Exposure to sublethal concentrations of chlorine enhances the cytotoxicity of Acanthamoeba castellanii.

Free-living amoebae belonging to the genus Acanthamoeba are the causative agents of infections in humans and animals. Many studies are being conducted to find effective compounds against amoebae, but their sublethal concentration effects on surviving amoebae seem to have been overlooked. Chlorine is a common disinfection agent commonly added to public water facilities and supplies. In this study, the cytopathic and phagocytic properties of Acanthamoeba castellanii trophozoites following exposure to sublethal concentrations of chlorine were examined. Two hours of exposure to 5 ppm hypochlorite calcium was considered the sublethal concentration for A. castellanii trophozoites. To compare the pathogenic potential of treated and untreated Acanthamoeba trophozoites, cytotoxicity, adhesion assays in RAW 264.7 macrophages, osmo, and thermotolerance tests were carried out. Bacterial uptake was assessed in treated cells to evaluate their phagocytic characteristics. Oxidative stress biomarkers and antioxidant activities were compared in treated and untreated trophozoites. Finally, the mRNA expression of the mannose-binding protein (MBP), cysteine protease 3 (CP3), and serine endopeptidase (SEP) genes was determined in cells. In all the experiments, untreated trophozoites were considered the control. In comparison to untreated trophozoites, in chlorine-treated trophozoites, cytopathic effects were more extensive and resulted in the detachment of macrophage monolayers. Treated trophozoites could not grow at high temperatures (43 °C). Besides, they showed osmotolerance to 0.5 M D-mannitol but not to 1 M. Results demonstrated a higher bacterial uptake rate by chlorine-treated trophozoites than untreated cells. The treated and untreated cells had significantly different glutathione and glutathione/glutathione disulfide ratios. Antioxidant enzyme activities, total antioxidant capacity, and malondialdehyde levels were increased significantly in chlorine-treated cells. Quantifying mRNA expression in chlorine-treated trophozoites revealed that virulence genes were upregulated. Chlorine can form resistance and virulent amoebae if it is not used at a proper concentration and exposure time. Identification of stress responses, their mechanisms in Acanthamoeba, and their relation to amoeba virulence would give us a better perception of their pathophysiology.

First molecular survey of Anaplasma phagocytophilum in hard ticks (Ixodidae) from Southwestern Iran.

Anaplasma phagocytophilum is a gram-negative obligate intracellular tick-borne rickettsia with veterinary and public health importance worldwide. This organism is an etiologic agent of tick-borne fever (TBF) in domesticated animals and human granulocytic anaplasmosis (HGA) as well. Hard ticks (Ixodida: Ixodidae) are incriminated as the main biologic vectors for Anaplasma spp. Studies represent that Ixodes spp. are the main vectors for A. phago-cytophilum and few reports hinted that other tick species may play this role. So, the goal of the presented work was to investigate the A. phagocytophilum in 2000 hard ticks in Khuzestan province of Iran by specific nested-PCR performing two consecutive amplifications of 16SrRNA gene fragment with highly variable nucleotide region. Each reaction included 10 salivary glands of distinct tick species. Specific nested-PCR on accumulated salivary glands detected specific bands in 15.5% of reactions (31 of 200) in electrophoresis only in Rhipicephalus sanguineous and Hyalomma marginatum ticks. We concluded that the broad distribution of A. phagocytophilum infection is not only is due to the existence of Ixodes spp. but other hard ticks may also play a role in this issue.

Effect of natural neosporosis on bull sperm quality.

Neospora is one of the protozoans that can infect the male and female's reproduction system. Despite the existence of N. caninum in the genitalia, its effect on sperm characteristics was not studied yet. Therefore, the aim of this study was to investigate the influence of natural neosporosis on the sperm parameters of bulls. Using 30 bulls with neosporosis diagnosed by modified agglutination test and enzyme-linked immunosorbent assay (ELISA) and 15 healthy bulls, some sperm parameters such as sperm concentration, viability, motility, and morphology were studied and compared. Also, the activity of super oxide dismutase (SOD), glutathione peroxidase (GPX), and malondialdehyde (MDA) level as the biomarker of lipid peroxidation was investigated. Results showed that sperm concentration, viability, and motility were significantly lower in bulls with neosporosis in the present study. There were no significant differences in activities of SOD and MDA level but GPX activity was significantly increased in infected bulls.

FIRST MOLECULAR DETECTION OF ANAPLASMA PHAGOCYTOPHILUM IN DROMEDARIES ( CAMELUS DROMEDARIUS).

Anaplasma phagocytophilum infects a wide variety of wild and domestic animals and causes an emerging zoonotic tick-borne disease. There are no available data regarding the presence of A. phagocytophilum in camels ( Camelus dromedarius). Therefore, the objective of this study was to investigate the prevalence of A. pagocytophilum in Iranian camels. Whole blood of 207 camels from five geographical regions of Iran was tested for A. phagocytophilum using polymerase chain reaction (PCR), nested PCR, and specific nested PCR based on 16S rRNA. The overall prevalence of infection in tested animals was 34.2% (71/207). Sex was not identified as a risk factor for A. phagocytophilum infection, but analysis revealed significant differences in age and region. In conclusion, Iranian camels can be potential reservoirs for A. phagocytophilum, and Iran must be considered an enzootic area for this infection as indicated by the high subclinical infection rate in camels.

Molecular and haematological investigations on Anaplasma ovis infection in goats in Ahvaz, Iran: Insights into infection rate, haemolytic effect and breed influences.

BACKGROUND: Anaplasma ovis (A. ovis) is the predominant causative agent of anaplasmosis in goats and sheep in most tropical and subtropical regions of the world. However, there is considerable variation in reported infection rates, breed susceptibility, and controversial findings regarding the haemolytic effects of A. ovis infection in goats. OBJECTIVES: Thus, we investigated the molecular and haematological aspects of A. ovis infection in goats from Ahvaz city. METHODS: One hundred and fifty apparently healthy goats (74 blacks and 76 Najdi goats) were randomly sampled from six flocks in the Ahvaz suburb during ticks' activity season. Haematological evaluation, smear microscopic (SM) examination and PCR assay were performed to assess A. ovis infection. Additionally, the percentage of parasitemia was determined from blood smears. RESULTS: SM examination revealed that 25.7% of the goats displayed erythrocyte Anaplasma-like inclusion bodies. PCR analysis indicated that 54% of the goats were positive for A. ovis infection (44.6% of blacks and 63.2% of Najdi goats). No significant difference in haematological values was observed between healthy and infected goats based on PCR testing. However, a significant difference in haematological indices was observed between the group with parasitemia level of 0.01-0.02% (SM and PCR positive) compared to the healthy goats (SM and PCR negative), particularly concerning Hb, PCV and RBC count (p < 0.01). CONCLUSIONS: When the parasitemia exceeds 0.01%, A. ovis infection may disrupt haematological parameters in infected goats. The high prevalence of A. ovis infection (54%) among the studied goats underscores the importance of giving special attention to implementing necessary measures for disease control in the Ahvaz suburb.

Inhibitory Effect of Hemiscorpius lepturus Scorpion Venom Fractions on Tachyzoites of Toxoplasma gondii.

Background: The present study determined the effect of the fractions obtained from Hemiscorpius lepturus scorpion venom on the tachyzoite of Toxoplasma gondii. Methods: The fractions of dried venom of He. lepturus scorpion of Khuzestan Province, southern Iran in 2019 were isolated through gel filtration chromatography, and then tachyzoites were exposed to fractions of venom at different concentrations. Trypan blue counting and MTT were applied to assay tachyzoite viability, and the inhibition of the cellular growth of fractions in Vero cells was evaluated. Results: The maximum effect on tachyzoite was observed in fraction 5 of venom. To further separate the protein, fraction 5 was used in high-performance liquid chromatography assay to purify its proteins. Based on the results of HPLC of fraction 5, among which the second peak, a peptide with <10 KDa representing a more potent effect in eliminating the tachyzoite of T. gondii. Conclusion: The scorpion venom-purified fractions possess anti-parasitic activity against the tachyzoite of T. gondii and can be used in parasite-controlling studies.

Occurrence of anti-Toxoplasma gondii antibodies in female cattle in south-west of Iran.

Toxoplasmosis, caused by Toxoplasma gondii, is a significant disease in livestock and humans. In Iran, studies shows that T. gondii infection in humans is relatively high and prevalence is associated mainly with consumption of undercooked meat or meat products. We have examined 450 serum samples from female cattle distributed over all Ahvaz, the center of Khouzestan province, south-west of Iran. IgG antibodies to T. gondii were assayed by the modified agglutination test using whole tachyzoites of T. gondii, and found in 71 (15.77%) of 450 cattle with titers of 1:25 in 38, 1:50 in 18, 1:100 in 11, 1:200 in three and 1:400 in one. Titers of antibodies were decreased in cattle over 2 years old. These results indicate that T. gondii infection in cattle of Khouzestan is relatively considerable, but not very high and consumption of beef may be a source of infection for humans in south-west of Iran.

First Molecular Survey on Anaplasma phagocytophilum Revealed High Prevalence in Rural Dogs from Khuzestan Province, Iran.

BACKGROUND: Anaplasmosis due to Anaplasma phagocytophilum is an important tick-borne zoonotic disease, which affects dogs, horses, cattle and human as well. This study aimed to probe the existence of this organism by means of molecular biology techniques for the first time in rural dogs of Khuzestan province, Southwestern Iran. METHODS: During Sep 2014 to Apr 2015 blood samples of 103 apparently healthy rural dogs (60 males) were collected for A. phagocytophilum detection by light microscopical examination of Giemsa stained slides and Nested PCR on a fragment of 16S rRNA gene. RESULTS: From the examined slides, 11.65% were positive for A. morulae while 57.28% of infection was revealed by Nested PCR method. There was no statistical difference between ages and sexes of dogs and infection in molecular survey of A. phagocytophilum. CONCLUSION: Molecular prevalence of A. phagocytophilum was noticeably high. It may cause the incidence of disease in human population.

Molecular characterization of Theileria equi infection in horse populations belonging to West Azerbaijan, Iran: insights into the importance of Equine Merozoite Antigen (EMA)-1 in its diagnosis

The major agent of equine piroplasmosis (EP), Theileria equi, contributes to significant losses in the equine industry. This study was designed to evaluate T. equi infection among horses from West Azerbaijan by microscopy and molecular approaches. One hundred and twenty six blood samples were collected from the jugular vein and placed in sterile tubes containing EDTA; these tubes were either used immediately for blood smears or stored at ­20°C for later examination by PCR. T. equi was detected in 3.2% and 27.7% of the animals examined using light microscopy and PCR methods, respectively. The prevalence of T. equi was higher in older animals (30.4%) than young equines (24.6%). Also, the females (31%) demonstrated higher T. equi infection rates than the males (23.6%). Additionally, while 12 horses housed with other animals were positive for T. equi, 23 not housed with other animals were found to be infected. No significant difference was found between infection rate and associated risk factors (age, sex, and housing with other animals). The results confirm a relatively high prevalence of T. equi in horses in the study area and also suggest that Equine Merozoite Antigen (EMA)-1 could be a strong candidate to develop diagnostic methods for T. equi infection. Due to the importance of EP in the equine industry, and the ability of animals to be lifelong carriers of T. equi, accurate and early diagnosis of the disease, based on specific antigens, is critical. Diagnosis would provide basic information about its epidemiology, distribution and prevalence, especially in apparently healthy animals, and effective control and vaccine measures.

A seroepidemiological survey of Toxoplasma gondii infection in referred dogs to Veterinary Hospital of Ahvaz, Iran.

Toxoplasma gondii is an intracellular protozoan parasite, which is the cause of toxoplasmosis and can infect a wide variety of warm-blooded animals, including dogs and humans. The present study evaluated the seroprevalence of T. gondii infection in pet dogs in Ahvaz, southwest city of Iran, and investigated the related possible risk factors. A total of 180 serum samples were collected from dogs referred to Veterinary Hospital of Ahvaz. The samples were then tested by indirect enzyme-linked immunosorbent assay. The prevalence of T. gondii antibodies were 46.67%. Logistic regression and chi square tests were used for evaluating of risk factors. The positivity increased statistically significantly with dog's gender (56% females and 39% males, P = 0.001), age (18% in <2 years old, 96% in ≥4 years old, P = 0.001) and place of living (47% outdoor dogs 38% house hold dogs, P = 0.025). However, no statistically significant association was found with dogs' breed, deworming, food ingestion or contact with cats. Overall, the results showed a relatively high seroprevalence of T. gondii infection in dogs in southwest Iran (Ahvaz) and proved association of T. gondii prevalence rates with the dog's age, gender and place of living.

Blood Levels of Oxidant/Antioxidant Parameters in Rats Infected with Toxoplasma gondii.

Toxoplasmosis is a common parasitic infection in the world. Since increased free radicals and oxidative stress are reported in many parasitic diseases the purpose of the present study was to evaluate the oxidative stress in acute and chronic toxoplasmosis. RH strains of Toxoplasma tachyzoites were used in the present study. Twenty-five female rats were infected with the parasite while 25 other rats were as the control group that received normal saline. Zero-, 5-, 7-, 10-, and 45-day postinfection (DPI) blood samples were taken. Some parameters related to oxidant and antioxidants such as antioxidant enzymes, malondialdehyde, and total antioxidant capacity were measured. On day 7 after infection, GPX activity and GSH level were significantly increased and in the mentioned day the amount of total antioxidant capacity was significantly reduced. In other cases, there were no significant differences between the groups in different days. Overall, based on the results it seems that, on day 7 after infection, in infected rats responses to oxidative stress were triggered and led to decrease of total antioxidant capacity. Furthermore, glutathione was increased to cope with stress. It seems that probably antioxidant defense system entered the infection to the chronic phase and changed the parasites stage.

Prevalence of Cryptosporidium spp. in ruminants of Lorestan province, Iran.

Cryptosporidiosis is one of the important zoonotic diseases caused by an intracellular protozoan parasite called Cryptosporidium. This study aimed to investigate the prevalence of Cryptosporidium spp. infection on 1,115 ruminants, cattle, sheep and goats, in Lorestan province, Iran. Using formol-ether concentration technique and modified Ziehl-Neelsen staining method afterwards, the overall prevalence of Cryptosporidium spp. infection in ruminants of Lorestan province was 7.17 %. Prevalence of infection was 9.07 % (39 of 430), 5.80 % (20 of 345) and 6.18 % (21 of 340) for cattle, sheep and goats respectively. There was no significant difference between contamination of all examined animals and different geographical and climatic situations (P > 0.05) and diarrhea was not directly associated with Cryptosporidium infection (P > 0.05). In conclusion, the prevalence of cryptosporidiosis in Lorestan province was relatively low, but it should be noticed that this opportunistic parasite is zoonosis and also can make epidemics in ruminants as well as human population in suitable conditions.

Detection of Sarcocystis spp. in cattle (Bos taurus) and water buffaloes (Bubalus bubalis) in Iran by PCR-RFLP.

Sarcocystis species are cyst-forming intracellular protozoan parasites. Cattle are mainly infected with Sarcocystis cruzi, Sarcocystis hominis and Sarcocystis hirsuta. Water buffaloes are intermediate hosts for Sarcocystis fusiformis, Sarcocystis levinei (S. cruzi-like species), Sarcocystis dubeyi, Sarcocystis sinensis (S. hominis-like species) and Sarcocystis buffalonis (S. hirsuta- like species). The aim of this study was Identification of Sarcocystis spp. in slaughtered cattle and water buffaloes in Ahvaz, Khuzestan province by PCR-restriction fragment length polymorphism. Meat inspection was done on 124 cattle and 147 water buffaloes. From each animal tissue samples (each 50 g) from heart, esophagus, diaphragm and intercostal muscle were collected during meat inspection. Samples examined with digestion method. Genomic DNA of 80 positive samples was extracted and their 18S rRNA gene was amplified. PCR products were digested by restricted enzymes (FokI, SspI and DraI). S. cruzi in cattle and S. fusiformis in water buffaloes were identified. Our study clarified that sarcocystosis in cattle in Ahvaz district may be results acute infection according to determined species, but in buffaloes as S. fusiformis was detected we may expect only economic loss follow up slaughterhouse inspection.

Development of an Indirect ELISA Using Different Fragments of Recombinant Ncgra7 for Detection of Neospora caninum Infection in Cattle and Water Buffalo.

BACKGROUND: Dense granules are immunodominant proteins for the standardization of immunodiagnostic procedures to detect neosporosis. In the presented study different fragment of a dense-granule protein was evaluated for serodiagnosis of Neospora caninum in cattle and water buffalo. METHODS: NcGRA7, from N. caninum tachyzoites was amplified. PCR product and pMAL-c2X plasmid were digested with EcoR1 restriction enzyme and expressed in Escherichia coli to evaluate its competence for detection of anti- N. caninum antibodies with ELISA in comparison with commercial IDEXX ELISA. Furthermore, 230 sera of presumably healthy cattle and water buffaloes (108 cattle and 122 water buffaloes) were analyzed by both tests to determine the agreement of these two procedures. RESULTS: Sensitivities and specificities of NcGRA7-based ELISA were 94.64% and 90.38% respectively using sera of cattle, but were 98.57% and 86.54% in the case of buffaloes respectively. A good correlation between the results of IDEXX ELISA and ELISA based on recombinant NcGRA7 for detecting N. caninum antibodies was appeared. Analyzing by Mc Nemar's showed that NcGRA7-based ELISA has acceptable capability to differentiate the positive results in comparison with IDEXX ELISA. CONCLUSION: NcGRA7-based ELISA considering utilized new fragment of genomic DNA is a good tool for serodiagnosis of anti- N. caninum antibodies for screening and epidemiological purposes on cattle herd and water buffaloes as well.

Molecular detection of Sarcocystis species in slaughtered sheep by PCR-RFLP from south-western of Iran.

Sarcocystis spp. are the cyst forming protozoan parasites that prevalent in livestock all around the world. In the presented work, we examined 40 macroscopic and 40 microscopic sarcocysts from Khouzestan and Lorestan provinces, south-western Iran, utilizing PCR-RFLP based on amplification of 18S rRNA gene. Using AvaI, HindII, TaqI and EcoRI restriction enzymes the results represented Sarcocystis gigantea in both macroscopic and microscopic cysts. This result supports the importance of molecular investigations on characterization of Sarcocystis species when we aimed to assess the reliable control and preventive programs.

Molecular determination and genotyping of Cryptosporidium spp. in fecal and respiratory samples of industrial poultry in Iran.

OBJECTIVE: To identify the genotypes of prevalent Cryptosporidia in broiler chickens in Lorestan province, Iran. METHODS: A total of 1 000 fecal and 1 000 trachea samples were collected from chickens. Smears from both fecal and tracheal samples were stained with modified Ziehl-Neelsen method and nested PCR-RFLP according to amplification of 18S rRNA gene using Ssp I and Vsp I restriction enzymes and DNA sequencing. RESULTS: From the examined chickens 0.7% was positive for Cryptosporidium. Infection was present in 0.5% fecal samples and also in 0.5% trachea. Only 0.3% of simultaneous infections in fecal and tracheal samples were observed. Nested PCR of our isolates demonstrated Cryptosporidium baileyi. CONCLUSIONS: In our work, low rate of Cryptosporidium baileyi infection was detected, but in critical situations and our poor management circumstances, cryptosporidiosis occurs in serious feature especially in immune suppressed individuals.

Cloning and expression of Neospora caninum dense-granule 7 in E. coli.

Neospora caninum is an obligate intracellular protozoan which induces abortion, still birth and neuromuscular disorders in cattle and is an important problem in dairy and beef industry worldwide. The dense granule protein 7 (GRA7) of N. caninum is an immune-dominant protein shared by both tachyzoite and bradyzoites. This study was conducted to produce recombinant GRA7 of N. caninum using a plasmid with high level of expression of this protein in E. coli. For this purpose, a segment of N. caninum DNA corresponding to GRA7 was amplified using PCR. After sequencing, this fragment was cloned into expression vector pMAL-c2X under the control of the lac promoter. Expression of this plasmid in E. coli strain TG1 was identified by western blotting. In this study, pMAL-c2X had a strong promoter to produce high level expression of NcGRA7. This result revealed that this recombinant protein with pMAL-c2X vector may be suitable for developing of diagnostic procedures.

Development and Standardization of Dot - ELISA for Detection of Neospora caninum Antibodies in Cattle and Comparison with Standard Indirect ELISA and Direct Agglutination Test (DAT).

BACKGROUND: Neospora caninum is a protozoan parasite from phylum apicomplexa and an important agent causing abortion in cattle which produce notable economic loss all around the world. METHODS: Dot-Elisa was set up performing checker board procedure and then 178 sera of cattle examined with commercial indirect ELISA and direct agglutination test (DAT) were also evaluated by dot-ELISA afterwards. RESULTS: Kappa statistical analysis revealed that Dot-ELISA has good agreements with ELISA as well as the DAT and also, Mc Nemar's analyzing showed that this procedure has acceptable ability to discriminate positive results. Relative sensitivity and specificity of Dot-ELISA were respectively 92.63% and 89.16% and 93.4% and 90.8% in comparison with ELISA and DAT. CONCLUSION: Since the dot-ELISA is easy, inexpensive and not needed high experience to interpret the results, it is superior to ELISA and DAT when we aim to screen the cattle on the farm and slaughterhouses or when the laboratory equipment is not available.

Prevalence and distribution patterns of Sarcocystis in camels (Camelus dromedarius) in Yazd province, Iran.

Sarcocystis spp., are zoonotic cyst-forming coccidian parasites that cause sarcocystosis. This study was conducted to investigate the prevalence as well as distribution patterns of Sarcocystis spp. infection in slaughtered one-humped camels of Yazd province, Iran. Muscles of 130 camels were investigated for either macroscopic or microscopic sarcocysts during summer 2009. No macroscopic cyst was observed in the animals at naked eye inspection. Of examined camels, 67 (51.5 %) were positive for bradyzoites of the parasite by pepsin-digestion method. The infection rates of infected animals were 55.22, 50.75, 38.81, 34.33, and 28.36 %, in esophagus, heart, masseter muscle, intercostal muscle and limb muscle, respectively. Esophagus was the most commonly infected organ. No significant difference in the rate of infection between male (52.08 %) and female (51.22 %) camels was observed. Logistic regression analyses showed that infection rates' risk increased with increment in age of camels. This considerable prevalence of microscopic Sarcocystis spp. in Yazd province camels reflects a significant role played by wild and domestic carnivores in the transmission of these parasites.

Seroprevalence of Toxoplasma gondii in water buffaloes (Bubalus bubalis) in South-West of Iran.

The prevalence of antibodies to Toxoplasma gondii was conducted in 300 buffaloes from Ahvaz, Kouzestan province, southwest of Iran. Blood sera were screened using a Modified agglutination test (MAT) incorporating 2-mercaptoethanol. Positive reactions in sera dilutions above 1:25 were considered as indicative for the presence of T. gondii antibodies. The overall prevalence of infection in the animals was 14.33% with titers of 1:25 in 21, 1:50 in 12, 1:100 in 6, 1:200 in 2 and 1:400 in 2. The prevalence was different in relation to the sex with buffaloes with 19.7% and 7% in females and males respectively. These results indicate that T. gondii infection in water buffaloes of Khouzestan is relatively high and consumption of buffalo meat may be a risk factor for humans in Ahvaz, southwest of Iran.