Isolation of Free-Living Amoebae from Sarein Hot Springs in Ardebil Province,Iran

Background

Free-living amoebae (FLA) are a group of ubiquitous protozoan, which are distributed in the natural and artificial environment sources. The main aim of the current study was to identify the presence of FLA in the recreational hot springs of Sarein in Ardebil Province of Iran.

Methods

Seven recreational hot springs were selected in Sarein City and 28 water samples (four from each hot spring) were collected using 500 ml sterile plastic bottles during three month. Filtration of water samples was performed, and culture was done in non-nutrient agar medium enriched with Escherichia coli. Identification of the FLA was based on morphological criteria of cysts and trophozoites. Genotype identification of Acanthamoeba positive samples were also performed using sequencing based method.

Results

Overall, 12 out of 28 (42.9%) samples were positive for FLA which Acanthamoeba and Vahlkampfiid amoebae were found in one (3.6%) and 11 (39.3%) samples, respectively. Sequence analysis of the single isolate of Acanthamoeba revealed potentially pathogenic T₄ genotype corresponding to A. castellanii.

Conclusion

Contamination of hot springs to FLA, such as Acanthamoeba T₄ genotype (A. castellanii) and Vahlkampfiid amoebae, could present a sanitary risk for high risk people, and health authorities must be aware of FLA presence.     

Seroprevalence of Acanthamoeba Antibodies in Rheumatoid Arthritis Patients by IFAT,Tehran, Iran 2007

Background

This preliminary study was conducted to discriminate the prevalence of Acanthamoeba antibodies in rheumatoid arthritis (RA) patients and healthy controls to analyze the correlation between these two groups.

Methods

From October 2006 to August 2007 a total of 121 serum samples from RA patients attending the Rheumatolgy Department at Shariati Hospital in Tehran were obtained and stored at -20°C until using by indirect fluorescent-antibody test (IFAT). RA was diagnosed according to the American Collage of Rheumatology classification criteria. The organism used in this study was isolated from various water resources in Tehran, Iran cultured axenically and then went on a PCR assay based on 18S rRNA to identify the genus Acanthomoeba. Indirect immunofluorescence antibody (IFA) staining of serum samples was carried out to detect anti Acanthomoeba antibodies.

Results

In culture, out of 22 samples, 13(59%) were grown in xenic but only two in axenic medium. PCR amplified a 904bp fragment, specific for Acanthamoeba. Of examined serum samples, Acanthamoeba antibodies were present in 70 (57.8%) and 52 (41.2%), respectively. The highest titer of antibodies (1:320) was detected in one patient with RA.

Conclusion

Our study supports the hypothesis that some parasitic microorganisms can involve and contribute toward the development of rheumatoid syndromes.     

Axenic Cultivation and Pathogenic Assays of Acanthamoeba Strains Using Physical Parameters

Background

The main goal of the present study was to set up an axenic cultivation of Acanthamoeba and assess the pathogenic ability of T4 genotypes from different clinical and environmental strains of Acanthamoeba using two physical assays.

Methods

Sixteen Acanthamoeba isolates including 10 environmental and 6 clinical strains were cultured axenically. Axenic cultivation was performed using Proteosepepton, yeast extract and glucose medium and TY-I-S33culture. Pathogenic survey was done using osmotolerance and thermotolerance assay. Briefly, differentosmolarity (0.5 M and 1 M) of non-nutrient agar plates were performed. One hundred fifty µl of axenic culture were collected and were inoculated in 1% agar medium. For thermotolerance assay 150 µl of amoebas from axenic culture were divided into fresh culture mediums. Cultures were incubated at 37°C and 42 °C. All plates were monitored for 24 h, 48 h and 72 h.

Results

Overall, 16 strains of Acanthamoeba isolates previously genotyped as T4 were cultivated axenically after several months. Thermotolerance and osmotolerance assay revealed that all of clinical strains, soil and animal feces strains were highly pathogenic isolates. Two dust and water strains did not grow at high temperature (42 °C) and osmolarity (1.5 M) and thus they were classified as weak pathogens.

Conclusion

Most of T4 genotypes are highly pathogenic organisms. This is an important finding since Acanthamoeba belonging to T4 type is the predominate genotype in environmental and clinical samples. The presence of highly pathogenic Acanthamoeba may pose a risk within susceptible people.     

Acanthamoeba species in Swimming Pools of Cairo,Egypt

Background

The free-living amoebae Acanthamoeba spp. have been recognized as etiologic agents of amoebic encephalitis, keratitis, otitis, lung lesions and other skin infections mainly in immuno-compromised individuals. The purpose of this study is to detect the presence of Acanthamoeba in swimming pools in Egypt using a polymerase chain reaction (PCR) method.

Methods

Water samples were collected from 10 different swimming pools in Cairo, Egypt. Samples were cultured on non-nutrient agar for the detection of Acanthamoeba isolates that were confirmed by PCR amplification using genus specific primers. The molecularly confirmed Acanthamoeba isolates were morphologically identified to the species level.

Results

Members of genus Acanthamoeba were detected in 49.2% of the examined swimming-pool water samples. Morphologically, six Acanthamoeba species were isolated from the examined swimming pool water namely A. polyphaga, A.castellanii, A. rhysodes, A. mauritaniensis, A. royreba and A. triangularis. All the identified species of Acanthamoeba were molecularly confirmed to be related to the genus Acanthamoeba.

Conclusion

The isolated species of Acanthamoeba could provoke variable degrees of infections to the swimmers. The culture method is cheaper and easier than PCR techniques that are faster for the detection of free-living amoebae     

Morpho-Physiological and Biochemical Criteria of Acanthamoeba spp. Isolated from the Egyptian Aquatic Environment

Background

The free-living amoebae Acanthamoeba spp., have been recognized as etiologic agents of amoebic encephalitis, keratitis, otitis, lung lesions and other skin infections mainly in immuno-compromised individuals. In this study, morpho-physiological and biochemical characterization of Acanthamoeba strains isolated from the Egyptian aquatic environment were surveyed.

Methods

Some Acanthamoeba species were cultivated on non-nutrient agar. Isolated strains of Acanthamoeba were identification based on the morphology of trophic and cyst forms in addition to temperature and osmo-tolerance assays. Biochemical characterization of the isolated amoeba strains was performed using quantitative assay as well as qualitative determination of proteolytic activity in zymograph analysis.

Results

Potentially pathogenic Acanthamoeba species were isolated from all of the examined water sources. Colorimetric assays showed protease activity in the heat-tolerant isolates of Acanthamoeba. All pathogenic isolates of Acanthamoeba exhibited higher protease activity than did the non-pathogenic ones. The zymographic protease assays showed various banding patterns for different strains of Acanthamoeba.

Conclusion

The incidence and prevalence of the pathogenic Acanthamoeba species in the aquatic environment using parasitological and biochemical diagnostic tools will provide baseline data against which the risk factors associated with waterborne transmission can be identified.     

Current Status of Acanthamoeba in Iran: A Narrative Review Article

Background:

Free-living amoebae belonging to the genus Acanthamoeba have an environmental distribution. Amoebic keratitis due to these protozoan parasites continue to rise in Iran and worldwide. In Iran, there are various researches regarding both morphological and molecular identification of Acanthamoeba spp. in environmental and clinical samples. However, there is no thorough review about Acanthamoeba genotypes and their distribution in environmental sources such as water, dust and biofilm in Iran. Besides, according to increasing cases of Amoebic keratitis in the region awareness regarding the pathogenic potential of these sight-threatening amoebae is of utmost importance.

Methods:

We conducted a thorough review based on the database sources such as MEDLINE, PubMed and Google scholar. No restrictions were placed on study date, study design or language of publication. We searched all valuable and relevant information considering the occurrence of the Acanthamoeba in both environmental and clinical samples.

Results:

According to our thorough review Acanthamoeba belonging to T4 genotype is the most prevalent type strain in environmental and clinical samples in several regions in Iran and worldwide, however, there are reports regarding Acanthamoeba belonging to other genotypes such as T2, T3, T5, T6 and T11 and the mentioned point could leads us to more researches with the goal of presenting the real genotype dominance of Acanthamoeba and related disease in the country.

Conclusion:

Overall, the present review will focus on present status of genotypes of Acanthamoeba in Iran during recent years.     

Occurrence of Potentially Pathogenic Bacterial-Endosymbionts in Acanthamoeba Spp.

Background:

Acanthamoeba- bacteria interactions enable pathogenic bacteria to tolerate harsh conditions and lead to transmission to the susceptible host. The present study was aimed to address the presence of bacterial endosymbionts of Acanthamoeba isolated from recreational water sources of Tehran, Iran. To the best of our knowledge this is the first study regarding occurrence of bacteria in environmental Acanthamoeba spp. in Iran.

Methods:

A total of 75 samples of recreational water sources were collected. Samples were cultured on non- nutrient agar 1.5% plates. Positive Acanthamoeba spp. were axenically grown. DNA extraction and PCR reaction was performed using JDP1-2 primers. All positive samples of Acanthamoeba were examined for the presence of endosymbionts using staining and molecular methods. The PCR products were then sequenced in order to determine the genotypes of Acanthamoeba and bacteria genera.

Results:

Out of 75 samples, 16 (21.3%) plates were positive for Acanthamoeba according to the morphological criteria. Molecular analysis revealed that Acanthamoeba belonged to T4 and T5 genotypes. Five isolates (35.7%) were positive for bacterial endosymbionts using staining method and PCR test. Sequencing of PCR products confirmed the presence of Pseudomonas aeruginosa and Agrobacterium tumefasiens.

Conclusion:

The presence of Acanthamoeba bearing pathogenic endosymbionts in water sources leads us to public health issues including improved sanitation and decontamination measures in recreational water sources in order to prevent amoebae-related infection. To the best of our knowledge this is the first report regarding the isolation of A. tumefasiens from Acanthamoeba in Iran and worldwide.     

Prevalence of Toxocara Spp. eggs in Public Parks in Tehran City,Iran

Background

The objective of the present research was to determine the frequency of Toxocara spp. eggs in soil samples of public parks, in the city of Tehran, Iran.

Methods

A total of 600 soil samples were taken from 120 parks between Aprils to November, 2008. Soil samples were collected from 5 distinct sites in the parks. The samples were washed with saline solution and the collected sediment from each park were equally divided and examined by floatation and Petri dish methods for Toxocara eggs.

Results

Ten percent were contaminated with Toxocara spp. eggs. The number of observed Toxocara eggs in each microscopic field was varied from 1-3. No significant differences were observed between floatation and Petri dish methods.

Conclusion

Our public parks showed a high risk of toxocariasis and the need for preventive studies.     

Molecular Detection and Identification of Theileria Species by PCR-RFLP Method in Sheep from Ahvaz,Southern Iran

Background

The present study was carried out to investigate the accurate status of ovine Theileria infection in sheep from Ahvaz and surrounding region, a tropical area southwest Iran.

Methods

A PCR-RFLP method based on 18S ribosomal RNA gene was designed which could detect and differentiate Theileria and Babesia spp. and also differentiate main Theileria species in sheep at the same time. 119 sheep blood samples were collected from Ahvaz and surroundings.

Results

Microscopic examination of blood smears revealed 69.7% (83/119) infection with Theileria spp. Of the total samples subjected to PCR, 89% (106/119) were found to be positive, all of which were identified as Theileria by RFLP analysis using enzyme Hind II. In enzymatic digestion of PCR products by Vsp I, 91.5% (97/106) of Theileria positive samples were identified as T. ovis while mixed Theileria infections were found in 9 samples. The samples with mixed infections were analyzed with an additional nested PCR-RFLP method, by HpaII enzyme digestion. 3 samples with T. lestoquardi infection, 1 sample with T. ovis and T. annulata, 1 sample with T. lestoquardi and T. annulata, and 4 samples with T. ovis, T. lestoquardi and T. annulata mixed infections were detected.

Conclusion

Ovine theileriosis caused by T. ovis is highly prevalent in southwest Iran while T. lestoquardi and T. annulata infection can be detected in a lesser propor-tion of sheep in this region. The new PCR-RFLP method that was designed in this study, can serve as a beneficial diagnostic tool, especially in T. ovis prevalent re-gions.     

Prevalence of Giardia duodenalis Infection in Household Cats of Ahvaz District,South-West of Iran

Background

The occurrence of Giardia duodenalis in cats is of potential significance from both clinical and public health perspectives. The object of this study was antigenic detection of G. duodenalis in household cats of Ahvaz district, South-West of Iran.

Methods

The prevalence of G. duodenalis was determined in fecal samples by two techniques: centrifugation-flotation and a commercial Giardia Antigen Test Kit (immunochromatography assay) in 150 household cats of different ages among referred cases to Veterinary Hospital of Ahvaz University from January 2008 to February 2010.

Results

Five out of 150 fecal samples (3.33%) were positive for antigen of G. duodenalis by immunochromatography assay. The prevalence was significantly higher in young cats less than 6 months (15.79%) compared with adult cats 6 months – 3 years (1.37%) (P=0.027) and above 3 years (1.72%) (P=0.044). The infection had more prevalence in diarrheic cats (17.39%) compared with non-diarrheic cats (0.79%) and the difference was significant (P=0.02) as well. The prevalence was higher in male cats (3.41%) than females (3.23%) and in the season of autumn (6.06%), but the difference was not significant between the prevalence of infection relative to host gender and season (P>0.05). Microscopy examination on fecal samples showed that 2% of the studied cats were positive.

Conclusion

The parasite antigen was present as a zoonotic infection in Ahvaz district, South-west of Iran. More sensitive techniques, such as immunochromatography assay, might yield more reliable results, in the detection of low levels of Giardia in fecal samples of cats.     

Detection of Acanthamoeba and Toxoplasma in River Water Samples by Molecular Methods in Iran

Background:

Free-living amoebae such as Acanthamoeba species may act as carriers of Cryptosporidium and Toxoplasma oocysts, thus, may play an important role in the water-borne transmission of these parasites. In the present study, a loop mediated isothermal amplification (LAMP) method for detection of Toxoplasma and a PCR assay were developed for investigation of Acanthamoeba in environmental water samples.

Methods:

A total of 34 samples were collected from the surface water in Guilan Province. Water samples were filtrated with membrane filters and followed by DNA extraction. PCR and LAMP methods used for detection of the protozoan parasites Acanthamoeba and Toxoplasma respectively.

Results:

Totally 30 and 2 of 34 samples were positive for Acanthamoeba and Toxoplasma oocysts respectively. Two samples were positive for both investigated parasites.

Conclusion:

The investigated water supplies, are contaminated by Toxoplasma and Acanthamoeba (oo)cystes. Acanthamoeba may play an important role in water-borne transmission of Toxoplasma in the study area. For the first time in Iran, protocol of LAMP method was used effectively for the detection of Toxoplasma in surface water samples in Iran.     

Detection and Molecular Characterization of Cryptosporidium species in Recreational Waters of Chaharmahal va Bakhtiyari Province of Iran using nested-PCR-RFLP

Background

The aim of this study was to detect and characterize Cryptosporidium spp. in water samples collected from recreational ponds of Chaharmahal va Bakhtiyari Province of Iran.

Methods

Thirty water samples were collected from November 2009 to May 2010. Each sample contained 10 liters of water. We used the SSU rRNA-based PCR-RFLP technique.

Results

Out of thirty samples examined, 6 (20%) were positive for different Cryptosporidium spp. Restriction pattern analysis showed that C. parvum has been the most prevalent genotype, followed by C. hominis and C. canis, respectively. In this area, the higher prevalence of C. parvum compared with other genotypes is consistent with the distribution of cattle.

Conclusion

Farm animals, particularly cattle are the main source of cryptosporidial contamination for recreational waters in this area.     

Pathogenic Assays of Acanthamoeba Belonging to the T4 Genotype

Background

Acanthamoeba genus is introduced as opportunistic and cosmopolitan parasite. Monkey and wistar rat are appropriate models for experimental study on Acanthamoeba infection. In this study Acanthamoeba spp. were isolated from hot spring (HS), windows dust (WD) and a corneal sample of keratitis patient (KP) and their pathogenicity surveyed by in vitro and in vivo tests.

Methods

Isolates of Acanthamoeba were cultivated axenically for 12 months in PYG medium. Overall, 30 wistar rats, in 6 equal groups were used for developing experimental Acanthamoeba keratitis (AK) and Granulomatous Amoebic Encephalitis (GAE). The Keratitis and Granulomatous Encephalitis experiments were performed by intrastromal and intranasal inoculation of Acanthamoeba cysts, respectively. Pathogenicity of the three isolates was also evaluated by in vitro test using osmotolerance and temperature tolerance assays. Identification of genotypes were performed by PCR technique and sequencing.

Result

None of the isolates could perform AK and GAE in wistar rats, although all isolates were described as T4 genotype. Isolates obtained from KP and WD could grow only in 30 °C, but not in 37 °C and 40 °C. On the other hand, HS isolate grew in 30 °C and 37 °C but not in 40 °C. Moreover, all of isolate grew in 0.5 M mannitol but not in 1 M and 1.5 M.

Conclusion

T4 isolates with a long-term axenic culture and different factors related to host and parasite may play role in pathogenicity of these free-living amoebae.     

Potentially Pathogenic Free-Living Amoebae in Contact Lenses of the Asymptomatic Contact Lens Wearers

Background

Free-living amoebae (FLA) including Acanthamoeba spp. and Hartmannella spp. are the causative agents of serious corneal infection especially within contact lens wearers. Thus contact lenses and their storage case could be a suitable niche for potentially pathogenic amoebae. The main objective of the present study was to evaluate the contamination of contact lenses to free living amoebae using morphological and sequencing based methods.

Methods

Overall, 90 volunteers provided their contact lenses. All volunteers wore soft contact lenses. Both lenses were cultured in the same plate. Forty-eight of the volunteers were medical and dentistry student and 42 were ophthalmology attendees of hospitals in Tehran, Iran. All of the samples were inoculated to non-nutrient medium and monitored daily for the outgrowth of the amoebae. PCR and sequencing were performed using various primer pairs.

Results

Of the 90 volunteers, 9 (10%) were positive for free-living amoebae outgrowth. Morphological analysis revealed that 3 isolates were belonged to Hartmannella genus according to small round cysts and 6 isolates were belonged to Acanthamoeba genus based on the star shape of endocysts. Sequencing revealed that Acanthamoeba belonged to T4, T3 and T5 genotype. Hartmannella were also belonged to vermiformis species.

Discussion

The presence of potentially pathogenic free living amoebae including Acanthamoeba and Hartmannella could be a high risk for people using soft contact lenses. These results revealed that improved clarification and professional recommendations for contact lens wearers is of utmost importance.     

The Prevalence of Cryptosporidium spp. in Children,Taiz District,Yemen

Background

This is the first work done on cryptosporidiosis among the children in Taiz, Yemen.

Methods

A number of 712 samples were collected from children of different ages (ranging from 1 month to 12 years) from Dec 2006 to Aug 2007. The collected samples were examined by Sheather''s sugar floatation and Modified Ziehl- Neelsen stain as well as ELISA methods. The test results were statistically analyzed by SPSS software.

Results

The overall positive percentage was 43.7%. The higher incidence (36.2%) was occurred in males while the lowest incidence (32.7%) was observed in females (r=0.876; P=0.001). The correlation between infected cases and the type of drinking water was r =0.121. Among the cases examined by ELISA (92 cases), 26.1% were infected. The correlation between seropositivity and gender was r=0.652 (P=0.031).

Conclusion

Cryptosporidium spp. is a significant pathogen among children at Taiz. Fresh water supplies, education, eating habits and domestic animals are considered the main sources for transmission of cryptosporidiosis.     

A Preliminary Parasitological Survey of Hepatozoon Spp. Infection in Dogs in Mashhad,Iran

Background

We attempted to determine the prevalence of Hepatozoon spp. infection in Mashhad, northeast of Iran, via blood smear parasitology.

Methods

The prevalence was investigated by examination of blood smear parasitology, using blood samples collected from 254 dogs (51 strays and 203 privately owned-dogs).

Results

Two stray dogs (2/51; 3.92%) and two privately-owned dogs (2/203; 0.98%) were infected with Hepatozoon spp. Therefore, as per blood smear parasitology, the prevalence of Hepatozoon spp. infection was 1.57% (4/254). Sixteen out of 254 dogs (6.29%) were infested with ticks; all of which were Rhipicephalus sanguineus. One of the dogs infected with Hepatozoon spp. exhibited ticks at the time of examination. Concurrent infection with Ehrlichia canis and Leishmania infantum was not detected in the four Hepatozoon spp. infected dogs.

Conclusion

This is the first epidemiological study on the prevalence of Hepatozoon spp. infection in dogs in Iran.     

First Report of Vannellidae Amoebae (Vannella Spp.) Isolated From Biofilm Source

Background

Members of the Vannellidae family are free-living amoebae (FLA) distributed mainly in water and soil sources. The present study reports the first isolation of this genus in the biofilm source from hospital environment in Tehran, Iran.

Methods

Biofilm samples were collected from hospital environment. Cultivation was performed in non-nutrient agar covered with a heat-killed Escherichia coli. Cloning of the suspected amoebae was done. PCR amplification and Homology analysis using the Basic Local Alignment Search Tool (BLASTn) was performed to search for the most similar reference sequences.

Results

Microscopic examination showed numerous fan-shaped amoebae and peculiar cysts different to the usual shape of typical FLA. Sequence analysis of the PCR- product revealed that the suspected amoebae are highly homologous with Vannella spp. gene (99% identity and 100% query coverage) available in the gene bank database.

Conclusion

Although Vannella spp. is not proved to be pathogenic itself, but they are capable of harboring pathogenic intracellular organisms such as Microsporidian parasites. Thus, identification of such amoebae can be of clinical importance, as they could lead to transmission of other pathogens to human.     

Prevalence of Linguatula serrata Nymphs in Mesenteric Lymph Nodes of Cattle and Buffaloes Slaughtered in Ahvaz Abattoir,Iran

Background

Linguatula serrata, one of the parasitic zoonoses, inhabits the canine respiratory system (final hosts). The objective of this study was to determine the prevalence rate of L. serrata nymphs in mesenteric lymph nodes (MLNs) of cattle and buffaloes (intermediate hosts) that were processed in the Ahvaz, Iran abattoir.

Methods

During November 2010 to March 2011, 223 animals (119 cattle and 104 buffaloes), in different sex and three age groups (<2, 2–< 3 and 3-> 3 years old) were sampled randomly at Ahvaz abattoir. Up to 35 grams of their mesenteric lymph nodes were examined separately for nymphal stages of L. serrata by digesting the samples with acid- pepsin method, collected the nymphs and counted under stereomicroscope.

Results

Overall 37(16.6%) of 223 animals were infected with L. serrata nymphs in their mesenteric lymph nodes. Prevalence of the infection in cattle and buffaloes were 16.8% and 16.3% respectively. The number of collected nymphs of MLNs was ranged from 1 to 16. No significant differences were seen in the infection rates between males and females (sexes) and age groups in the cattle and buffaloes (P <0.05).

Conclusion

Linguatula serrata has an active life cycle in the studied area and a zoonotic potential for transmission between animal and human. Avoiding use of raw MLNs to dogs can help reduce the infection.     

Prevalence and Molecular Identification of Cryptosporidium Spp. in Pre-Weaned Dairy Calves in Mashhad Area,Khorasan Razavi Province,Iran

Background

Cryptosporidium parvum is a zoonotic pathogen transmissible from a variety of animals to humans and is a considerable public health concern. Dairy cattle have been identified in numerous reports as a major source of environmental contamination with this pathogen. The aim of study was to detect and isolate the Cryptosporidium spp. from fecal samples of naturally infected pre-wean calves in the Mashhad area

Methods

Overall, 300 fecal specimens from 1 to 30 days pre-weaned calves were collected from 10 farms in the Mashhad area the capital center of the Khorasan Razavi Province, Iran and microscopically examined for Cryptosporidium spp. All infected samples were also analyzed using nested –PCR. A polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analysis of the small-subunit (SSU) rRNA gene was also used to detect and identify Cryptosporidium spp. in PCR- positive samples.

Results

Eighty five (28.3%) of the specimens were positive for Cryptosporidium spp. The prevalence of Cryptosporidium spp. in 8-14 days old and diarrheic calves were significantly higher than other groups. Restriction digestion of the PCR products by SspI, VspI restriction enzymes and sequence analysis revealed the presence of C. parvum bovine genotype in all isolates.

Conclusions

Our results suggest that pre-weaned calves are likely to be an important reservoir of zoonotic C. parvum.