Prevalence and genotyping of Toxoplasma gondii in stray cats in Mashhad area, Iran.

DOI: https://doi.org/10.21203/rs.2.14736/v2

Abstract

Background: Cats as a definitive host have an important role in the epidemiology of toxoplasmosis in humans and animals. The aim of the study was to determine the frequency of Toxoplasma gondii infection and isolate and identify the genotypes of T. gondii in stray cats in Mashhad suburb. Methods: From April 2016 to August 2017, 175 fecal samples from stray cats and 31 brain samples from cats killed in driving accidents were collected. The fecal samples were examined by fecal flotation technique and T. gondii-specific PCR. The brain samples were investigated by T. gondii-specific PCR and consequently examined by mice bioassay. The DNA of T. gondii isolated was genotyped using SAG1, SAG2, SAG3, BTUB and GRA6 as PCR-restriction fragment length polymorphism (PCR-RFLP) markers. Results: In the present study, Toxoplasma-like oocysts were microscopically observed in 2.2% (4/175) fecal samples. The presence of Toxoplasma oocysts was confirmed in one microscopy-positive sample by PCR. In addition, T. gondii DNA was detected in 4% (7/175) microscopy-negative samples using PCR.  T. gondii was isolated from one brain PCR-positive sample by mice bioassay. The isolate was avirulent and many T. gondii cysts were observed in mice brain. The isolate was successfully genotyped by PCR-RLFP analysis .The isolated genotyped was type II.  Beside, eight Toxoplasma-positive fecal samples contained insufficient DNA and only amplified at SAG-3 locus in PCR. These samples were also showed type II pattern at this locus. Conclusions: Parasitological and molecular results showed low frequency of Toxoplasma infection in the stray cats, and identified the genotype of T. gondii isolate as type II, for the first time in Mashhad area, Khorasan Razavi Province.

Background

Toxoplasmosis is considered an important zoonotic disease caused by T. gondii, an obligate intracellular protozoan [1]. Sexual stage develops only in cat and other felids as the definitive hosts that excrete heavy walled oocysts in feces. It typically occurs in humans and other warm-blooded animals as intermediated hosts tachyzoites are formed first, followed by the formation of tissue cysts. T. gondii infection is also transmitted by different routes in humans and animals. Humans acquire Toxoplasma infection by eating undercooked or raw meat containing viable tissue cysts, or by direct ingesting of sporulated oocysts and or by congenital route [1, 2].

A large proportion of T. gondii infection is asymptomatic in humans, but may lead to acute and fatal toxoplasmosis in immunocompromised patients [3]. Congenital toxoplasmosis can cause abortion, stillbirths or fetal death [4]. The severity of toxoplasmosis is associated with genetics and immunity of host and Toxoplasma strains [1].

Based on the virulence levels of Toxoplasma strains in outbred mice, strains were classified into three genotypes: I, II and III [5]. Multilocus PCR-restriction fragment length polymorphism (PCR-RFLP) , microsatellite DNA analysis and multilocus DNA sequence typing of intron methods have been used to determine the T. gondii genotype in many studies [6, 7]. More genotyping studies used multilocus PCR-RLFP analysis of five to ten markers. Among these markers, SAG1, SAG2, SAG3, BTUB, GRA6 could clearly differentiate different genotypes by using nested PCR reactions followed by endonuclease digestion [8-11]. So far, many Toxoplasma types were identified that were genetically different with classical types and some have been categorized under unclonal genotypes [9, 12]. An infected cat as the definitive host may shed one billion oocysts during primary infection and have the main role in the epidemiology of toxoplasmosis. [1]. Many seroepidemiological studies have been performed on T. gondii infection in humans and animals in Iran [13]. The overall seroprevalence of Toxoplasma infection was estimated to be 22–86% in cats [13]. Despite a high seroprevalence of T. gondii in cats in Iran, there are few studies on genetic characterization of T. gondii isolates in cats. The present study was designed to determine the occurrence of T. gondii in cat feces and to isolate and identify T. gondii genotype by using mouse bioassay and PCR-RFLP.

Results

A total of 175 fecal samples, low number Toxoplasma-like oocysts with a diameter 9–12 µm, were microscopically observed in 2.2% (4/175) of fecal samples ,whereas, T. gondii DNA was detected in 4/5% ( 8/175) of fecal samples by nested-PCR. One infected fecal sample with Toxoplasma-like oocysts was positive only by nested-PCR. No significant statistical differences were identified between the prevalence of T. gondii infection in different age and gender groups of stray cats (Table 1) (p > 0.05). The DNA of T. gondii was detected in 3.2% (1/31) of the brain samples and 6.8% (2/31) fecal samples of dead cats by PCR. All brain samples were examined by mice bioassay, T. gondii was isolated only from the PCR-positive brain sample. Poor agreement was observed between parasitological and PCR methods (Kappa=0. 0.127).

Many T. gondii tissue cysts were microscopically observed at 6 wk PI in the brain smears of inoculated mice. The size of cysts range was 7–22 µm. The course of infection was without symptoms in all infected mice, thus indicating the isolation of an avirulent (murine) strain. The five multilocus PCR-RFLP analyses revealed that the isolate of brain mice gave restriction digest patterns consistent with infection with type II. Eight Toxoplasma-positive fecal samples were also genotyped using PCR-RLFP analysis. These samples contained insufficient DNA Toxoplasma genotyping and only amplified at SAG-3 locus in PCR. The type II pattern was also observed at this marker. The amplified B1 genes of the Toxoplasma isolate was sequenced and deposited in GenBank (NCBI) under accession no of MH673033.

Discussion

In the current study, T. gondii-like oocysts were microscopically detected in 2.2% (4/175) of fecal samples. The presence of Toxoplasma oocysts was confirmed in one microscopy-positive sample by PCR. Other samples may be infected with other T. gondii-like oocysts such as Hammondia spp. In the present study, no T. gondii-like oocysts were detected in 4% (7/175) of PCR positive samples. This result may be due to consumption of meat contaminated with Toxoplasma cysts or due to the small number of Toxoplasma oocysts in fecal specimens which are difficult to determine by fecal examination.  Similar to our results , a low prevalence of oocysts shedding in cats was determined at 1.2% in Iran [16], at 2.3% in Italy[17], at 0.3% in Japan [18] , at 0.14 % in Germany [19], at 0.4% in Switzerland [20], at 4.7% in South Korea [21], at 0.9 in the USA [22], at 0.8% in Thailand [23], at 0.76 % in Finland [24] by microscopy and PCR methods. In contrast to serological studies, examining feces did not show any information about age and gender as risk factors, due to the low prevalence of oocyst shedding by the cats [13, 25].

Toxoplasma gondii was isolated from the brain of a PCR-positive stray cat by mice bioassay. All inoculated mice survived and developed antibodies against T. gondii until sacrifice time, thus indicating the isolate belongs to avirulent strain [5, 26, 27]. In the previous study, a non-virulent strain was isolated from aborted ovine fetuses in Mashhad, Iran, by bioassay method [28].

The PCR-RFLP assay at five markers revealed that the avirulent isolate in this study belongs to type II clonal lineage. These five markers have been successfully used in T. gondii genotypes in cats in previous studies and proved to identify the genotype of isolates in cats [29-31]. Although, ten genetic markers allow isolates with high resolution [32]. In Iran, types II and III isolates have been detected by RLFP analysis at GRA6 locus [33, 34] and type I and III by RLFP analysis at SAG 2 locus in stray cats [35]. In addition, type II has been detected in humans, sheep and birds by PCR- RLFP assay at GRA6 and in wild rats by SAG 1 locus [36, 37]. However, a single marker was used to genotype Toxoplasma in these studies but it does not allow identification of nonclonal strains, and to determine more precisely the presence of polymorphisms in the population [7, 38]. Our result also agreed with similar studies in other countries that type II was detected in stray cats using PCR-RLFP [22, 25, 31, 39-43]. In Europe and North America, type II is the most prevalent strain isolated from both humans and animals [44, 45].

Conclusions

Based on our results, the presence of T. gondii oocysts was identified in one fecal sample of stray cats. In addition, T. gondii type II genotype was identified for the first time in stray cats in Mashhad area. Further, similar studies with more markers are required to provide wider insight about the different Toxoplasma strains in cats in different parts of Iran.

Methods

Research location

The study was performed in Mashhad area as the center city of Khorasan Razavi province from April 2016 to August 2017. The city is located at 36.20º North latitude and 59.35º East longitude, in the valley of the Kashafrud River near Turkmenistan, between the two mountain ranges of Binalood and Hezar Masjed Mountains. The city benefits from the proximity of the mountains, having cool winters, pleasant springs, and mild summers. (Fig.1)

Sampling

One hundred seventy five stray cats were trapped from different areas of the Mashhad in with the help of local municipality. Furthermore, Thirty-one samples were from killed stray cats during driving accidents. The trapped cats and carcasses were transferred to the diagnostic laboratory of the parasitology department for laboratory examination. They were sedated by premedication with intramuscular (IM) Ketamine hydrochloride (6mg/kg). The approximate age of cat was determined by teeth examination. All of the adult teeth are in place by 6 months of age, and the growth is no longer useful in determining a cat's age. In older cats, the amount of staining, or tartar, on a cat's teeth is also an indicator of age. Then, the data related to their age, the sex were recorded and collected feces from each cat. The killed cats were necropsied and feces and the brain were collected. Collected fecal and brain samples were kept in a refrigerator at 4 ˚C for further examination. The trapped cats were released after sampling with help of the Mashhad municipality.

Parasitological method

Feces (1g) of each animal were emulsified in sucrose solution (specific gravity 1.203), filtered through gauze, and centrifuged in a 15 mL tube at 400g for 10 min. A drop of the float from the meniscus was examined microscopically at × 400 magnifications for the presence of T. gondii oocysts [1]. The size of oocysts was measured by a calibrated ocular micrometer (Zeiss Company, Germany).

Nested-PCR amplification

Oocysts of fecal samples were repeatedly washed in PBS and homogenized by grinding with 0.5mm glass beads for 30min. DNA of  homogenized oocysts, fecal and brain samples was extracted  by commercial  kit ( Molecular and Biological Transmission Systems (MBST), Tehran, Iran) as per manufacturer’s recommendations. T. gondii B1 gene PCR amplification was carried out using a nested-PCR as previously described by Burg et al., 1989[14]. Amplification in 25 μL reaction volumes (Accupower PCR premix kit, Bioneer®, South Korea) in first reaction contained:  250 μM of each dNTP, 10mM Tris-HCl pH 9.0, 30mM KCl and 2mM MgCl2, 1U Taq DNA polymerase and 10 pmol of each PCR primer (Denazist, Mashhad, Iran). Then 1 μL of DNA template (250-500 ng) was added to each reaction and the remaining 25 μL reaction volume was filled with sterile distilled water.

After 3 minutes of initial denaturation at 94°C, 38 cycles of amplification (each cycle: 1 minute at 94°C, 1 minute at 50°C, and 1 minute  at 72°C) and a final extension step for 7 minutes at 72°C were performed in an automated thermocycler (MJ Mini Thermal Cycler, Bio-Rad Co , USA). The PCR products were visualized by electrophoresis on a 1.5% agarose gel. One μL of  the diluted  (1:10) each reaction is then used in second reaction in the same mixture and cycling condition, except for the annealing temperature which was 52 °C; the number of cycles was 30.  The presence of specific bands of 193 bp in primary PCR and 96 bp in nested -PCR on agarose gel was considered a positive sample [14].Distilled water was used as a negative control and T. gondii strains (RH) was used as positive controls. 

Isolation of T.gondii

The tissue homogenates were prepared from the brain tissue of the cats as the method that described by Dubey [1]. Briefly, 100 grams of brain samples were homogenized in 0.5 L of normal saline (0.85%) with penicillin 100 IU/mL and streptomycin 1mg/mL by the electrical mixture. The tissue homogenate was strained through 2 layers of gauze to remove coarse material. The homogenate was kept at room temperature for three hours and centrifuged at 1.500 g for 5 min. The homogenate (0.5 mL per each mouse) was inoculate subcutaneously to Swiss Webster mice (Razi Vaccine& serum research institute, Mashhad, Iran).None inoculated mice were shown clinical signs.  Six weeks after inoculation, blood samples were collected from the tail of mice. Serum samples were separated and analyzed for the presence of antibodies against T. gondii by ELISA test (ID.vet Innovative Diagnostics, Grabels, France). Seropositive mice were killed at 42 days post-infection by chloroform-inhalation. Then, mouse brain was homogenized with an equal volume of sterile normal saline by passing through a 16 g needle ten times by mean a syringe. One drop of given suspension placed on a slide and spread out covered with a slip and microscopically examined.  At least five slides should be examined. The isolation of T.gondii was successful if Toxoplasma cysts were found in the mouse brain.

PCR-RFLP analysis

PCR-RFLP with SAG1, SAG2, SAG3, BTUB and GRA6 markers were performed to determine the T. gondii genotype in fecal and brain samples according to described methods [8-11]. Briefly, the PCR reaction was performed in a 25 µl reaction mixture containing containing 1 µl of extracted DNA, 75 mM Tris-HCl (pH 8.5), 20 mM (NH4)2SO4, 1.5 mM MgCl2, 0.1% Tween 20, 0.2 mM dNTPs, 0.025 U/µl amplicon Taq DNA polymerase, inert red dye, a stabilizer  and 10 pmol of each primer described in Table.1  After 5 minutes of initial denaturation at 95°C, 35 cycles of amplification followed by 30 sec at 94˚C, 1 min at 60˚C, 2 min at 72˚C, and a final extension of 72˚C for 10 min ( MJ Mini Thermal Cycler, Bio-Rad Co , USA). 

After that, 1.5 U of enzymes endonuclease with 2 U buffers was added to 15-mL of each PCR product and incubated as the manufacturer’s protocol. The digested products were electrophoresed to separate restriction fragments in 1.6% agarose gel. Finally the agarose gel was stained with ethidium bromide and visualized under UV. Extracted DNA of RH strain was used as a positive control.

DNA sequencing

The purified PCR products of B1 with primers were sent to DNA sequencing in the Bioneer Inc. (Bioneer Company, Seoul, Company).The assembling and editing of nucleotide sequences were used by CLc bio software.

Statistical analysis

The relationship between infection rate and variables such as age and gender was analyzed by the chi-square test. A significant association was identified when a p-value of less than 0.05 was observed [15]. The agreement between the different tests was showed as k-value. The agreement as poor if k-values between 0.2 and 0.4, moderate if k-values between 0.4 and 0.6, substantial if 0.6 and 0.8 and good if it exceeds 0.8 and 1.3 [15].

Abbreviations

PCR: Polymerase chain reaction

PCR-RFLP:  PCR-restriction fragment length polymorphism

ELISA: Enzyme-linked immunosorbent assay

SAG-1-3: Surface antigen-1-3

GRA-6: Dense granule protein-6

BTUB: Beta-tubulin

NCBI: National Center for Biotechnology Information

Declarations

Acknowledgments 

We thank the municipality of Mashhad for cooperation and assistance in collecting stray cats. We would like to thank Mr. Amin Bakhshani for his help during sampling. 

Authors’ contributions 

"G.R.R was the supervisor of project and analyzed the data and was a major contributor in writing the manuscript. MK collected samples and performed the experiments. All authors read and approved the final manuscript" 

Funding 

The research leading to these results was funded by a grant (No.3.40334) from the Research council of the Ferdowsi University of Mashhad. 

Availability of data and materials 

The datasets used and/or analyzed (Persian language) during the current study are available from the corresponding author on reasonable request. 

Ethics approval and consent to participate 

The experiment on animals in the present study was approved by Ethics Committee of Ferdowsi University of Mashhad (Approval ID: IR.UM.REC.1398.083)

Consent to publish

Not applicable

Competing interests

The authors declare that they have no competing interests.

References

  1. Dubey JP. Toxoplasmosis of Animals and Humans. Second ed. CRC Press; Boca Raton; Florida; 2010.
  2. Tenter IS, Heckeroth AR, Weiss LM. Toxoplasma gondii: from animals to humans. Inter J Parasitol. 2010; 30: 1217-1258
  3. Cuomo G, D’Abrosca V, Rizzo V, et al. Severe polymyositis due to Toxoplasma gondii in an adult immunocompetent patient: a case report and review of the literature. Infect. 2013; 41:859-862
  4. Montoya J, Liesenfeld O. Toxoplasmosis. Lancet. 2004; 363: 1965–1976.
  5. Howe DK, L.D. Sibley LD, Toxoplasma gondii comprises three clonal lineages: correlation of parasite genotype with human disease, J Infect Dis.1995; 172:1561-1566.
  6. Ajzenberg D, Year H, Marty P, Paris L, et al. Genotype of 88 Toxoplasma gondii isolates associated with toxoplasmosis in immunocompromised patients and correlation with clinical findings. J Infect Dis. 2009; 199:1155-67
  7. Su C, Zhang X, Dubey J. Genotyping of Toxoplasma gondii by multilocus PCR-RFLP markers: a high resolution and simple method for identification of parasites. Inter J Parasitol. 2006; 36:841-848.
  8. Fazaeli A, Carter P, Darde M, Pennington T. Molecular typing of Toxoplasma gondii strains by GRA6 gene sequence analysis. Inter J Parasitol. 2006; 30: 637-42
  9. Grigg ME, Ganatra J, Boothroyd JC, Margolis TP. Unusual abundance of atypical strains associated with human ocular toxoplasmosis. J Infect Dis. 2001; 184:633-639
  10. Khan A, Su C, German M, Storch G, Clifford D, Sibley LD. Genotyping of Toxoplasma gondii strains from immunocompromised patients reveals high prevalence of type I strains. J Clin Microbiol. 2005; 43: 5881-5887
  11. Su C, Shwab E, Zhou P, Zhu X, Dubey JP.Moving towards an integrated approach to molecular detection and identification of Toxoplasma gondii. Parasitol. 2010; 137: 1-11.
  12. Khan A, Dubey J, Su C, Ajioka JW., Rosenthal BM, Sibley LD. Genetic analyses of atypical Toxoplasma gondii strains reveal a fourth clonal lineage in North America. Inter J Parasitol. 2011; 41:645-655
  13. Rahimi MT, Daryani A, Sarvi1 S, et al . Cats and Toxoplasma gondii: A systematic review and meta-analysis in Iran. Onderst J Vet Res. 2015; 82: 1-10.
  14. Burg JL, Grover CM, Pouletty P, Boothroyd JC. Direct and sensitive detection of a pathogenic protozoan, Toxoplasma gondii, by polymerase chain reaction. J Clin Microbiol.1989; 27: 1787–1792
  15. Petrie A, Watson P. Statistic for veterinary and animal science. Oxford: Blackwell publishing; 2006.
  16. Razmi, G.R. Prevalence of feline coccidia in Khorasan province of Iran, J Appl Anim Res.2000; 17: 301–303.
  17. Veronesi F, Santoro A, Milardi GL, Diaferia M, Morganti G, Ranucci D, Gabrielli S. Detection of Toxoplasma gondii in feces of privately owned cats using two PCR assays targeting the B1 gene and the 529-bp repetitive element. Parasitol Res. 2017; 116: 1063-1069.
  18. Salman D, Pumidonming W, Oohashi E, Igarashi M. Prevalence of Toxoplasma gondii and other intestinal parasites in cats in Tokachi sub-prefecture, Japan. J Vet Med Sci. 2018; 80: 960-967.
  19. Schares G, Vrhovec MG, Pantchev N, Herrmann DC, Conraths FJ. Occurrence of Toxoplasma gondii and Hammondia hammondi oocysts in the feces of cats from Germany and other European countries. Vet Parasitol. 2008; 152: 34-45.
  20. Berger-Schoch AE, Herrmann DC, Schares G, Müller N, Bernet D, Gottstein B, Frey CF. Prevalence and genotypes of Toxoplasma gondii in feline feces (oocysts) and meat from sheep, cattle and pigs in Switzerland. Vet Parasitol. 2011; 177: 290-297
  21. Jung BK, Lee SE, Lim H, et al. Toxoplasma gondii B1 gene detection in feces of stray cats around Seoul, Korea and genotype analysis of two laboratory-passaged isolates. Korean J Parasitol. 2015; 53: 259-263.
  22. Dabritz HA, Miller MA, Atwill ER, Gardner IA, Leutenegger CM, Melli AC, Conrad
  23. Detection of Toxoplasma gondii-like oocysts in cat feces and estimates of the Environmental oocyst burden. J Am Vet Med Assoc. 2007; 231:1676-84.
  24. Chemoh W, Sawangjaroen N, Nissapatorn V, Sermwittayawong N. Molecular investigation on the occurrence of Toxoplasma gondii oocysts in cat feces using TOX-element and ITS-1 region targets. Vet J. 2016; 215:118-22.
  25. Jokelainen P, Simola O, Rantanen E, Näreaho A, Lohi H, Sukura A. Feline toxoplasmosis in Finland: cross-sectional epidemiological study and case series study. J Vet Diagn Invest. 2012; 24:1115-24.
  26. Ding H, Gao YM, Deng Y, Lamberton PH, Lu DB. A systematic review and meta-analysis of the seroprevalence of Toxoplasma gondii in cats in mainland China. Parasit Vectors. 2017; 10:27. doi: 10.1186/s13071-017-1970-6.
  27. Dubey JP, Lindsay D, Speer C. Structures of Toxoplasma gondii tachyzoites, bradyzoites, and sporozoites and biology and development of tissue cysts, Clinic Microbiol Rev.1998; 11 : 267-299.
  28. Hooshyar H, Rostamkhani P, Arbabi M. Study on growth of Toxoplasma gondii tissue cyst in laboratory mouse, Jundi J Microbiol. 2009; 2:140-143.
  29. Razmi GR, Ghezi K, Mahooti A, Naseri Z. A Serological Study and Subsequent Isolation of Toxoplasma gondii from aborted ovine fetuses in Mashhad area, Iran. J Parasitol. 2010; 96:812-814.
  30. Dubey JP, Su C, Cortés JA, Sundar N, Gomez-Marin JE, Polo LJ, Zambrano L, Mora LE, Lora F, Jimenez J, Kwok OC, Shen SK, Zhang X, Nieto A, Thulliez P. Prevalence of Toxoplasma gondii in cats from Colombia, South America and genetic characterization of T. gondii isolates. Vet Parasitol. 2006 b; 141:42-7
  31. Dubey JP, Su C, Oliveira J, Morales JA, Bolaños RV, Sundar N, Kwok OC, Shen SK. Biologic and genetic characteristics of Toxoplasma gondii isolates in free-range chickens from Costa Rica, Central America. Vet Parasitol. 2006 a; 139:29-36.
  32. Chemoh W, Sawangjaroen N, Nissapatorn V, Sermwittayawong N. Molecular investigation on the occurrence of Toxoplasma gondii oocysts in cat feces using TOX-element and ITS-1 region targets. Vet Parasitol: Vet Parasitol Reg Stud Reports; 2018; 13: 105-109.
  33. Dubey JP, Sundar N, Gennari SM, Minervino AH, Farias NA, Ruas JL, dos Santos TR, Cavalcante GT, Kwok OC, Su C. Biologic and genetic comparison of Toxoplasma gondii isolates in free-range chickens from the northern Pará state and the southern state Rio Grande do Sul, Brazil revealed highly diverse and distinct Parasite populations. Vet Parasitol. 2007; 143:182-8.
  34. Zia-Ali N, Fazaeli A, Khoramizadeh M, Ajzenberg D, Dardé M, Keshavarz-Valian H. Isolation and molecular characterization of Toxoplasma gondii strains from different hosts in Iran.Parasitol Res. 2007; 101:111-115.
  35. Tavalla M, Oormazdi H, Akhlaghi L, Shojaee S, Razmjou E, Hadighi R, Meamar A. Genotyping of Toxoplasma gondii isolates from soil samples in Tehran, Iran, Iranian J Parasitol. 2013; 8 : 227-233.
  36. Khademvatan S, Saki J, Yousefi E, Abdizadeh R. Detection and genotyping of Toxoplasma gondii strains isolated from birds in the southwest of Iran, Br Poult Sci. 2013; 54:76-80.
  37. Kareshk TA, Mahmoudvand H, Keyhani A, Oliaee TR, Mohammadi M, Babaei Z, Hajhosseini M, Zia-Ali N. Molecular detection and genetic diversity of Toxoplasma gondii in different tissues of sheep and goat in Eastern Iran, Trop Biomed. 2017; 34:681-690.
  38. Shariat Bahadori E, Dalimi A, Namroodi S, Pirestan M. Phylogenetic Analysis of Toxoplasma gondii Type II and Type III by PCR-RFLP Plus Sequencing on Wild-Rats of Golestan Forest, Iran.Vet Sci Technol. 2018; 9: 3. DOI: 10.4172/2157-7579.1000541.
  39. Ajzenberg D, Dumètre A, Dardé ML. Multiplex PCR for typing strains of Toxoplasma gondii. J Clin Microbiol. 2005; 43: 1940-1943.
  40. Dubey JP, Gennari SM, Labruna MB, Camargo LM, Vianna MCB, Marcet PL, Lehmann T. Characterization of Toxoplasma gondii isolates in free-range chickens from Amazon, Brazil, J Parasitol. 2006; 92 :36-40.
  41. Dubey JP, Moura L, Majumdar D, Sundar N, Velmurugan G, Kwok O, Kelly P, Krecek R, Su C. Isolation and characterization of viable Toxoplasma gondii isolates revealed possible high frequency of mixed infection in feral cats (Felis domesticus) from St Kitts, West Indies, Parasitol.2009; 136: 589-594.
  42. Chen Z, Gao J, Huo X, Wang L, Yu L, Halm-Lai F, Xu Y, Song W, Hide G, Shen J. Genotyping of Toxoplasma gondii isolates from cats in different geographic regions of China, Vet Parasitol. 2011;183 : 166-170.
  43. Vilares A, Gargaté MJ, Ferreira I, Martins S, Júlio C, Waap H, Ângelo H, Gomes JP. Isolation and molecular characterization of Toxoplasma gondii isolated from pigeons and stray cats in Lisbon, Portugal, Vet Parasitol. 2014; 205: 506-511.
  44. Yekkour F, Aubert D, Mercier A, Murat JB, Khames M, Nguewa P, Ait-Oudhia K, Villena I, Bouchene Z. First genetic characterization of Toxoplasma gondii in stray cats from Algeria. Vet Parasitol. 2017; 239 : 31-36.
  45. Ajzenberg D, Year H, Marty P, Paris L, Dalle F, Menotti J, Aubert D, Franck J, Bessières MH, Quinio D. Genotype of 88 Toxoplasma gondii isolates associated with toxoplasmosis in immunocompromised patients and correlation with clinical findings, J Infect Dis. 2009; 199: 1155-1167.
  46. Robert-Gangneux F, Dardé ML. Epidemiology of and diagnostic strategies fortoxoplasmosis. Clin Microbiol Rev. 2012; 25:264-96.

Tables

Table.1. Results of Fecal flotation technique and PCR examination of fecal samples of stray cats in Mashhad area

Variable

 

Fecal flotation technique

 

PCR of Feces

 

 

Total

 

 

Negative

No

Positive

No (%)

Negative

No

 

Positive

No (%)

 

Gender

 

 

 

 

Male

 

58

3(4.9)

58

3(4.9)

61

Female

113

1(0.9)

109

5(4.8)

114

Age (year)

 

 

 

<1

32

1(3)

32

1 (3)

33

1-3

53

1(1.8)

53

1(1.5)

54

> 3

86

2(2.2)

82

6 (6.8)

88

Total

 

171

4 (2.2)*

167

8 (4.5)*

175

* only one sample was positive both parasitology  and PCR