. The results indicate that ZEO nano-emulsions have a higher protoscolicidal effect than its emulsion, but these two compounds had similar effects on microcysts.

Efficiency of flubendazole-loaded mPEG-PCL nanoparticles: A promising formulation against the protoscoleces and cysts of Echinococcus granulosus.

None of the existing drugs can effectively treat the human cystic echinococcosis. This study aimed to improve the efficacy of flubendazole (FLBZ) against the protoscoleces and cysts of Echinococcus granulosus by preparing polymeric FLBZ-loaded methoxy polyethylene glycol-polycaprolactone (mPEG-PCL) nanoparticles. The protoscoleces and microcysts were treated with FLBZ-loaded mPEG-PCL nanoparticles (FLBZ-loaded nanoparticles) and free FLBZ at the final concentrations of 1, 5, and 10 µg/mL for 27 and 14 days, respectively. The chemoprophylactic efficacy of the drugs was evaluated in experimentally infected mice. The nanoparticles were stable for 1 month, with an average size of 101.41 ± 5.14 nm and a zeta potential of -19.13 ± 2.56 mV. The drug-loading and entrapment efficiency of the FLBZ-loaded nanoparticles were calculated to be 3.08 ± 0.15% and 89.16 ± 2.93%, respectively. The incubation of the protoscoleces with the 10 µg/mL nano-formulation for 15 days resulted in 100% mortality, while after incubation with the 10 µg/mL free FLBZ, the viability rate of the protoscoleces was only 44.0% ± 5.22%. Destruction of the microcysts was observed after 7 days' exposure to the FLBZ-loaded nanoparticles at a concentration of 10 µg/mL. The in vivo challenge showed a significant reduction in the weight and number of the cysts (P < 0.05) in the mice treated with the FLBZ-loaded nanoparticles, yielding efficacy rates of 94.64% and 70.21%, correspondingly. Transmission electron microscopy revealed extensive ultrastructural damage to the cysts treated with the FLBZ-loaded nanoparticles. The results indicated that the FLBZ-loaded nanoparticles were more effective than the free FLBZ against the protoscoleces and cysts of E. granulosus both in vitro and in vivo.

In vitro and in vivo effects of chitosan-praziquantel and chitosan-albendazole nanoparticles on Echinococcus granulosus Metacestodes.

Cystic echinococcosis (CE), which is caused by the metacestode of Echinococcus granulosus, is one of the most important zoonoses affecting humans. Benzimidazoles (in particular albendazole) and praziquantel (PZQ) are effective against CE, but poor water solubility of these agents often leads to inadequate results. Here, we evaluate the effects of chitosan-albendazole (ChABZ) and chitosan-praziquantel (ChPZQ) nanoparticles as a new formulation on hydatid cysts both in vitro and in vivo. Developed microcysts in culture were treated with different concentrations of ChABZ and ChPZQ nanoparticles (either alone or in combination), and ABZ + PZQ suspension. The viability rate of microcysts was used to evaluate the drug efficacies. In addition, the prophylactic and therapeutic effects of the drugs were studied on infected DBA/2 mice. Transmission electron microscopy was used to observe the ultra-structural changes. The viability rate of microcysts and differences in cyst weights were compared by ANOVA, and the cyst numbers were compared using the Kruskal-Wallis test. The combination of ChABZ + ChPZQ nanoparticles was more effective than the ABZ + PZQ suspension in vitro (p < 0.05). In prophylaxy, a significant reduction was observed both in size and in number of the cysts in ChABZ + ChPZQ nanoparticle groups compared with the control group (p < 0.05). In the therapeutic stage, however, this treatment only reduced the cyst numbers. Degeneration of the microcysts treated with the drugs was evident in the ultra-structural imaging. Overall, the nanoparticulate drugs were more effective than their suspension counterparts, but further studies are recommended to evaluate the full potential of these nanoparticles in the treatment of human CE.

Dogs' Gastrointestinal Parasites and their Association with Public Health in Iran.

INTRODUCTION: Dogs harbour zoonotic parasites that cause serious infections in humans, such as visceral larva migrans, ocular larva migrans, cystic echinococcosis, and alveolar echinococcosis. Studies on dogs' gastrointestinal parasites in different geographical locations are required to increase knowledge of the risk of canine zoonoses in human populations. MATERIAL AND METHODS: The presence of parasites was examined in 450 faecal samples collected from eight zones of Zanjan province, northwest Iran from June to November 2015. The samples were examined using the sedimentation concentration method and modified Ziehl-Neelsen staining. RESULTS: Gastrointestinal parasites were found in 86 (19.1%) faecal samples. Sarcocystis spp. (7.3%), Taenia/Echinococcus spp. (5.6%), Toxocara spp. (1.8%), and Cystoisospora spp. (1.6%) were the most common parasites observed. The other detected parasites consisted of Dicrocoelium dendriticum (0.7%), Eimeria spp. (0.7%), Cryptosporidium spp. (0.4%), Physaloptera spp. (0.4%), Giardia spp. (1.3%), and Spirocerca lupi (1.3%). The lowest parasite infection rates belonged to Trichuris vulpis and Acanthocephalans (0.2% each). CONCLUSION: This study provides current information on the infection rates in dog populations in Zanjan Province. Furthermore, the study shows a high prevalence of gastrointestinal parasitic infections, including zoonotic ones and particularly Taenia/Echinococcus spp., potentially transmissible to humans and thus relevant to public health.

Genetic characterization of livestock and human hydatid cyst isolates from northwest Iran, using the mitochondrial cox1 gene sequence.

Cystic echinococcosis (CE), caused by larval stages of the tapeworm Echinococcus granulosus, is one of the most important zoonoses distributed worldwide. Genotype analysis of the parasite isolates from various hosts is required to better understand the host specificity and transmission routes. The aim of this study was to identify the genotypes of E. granulosus isolated from humans and domestic animals from northwest of Iran (Zanjan Province) using the mitochondrial cox1 gene sequence. A total of 86 hydatid cysts including 49 sheep and 28 cattle isolates from the slaughterhouse and nine human isolates from surgical wards of local hospitals were collected. The isolates were subjected to DNA extraction, PCR amplification, and sequence. Eighty-two (95.35 %) isolates, including 47 sheep, 26 cattle, and all nine human isolates, were determined as G1 genotype, and the remaining four (4.65 %), including two sheep and two cattle isolates, were identified as G3 genotype. From the cox1 sequence data, 13 different haplotypes (10 G1s and three G3s) were detected and named as EGH1-EGH13 (GenBank accession numbers, KP859559-KP859571). EGH1 was the major variant among the haplotypes, and it was identified in 46 (53.49 %) isolates (31 sheep, 14 cattle, and one human). Alignment of the partial cox1 sequences showed 12 point mutations including seven (58.3 %) synonymous and five (41.7 %) non-synonymous substitutions. Based on the results, G1 was the major genotype of E. granulosus in northwest of Iran affecting sheep, cattle, and humans. In addition, a minor group of G3 genotype was found to be circulating in this region.