Molecular detection of Toxoplasma gondii in snakes

Accepted Manuscript Molecular detection of Toxoplasma gondii in snakes Vahid Nasiri, Shohreh Teymurzadeh, Gholamreza Karimi, Mehdi Nasiri PII:

S0014-4894(16)30160-6

DOI:

10.1016/j.exppara.2016.08.002

Reference:

YEXPR 7284

To appear in:

Experimental Parasitology

Received Date: 25 May 2016 Revised Date:

23 July 2016

Accepted Date: 9 August 2016

Please cite this article as: Nasiri, V., Teymurzadeh, S., Karimi, G., Nasiri, M., Molecular detection of Toxoplasma gondii in snakes, Experimental Parasitology (2016), doi: 10.1016/j.exppara.2016.08.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Molecular detection of Toxoplasma gondii in snakes

Vahid Nasiri a,*, Shohreh Teymurzadeh b, Gholamreza Karimi a, Mehdi

Department of Parasitology, Razi Vaccine and Serum Research Institute,

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a

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Nasiri c

Karaj, Alborz, Iran

Department of Venomous Animals and Antivenom Production, Razi Vaccine

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b

and Serum Research Institute, Alborz, Karaj, Iran

Faculty of physics , Iran University of Science and Technology , Tehran, Iran

*Correspondence:

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c

Vahid Nasiri, PhD of medical parasitology, Department of Parasitology, Razi

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E-mail:

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Vaccine and Serum Research Institute, Karaj, Alborz, Iran.

[email protected]

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Abstract Toxoplasma gondii, an obligate intracellular protozoan parasite, is responsible for one of the most common zoonotic parasitic diseases in almost all warm-blooded vertebrates worldwide, and it is estimated that about one-third of the world human population is chronically infected

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with this parasite. Little is known about the circulation of T. gondii in snakes and this study for the first time aimed to evaluate the infection rates of snakes by this parasite by PCR methods. The brain of 68 Snakes, that were collected between May 2012 and September 2015

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and died after the hold in captivity, under which they were kept for taking poisons, were examined for the presence of this parasite. DNA was extracted and Nested-PCR method was

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carried out with two of pairs of primers to detect the 344 bp fragment of T. gondii GRA6 gene. Five positive nested-PCR products were directly sequenced in the forward and reverse directions by Sequetech Company (Mountain View, CA). T. gondii GRA6 gene were detected from 55 (80.88%) of 68 snakes brains. Sequencing of the GRA6 gene revealed 98–

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100% of similarity with T. gondii sequences deposited in GenBank. To our knowledge, this is the first study of molecular detection of T. gondii in snakes and our findings show a higher frequency of this organism among them.

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Keywords: Toxoplasma gondii, snake, PCR, GRA6 gene.

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ACCEPTED MANUSCRIPT 1. Introduction Toxoplasma gondii ,an important protozoan parasite with major public health, is found worldwide and according to the literatures it infects virtually all warm-blooded vertebrates

human population (Dubey, 2009, 2008; Gazzonis et al., 2015).

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,including birds , livestock , marine mammals and humans and it affects one-third of the

In wildlife, many animals also were infected by T. gondii and some studies reported high infection rate of T. gondii in zoo animals and wild birds (Chen et al., 2015). Murata in early

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1989 just showed 29.28% (53/181) and 16.94% (61/179) of T. gondii IgG in mammals and wild birds, respectively, by serological survey(Murata K., 1989). In some researches,

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reported that there had 25% (4/16) of T. gondii antibody in primates, 69.4% (25/36) in carnivores, 27.6% (8/29) in herbivores (Zhang et al., 2000) and 36.1% (73/202) of wild birds captured from the wild environment(Gennari et al., 2014).

Species of these exclusively parasitic families are known from many birds and mammals but

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they are rarely found in poikilothermic vertebrates(Cooper, 2007). Munday et al mentioned for the first time that oocysts/sporocysts which may belong to either Sarcocystis or Frenkelia were found in the intestinal tract of three Australian reptiles (Munday et al., 1979). From

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amphibians, Levine and Nye recorded three species of the genus Toxoplasma from anurans

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(Levine and Nye, 1977, 1976), but Dubey(Dubey, 1977) termed them as "sporozoans of unknown taxonomic position". The investigations by Munday et al(Munday et al., 1979)could not reveal these apicomplexan families in 90 Australian amphibians belonging to nine species in two families. The distribution of genetic diversity of T. gondii in wildlife animals is of great importance to understand the transmission of this parasite in the environment(Chen et al., 2015). For example, In a study, they identified the T. gondii infection in Black-capped, Wild Red Dog, Lemur, zebra, Red panda, Pea-cock, Red-crowned Crane, Budgerigar, Black-billed Magpie, 3

ACCEPTED MANUSCRIPT Zebra Finch in China, which indicated these animals could be served as a potential source of infection for other animals and even humans(Chen et al., 2015). Through molecular screening by PCR, parasites can be quickly and efficiently detected in host samples, and sequencing provides abundant characters for phylogenetic analysis

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(Queirós, 2012) .

The dense granule antigens (GRA) are parasitic molecules secreted to the parasitophorous vacuole and are immunogenic and are responsible for the intracellular survival of the

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parasites (Decoster et al., 1988; Lecordier et al., 1995; Sibley et al., 1991). GRA6 is a single copy gene (Lecordier et al., 1995) and we have used the coding region of this protein gene for

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PCR analysis and assessed its application for confirmation of presence of T. gondii parasite. There is no information about molecular detection of T. gondii in snakes worldwide and in this study, for the first time, the presence of this parasite in native species of Iranian snakes that taken from the wild were investigated and determined by molecular study.

2.1. Sample collection

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2. Materials and Methods

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A total of 68 snakes representing 5 species that were collected between May 2012 and September 2015 from various provinces of Iran sent to the department of Venomous Animals

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and Antivenom Production, Razi Vaccine and Serum Research Institute (table 1).These snakes were kept under captivity and after ding transferred immediately to the Parasitology laboratory of Razi Vaccine and Serum Research Institute. Whole brains of snakes were removed from the heads (Fig. 1) and kept at -20°C until used. All animal experimentation protocols were approved by Animal Care Committee of Razi Vaccine and Serum Research Institute, Alborz, Iran.

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2.2. DNA extraction All parts of Brain tissue of each snake were removed and homogenized and used for DNA

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extraction by phenol-chloroform extraction method. Briefly, all of the homogenized samples diluted with 1 ml of lysis buffer extraction solution (50 mM Tris-HCL, pH8.0; 25 mM EDTA and 400 mM NaCl), 100 µL 10% SDS (Biase et al. 2002), and Proteinase K (100 µg/ml)

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(Fermentas, USA) in 2 mL microtube and incubated at 55 ºC for overnight. For precipitation of undissolved debris and proteins, 300 µL 5M NaCl was added to the suspension and kept at

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4 °C for 15 min, then centrifugation was done at 13,000 g for 15 min and the supernatant were transferred to a new 2 mL microtube (Biase, et al., 2002). Then, the samples were extracted with phenol–chloroform–isoamyl alcohol (25:24:1). DNA was precipitated by adding equal volume of cold isopropanol and 1/10 volume of sodium acetate solution (3M, pH

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5.2), kept at -20°C for overnight, followed by centrifugation at 13,000 g for 5 min. Then the pellet washed twice with 70% ethanol and resuspended in 50 µL of distilled water. Concentration of DNA was determined by spectrophotometric analysis at 280/260 nm .The

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extracted DNA was stored at -20 °C until used. A mock control (DNA extracted from the brain of the inbreed BALB/c mice, which serologically confirmed that they were negative for

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toxoplasmosis) isolated simultaneously with the samples as the negative control for confirmation of procedures accuracy. 2.3. Nested-PCR

First round of PCR was performed using a pair of T.gondii –GRA6 gene specific primers: GRA6-F1

(5´-ATTTGTGTTTCCGAGCAGGT-3´)

and

GRA6-R1

(5´-

GCACCTTCGCTTGTGGTT -3´) and then Nested-PCR were performed with a second pair of

primers:

GRA6-F2

(5´TTTCCGAGCAGGTGACCT-3´) 5

and

GRA6-R2

(5´-

ACCEPTED MANUSCRIPT TCGCCGAAGAGTTGACATAG -3´)(Khan et al., 2005). The 1st round reaction was carried out in 20 µL reaction mixtures containing 10 µL of 2 x PCR master mixes (Sinaclon, Iran), 20 pmol of each primer(2 µL), 5 µL of template DNA and 3 µL of distilled water. Amplification of 1st round reaction was conducted with initial denaturation for 5 minutes at 94°C, followed

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by 35 cycles of 94°C for 45 seconds (denaturation), annealing at 58°c for 45 seconds, extension at 72°c for 60 seconds and final extension at 72° c for 10 minutes. The nested-PCR (The 2st round reaction) was carried out in 20 µL reaction mixtures containing 10 µL of 2 x

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PCR master mixes (Sinaclon, Iran), 20 pmol of each primer(2 µL), 1 µL of 1st round PCR product as the template DNA and 7 µL of distilled water. Amplification of 2st round reaction

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was conducted with initial denaturation for 5 minutes at 94°C, followed by 35 cycles of 94°C for 45 seconds (denaturation), annealing at 57.5°c for 45 seconds, extension at 72°c for 45 seconds and final extension at 72° c for 10 minutes. For each reaction, a positive control (DNA extracted from the RH strain of T. gondii) and a negative control (DNA extracted from

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the brain of the inbreed BALB/c mice, which serologically confirmed that they were negative for toxoplasmosis) were included. Following PCR amplifications DNA fragment was

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identified by agarose gel electrophoresis of five microliters of the PCR products. 2.4. Nucleotide sequence analysis

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Five positive nested-PCR products, that were detected from four different species of snakes (Naja oxiana, Agkistrodon intermedius, Pseudocerastes persicus fieldi, Vipera lebetina obtusa), were extracted from the gel using Gel Purification kit (Vivantis, Selangor Darul Ehsan, Malaysia) according to the manufacturer’s protocols and the nested- PCR products were directly sequenced in the forward and reverse directions by Sequetech Company (Mountain View, CA). The sequences results were edited and aligned with GRA6 partial sequences of T. gondii from other hosts and compared with sequences available in GenBank by BioEdit Sequence Alignment Editor (Hall, 1999). Phylogenetic tree and evolutionary 6

ACCEPTED MANUSCRIPT analyses were conducted using the neighbour-joining and maximum-likelihood methods with MEGA6 software (Tamura et al., 2013). The bootstrap scores were calculated for 2000 replicates. 3. Results

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Out of 68 snakes, T.gondii GRA6 gene was detected in 55 snakes (80.88%) (table 2) and following nested-PCR amplification, a 344 bp DNA fragment for the GRA6 gene were identified by agarose gel electrophoresis (Fig. 2). After sequencing, five selected nucleotide

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sequences of the GRA6 gene with a length of 344 bp were submitted to GenBank database (GenBank accession no. KU523403, KU523404, KU523405, KU523406, KU523407).After

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alignment, the results revealed that our sequences shared 99-100% similarity with each other and 98-99% similarity for T.gondii GRA6 gene from other hosts (Supplementary Table S1).Phylogenetic trees using neighbour joining method (Fig. 3) and Maximum Likelihood (Supplementary Fig. S1) showed intra-specific variations among T.gondii isolates in this

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study and some other T. gondii deposited in GenBank. Analysis of the GRA6 gene sequence in four of our sample that were detected from Naja oxiana (KU523403),Agkistrodon intermedius

(KU523404),Pseudocerastes

persicus

fieldi

(KU523405),

Agkistrodon

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intermedius (KU523406) showed the highest similarity (100%) with each other and (99-

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100%) with T. gondii GRA6 gene isolated from Homo sapiens (KT735111.1, KT735112.1), Ovis aries (KT735113.1, KT735119.1), sparrow (KR809554.1, KR809558.1, KT809309.1), Rattus rattus (KP792610.1, KP792614.1), Felis catus (KP792615.1, KP792621.1), Corvus frugilegus (KP792600.1, KP792604.1), Columba livia (KP792605.1, KP792609.1).Our other sequence that was detected from Vipera lebetina obtusa (KU523407) showed 98% similarity with T. gondii GRA6 gene isolated from above mentioned isolates (Supplementary Table S1).

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ACCEPTED MANUSCRIPT 4. Discussion Intracellular parasites, such as the members of the phylum Apicomplexa are a highly diverse group of organisms with essential roles in the ecosystems they inhabit but, regardless, they remain one of the most poorly studied groups of organisms, especially in wild hosts (Queirós,

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2012) .

The class Reptilia, consisting of more than 6,000 species, is host to a wide variety of protozoan and metazoan parasites and virtually 100% of free-ranging reptiles harbour some

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kind of parasites that these animals may serve as definitive, intermediate, accidental or paratenic hosts(Barnard and Upton, 1994; Cooper and Jackson, 1981; Divers and Mader,

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2005; Frye, 1991; Hernandez-Divers, 2006; McArthur et al., 2008; McFarlen, 1991). In Iran, 69 species of snakes assigned to 37 genera in 6 families have been identified, of which 36 species are non-venomous, 25 species are venomous and 8 species are semi-venomous(Latifi, 2000; Nasiri et al., 2014; Zare Mirakabadi and Teimourzadeh, 2008).

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The reptiles have important impacts on different aspects of their ecology and thus demands deep research regarding the influence of these animals and their flora on people and autochthonous animal species. The varieties of different pathogens in these species are very

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large (Nasiri et al., 2014) and thus the present study was carried out to evaluate the presence

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of one of these pathogens, T. gondii, in snakes from wildlife. Experimental transmission of T. gondii from mice to reptiles failed under normal temperatures(Hoff, 2012). However, Stone and Manwell have shown that Toxoplasma may be successfully transferred to reptiles and even to amphibians especially when these animals were kept at higher temperatures(Stone and Manwell, 1969). The parasites persisted at least 6 days or longer in some individuals, but more often when the animals were kept at 37º C rather than room temperature(Hoff, 2012). Levit was able to infect reptiles with the low virulence Toxoplasma strain FCL if the reptiles were maintained at higher temperatures (31

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ACCEPTED MANUSCRIPT to 35º C). A variation in susceptibility was observed with Agama and Vipera being most susceptible and Testudo and Natrix most resistant(Hoff, 2012). Moreover, Levine grouped some

species

of

protozoans

from

cold-blooded

vertebrates

under

the

genus

Toxoplasma(Norman D.Levine et al., 1977). The species were: 1) T. alencari, from the brain

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of the Brazilian frog Leptodactylus ocellatus(Hoff, 2012); 2) T. ranae ,Levine and Nye, 1976, from brain pseudocysts of North American Rana pipiens; 3) T. serpai ,Scorza, Dagert and Itturriza Arocha, 1956, from the erythrocytes, liver, spleen, kidneys and brain of Venezuelan

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Bufo marinus; 4) T. brumpti ,Coutelen, 1932, from mononuclear-leucocytes of Iguana tuberculata from Trinidad; and, 5) T. colubri ,Tibaldi, 1921, from white blood corpuscles of

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Coluber viridiflavus from Sardinia(Hoff, 2012).On that time, Dubey doubted these classifications since nothing is known concerning the life cycles and very little of the structure(Dubey, 1977).

The study was proposed because we previously observed that, in captivity, snakes presented a

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high prevalence (above 70%) of infection by different parasite species (Nasiri et al., 2014) and the present investigation showed that 55 (80.88%) of 68 analyzed snakes arrived for

5. Conclusion

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captivity already infected by T. gondii.

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In conclusion, to our knowledge, this is the first data on the molecular detection of T. gondii in snakes and our findings show a high prevalence of this organism among them. Control and prevention of many of parasitic diseases is associated with breaking the cycle of transmission, and there is no shadow of doubt that precise information about diseases and their causative agents is the major key to control of them and thus, further precise parasitological investigations are required due to the noticeable unexplored area of snake toxoplasmosis in order to ascend our knowledge concerning parasites of snakes and probable zoonotic and veterinary importance of them.

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ACCEPTED MANUSCRIPT Acknowledgements The authors would like to thank Dr. Abbas Zare Mirakabadi for the help in preparation of the samples and for helpful advice and feedback.

We declare that we have no conflict of interest. References

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Conflict of interest statement

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Plenum Press, New York, New York and London. doi:10.1163/157075407782424502 Cooper, J.E., Jackson, O.F., 1981. Diseases of the Reptilia. Volumes 1 and 2. Academic Press Inc.(London) Ltd.

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ACCEPTED MANUSCRIPT Frye, F.L., 1991. Biomedical and surgical aspects of captive reptile husbandry. 2 vols Krieger Publ. Co., Malabar, Florida. Gazzonis, A.L., Veronesi, F., Di Cerbo, A.R., Zanzani, S.A., Molineri, G., Moretta, I., Moretti, A., Piergili Fioretti, D., Invernizzi, A., Manfredi, M.T., 2015. Toxoplasma

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Murata K., 1989. A serological survey of Toxoplasma gondii infection in zoo animals and

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aids and Treatment. Tehran: Teimourzadeh and Taieb.

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Zare Mirakabadi, A., Teimourzadeh, S., 2008. Venomous Snakes of Iran, Prevention, First

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People’s Republic of China. Parasitol. Int. 49, 171–174.

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344 bp). Fig.3.Phylogenetic relationships among Toxoplasma gondii based on a partial GRA6 gene sequence. The evolutionary history was inferred using the neighbour-joining method,

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supported by 2000 bootstrap replicates. Samples isolated in the present study (diamond dots) (GenBank accession no. KU523403, KU523404, KU523405, KU523406, KU523407) were

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compared to isolates selected from GenBank.

Supplementary Fig. S1.Phylogenetic relationships among Toxoplasma gondii based on a partial GRA6 gene sequence. The evolutionary history was inferred using the Maximum Likelihood method, supported by 2000 bootstrap replicates. Samples isolated in the present

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study (diamond dots) (GenBank accession no. KU523403, KU523404, KU523405,

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KU523406, KU523407) were compared to isolates selected from GenBank.

ACCEPTED MANUSCRIPT Table1. The taxonomic characterization of examined snakes Number of examined scientific name of snakes

Common name

Persian Horned Viper

Naja oxiana

Central Asian Cobra

Vipera albicornuta

zigzag mountain viper

Vipera lebetina obtusa

West-Asian blunt-nosed viper

Agkistrodon intermedius

Caucasian Pit viper

caucasicus

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Total

10

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Pseudocerastes persicus fieldi

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snakes

4

7

9

38

68

ACCEPTED MANUSCRIPT Table2. Positive Number and percentage of examined snakes in different species.

Number

percentage of Number of

of scientific name of snakes

positive snakes positive (in species/in snakes

snakes

10 4

Vipera albicornuta

7

Vipera lebetina obtusa

Agkistrodon intermedius caucasicus

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100(14.70)

4

100(5.89)

5

71.42(7.35)

9

7

77.77(10.29)

38

29

76.31(42.65)

68

55

80.88

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Total

10

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Naja oxiana

all)

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Pseudocerastes persicus fieldi

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examined

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Little is known of the snakes' toxoplasmosis in the world.

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This study demonstrates the high prevalence of toxoplasmosis among Iranian snakes.

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This is the first data on the cold blooded animals' toxoplasmosis.

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