Toxoplasma gondii in Captive Wild Felids of Mexico: Its Frequency and Capability to Eliminate Oocysts

Abstract

There is little information about Toxoplasma gondii in wild felids, even when these species have been associated with cases of toxoplasmosis in humans. In this study, samples of serum and whole blood were collected from 42 felids from 10 different species, in 4 Mexican zoos. Stool samples from 36 animals were also collected, corresponding to 82% of the felids included in the study. Stool samples were used for the search of oocysts by light field microscopy and PCR. Serum samples were analyzed by indirect immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) and indirect fluorescent antibody test (IFAT). DNA samples were purified from whole blood and stool for the amplification of a fragment of the SAG1 gene of T. gondii by a nested PCR (nPCR). The seroprevalence of IgG anti-T. gondii-specific antibodies by means of the ELISA was 100% (42/42) and 52.4% (22/42) by IFAT. The titers obtained varied from 1:80 to 1:2560. DNA of T. gondii was detected in 9.5% (4/42) of the blood samples by using nPCR. No oocysts were observed in the stool samples analyzed by light field microscopy. However, the DNA of the parasite was identified in 14.3% (5/35) of the stool samples evaluated. These results indicate a high prevalence of T. gondii in the studied populations of wild felids in captivity, with evidence of parasitemia and elimination of few oocysts even in adult hosts.

Introduction

T oxoplasma gondii is an intracellular protozoan of worldwide distribution, with a life cycle that includes members of family Felidae as definitive hosts and, as intermediate hosts, a wide range of birds and mammals (Dubey 2009). A susceptible wild or domestic felid can excrete millions of oocysts after ingesting raw meat containing T. gondii cysts; excreted oocysts can survive in the soil even for years (Yang et al. 2017), can also be transported through fresh water runoff of main cities (Hernandez-Cortazar et al. 2017). Humans are usually infected by the ingestion of tissue cysts in poorly cooked meat, or by the consumption of food or beverages contaminated with T. gondii oocysts (Dubey et al. 2014).

There is little information on parasite load, genotypes, and T. gondii cycle in wild felid species. In these wild hosts, T. gondii may negatively affect their conservation, reintroduction, and/or translocation projects (Ullmann et al. 2010). Likewise, cases of toxoplasmosis have been reported in humans associated with wild felids (either by direct or indirect contact) (Gómez-Marín et al. 2012); in the 1990s an outbreak of human toxoplasmosis was reported in Victoria, British Columbia, Canada, associated with water supply contaminated most probably with oocysts from cougars and domestic cats (Bowie et al. 1997).

In Mexico, few studies describe the presence of T. gondii in wild felids (Kikuchi et al. 2004, Rendón-Franco et al. 2012, Dubey et al. 2013). The last study in Mexico reported a high seroprevalence (81.4%) from three zoos in Mexico City (Alvarado-Esquivel et al. 2013). Molecular studies of T. gondii have been used in organ tissues of wild felids in captivity with suggestive signs of acute toxoplasmosis (Kenny et al. 2002, Lloyd and Stidworthy 2007, Dubey et al. 2010) and free-living felids that died from various causes (Miller et al. 2008, Dubey et al. 2013, Vitaliano et al. 2014).

The objective of this study was to determine the serological and molecular frequency of T. gondii in wild felids of four Mexican zoos and to evaluate their capability to excrete oocysts.

Materials and Methods

Studied area

Between July 2015 and November 2016, samples of peripheral blood and feces were collected from felids belonging to four Mexican zoos (named 1, 2, 3, and 4), located in the states of Quintana Roo, Yucatan, Chiapas, and Morelos, respectively. The species included in the study were Yaguarundi (Puma yagouarundi), Cougar (Puma concolor), Tigrillo (Leopardus tigrinus), Ocelot (Leopardus pardalis), Lynx (Caracal caracal), Red lynx (Lynx rufus), Jaguar (Panthera onca), Leopard (Panthera pardus), Tiger (Panthera tigris), and Lion (Panthera leo). The felids included in this study were usually fed with raw meat of chicken, horse, and occasionally rabbit; in all the zoos, the meat was frozen at −10°C for a maximum of 2 days before feeding.

Collection and processing of samples

Blood samples (with ethylenediaminetetraacetic acid anticoagulant to obtain whole blood and without anticoagulant to obtain serum) were obtained by venipuncture of the cephalic, saphenous, femoral, jugular, or caudal veins of chemically immobilized animals during routine checkups or those performed for clinical purposes. From each animal, 2 g of feces was collected, preferably on the same day of the anesthetic management of the specimens or during the 24 h before or after handling; samples were placed in tubes containing 70% ethanol (3 mL/g of feces) and refrigerated until processing (Jongwutiwes et al. 2002, Piggott and Taylor 2003).

Blood samples were centrifuged for 10 min at 448 g to obtain the serum and, subsequently, these sera were individually labeled and stored at −20°C until analysis.

Enzyme-linked immunosorbent assay

The presence of anti-T. gondii immunoglobulin G (IgG) antibodies was determined using an indirect enzyme-linked immunosorbent assay (ELISA) test (Human-GmbH, Wiesbaden, Germany; catalog number ab108776) following the modifications described by Figueroa-Castillo et al. (2006), using as secondary antibody an anti-cat IgG coupled to horseradish peroxidase (Santa Cruz, Inc., CA).

Indirect fluorescent antibody test

Likewise, the samples were analyzed and titrated using the indirect fluorescent antibody test (IFAT), for which 10 μL of serum was placed in plates previously sensitized with 10 μL of T. gondii tachyzoites from axenic cultures (at a concentration of 7 × 10⁶ parasites/mL), fixed with methanol and blocked with 50% horse serum in phosphate-buffered saline (PBS). The titration began with a 1:40 dilution and was exponentially doubled to 1:10,240. As secondary antibody, anti-IgG-cat-fluorescein isothiocyanate (Bethyl, Montgomery, TX) was used at a dilution of 1:80 and 0.01% Evans blue added to the blocking solution (50% horse serum in PBS), subsequently it was incubated for 1 h in a humid chamber and in the dark. Finally, the plates were mounted using 9 μL of Vectashield^® (Vectorlabs) and examined under a fluorescence microscope (epi-illumination system). As a positive control, domestic cat serum previously diagnosed by ELISA and PCR was used. The highest serum dilution in which fluorescence was detected in the periphery of the parasite was considered as a positive titer; in cases in which there was no fluorescence or it was located only at one end of the parasite, the titer was considered negative (Domingues and Machado 1998).

Stool evaluation

Before oocyst flotation, stool samples were washed repeatedly with double-distilled water. The flotation was carried out according to the methodology described by Dubey and Beattie (1988); in brief, Sheather's solution (106 g of glucose, 100 mL of double-distilled water, and 0.8 mL of liquid phenol, with a density of 1.27) was added to an equal volume of the stool samples, vortexed briefly and centrifuged at 1000 g for 15 min. Examination of oocysts was performed by using light microscopy. A wire loop (diameter = 6 mm) was used to touch the surface of the sample flotate after centrifugation, and then the samples were examined with a light microscope at a magnification of 40 × .

DNA extraction and evaluation of inhibitors from the samples

From the whole blood samples, 1 mL was taken to purify genomic DNA, following the methodology described by Jalal et al. (2004) and using the DNeasy Blood and Tissue commercial kit (QIAGEN; catalog number 69506). Subsequently, to demonstrate the absence of inhibitors in the purified blood samples, the amplification of the gene coding for the enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was carried out, the primers used were the sense 5′-AGCGCGCGTCTAAGACTTACA-3′ and the antisense 5′-TGGAGCTGCGGTTGTCAATT-3′. The amplification conditions were initial denaturation of 95°C for 3 min followed by 39 cycles at 95°C for 10 s and, finally 55°C for 30 s (Dzib-Paredes et al. 2016).

In the case of stool samples, the floating supernatant with Sheather's solution was used to obtain DNA. A commercial QIAamp^® DNA Stool Mini Kit (QIAGEN; catalog number 51504) was used following the extraction protocol described by Salant et al. (2010). The purified DNA samples were kept frozen at −20°C until analysis.

To evaluate the absence of inhibitors in the purified stool samples, the constitutive 18S gene (ribosomal RNA) was amplified, with the sense primers 5′-ACCGCAGCTAGGAATAATGGA-3′ and antisense 5′-GCCTCAGTTCCGAAAACCA-3′ (Shi et al. 2013). The conditions were initial denaturation of 95°C, 3 min, followed by 40 cycles at 95°C for 15 s, 60°C for 30 s, and, finally, 55°C for 30 s. The amplifications for both constitutive genes were performed in a C1000 CFX 96TM C1000 real-time thermocycler (Bio-Rad). The samples of blood and stool that amplified GAPDH and 18S, respectively, were analyzed to determine the presence of T. gondii DNA by means of a nested PCR (nPCR).

nPCR for the detection of T. gondii DNA

The nPCR was used to amplify a fragment of 390 bp of the SAG1 gene that codes for the main protein of surface of T. gondii (Su et al. 2010). The amplification was performed in a Veriti thermocycler (Applied Biosystems), using the external primers sense 5′-GTTCTAACCACGCACCCTGAG-3′ and antisense 5′-AAGAGTGGGAGGCTCTGTGA-3′; in the second amplification the internal primers were sense 5′-CAATGTGCACCTGTAGGAAGC-3′ and antisense 5′-GTGGTTCTCCGTCGGTGTGAG-3′. The first amplification reaction was carried out with the PCR buffer IX (PROMEGA), MgCl₂ 2 mM, 0.8 mM dNTPs, 0.5 μM for both primers, 1.5 U of the Taq polymerase, and 4 μL of DNA from the samples in a final volume of 25 μL per reaction. The second run had the same concentrations as the first reaction, but the concentration of the internal primers was 0.3 μM and 2 μL of the PCR product of the first run was used as tempering. The conditions of the nPCR in the first run were 95°C for 5 min, followed by 30 cycles of 94°C for 30 s, 55°C for 1 min, and 72°C for 2 min; and in the second run, 95°C for 5 min, followed by 35 cycles of 94°C for 30 s, 60°C for 1 min, and 72°C for 1:30 min. Positive controls (T. gondii DNA) and negative (reaction without DNA) were included in each run of nPCR. The amplification products were visualized in 1.5% agarose gels stained with ethidium bromide (10 mg/mL in H₂O).

Results

A total of 42 viable serum and whole blood samples were collected from 10 species of wild felids (Table 1) of which 2 samples belong to zoo 1, 9 to zoo 2, 10 to zoo 3, and 21 to zoo 4. Regarding the stool samples, 36 samples were collected (85.7% of the felids included in the study). However, on three occasions it was necessary to pool feces samples since two felids were kept in the same space.

Table 1.

Toxoplasma gondii in Samples of Serum and Feces of Felines in Captivity in Mexico

Species	Anti-IgG Toxoplasma gondii		nPCR SAG1
Species	ELISA	IFAT	Blood	Stool
Leopardus tigrinus	3/3	2/3 (1:320)	1/3	0/3
Puma yagouarundi	2/2	0/2	0/2	0/1
Lynx rufus	2/2	1/2 (1:2560)	0/2	0/1^a
Caracal caracal	1/1	1/1 (1:160)	0/1	0/1
Leopardus pardalis	2/2	0/2	0/2	1/2
Puma concolor	2/2	0/2	1/2	1/3
Panthera pardus	1/1	1/1 (1:320)	0/1	1/1
Panthera onca	12/12	5/12	0/12	1/9^a
Panthera tigris	9/9	6/9 (1:2560)	1/9	1/8^a
Panthera leo	8/8	6/8 (1:320)	1/8	0/6
Total	42/42 (100%)	22/42 (52%)	4/42 (9.5%)	5/35 (14.3%)

Considering a combined sample.

nPCR, nested PCR; ELISA, enzyme linked immunosorbent assay; IFAT, indirect fluorescent antibody test; IgG, immunoglobulin G.

The seroprevalence of anti-T. gondii IgG antibodies was 100% (42/42) with the ELISA test and 52.38% (22/42) with the IFAT test (Table 1). The antibody titers for IFAT were from 1:40 (n = 1) to 1:2560 (n = 3), with the majority of the specimens being titrated between 1:80 (n = 8) and 1: 160 (n = 5). No T. gondii oocysts were found in any of the stool samples analyzed by light field microscopy. All blood samples were positive for the constitutive gene GAPDH (42/42) and, in the case of the stool, 35 out of 36 samples were positive for the constitutive 18S gene. DNA of T. gondii was detected in 9.5% (4/42) of the blood samples and in 14.3% (5/35) of the stool samples, amplifying a fragment of 390pb of the SAG 1 gene (Fig. 1).

FIG. 1.

Electrophoresis in 1.5% agarose gel stained with ethidium bromide of the amplification product with the primers for SAG1 of Toxoplasma gondii. Lanes: MP, molecular weight marker (100 bp DNA ladder, reference: G210A, Promega); CN1, first reaction negative control (master mix without tempering); CN2, second reaction negative control (master mix without tempering); CP, 390 bp positive control (DNA of T. gondii tachyzoites from axenic culture); M1, positive sample of a lion; M2, negative samples of an oncilla; M3–M4, positive sample of oncillas; M5, positive sample of a jaguar; M6, negative samples of a jaguar; M7, negative sample of an ocelot. bp, base pairs.

Discussion

The seroprevalence found in this study has been one of the highest described so far in wild felids in captivity, using an indirect ELISA test. In another study carried out in three zoos from Mexico City, a seropositivity of 81.4% (n = 43) was reported, using the modified agglutination test (MAT) (Alvarado-Esquivel et al. 2013). Studies conducted in Brazil indicate a seroprevalence of 63.4% in 161 wild felids of 14 species using the IFAT technique (André et al. 2010), and 54.6% in 865 specimens of 8 species of felids kept in captivity (including 71 zoos and 15 private breeders) with the MAT (Silva et al. 2001). The high frequency of seropositive observed in this study is similar to those found in domestic cats in the different regions of Mexico where the detection of IgG antibodies varies with a prevalence that can reach up to 91.8% (Alvarado-Esquivel et al. 2007, García-Márquez et al. 2007, Besné-Mérida et al. 2008, Castillo-Morales et al. 2012), which may suggest high environmental contamination with infectious oocysts.

Although high seroprevalences are expected in felids, it is important to determine the origin of this, since it may be due to particular conditions of the zoos where risk factors such as feeding with raw meat can be an important route of infection and reinfection (Ramos Silva et al. 2007); the presence of domestic cats in the zoos may also contribute to the spread of infective oocytes. The captive felids included in this study were fed with chicken, horse, and occasionally rabbit meat, species in which anti-T. gondii antibodies have been identified in Mexico (Figueroa-Castillo et al. 2006, Alvarado-Esquivel et al. 2012a, 2012b).

The oral route of infection was probably responsible for the high prevalence of seropositive animals. T. gondii cysts present in the meat can be inactivated when frozen at −10°C for 3 days or at −20°C for 2 days (El-Nawawi et al. 2008). This situation may favor the first infection and/or reinfection of the animals in captivity; however, reinfection appears very rare. To determine this, it is necessary to titrate the antibodies with an interval of 2–4 weeks to establish if a reinfection appears or is an active infection (Lappin and Powell 1991). Although ELISA and IFAT have been used in several studies of toxoplasmosis, it has not been adequately validated in wild felids. However, the results of the IFAT when demonstrating very high titers in some specimens may be indicative of an active toxoplasmosis, or of a reactivation of the infection due to an immunosuppression (Robert et al. 1981).

Direct microscopy to detect T. gondii oocysts excretion in feces is a simple and economical technique, but with a low sensitivity (depending on the experience of the observer, the time of collection of the sample, and the number of excreted oocysts per gram of feces [OPG]). Therefore, the monitoring of feces for >3 weeks is required and must be complemented with bioassay techniques to confirm the presence of the parasite (Jewell et al. 1972, Miller et al. 1972, Aramini et al. 1998); this is why probably no oocysts were found in any of the samples evaluated in our study. Salant et al. (2010) concluded that copro-PCR correlates with the bioassay and that it is much more sensitive than microscopy alone (even than bioassays). In this study it was possible to detect DNA of the parasite in 14.3% of the stool samples, indicating that PCR-positive animals were in an active phase of oocyst excretion, but probably with a small number of OPG that could not be detected by coprological analysis. However, it is possible that more specimens eliminated oocysts before or after the sampling.

As for the blood PCR positive animals, these results indicate a parasitemia that could be due to a prime infection, a reactivation of a latent toxoplasmosis, or a reinfection with different T. gondii genotype, since this can be established and circulate in the bloodstream until specific immunity develops against it; so it is important to determine if these felids eliminated oocysts at any time or if they had any change in antibody titers. Dubey (2014) mentions that the excretion period and the OPG eliminated by wild felids have not been determined for all species, but it is assumed that they are similar to the domestic cat and that, after the first infection, these definitive wild hosts could have periods of elimination shorter and with significantly lower number of OPG. However, this has not been completely confirmed and, therefore, it is possible that there are variations of host species. Similarly, like the domestic cat, wild felids can be affected by immunosuppressive factors, which can favor the intra-organic recirculation and fecal excretion of the parasite. Some studies in wild felids sharing a coinfection of T. gondii with leukemia virus, immunodeficiency, calicivirus, and feline panleukopenia, as well as canine distemper have been reported (Ketz-Riley et al. 2003, Buddhirongawatr et al. 2006, Fiorello et al. 2006, 2007, Bevins et al. 2012), which may favor a reactivation of parasitemia and/or elimination of oocysts. In Mexico, there are no reports on the prevalence of these viruses in wild felids. However, recent studies have showed a prevalence of feline viral leukemia in domestic cats of 7.5% (ELISA) (Ortega-Pacheco et al. 2014) to 75% (PCR) (Ramírez et al. 2016). Moreover, in the case of the feline immunodeficiency virus, a seroprevalence of 2.5% (ELISA) has been reported (Ortega-Pacheco et al. 2014), and 52% of seroprevalence of the canine distemper virus in dogs (Ortiz-Amador 2015). Owing to the mentioned, it is advisable to determine the presence of these viruses in the populations included in this study, since in nine specimens (seven of them healthy adults) the presence of the parasite genotype was detected.

The positive copro-PCR specimens were clinically healthy at the time of sampling, except in two cases: a 7-month-old ocelot that developed diarrhea and an adult cougar that suffered hemorrhagic gastroenteritis with fatal outcome. The etiological agent associated with the deceases was not determinate. Both specimens belonged to zoos with different environments and nutritional management. Previously, the presence of T. gondii oocysts in diarrheal feces has been reported in cheetah (Acinonyx jubatus) and Siberian tiger (Panthera tigris altaica) in captivity (Dorny and Fransen 1989, Lloyd and Stidworthy 2007).

Conclusion

There is a high seroprevalence of T. gondii in captive wild felid populations studied with probable phases of parasitemia and elimination of oocysts. Prospective studies are needed to titrate the antibodies to discern identified type of infection, as well as to determine the evolution of the number of oocyst excreted per gram of feces and the excretion period in these populations contemplating the necessary strategies for the identification of the genotypes involved. As contamination in the zoo cannot be ruled out, meat should be frozen for >2 days before feeding the animals.

Footnotes

Acknowledgments

The authors thank the veterinary and administrative personnel of the zoos: Epigmenio Cruz Aldán, Angel D. Alvarado Díaz, Jorge A. Santiago Vazquez, Romeo Morales Espinoza, Angel J. Tzuc Salinas, Odeisi Mora Camacho, Antonio Avila Ruiz, Rosa Reyes Valle, and Geovanni Alcoser Conde.

Author Disclosure Statement

No conflicting financial interests exist.

References

Alvarado-Esquivel

, Gayosso-Dominguez

, Villena

, Dubey

. Seroprevalence of Toxoplasma gondii infection in captive mammals in three zoos in Mexico City, Mexico. J Zoo Wildl Med, 2013; 44:803–806.

Alvarado-Esquivel

, González-Salazar

, Alvarado-Esquivel

, Ontiveros-Vázquez

, et al. Seroprevalence of Toxoplasma gondii infection in chickens in Durango state, Mexico. J Parasitol, 2012a; 98:431–432.

Alvarado-Esquivel

, Liesenfeld

, Herrera-Flores

, Ramírez-Sánchez

, et al. Seroprevalence of Toxoplasma gondii antibodies in cats from Durango city, Mexico. J Parasitol, 2007; 93:1214–1216.

Alvarado-Esquivel

, Rodríguez-Peña

, Villena

, Dubey

. Seroprevalence of Toxoplasma gondii infection in domestic horses in Durango state, Mexico. J Parasitol, 2012b; 98:944–945.

André

, Adania

, Teixeira

RHF

, Silva

, et al. Antibodies to Toxoplasma gondii and Neospora caninum in captive neotropical and exotic wild canids and felids. J Parasitol, 2010; 96:1007–1009.

Aramini

, Stephen

, Dubey

. Toxoplasma gondii in Vancouver Island cougars (Felis concolor vancouverensis): serology and oocyst shedding. J Parasitol, 1998; 84:438–440.

Besnacute-Mérida

, Figueroa-Castillo

, Martínez-Maya

, Luna-Pastén

, et al. Prevalence of antibodies against Toxoplasma gondii in domestic cats from Mexico City. Vet Parasitol, 2008; 157:310–313.

Bevins

, Carver

, Boydston

, Lyren

, et al. Three pathogens in sympatric populations of pumas, bobcats, and domestic cats: Implications for infectious disease transmission. PLoS One, 2012; 7:e31403.

Bowie

, King

, Werker

, Issac-Ramon

, et al. Outbreak of toxoplasmosis associated with municipal drinking water. Lancet, 1997; 350:173–177.

10.

Buddhirongawatr

, Tungsudjai

, Chaichoune

, Sangloung

, et al. Detection of Toxoplasma gondii in captive wild felids. Southeast Asian J Trop Med Public Health, 2006; 37:15–17.

11.

Castillo-Morales

, Acosta-Viana

, Guzmán-Marín

EDS

, Jiménez-Coello

, et al. Prevalence and risk factors of Toxoplasma gondii infection in domestic cats from the tropics of Mexico using serological and molecular tests. Interdiscip Perspect Infect Dis, 2012; 529108.

12.

Domingues

, Machado

. Canine toxoplasmosis: A comparative evaluation of the detection of anti-Toxoplasma gondii antibodies by the indirect immunoenzymatic assay (ELISA). Braz J Vet Parasitol, 1998; 7:79–85.

13.

Dorny

, Fransen

. Toxoplasmosis in a Siberian tiger (Panthera tigris altaica). Vet Rec, 1989; 125:647.

14.

Dubey

. History of the discovery of the life cycle of Toxoplasma gondii . Int J Parasitol, 2009; 39:877–882.

15.

Dubey

. The history and life cycle of Toxoplasma gondii. In: Weiss LM, ed. Toxoplasma gondii: The Model Apicomplexan—Perspectives and Methods . Elsevier,, 2014:1–17.

16.

Dubey

, Alvarado-Esquivel

, Herrera-Valenzuela

, Ortiz-Diaz

, et al. A new atypical genotype mouse virulent strain of Toxoplasma gondii isolated from the heart of a wild caught puma (Felis concolor) from Durango, Mexico. Vet Parasitol, 2013; 197:674–677.

17.

Dubey

, Beattie

. Toxoplasmosis of Animals and Man. Boca Raton, FL: CRC Press,, 1988:1–220.

18.

Dubey

, Pas

, Rajendran

, Kwok

OCH

, et al. Toxoplasmosis in Sand cats (Felis margarita) and other animals in the Breeding Centre for Endangered Arabian Wildlife in the United Arab Emirates and Al Wabra Wildlife Preservation, the State of Qatar. Vet Parasitol, 2010; 172:195–203.

19.

Dubey

, Van Why

, Verma

, Choudhary

, et al. Genotyping Toxoplasma gondii from wildlife in Pennsylvania and identification of natural recombinants virulent to mice. Vet Parasitol, 2014; 200:74–84.

20.

Dzib-Paredes

, Rosado-Aguilar

, Acosta-Viana

, Ortega-Pacheco

, et al. Seroprevalence and parasite load of Toxoplasma gondii in Mexican hairless pig (Sus scrofa) tissues from the Southeast of Mexico. Vet Parasitol, 2016; 229:45–49.

21.

El-Nawawi

, Tawfik

, Shaapan

. Methods for inactivation of Toxoplasma gondii cysts in meat and tissues of experimentally infected sheep. Foodborne Pathog Dis, 2008; 5:687–690.

22.

Figueroa-Castillo

, Duarte-Rosas

, Juárez-Acevedo

, Luna-Pastén, et al. Prevalence of Toxoplasma gondii antibodies in rabbits (Oryctolagus cuniculus) from Mexico. J Parasitol, 2006; 92:394–395.

23.

Fiorello

, Noss

, Deem

, Maffei

, et al. Serosurvey of small carnivores in the Bolivian Chaco. J Wildl Dis, 2007; 43:551–557.

24.

Fiorello

, Robbins

, Maffei

, Wade

. Parasites of free-ranging small canids and felids in the Bolivian Chaco. J Zoo Wildl Med, 2006; 37:130–134.

25.

García-Márquez

, Gutiérrez-Díaz

, Correa

, Luna-Pastén

, et al. Prevalence of Toxoplasma gondii antibodies and the relation to risk factors in cats of Colima, Mexico. J Parasitol, 2007; 93:1527–1528.

26.

Gómez-Marín

, De-la-Torre

, Barrios

, Cardona

, et al. Toxoplasmosis in military personnel involved in jungle operations. Acta Trop, 2012; 122:46–51.

27.

Hernandez-Cortazar

, Acosta-Viana

, Guzman-Marin

, Ortega-Pacheco

, et al. Presence of Toxoplasma gondii in drinking water from an endemic region in Southern Mexico. Foodborne Pathog Dis, 2017; 14:288–292.

28.

Jalal

, Nord

, Lappalainen

, Evengard

. Rapid and sensitive diagnosis of Toxoplasma gondii infections by PCR. Clin Microbiol Infect, 2004; 10:937–939.

29.

Jewell

, Frenkel

, Johnson

, Reed

, et al. Development of Toxoplasma oocysts in neotropical felidae. Am J Trop Med Hyg, 1972; 21:512–517.

30.

Jongwutiwes

, Tiangtip

, Yentakarm

, Chantachum

. Simple method for long-term copro-preservation of Cryptosporidium oocysts for morphometric and molecular analysis. Trop Med Int Health, 2002; 7:257–264.

31.

Kenny

, Lappin

, Knightly

. Toxoplasmosis in Pallas' cats (Otocolobus felis manul) at the Denver Zoological Gardens. J Zoo Wildl Med, 2002; 33:131–138.

32.

Ketz-Riley

, Ritchey

, Hoover

, Johnson

, et al. Immunodeficiency associated with multiple concurrent infections in captive Pallas' cats (Otocolobus manul). J Zoo Wildl Med, 2003; 34:239–245.

33.

Kikuchi

, Chomel

, Kasten

, Martenson

, et al. Seroprevalence of Toxoplasma gondii in American free-ranging or captive pumas (Felis concolor) and bobcats (Lynx rufus). Vet Parasitol, 2004; 120:1–9.

34.

Lappin

, Powell

. Comparison of latex agglutination, indirect hemagglutination, and ELISA techniques for the detection of Toxoplasma gondii-specific antibodies in the serum of cats. J Vet Intern Med, 1991; 5:299–301.

35.

Lloyd

, Stidworthy

. Acute disseminated toxoplasmosis in a juvenile cheetah (Acinonyx jubatus). J Zoo Wildl Med, 2007; 38:475–478.

36.

Miller

, Miller

, Conrad

, James

, et al. Type X Toxoplasma gondii in a wild mussel and terrestrial carnivores from coastal California: New linkages between terrestrial mammals, runoff and toxoplasmosis of sea otters. Int J Parasitol, 2008; 38:1319–1328.

37.

Miller

, Frenkel

, Dubey

. Oral infections with Toxoplasma cysts and oocysts in felines, other mammals, and in birds. J Parasitol, 1972; 58:928–937.

38.

Ortega-Pacheco

, Aguilar-Caballero

, Colin-Flores

, Acosta-Viana

, et al. Seroprevalence of feline leukemia virus, feline immunodeficiency virus and heartworm infection among owned cats in tropical Mexico. J Feline Med Surg, 2014; 16:460–464.

39.

Ortiz-Amador

Epidemiological investigation of canine distemper virus in domestic dogs, jaguars and pumas in the surroundings of the Calakmul Biosphere Reserve in Southern Mexico. MSc Thesis, National Autonomous University of Mexico (UNAM), 2015.

40.

Piggott

, Taylor

. Extensive evaluation of faecal preservation and DNA extraction methods in Australian native and introduced species. Aust J Zool, 2003; 51:341–355.

41.

Ramírez

, Autran

, García

, Carmona

MÁ

, et al. Genotyping of feline leukemia virus in Mexican housecats. Arch Virol, 2016; 161:1039–1045.

42.

Ramos Silva

, Marvulo

MFV

, Dias

, Ferreira

, et al. Risk factors associated with sero-positivity to Toxoplasma gondii in captive neotropical felids from Brazil. Prev Vet Med, 2007; 78:286–295.

43.

Rendón-Franco

, Caso-Aguilar

, Jiménez-Sanchéz

, Brousset Hernandez-Jauregui

, et al. Prevalence of anti-Toxoplasma gondii antibody in free-ranging ocelots (Leopardus pardalis) from Tamaulipas, Mexico. J Wildl Dis, 2012; 48:829–831.

44.

Robert

, Chabasse

, Hocquet

. Anti-Toxoplasma IgM studied by indirect immunofluorescence and hemagglutination elimination of false positives and negatives by adsorption of IgG on immobilized protein A. Biomedicine, 1981; 35:61–65.

45.

Salant

, Spira

, Hamburger

. A comparative analysis of coprologic diagnostic methods for detection of Toxoplama gondii in cats. Am J Trop Med Hyg, 2010; 82:865–870.

46.

Shi

, Cui

, Engel

, Tanabe

. Lysine-specific demethylase 1 is a therapeutic target for fetal hemoglobin induction. Nat Med, 2013; 19:291–294.

47.

Silva

, Ogassawara

, Adania

, Ferreira

, et al. Seroprevalence of Toxoplasma gondii in captive neotropical felids from Brazil. Vet Parasitol, 2001; 102:217–224.

48.

, Shwab

, Zhou

, Zhu

, et al. Moving towards an integrated approach to molecular detection and identification of Toxoplasma gondii . Parasitology, 2010; 137:1–11.

49.

Ullmann

, da Silva

, de Moraes

, Cubas

, et al. Serological survey of Toxoplasma gondii in captive Neotropical felids from Southern Brazil. Vet Parasitol, 2010; 172:144–146.

50.

Vitaliano

, Soares

, Minervino

AHH

, Santos

, et al. Genetic characterization of Toxoplasma gondii from Brazilian wildlife revealed abundant new genotypes. Int J Parasitol Parasites Wildl, 2014; 3:276–283.

51.

Yang

, Feng

, Lu

, Dong

, et al. Antibody detection, isolation, genotyping, and virulence of Toxoplasma gondii in captive felids from China. Front Microbiol, 2017; 8:1414.