Veterinary Research Forum

Abstract There is an increasing interest in exploring plant extracts as potential new treatment options for these ailments. Ziziphus vulgaris L. and Camellia sinensis (L.) Kuntze are medicinal plants widely used in different traditional medical practices, especially in Iranian Traditional Medicine, for addressing a range of health issues. Although their biological properties are well-established, their effectiveness against Giardia duodenalis cysts is still uncertain. This research aimed to assess the impact of Z. vulgaris and C. sinensis extracts on G. duodenalis cysts in vitro and to compare their effectiveness with metronidazole. Cysts were extracted from stool samples and concentrated using a 0.85 M sucrose solution. Extracts of Z. vulgaris and C. sinensis were prepared at concentrations of 25, 50, and 100 mg mL-1. The impacts of these extracts at different concentrations were evaluated at 10, 15, 30, 60, and 180 min, and the results were compared to control groups. The collected data were documented and statistically analyzed. The findings revealed that Z. vulgaris extract at a concentration of 100 mg mL-1and C. sinensis extract at a concentration of 50 mg mL-1 showed similar effectiveness to metronidazole in eliminating Giardia cysts. In addition, the cytotoxic effects of Z. vulgaris and C. sinensis extracts, in comparison to metronidazole, indicated a rise in fatality rates with prolonged exposure times and higher extract concentrations (p < 0.05). Therefore, it can be concluded that extracts of Z. vulgaris and C. sinensis are as effective as metronidazole for killing Giardia cysts in vitro.

Abstract The current finding reports the development of iELISA with recombinant AMA-1 (rAMA-1) to identify an infection of Babesia in naturally infected cattle. The 48 kDa protein-encoding recombinant AMA-1 gene was cloned into the pET-28a (+) expression vector and expressed in E. coli. The resulting congregate protein was refined under native settings. Towards the evaluation of the diagnostic potential of AMA-1 as a sero-diagnostic reagent, a panel of sera samples from Babesia infected cattle; uninfected sera as well as Babesia positive samples with other species including B. bigemina, B. divergens, B. major, B. occultans were utilized. Additionally, the efficacy of rAMA-1-based serological assays was compared with commercially available kits using 200 samples taken from cattle suspected of babesiosis. The results demonstrated that the iELISA using rAMA-1 exhibited a diagnostic sensitivity of 88.89%, when compared to commercially available ELISA kit as the reference test. The specificity of this assay was 76.66%. These findings suggest that the iELISA employing rAMA-1 can be utilized on large-scale epidemiological surveys and clinical detection of Babesia infection in cattle.

Abstract Selenium (Se) and gold (Au) nanoparticles (NPs) possess unique properties such as high absorption ability, low toxicity and minimal environmental persistence. These properties, combined with demonstrated anti-parasitic effects against certain parasites, suggest their potential as candidate for house fly control. This study aimed to evaluate the effectiveness of Se and AuNPs in controlling house flies. We investigated the insecticidal effect of Se and AuNPson Musca domestica using a dipping method. Several concentrations (20.00, 60.00, 100, 400, and 1,000 ppm) were prepared using deionized water. Fifteen larvae per concentration were tested, with each test repeated three times. The larvae were dipped in the NP solutions for 30, 60 and 90 sec in 10.00 mL of the tested concentrations. Distilled water was used for the control group. Larval mortality was recorded 24 hr post-treatment. No mortality was observed in house fly larvae 24 hr after exposure to different concentrations of Se and AuNPs, regardless of the incubation time. Thus, Se and AuNPs showed no insecticidal effect on house fly larvae. In conclusion, Se and AuNPs cannot be used to control house flies. Due to the issues associated with chemical control, it is crucial to search for alternative pesticides.

Abstract Physalopteridae nematodes pose a threat to a broad spectrum of animals including amphibians, reptiles, birds and mammals. The current study was the first molecular and phylogenetic characterization of Physaloptera clausa parasitizing long-eared hedgehogs (Hemiechinus auritus) in Iran. A male road-killed H. auritus was collected from Shahriar City, Tehran province in May 2022 and it was subjected to necropsy. After isolating parasites, they underwent morphological analysis using a light microscope and an identification key. For molecular analysis, the genomic DNA was isolated using the FavorPrepTM Tissue Genomic DNA Extraction Mini Kit. The PCR products were sequenced, the sequence data were analyzed and multiple alignments were conducted using the Clustal Omega. For phylogenetic analysis, these nucleotide sequences were aligned in MEGA 11 based on the lowest bayesian information criterion score. A cluster of parasites was found on the mucosa of the hedgehog stomach. All the nematodes were identified as P. clausa. The sequence obtained in this study has been submitted to GenBank^® with the accession number OR088573. The phylogeny analysis revealed that the genus Physaloptera formed a major clade where P. clausa was clustered with various Physaloptera species, closely related to Turgida, Physalopteroides and Skrjabinoptera genera. Our study specified the necessity for additional taxonomic and phylogenetic research on Physaloptera species and related genera to fully understand the evolutionary dynamics and ecological significance of these parasites.

Abstract Dicrocoeliasis is a globally significant condition impacting both economic and public health. The lack of effective vaccines and emergence of drug-resistant flukes have prompted research into alternative treatments. Metallic nanoparticles have recently been studied for their potential as anthelmintic agents. This research examined the in vitro anthelmintic activity of copper oxide (CuO-NPs) and zinc oxide nanoparticles (ZnO-NPs) against Dicrocoelium dendriticum. Using adult motility inhibition tests and oxidative stress biomarkers, including glutathione peroxidase , glutathione S-transferase , superoxide dismutase , and malondialdehyde , this study evaluated the effects of CuO-NPs and ZnO-NPs. Flukes were treated with various concentrations of nanoparticles (1.00, 4.00, 8.00, 12.00, and 16.00 ppm) for 24 hr. The CuO-NPs and ZnO-NPs demonstrated concentration- and time-dependent anthelmintic activity. Higher concentrations (12.00 and 16.00 ppm of CuO-NPs, and 16.00 ppm of ZnO-NPs) significantly inhibited worm motility compared to the controls. The nanoparticles induced oxidative stress in the flukes, with decreased superoxide dismutase, glutathione S-transferase, and glutathione peroxidase activities and increased malondialdehyde levels. Based on these findings, CuO-NPs and ZnO-NPs exhibit potential as therapeutic agents for controlling and treating D. dendriticum. However, further studies are necessary to assess their safety and efficacy in vivo for managing parasitic infections.

Abstract Leishmaniasis comprises a spectrum of clinical manifestations caused by protozoan parasites of the genus Leishmania, order Trypanosomatida. Cutaneous leishmaniasis remains a significant zoonotic disease prevalent in tropical and subtropical regions, particularly in developing countries. Despite ongoing research, a definitive cure for this parasitic infection is still needed. This study explored the potential of activatable melittin (AM) as a selective treatment for cutaneous leishmaniasis caused by Leishmania major. The AM was designed using PepFold and ExPASy servers, incorporating a matrix metalloproteinase -2/9 cleavable linker to target L. major-infected macrophages selectively. To enhance drug delivery and reduce potential toxicity, AM was encapsulated within albumin nanoparticles and liposomes. The anti-leishmanial efficacy of these formulations was evaluated at AM concentrations ranging from 25.00 to 100 µg mL^-1 over 48 hr, with each experiment performed in 10 independent replicates (n = 10 per group). Statistical analysis using one-way ANOVA followed by Tukey's post-hoc test revealed a significant reduction in the average number of intracellular amastigotes per macrophage in the liposome-treated and albumin nanoparticle-treated groups (7.00 ± 1.50 amastigotes per macrophage) compared to the untreated infected control group (35.00 ± 3.20 amastigotes per macrophage). Treatment with 25.00 µg mL^-1 of AM encapsulated in non-toxic albumin nanoparticles and liposomes demonstrated the most promising anti-leishmanial effect, resulting in an approximately 80.00% reduction in intracellular L. major amastigotes (compared to control).

Abstract The complete mitochondrial genome sequence of Dermacentor marginatus isolated from Hainan Island, a tropical island in China, has recently been elucidated. This ornate sheep tick is a three-host species, meaning it requires feeding on different hosts throughout its larval, nymph, and adult stages. This broad host range contributes significantly to its role in the transmission of various pathogens including spotted fever group rickettsia. The mitochondrial genome of D. marginatus was sequenced and found to be a circular, double-stranded DNA molecule with a total length of 15,005 bp. The genome exhibited a guanine and cytosine (GC) content of 21.23%, reflecting its unique genetic composition. Annotation of the genome revealed the presence of 37 genes, comprising 22 tRNA genes, 13 mRNA genes as protein-coding genes, (PCGs) and two rRNA genes. Phylogenetic analysis placed D. marginatus closest to D. silvarum from Heilongjiang province in China, clustering with D. sinicus from Hubei province in China. D. marginatus also exhibited proximity to D. species from China and other countries forming a distinct cluster. The availability of the complete mitochondrial genome sequence of D. marginatus marked a notable advancement in deciphering the genetic diversity and evolutionary path of this tick species. The study concentrated on the mitochondrial genome of D. marginatus from Hainan Island, examining its genetic and phylogenetic features, and assessing genomic variations that may influence mitochondrial function and tropical adaptation. The investigation into the genetic adaptations of D. marginatus to its tropical niche yielded vital information for forecasting how tick populations may react to changes in climate and diverse environmental conditions.

Abstract Trichinella britovi is a species circulating in Iran and has already been observed in wild and domestic carnivores. The present study aims to determine the occurrence of Trichinella species in Iran's most important provinces, including the northern, northeastern, and northwest regions. In total, 93 carcasses of golden jackals, collected due to road accidents between 2019 and 2022, have been obtained from Ardabil, Mazandaran, Golestan, North Khorasan, and Khorasan Razavi provinces, Iran. Genomic DNA (four positives out of 93 carcasses) from each Trichinella genotype was amplified in a multiplex polymerase chain reaction (PCR) reaction. To confirm the multiplex PCR results, the sequences of the PCR products with cytochrome oxidase subunit I were sent to a reference laboratory within the European Union. Bioinformatics software was used to verify the sequencing results of primer fragments and to compare and analyze the results obtained with the sequences stored in the NCBI genetic database. Following multiplex PCR amplification, a two-band pattern was observed. Trichinella britovi larvae displayed the expected band pattern of 127 bp and 253 bp. The PCR products amplified using primers specific for the cytochrome oxidase subunit I gene showed 861 base pair bands in all four samples analyzed. The obtained sequences of cytochrome oxidase subunit I from all four individual muscle larvae had 100% identical haplotypes except for one nucleotide transversion in one sequence. As a result of this study, valuable insights into T. britovi prevalence and phylogenetic distribution among Iranian golden jackals have been gained in terms of prevalence and phylogenic distribution.

Abstract Dogs serve as definitive or intermediate hosts for some protozoan and helminth parasites, some of which are of zoonotic, and others cause severe infectious diseases in other animals. The aim of this study was to determine the prevalence of intestinal parasites in shelter dogs in Khorasan Razavi province, Iran. A total of 323 fecal samples were randomly collected from shelter dogs in Mashhad and Neyshabur areas. All samples were then transferred to the Parasitology Laboratory and examined detect intestinal protozoa and helminths using the formalin-ether technique. Out of the 323 fecal samples, 92 (28.48%) tested positive for intestinal parasites. The overall prevalence of intestinal parasites in shelter dogs in Mashhad and Neyshabur areas were 23.83 and 35.38%, respectively. Monospecific infection was found in 19.50% of dogs, while concurrent infection with two or more species was seen in 8.97% of dogs. The frequencies of the nine detected parasites were as follows: Sarcocystis sp. 12.38%, Giardia sp. 4.64%, Cystoisospora sp. 2.78%, Hammondia Neospora-like oocysts 2.16%, Taenia sp. 6.19%, Toxascaris leonina 7.43%, Dicrocoelium dendriticum 1.54%, Toxocara canis 0.92% and Capillaria sp. 0.30%. The results of this study showed the high frequency of intestinal parasites in shelter dogs. To control important zoonotic helminthic diseases such as echinococcosis and toxocariasis, it is recommended to eliminate stray dogs in cooperation with municipalities and veterinary departments in Iran. If stray dogs are transferred to shelters, they should receive regular treatment with effective anti-helminthic drugs.

Abstract To date, routine diagnosis of canine heartworm disease relies on detecting Dirofilaria immitis antigens in blood or the microscopic examination of blood smears. However, each method has limitations, potentially leading to life-threatening situations for infected dogs. This study aimed to determine the prevalence of filarial infection, risk factors, and appropriate detection methods in practical clinics. A total of 113 dog blood samples from two provinces in Thailand (Chonburi = 73 and Nakhon Nayok = 40) were analyzed for D. immitis infection using buffy coat smears, commercial immunochromatographic tests (SNAP 4Dx Plus), and polymerase chain reaction. Overall prevalence was 51.53% (58/113) across all methods. The positivity rates were 15.38% (12/78) for buffy coat smears, 8.00% (4/50) for SNAP 4Dx Plus, and 45.43% (51/113) for polymerase chain reaction. All positives from the test kits correlated with other methods. A significantly high prevalence was observed in dogs under 2 years old. Accessibility to pet care services in urban areas appeared to have a protective effect. Positive commercial test results could confirm D. immitis infection. However, selecting more than one diagnostic technique in clinics, including morphological examination, immunochromatography, or molecular methods, is recommended for early and more accurate detection, along with the promotion of heartworm prevention strategies.

Abstract Infections causing diarrhea in cattle breeding pose a substantial threat to calf breeding and health, causing considerable economic losses worldwide, including in Türkiye. The primary causative agents of calf diarrhea include bovine group A rotavirus, bovine coronavirus (BCoV), Escherichia coli K99, Cryptosporidium spp., and Giardia spp. The objective of this study was to diagnose the pathogens present in samples taken from calves exhibiting diarrhea symptoms in the Elaziğ province of Türkiye. The study material consisted of 85 fecal samples from calves up to 30 days of age with diarrhea symptoms and 42 small intestine samples from calves died as a result of diarrhea symptoms after necropsy. Following total RNA isolation from the samples, one-step duplex reverse transcription polymerase chain reaction (RT-PCR) analysis was conducted to identify the bovine rotavirus (BRV) and BCoV. Our results indicated that 24 of the samples were positive for BRV, 13 were positive for BCoV, and 25 were positive for BRV and BCoV in the form of mixed infection. The immunochromatographic analysis revealed that three samples were positive for E. coli K99 antigens, 12 for Cryptosporidium spp. antigens, and 28 for Giardia spp. antigens. Our results indicate that one-step duplex RT-PCR, immunohistochemistry, and immunochromatography methods can be valuable tests to be used in routine diagnostic laboratories for the detection of pathogens associated with calf diarrhea.

Abstract Crimean-Congo hemorrhagic fever (CCHF) is indeed to be considered as one of the most significant vector-borne diseases globally. The virus responsible for CCHF can persist in various animals and lead to severe infections in humans. Ticks of the Ixodidae family are the acknowledged vectors of CCHF virus (CCHFV) transmission to humans. In this review, different tick species from Iran and its neighbors and their roles in the transmission of CCHFV were evaluated. However, the transmission patterns of CCHF to humans primarily involve tick bites, direct contact with the blood, organs, or tissues of contaminated animals, and exposure to CCHF patients are important in virus transmission. The results of this review confirm that the tick vectors in Iran and its neighbors are very common. Even though tick bite is one of the most important risk factors for CCHF, it cannot explain all cases, and there are other important risk factors, such as high-risk occupations and having contact with livestock. Ticks that have been proven to transmit the disease, especially Hyalomma marginatum, are scattered in Iran and neighboring countries. Transporting passengers and livestock and sometimes smuggling livestock are the means of transmitting the virus between neighboring countries. Therefore, there is a need for joint preparedness and response programs to prevent and manage CCHF between Iran and its neighbors.

Abstract Rhipicephalus sanguineus, a repulsive obligate blood feeder, is a three-host tick inflicting tremendous damage. Blood-sucking initiates tick-pathogen-host interactions along with alterations in the expression levels of numerous bioactive ingredients. Key molecules regulating blood meals were identified using the transcriptomic approach. A total number of 744 transcripts showed statistically significantly differential expression including 309 significantly upregulated transcripts and 435 significantly downregulated transcripts in semiengorged female ticks compared to unfed ticks, all collected in 2021. The top 10 differentially upregulated transcripts with explicit functional annotations included turripeptide OL55-like protein, valine tRNA ligase-like protein and ice-structuring glycoprotein-like protein. The top 10 differentially down-regulated transcripts were uncharacterized proteins. Gene Ontology (GO) enrichment analysis revealed four associated terms in the cellular component category and 16 in the molecular function category among the top 20 terms. Differentially expressed genes (DEGs) were enriched in GO terms ID 0000323 (lytic vacuole) and ID 0005773 (vacuole).  The top 20 enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways included metabolism, cellular processes, organismal systems and human diseases. The DEGs were enriched in the KEGG term ID: ko-04142 (lysosome pathway) associated with intracellular digestion in the tick midgut epithelium. Molecular markers annotated via comparative transcriptomic profiling were expected to be candidate markers for the purpose of tick control.

Abstract The over-use of anti-parasitic compounds as a method of control has led to insufficient effectiveness and widespread drug resistance worldwide. The aim of this study was to investigate the efficacy of albendazole and levamisole as anti-parasitic agents in a lung worm control program in goat flocks. During 2021 and 2022, a total of 110 goats (age of four months and above) were randomly selected from 11 herds in the north-western region of Iran including Saanen breed (both sexes of the same age). The results indicated that 3.60, 50.80 and 41.90% were respectively infected with Dictyocaulus filaria, Muellerius capillaris and Proto-strongylus rufescens, and generally all the lung parasites in goats of this region were resistant to albendazole and levamisole. Due to clinical importance of D. filaria in goats, the molecular analysis of two samples was also done. Sequencing results showed that the identified parasites were 100% similar to the reference sequences registered in the GenBank^®. The results of this research showed low level of these anthelmintics efficacy against Dictyocaulus and Muellerius. Generally, the lung parasites in goats of this region are resistant to albendazole and levamisole. The P. rufescens showed high resistance to these drugs. Totally, it can be concluded that the level of drug resistance varies in different parts of the world; but, the frequencies of drug resistance in different parts of the world are not the same, requiring more studies.

Abstract A 6-year-old male domestic short-haired cat (Felis catus) was presented with weakness, anorexia, fever, icterus, a painful abdomen, ruffled hair and a tick infestation, and it had no prior surgery. Laboratory analysis revealed left-shifted neutrophils, thrombocytopenia, low albumin content and high serum bilirubin concentration as well as activities of hepatic enzymes including alanine aminotransferase and aspartate aminotransferase. Azotemia and increased serum levels of creatinine and urea were also recorded. In Giemsa-stained blood smear, Hepatozoon gamonts were observed within neutrophils. Species-specific polymerase chain reaction assay was used to amplify an approximately 590 bp fragment of 18S rRNA gene and confirmed Hepatozoon felis infection. The cat was treated with imidocarb dipropionate and doxycycline and recovered completely. Six-month follow-up showed no recurrence. This study reveals the presence of H. felis in Iran and it should be considered in differential diagnosis in febrile and icteric cats. To the authors' knowledge, this is the first description of H. felis infection in a cat in Iran.

Abstract Tropical or Mediterranean theileriosis in dairy cattle is widely distributed in many tropical regions of the world. The purpose of this study was to evaluate the proliferation status of mononuclear cells infected with Theileria annulata schizonts in different tissues and its relationship with the pathogenesis of the parasite in cattle by histopathology, immuno-histochemistry and polymerase chain reaction (PCR). Blood and tissue samples of eight Holstein cattle that had been lost due to theileriosis and eight healthy slaughtered cattle of the same breed were collected as a control group after necropsy. The piroplasms in the blood smears and the schizonts in the cytoplasm of the lymphocytes and macrophages of the lymph nodes were microscopically detected. Histopathologically, the proliferation of macrophages, lymphocytes, and plasma cells in lymph nodes and the heart, congestion, and bleeding in the red pulp of the spleen, portal tracts of the liver, interstitial tissue of the kidneys, multifocal necrosis and ulceration in the abomasum together with hyperemia and hemorrhages and lymphoblastic infiltration in the submucosa and lamina propria adjacent to these lesions and emphysema with ecchymotic hemorrhage in the lungs were evident. Immunohistochemistry identified the proliferated cells as mostly Cluster of Differentiation 3- Positive T lymphocytes and macrophage marker antibody 387- positive macrophages. Positive results of PCR for the Tams1 30.00 kDa gene were observed in lymph nodes, liver, lung and abomasum. It was concluded that the pathological changes were the result of schizont-infected macrophage proliferation leading to severe uncontrolled proliferation of uninfected T lymphocytes.

Abstract Giardia duodenalis is a protozoan parasite found in humans and several mammals. This parasite spreads worldwide and is generally recognized as a zoonotic agent being reported to be one of the most common causes of diarrhea in humans and animals. In this study, it was aimed to determine the prevalence and genotypes of G. duodenalis in shelter dogs in Diyarbakır province being located in the southeastern Anatolia region of Türkiye. Native-Lugol method and nested polymerase chain reaction analyses of 100 fecal samples showed a prevalence of 3.00 and 4.00%, respectively. The prevalence was higher in females and in those younger than 1 year. Sequence analysis revealed the presence of zoonotic assemblage B, assemblage D and assemblage E. The detection of zoonotic assemblage B in this study suggests that dogs may be a reservoir for human giardiasis. Further molecular research is needed to determine the genotype diversity of Giardia as well as its possible role in the transmission of this parasite to humans.