Accepted Manuscript Title: First isolation of Toxoplasma gondii from cats of Colima, Mexico: Tissue distribution and genetic characterization Author: Claudia Patricia Rico-Torres Alejandra Del Viento-Camacho Heriberto Caballero-Ortega Alejandro Besn´e-M´erida H´ector Luna-Past´en Dolores Correa Jos´e Manuel Palma-Garc´ıa PII: DOI: Reference:
S0304-4017(15)00049-7 http://dx.doi.org/doi:10.1016/j.vetpar.2015.02.004 VETPAR 7528
To appear in:
Veterinary Parasitology
Received date: Revised date: Accepted date:
20-8-2014 4-2-2015 8-2-2015
Please cite this article as: Rico-Torres, C.P., Del Viento-Camacho, A., CaballeroOrtega, H., Besn´e-M´erida, A., Luna-Past´en, H., Correa, D., Palma-Garc´ia, J.M.,First isolation of Toxoplasma gondii from cats of Colima, Mexico: tissue distribution and genetic characterization, Veterinary Parasitology (2015), http://dx.doi.org/10.1016/j.vetpar.2015.02.004 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.
2
RESEARCH HIGHLIGHTS
-
3 4 5
We isolated viable T. gondii tachyzoites (ToxoDB PCR-RFLP genotype #28) from one cat of Colima, Mexico.
-
The brachiocephalic muscle, the brain, and the spleen were the tissues where T.
ip t
1
gondii DNA was more frequently detected.
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Page 1 of 14
1
First isolation of Toxoplasma gondii from cats of Colima, Mexico: tissue distribution
2
and genetic characterization
3
Claudia Patricia Rico-Torresa, Alejandra Del Viento-Camachob, Heriberto Caballero-
5
Ortegaa, Alejandro Besné-Méridaa, Héctor Luna-Pasténa, Dolores Correaa, José Manuel
6
Palma-Garcíab*
cr
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4
9
a. Laboratorio de Inmunología Experimental. Instituto Nacional de Pediatría, Secretaría de Salud, México D.F., C.P. 04530, México.
an
8
us
7
b. Centro Universitario de Investigación y Desarrollo Agropecuario, Facultad de
11
Medicina Veterinaria y Zootecnia, Universidad de Colima, México. Av. Gonzalo de
12
Sandoval # 444, Colima, CP 28045 AP 22, México.
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te
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*Corresponding author:
15
José Manuel Palma García, DVM, PhD
16
Address: Avenue Gonzalo de Sandoval # 444, Colima, CP 28045 AP 22, México.
17
Tel.: +52 (312) 316 10 00 ext. 40011
18
Fax: +52 (312) 312 75 81
19
E-mail: [email protected]
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1 Page 2 of 14
ABSTRACT
22
Toxoplasma gondii is among the commonest zoonotic infectious agents worldwide. It
23
infects many warm-blooded animals, including felines, the definitive hosts. This parasite is
24
now classified in 15 haplogroups spread out around the world. Few reports reveal a
25
predominance of genotypes I and III in Mexico, although recombinant and atypical variants
26
have also been found in humans and animals. The aim of this study was to detect, isolate
27
and genotype T. gondii from cats of Colima Mexico, and to analyze tissue distribution of
28
the parasite. IgG specific antibodies were investigated in 48 serum samples from unwanted
29
and stray cats by indirect ELISA. Isolation in mice and molecular characterization by PCR-
30
RFLP and sequencing were attempted using pools of brain, heart, liver, lung, spleen and
31
brachiocephalic muscle samples of seropositive cats. Fourteen animals (29.2%) were
32
seropositive, the frequency ranged between 27.3% and 40% among the different localities.
33
Ten seropositive animals were euthanized, eight of them were positive for the B1 gene by
34
conventional PCR. More frequently infected tissues were the brachiocephalic muscle
35
(75.0%) the brain (63.0%) and the spleen (63.0%). Genotype III was determined for the
36
SAG3 locus of the parasite infecting an unwanted cat. Tachyzoites were isolated from the
37
peritoneal cavity of two mice inoculated with the tissue pool of one kitten. Type I alleles
38
were found in SAG1, SAG2, SAG3, BTUB, GRA6, c29-2 and PK1 loci, while c22-8 was
39
type II, and L358 and Apico were type III. This genotype corresponds to ToxoDB genotype
40
#28. This is the first T. gondii isolate genetically characterized in Colima, Mexico and is
41
different to other isolations of the country.
42
Keywords: Toxoplasma gondii; cats; molecular characterization; isolation; genotype;
43
Mexico
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44 2 Page 3 of 14
1. INTRODUCTION
46
Toxoplasma gondii is an obligate intracellular parasite that infects a broad range of warm-
47
blooded animals. Felines play a critical role in the epidemiology of this parasitosis, because
48
they release oocysts with their feces, which survive several months in the environment,
49
especially in warm and humid zones (Dubey, 2004).
50
Currently T. gondii is classified into 15 haplogroups with a world-wide distribution (Su et
51
al., 2012). In Mexico a predominance of type I and III has been supported from studies
52
performed in several species, including humans, although atypical variants have also been
53
found (Dubey et al., 2004; 2009; Alvarado-Esquivel et al., 2011; Cedillo-Peláez et al.,
54
2011; Rico-Torres et al., 2012). Nevertheless, little is known about genotypes of T. gondii
55
circulating in cats; only atypical and recombinant variants have been reported in domestic
56
cats and one puma of Durango (Dubey et al., 2009; 2013).
57
The presence of T. gondii in cats of Colima has been documented, but no genotyping has
58
been performed in this zone (García-Marquez et al., 2007). Thus, the aim of this study was
59
to detect, isolate and genotype T. gondii which infects cats in Colima and to analyze tissue
60
distribution of the parasite.
61
2. MATERIALS AND METHODS
62
2.1. Bioethics
63
Animals were handled according to the Mexican Official Standards for animal care (NOM-
64
033-ZOO-1995 and NOM-062-ZOO-1999). The Committee for Care and Use of
65
Laboratory Animals of the Instituto Nacional de Pediatría (INP) approved the project
66
(013/2012).
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67 68 3 Page 4 of 14
2.2. Collection of samples and serology
70
A total of forty-eight cats -three months to four years old- from Colima, Manzanillo and
71
Villa de Álvarez municipalities of Colima State, Mexico, were studied from February to
72
July 2008. Blood samples from the jugular vein or the heart were obtained and sera were
73
tested using an in-house indirect ELISA as previously reported (Besné-Merida et al., 2008).
74
Samples from the brain, heart, liver, lung, spleen and brachiocephalic muscle of
75
seropositive cats were sent to INP within 12h of their collection at 2-4°C for bioassays in
76
mice and molecular analysis.
77
2.3. Bioassay in mice
78
Pools of tissues (200 mg of each) were manually homogenized using a mortar in 1 mL of
79
sterile PBS; then, 0.25 mL of the supernatant were inoculated intraperitoneally into each of
80
two Balb/c mice. They were bled before infection and on days 10, 20, 30, 40, 50 and 60
81
after inoculation and tested by indirect ELISA; serum samples were diluted 1:150 and
82
1:250 for IgM and IgG anti-T. gondii antibodies (abs), respectively, and the reaction was
83
developed using anti-mouse IgM (1:250) or IgG (1:1000) peroxidase conjugates (Sigma-
84
Aldrich Corp., St Louis, MO, USA). Serocoverted mice were euthanized eight weeks after
85
inoculation and a pool of tissues (brain, heart, liver, lung and spleen) was used to test for T.
86
gondii by PCR.
87
2.4. Detection of repetitive B1 gene of T. gondii for molecular diagnosis
88
DNA was extracted with the GENTRA® commercial kit (Minneapolis, MN, USA) using
89
200 mg (cats) or 20 mg of each tissue/organ (mice) according to the manufacturer’s
90
instructions. The repetitive B1 gene was specifically amplified to obtain 364 and 62 bp
91
fragments by conventional PCR and real time PCR, respectively (Rico-Torres et al., 2012).
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2.5. Genetic characterization
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In order to determine the genotype of T. gondii, SAG1, Alt.SAG2, SAG3, BTUB, GRA6,
95
c22-8, c29-2, L358, PK1 and Apico genes were analyzed by PCR-RFLP and sequencing
96
(Su et al., 2010). RH, (type I), VEG (type III) and Wiktor (ToxoDB #6) strains were used
97
for reference restriction patterns. Sequences were analyzed with Chromas Lite 2.1.1
98
software and aligned with those reported in GenBank and Toxo.DB.org web pages by
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Clustal W2 and NCBI/BLAST websites.
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3. RESULTS
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Anti-T. gondii abs were found in 14 out of 48 (29.2%) cats of Colima. The frequency was
103
28.1%, 27.3% and 40%, for Colima, Manzanillo and Villa de Álvarez municipalities,
104
respectively (Fig. 1). Ten of the fourteen seropositive cats were euthanized and eight of
105
them (80.0%) were positive by conventional PCR.
106
Parasite DNA was more frequently detected in brachiocephalic muscle, brain and spleen
107
(Table 1). The genotype could only be determined at the SAG3 locus in cat #6 (brain) being
108
type III. At least one mouse inoculated with homogenates from cats #1, #2, and #7
109
seroconverted for abs. Mice injected with tissues of cats #1 and #7 were euthanized eight
110
weeks later and resulted positive for the B1 and SAG3 loci; however, bands obtained were
111
faint, so the genotype could not be determined. Mice inoculated with tissues of cat #2
112
seroconverted at day 21 after inoculation and were euthanized because they presented
113
clinical signs compatible with toxoplasmosis. Tachyzoites were recovered from the
114
peritoneal cavity and the DNA was positive by conventional and real time PCR. The
115
analysis by PCR-RFLP and sequencing showed that all loci were type I except for c22-8
116
(type II) and L358 and Apico (type III) corresponding to genotype #28, according to
117
ToxoDB.
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4. DISCUSSION
120
There are few studies about T. gondii genotypes in Mexico and none in Colima, a state with
121
warm humid climate where transmission has been documented. Through serological
122
screening we found candidate cats for genotyping and tissue distribution analyses. The
123
brachiocephalic muscle was the tissue more frequently infected, followed by the brain and
124
the spleen; surprisingly, T. gondii DNA was less frequent in the heart, which is different to
125
data reported elsewhere (Dubey el al., 2009).
126
Parasite genotype could only be determined at the SAG3 in the brain sample of a cat of the
127
coast, being type III. Tachyzoites were isolated from mice inoculated with tissues of a
128
kitten of Colima city and presented a combination of type I, II and III, corresponding to
129
genotype ToxoDB #28, which has previously been reported in USA (humans), Brazil
130
(chickens) and Colombia (cats and chickens) (Howe and Sibley, 1995; Dubey et al., 2006;
131
2007). In Durango State, in the Northwest region of Mexico, genotypes #2, #9, #73, #74,
132
#155 and #222 have been demonstrated in cats, dogs, chickens and one puma (Dubey et al.,
133
2009; 2013). Samples from congenitally infected children and squirrel monkeys with
134
toxoplasmosis acquired around Mexico city revealed type I alleles for Alt.SAG2 and SAG3
135
genes, as for the cat isolate, but u-1 alleles were found in other two congenital cases at
136
Alt.SAG2 and BTUB loci (Cedillo-Peláez et al., 2011; Rico-Torres et al., 2012).
137
conclusion, T. gondii varies within the State of Colima for the SAG3 locus and in relation to
138
other regions of the country.
139
CONFLICT OF INTEREST STATEMENT
140
The authors declare no conflict of interests.
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ACKNOWLEDGMENTS
144
The authors are most thankful to Dr. Chunlei Su for his kind advice on multilocus PCR-
145
RFLP technical details. This work was partially financed by grant 013/2012 from Instituto
146
Nacional de Pediatría.
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8 Page 9 of 14
REFERENCES
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Alvarado-Esquivel, C., Rajendran, C., Ferreira, L.R., Kwok, O.C., Choudhary, S.,
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Alvarado-Esquivel, D., Rodríguez-Peña, S., Villena, I., Dubey, J.P., 2011.
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Prevalence of Toxoplasma gondii infection in wild birds in Durango, Mexico. J.
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Parasitol. 97, 809–812.
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Besné-Mérida, A., Figueroa-Castillo, J.A., Martínez-Maya, J.J., Luna-Pastén, H., Calderón-
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Segura, E., Correa, D., 2008. Prevalence of antibodies against Toxoplasma gondii in
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domestic cats from Mexico City. Vet. Parasitol. 157, 310-313.
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Cedillo-Peláez, C., Rico-Torres, C.P., Salas-Garrido, C.G., Correa, D., 2011. Acute
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toxoplasmosis in squirrel monkeys (Saimiri sciureus) in Mexico. Vet. Parasitol.
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180, 368–371.
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Dubey, J.P., 2004. Toxoplasmosis - a waterborne zoonosis. Vet Parasitol. 126, 57-72.
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Dubey, J.P., Morales, E.S., Lehmann, T., 2004. Isolation and genotyping of Toxoplasma
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gondii from free-ranging chickens from Mexico. J. Parasitol. 90, 411–413. Dubey, J.P., Su, C., Cortes, J.A., Sundar, N., Gomez-Marin, J.E., Polo, L.J., Zambrano, L.,
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Mora, L.E., Lora, F., Jimenez, J., Kwok, O.C.H., Shen, S.K., Zhang, X., Nieto, A.
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and Thulliez, P., 2006. Prevalence of Toxoplasma gondii in cats from Colombia, South America and genetic characterization of T. gondii isolates. Vet. Parasitol. 141, 42-47.
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Dubey, J.P., Sundar, N., Gennari, S.M., Minervino, A.H.H., Farias, N.A.d.R., Ruas, J.L.,
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dos Santos, T.R.B., Cavalcante, G.T., Kwok, O.C.H., and Su, C., 2007. Biologic
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and genetic comparison of Toxoplasma gondii isolates in free-range chickens from
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the northern Para state and the southern state Rio Grande do Sul, Brazil revealed
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highly diverse and distinct parasite populations. Vet. Parasitol. 143, 182-188. 9 Page 10 of 14
Dubey, J.P., Velmurugan, G.V., Alvarado-Esquivel, C., Alvarado-Esquivel, D., Rodríguez-
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Peña, S., Martínez-García, S., González-Herrera, A., Ferreira, L.R., Kwok, O.C.,
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Su, C., 2009. Isolation of Toxoplasma gondii from animals in Durango, Mexico. J.
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Parasitol. 95, 319–322.
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Dubey, J.P., Alvarado-Esquivel, C., Herrera-Valenzuela, V.H., Ortiz-Diaz, J.J., Oliveira, S.,
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Verma, S.K., Choudhary, S., Kwok, O.C., Su, C., 2013. A new atypical genotype
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mouse virulent strain of Toxoplasma gondii isolated from the heart of a wild caught
179
puma (Felis concolor) from Durango, Mexico. Vet. Parasitol. 197, 674-677.
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García-Márquez, L.J., Gutiérrez-Díaz, M.A., Correa, D., Luna-Pastén, H., Palma, J.M.,
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2007. Prevalence of Toxoplasma gondii antibodies and the relation to risk factors in
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cats of Colima, Mexico. J. Parasitol. 93, 1527-1528.
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Howe, D.K., and Sibley, L.D., 1995. Toxoplasma gondii comprises three clonal lineages: correlation of parasite genotype with human disease. J. Infect. Dis. 172, 1561-1566.
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Rico-Torres, C.P., Figueroa-Damián, R., López-Candiani, C., Macías-Avilés, H.A.,
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Cedillo-Peláez, C., Cañedo-Solares, I., Luna-Pastén, H., Tecuatl-Herrada, B.L.,
187 188
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Correa, D., 2012. Molecular diagnosis and genotyping of cases of perinatal toxoplasmosis in Mexico. Pediatr. Infect. Dis. J. 31, 411-413.
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Su, C., Shwab, E.K., Zhou, P., Zhu, X.Q., Dubey, J.P., 2010. Moving towards an integrated
190
approach to molecular detection and identification of Toxoplasma gondii. Parasitol.
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137, 1-11.
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Su, C., Khan, A., Zhou, P., Majumdar, D., Ajzenberg, D., Dardé, M.L., Zhu, X.Q., Ajioka,
193
J.W., Rosenthal, B.M., Dubey, J.P., Sibley, L.D., 2012. Globally diverse
194
Toxoplasma gondii isolates comprise six major clades originating from a small
195
number of distinct ancestral lineages. Proc. Natl. Acad .Sci. U S A. 109, 5844-5849. 10 Page 11 of 14
FIGURE CAPTIONS
197
Figure 1. Map of Colima, Mexico showing the climatic characteristics of the three
198
municipalities studied, the frequency of IgG antibodies and the detection of Toxoplasma
199
gondii DNA in tissues from seropositive cats and the genotypes obtained by PCR-RFLP of
200
Colima, Mexico. masl = meters above sea level; *parasite genotype could only be
201
determined at the SAG3 locus.
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11 Page 12 of 14
ip t
Table
Table 1. Molecular detection of Toxoplasma gondii in sample tissues from cats of Colima, Mexico
Cat No. (n=8)
Municipality
1
Colima
Unwanted
18
2
Colima
Unwanted
3
3
Colima
Stray
12
4
Colima
Stray
12
5
Manzanillo
Unwanted
12
6
Manzanillo
Unwanted
7
Manzanillo
Unwanted
8
Villa de Álvarez
Frequency (%)
Stray
Brachiocephalic muscle
Heart
Liver
Lung
–
B1
–
–
–
–
B1
–
B1
B1
B1
B1, SAG3
B1, SAG3
–
–
–
B1
–
–
B1
–
–
B1, SAG3
–
–
–
–
–
B1
B1
30
B1, SAG3a
–
–
–
B1
B1
30
B1
–
B1, SAG3
B1
B1
B1
B1
–
–
–
–
B1
5
2
2
2
6
5
63
25
25
25
75
63
M an
ed 24
us
Brain
Spleen
Genotype III could only be determined with SAG3 locus.
Ac
a
Age (months)
ce pt
Total
Origin
cr
Conventional PCR amplification of locus:
Page 13 of 14
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ce
pt
ed
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Figure
Page 14 of 14
S0304-4017(15)00049-7 http://dx.doi.org/doi:10.1016/j.vetpar.2015.02.004 VETPAR 7528
To appear in:
Veterinary Parasitology
Received date: Revised date: Accepted date:
20-8-2014 4-2-2015 8-2-2015
Please cite this article as: Rico-Torres, C.P., Del Viento-Camacho, A., CaballeroOrtega, H., Besn´e-M´erida, A., Luna-Past´en, H., Correa, D., Palma-Garc´ia, J.M.,First isolation of Toxoplasma gondii from cats of Colima, Mexico: tissue distribution and genetic characterization, Veterinary Parasitology (2015), http://dx.doi.org/10.1016/j.vetpar.2015.02.004 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.
2
RESEARCH HIGHLIGHTS
-
3 4 5
We isolated viable T. gondii tachyzoites (ToxoDB PCR-RFLP genotype #28) from one cat of Colima, Mexico.
-
The brachiocephalic muscle, the brain, and the spleen were the tissues where T.
ip t
1
gondii DNA was more frequently detected.
Ac ce p
te
d
M
an
us
cr
6
Page 1 of 14
1
First isolation of Toxoplasma gondii from cats of Colima, Mexico: tissue distribution
2
and genetic characterization
3
Claudia Patricia Rico-Torresa, Alejandra Del Viento-Camachob, Heriberto Caballero-
5
Ortegaa, Alejandro Besné-Méridaa, Héctor Luna-Pasténa, Dolores Correaa, José Manuel
6
Palma-Garcíab*
cr
ip t
4
9
a. Laboratorio de Inmunología Experimental. Instituto Nacional de Pediatría, Secretaría de Salud, México D.F., C.P. 04530, México.
an
8
us
7
b. Centro Universitario de Investigación y Desarrollo Agropecuario, Facultad de
11
Medicina Veterinaria y Zootecnia, Universidad de Colima, México. Av. Gonzalo de
12
Sandoval # 444, Colima, CP 28045 AP 22, México.
d
M
10
te
13
*Corresponding author:
15
José Manuel Palma García, DVM, PhD
16
Address: Avenue Gonzalo de Sandoval # 444, Colima, CP 28045 AP 22, México.
17
Tel.: +52 (312) 316 10 00 ext. 40011
18
Fax: +52 (312) 312 75 81
19
E-mail: [email protected]
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14
20
1 Page 2 of 14
ABSTRACT
22
Toxoplasma gondii is among the commonest zoonotic infectious agents worldwide. It
23
infects many warm-blooded animals, including felines, the definitive hosts. This parasite is
24
now classified in 15 haplogroups spread out around the world. Few reports reveal a
25
predominance of genotypes I and III in Mexico, although recombinant and atypical variants
26
have also been found in humans and animals. The aim of this study was to detect, isolate
27
and genotype T. gondii from cats of Colima Mexico, and to analyze tissue distribution of
28
the parasite. IgG specific antibodies were investigated in 48 serum samples from unwanted
29
and stray cats by indirect ELISA. Isolation in mice and molecular characterization by PCR-
30
RFLP and sequencing were attempted using pools of brain, heart, liver, lung, spleen and
31
brachiocephalic muscle samples of seropositive cats. Fourteen animals (29.2%) were
32
seropositive, the frequency ranged between 27.3% and 40% among the different localities.
33
Ten seropositive animals were euthanized, eight of them were positive for the B1 gene by
34
conventional PCR. More frequently infected tissues were the brachiocephalic muscle
35
(75.0%) the brain (63.0%) and the spleen (63.0%). Genotype III was determined for the
36
SAG3 locus of the parasite infecting an unwanted cat. Tachyzoites were isolated from the
37
peritoneal cavity of two mice inoculated with the tissue pool of one kitten. Type I alleles
38
were found in SAG1, SAG2, SAG3, BTUB, GRA6, c29-2 and PK1 loci, while c22-8 was
39
type II, and L358 and Apico were type III. This genotype corresponds to ToxoDB genotype
40
#28. This is the first T. gondii isolate genetically characterized in Colima, Mexico and is
41
different to other isolations of the country.
42
Keywords: Toxoplasma gondii; cats; molecular characterization; isolation; genotype;
43
Mexico
Ac ce p
te
d
M
an
us
cr
ip t
21
44 2 Page 3 of 14
1. INTRODUCTION
46
Toxoplasma gondii is an obligate intracellular parasite that infects a broad range of warm-
47
blooded animals. Felines play a critical role in the epidemiology of this parasitosis, because
48
they release oocysts with their feces, which survive several months in the environment,
49
especially in warm and humid zones (Dubey, 2004).
50
Currently T. gondii is classified into 15 haplogroups with a world-wide distribution (Su et
51
al., 2012). In Mexico a predominance of type I and III has been supported from studies
52
performed in several species, including humans, although atypical variants have also been
53
found (Dubey et al., 2004; 2009; Alvarado-Esquivel et al., 2011; Cedillo-Peláez et al.,
54
2011; Rico-Torres et al., 2012). Nevertheless, little is known about genotypes of T. gondii
55
circulating in cats; only atypical and recombinant variants have been reported in domestic
56
cats and one puma of Durango (Dubey et al., 2009; 2013).
57
The presence of T. gondii in cats of Colima has been documented, but no genotyping has
58
been performed in this zone (García-Marquez et al., 2007). Thus, the aim of this study was
59
to detect, isolate and genotype T. gondii which infects cats in Colima and to analyze tissue
60
distribution of the parasite.
61
2. MATERIALS AND METHODS
62
2.1. Bioethics
63
Animals were handled according to the Mexican Official Standards for animal care (NOM-
64
033-ZOO-1995 and NOM-062-ZOO-1999). The Committee for Care and Use of
65
Laboratory Animals of the Instituto Nacional de Pediatría (INP) approved the project
66
(013/2012).
Ac ce p
te
d
M
an
us
cr
ip t
45
67 68 3 Page 4 of 14
2.2. Collection of samples and serology
70
A total of forty-eight cats -three months to four years old- from Colima, Manzanillo and
71
Villa de Álvarez municipalities of Colima State, Mexico, were studied from February to
72
July 2008. Blood samples from the jugular vein or the heart were obtained and sera were
73
tested using an in-house indirect ELISA as previously reported (Besné-Merida et al., 2008).
74
Samples from the brain, heart, liver, lung, spleen and brachiocephalic muscle of
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seropositive cats were sent to INP within 12h of their collection at 2-4°C for bioassays in
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mice and molecular analysis.
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2.3. Bioassay in mice
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Pools of tissues (200 mg of each) were manually homogenized using a mortar in 1 mL of
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sterile PBS; then, 0.25 mL of the supernatant were inoculated intraperitoneally into each of
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two Balb/c mice. They were bled before infection and on days 10, 20, 30, 40, 50 and 60
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after inoculation and tested by indirect ELISA; serum samples were diluted 1:150 and
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1:250 for IgM and IgG anti-T. gondii antibodies (abs), respectively, and the reaction was
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developed using anti-mouse IgM (1:250) or IgG (1:1000) peroxidase conjugates (Sigma-
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Aldrich Corp., St Louis, MO, USA). Serocoverted mice were euthanized eight weeks after
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inoculation and a pool of tissues (brain, heart, liver, lung and spleen) was used to test for T.
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gondii by PCR.
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2.4. Detection of repetitive B1 gene of T. gondii for molecular diagnosis
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DNA was extracted with the GENTRA® commercial kit (Minneapolis, MN, USA) using
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200 mg (cats) or 20 mg of each tissue/organ (mice) according to the manufacturer’s
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instructions. The repetitive B1 gene was specifically amplified to obtain 364 and 62 bp
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fragments by conventional PCR and real time PCR, respectively (Rico-Torres et al., 2012).
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2.5. Genetic characterization
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In order to determine the genotype of T. gondii, SAG1, Alt.SAG2, SAG3, BTUB, GRA6,
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c22-8, c29-2, L358, PK1 and Apico genes were analyzed by PCR-RFLP and sequencing
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(Su et al., 2010). RH, (type I), VEG (type III) and Wiktor (ToxoDB #6) strains were used
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for reference restriction patterns. Sequences were analyzed with Chromas Lite 2.1.1
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software and aligned with those reported in GenBank and Toxo.DB.org web pages by
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Clustal W2 and NCBI/BLAST websites.
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3. RESULTS
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Anti-T. gondii abs were found in 14 out of 48 (29.2%) cats of Colima. The frequency was
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28.1%, 27.3% and 40%, for Colima, Manzanillo and Villa de Álvarez municipalities,
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respectively (Fig. 1). Ten of the fourteen seropositive cats were euthanized and eight of
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them (80.0%) were positive by conventional PCR.
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Parasite DNA was more frequently detected in brachiocephalic muscle, brain and spleen
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(Table 1). The genotype could only be determined at the SAG3 locus in cat #6 (brain) being
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type III. At least one mouse inoculated with homogenates from cats #1, #2, and #7
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seroconverted for abs. Mice injected with tissues of cats #1 and #7 were euthanized eight
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weeks later and resulted positive for the B1 and SAG3 loci; however, bands obtained were
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faint, so the genotype could not be determined. Mice inoculated with tissues of cat #2
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seroconverted at day 21 after inoculation and were euthanized because they presented
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clinical signs compatible with toxoplasmosis. Tachyzoites were recovered from the
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peritoneal cavity and the DNA was positive by conventional and real time PCR. The
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analysis by PCR-RFLP and sequencing showed that all loci were type I except for c22-8
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(type II) and L358 and Apico (type III) corresponding to genotype #28, according to
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ToxoDB.
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4. DISCUSSION
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There are few studies about T. gondii genotypes in Mexico and none in Colima, a state with
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warm humid climate where transmission has been documented. Through serological
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screening we found candidate cats for genotyping and tissue distribution analyses. The
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brachiocephalic muscle was the tissue more frequently infected, followed by the brain and
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the spleen; surprisingly, T. gondii DNA was less frequent in the heart, which is different to
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data reported elsewhere (Dubey el al., 2009).
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Parasite genotype could only be determined at the SAG3 in the brain sample of a cat of the
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coast, being type III. Tachyzoites were isolated from mice inoculated with tissues of a
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kitten of Colima city and presented a combination of type I, II and III, corresponding to
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genotype ToxoDB #28, which has previously been reported in USA (humans), Brazil
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(chickens) and Colombia (cats and chickens) (Howe and Sibley, 1995; Dubey et al., 2006;
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2007). In Durango State, in the Northwest region of Mexico, genotypes #2, #9, #73, #74,
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#155 and #222 have been demonstrated in cats, dogs, chickens and one puma (Dubey et al.,
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2009; 2013). Samples from congenitally infected children and squirrel monkeys with
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toxoplasmosis acquired around Mexico city revealed type I alleles for Alt.SAG2 and SAG3
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genes, as for the cat isolate, but u-1 alleles were found in other two congenital cases at
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Alt.SAG2 and BTUB loci (Cedillo-Peláez et al., 2011; Rico-Torres et al., 2012).
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conclusion, T. gondii varies within the State of Colima for the SAG3 locus and in relation to
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other regions of the country.
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CONFLICT OF INTEREST STATEMENT
140
The authors declare no conflict of interests.
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ACKNOWLEDGMENTS
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The authors are most thankful to Dr. Chunlei Su for his kind advice on multilocus PCR-
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RFLP technical details. This work was partially financed by grant 013/2012 from Instituto
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Nacional de Pediatría.
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8 Page 9 of 14
REFERENCES
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Alvarado-Esquivel, C., Rajendran, C., Ferreira, L.R., Kwok, O.C., Choudhary, S.,
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Alvarado-Esquivel, D., Rodríguez-Peña, S., Villena, I., Dubey, J.P., 2011.
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Prevalence of Toxoplasma gondii infection in wild birds in Durango, Mexico. J.
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Parasitol. 97, 809–812.
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Besné-Mérida, A., Figueroa-Castillo, J.A., Martínez-Maya, J.J., Luna-Pastén, H., Calderón-
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Segura, E., Correa, D., 2008. Prevalence of antibodies against Toxoplasma gondii in
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domestic cats from Mexico City. Vet. Parasitol. 157, 310-313.
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Cedillo-Peláez, C., Rico-Torres, C.P., Salas-Garrido, C.G., Correa, D., 2011. Acute
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toxoplasmosis in squirrel monkeys (Saimiri sciureus) in Mexico. Vet. Parasitol.
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Dubey, J.P., 2004. Toxoplasmosis - a waterborne zoonosis. Vet Parasitol. 126, 57-72.
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Dubey, J.P., Morales, E.S., Lehmann, T., 2004. Isolation and genotyping of Toxoplasma
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Mora, L.E., Lora, F., Jimenez, J., Kwok, O.C.H., Shen, S.K., Zhang, X., Nieto, A.
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and Thulliez, P., 2006. Prevalence of Toxoplasma gondii in cats from Colombia, South America and genetic characterization of T. gondii isolates. Vet. Parasitol. 141, 42-47.
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Dubey, J.P., Sundar, N., Gennari, S.M., Minervino, A.H.H., Farias, N.A.d.R., Ruas, J.L.,
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dos Santos, T.R.B., Cavalcante, G.T., Kwok, O.C.H., and Su, C., 2007. Biologic
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and genetic comparison of Toxoplasma gondii isolates in free-range chickens from
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the northern Para state and the southern state Rio Grande do Sul, Brazil revealed
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Dubey, J.P., Velmurugan, G.V., Alvarado-Esquivel, C., Alvarado-Esquivel, D., Rodríguez-
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Peña, S., Martínez-García, S., González-Herrera, A., Ferreira, L.R., Kwok, O.C.,
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Su, C., 2009. Isolation of Toxoplasma gondii from animals in Durango, Mexico. J.
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Dubey, J.P., Alvarado-Esquivel, C., Herrera-Valenzuela, V.H., Ortiz-Diaz, J.J., Oliveira, S.,
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Verma, S.K., Choudhary, S., Kwok, O.C., Su, C., 2013. A new atypical genotype
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mouse virulent strain of Toxoplasma gondii isolated from the heart of a wild caught
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puma (Felis concolor) from Durango, Mexico. Vet. Parasitol. 197, 674-677.
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García-Márquez, L.J., Gutiérrez-Díaz, M.A., Correa, D., Luna-Pastén, H., Palma, J.M.,
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2007. Prevalence of Toxoplasma gondii antibodies and the relation to risk factors in
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Howe, D.K., and Sibley, L.D., 1995. Toxoplasma gondii comprises three clonal lineages: correlation of parasite genotype with human disease. J. Infect. Dis. 172, 1561-1566.
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Rico-Torres, C.P., Figueroa-Damián, R., López-Candiani, C., Macías-Avilés, H.A.,
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Cedillo-Peláez, C., Cañedo-Solares, I., Luna-Pastén, H., Tecuatl-Herrada, B.L.,
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Correa, D., 2012. Molecular diagnosis and genotyping of cases of perinatal toxoplasmosis in Mexico. Pediatr. Infect. Dis. J. 31, 411-413.
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Su, C., Shwab, E.K., Zhou, P., Zhu, X.Q., Dubey, J.P., 2010. Moving towards an integrated
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Su, C., Khan, A., Zhou, P., Majumdar, D., Ajzenberg, D., Dardé, M.L., Zhu, X.Q., Ajioka,
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J.W., Rosenthal, B.M., Dubey, J.P., Sibley, L.D., 2012. Globally diverse
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Toxoplasma gondii isolates comprise six major clades originating from a small
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number of distinct ancestral lineages. Proc. Natl. Acad .Sci. U S A. 109, 5844-5849. 10 Page 11 of 14
FIGURE CAPTIONS
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Figure 1. Map of Colima, Mexico showing the climatic characteristics of the three
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municipalities studied, the frequency of IgG antibodies and the detection of Toxoplasma
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gondii DNA in tissues from seropositive cats and the genotypes obtained by PCR-RFLP of
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Colima, Mexico. masl = meters above sea level; *parasite genotype could only be
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determined at the SAG3 locus.
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Table
Table 1. Molecular detection of Toxoplasma gondii in sample tissues from cats of Colima, Mexico
Cat No. (n=8)
Municipality
1
Colima
Unwanted
18
2
Colima
Unwanted
3
3
Colima
Stray
12
4
Colima
Stray
12
5
Manzanillo
Unwanted
12
6
Manzanillo
Unwanted
7
Manzanillo
Unwanted
8
Villa de Álvarez
Frequency (%)
Stray
Brachiocephalic muscle
Heart
Liver
Lung
–
B1
–
–
–
–
B1
–
B1
B1
B1
B1, SAG3
B1, SAG3
–
–
–
B1
–
–
B1
–
–
B1, SAG3
–
–
–
–
–
B1
B1
30
B1, SAG3a
–
–
–
B1
B1
30
B1
–
B1, SAG3
B1
B1
B1
B1
–
–
–
–
B1
5
2
2
2
6
5
63
25
25
25
75
63
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Brain
Spleen
Genotype III could only be determined with SAG3 locus.
Ac
a
Age (months)
ce pt
Total
Origin
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Conventional PCR amplification of locus:
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Figure
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