J. Parasitol., 97(4), 2011, pp. 616–619 F American Society of Parasitologists 2011

SEROPREVALENCE OF TOXOPLASMA GONDII INFECTION IN DOMESTIC PIGS IN STATE,

C. Alvarado-Esquivel, C. Garcı´a-Machado*, D. Alvarado-Esquivel, A. M. Gonza´lez-Salazar*, C. Briones-Fraire, J. Vitela-Corrales*, I. Villena`, and J. P. Dubey§I Faculty of Medicine, Jua´rez University of Durango State, Avenida Universidad S/N esquina Fanny Anitua, 34000 Durango, Durango State, Mexico. e-mail: [email protected]

ABSTRACT: Little is known concerning the prevalence of Toxoplasma gondii infection in pigs in Mexico. Accordingly, antibodies to T. gondii were determined in 1,074 domestic pigs in Durango, Mexico using the modified agglutination test. Two groups (A, B) of pigs were sampled: Group A pigs (n 5 555) were raised in 3 geographical regions in Durango State and Group B pigs (n 5 519) were from State but slaughtered in Durango . Overall, antibodies to T. gondii were found in 136 (12.7%) of 1,074 pigs with titers of 1:25 in 29, 1:50 in 23, 1:100 in 18, 1:200 in 22, 1:400 in 12, 1:800 in 8, 1:1,600 in 2, and 1:3,200 or higher in 22. Of the pigs raised in Durango State, seroprevalence varied with age, management, and the geographic region; pigs raised in backyards in the mountainous region had a significantly higher seroprevalence (32.1%) than those raised in the valley (13.0%) and the semi-desert regions (14.0%). In Group A pigs from Durango, seroprevalence of T. gondii infection was significantly higher in pigs older than 8 mo (19.5%) than in younger pigs (10.9%). In the whole pig population (Groups A and B together), seroprevalence was higher in pigs raised in Durango (16.0%) than in those raised in Sonora (9.1%) and higher in mixed-breed pigs (15.7%) than in pure-bred pigs (10.3%). This is the first, in-depth study on the seroprevalence of T. gondii infection of pigs in Mexico and the first report on pigs from Durango State, Mexico. Results indicate that infected pork is likely an important source of T. gondii infection for humans in Durango State.

Toxoplasma gondii infections in pigs (Sus scrofa) occur MATERIALS AND METHODS worldwide (Dubey, 2009a, 2009b). Toxoplasmosis can cause Pigs surveyed mortality and morbidity in pigs, especially in neonates (Dubey and Beattie, 1988). Importantly, the consumption of raw or Group A: These pigs (n 5 555) were from 12 municipalities in 3 geographical regions of Durango State, Mexico (Table I). Most (n 5 539) undercooked meat from T. gondii-infected pigs represents a risk for were raised in backyards in 191 homes in the 12 municipalities; the remaining T. gondii infection in humans (Choi et al., 1997; Dubey, 2009a). 16 pigs were from a farm inside the municipality of Durango. The number of We have been studying the epidemiology of T. gondii infection in pigs per property surveyed ranged from 1 to 18 (median 3). Of these, 221 humans (Alvarado-Esquivel et al., 2006, 2008, 2009; Alvarado- (39.8%) were juveniles (4- to 8-mo-old) and 334 (60.2%) were adults (9- to 4- yr-old). The juvenile pigs included 114 (51.6%) females and 107 (48.4%) Esquivel, Liesenfeld, Ma´rquez-Conde et al., 2010; Alvarado-Esquivel, males; 39 (17.6%) were pure breed Yorkshire and 182 were mixed breed Liesenfeld, Torres-Castorena et al., 2010; Alvarado-Esquivel, Rojas- (82.4%). The older pigs consisted of 272 (81.4%)femalesand62(18.6%) Rivera et al., 2010; Alvarado-Esquivel, Estrada-Martinez et al., 2011; males; 78 (23.4%) were pure breed (Yorkshire or Landrace) and 256 (76.6%) Alvarado-Esquivel, Urbina-A´ lvarez et al., 2011) and in other animals were mixed breed. All pigs were apparently healthy. Background data were obtained by attending veterinarians and blood was collected from a jugular (Alvarado-Esquivel et al., 2007; Dubey et al., 2009) in Durango, vein of these pigs in August 2009 to November 2010. Mexico. Durango, with a temperate climate, is in the north of Mexico Group B: These pigs (n 5 519) were raised in Sonora State but slaughtered and is located in the valley region in the south-central in an abattoir in Durango City. Sonora State is located in northwest Mexico part of Durango State at 1,880 m above sea level. In a recent and has a common border with the states of and in the . Sonora State is the main provider of pigs for slaughtering in epidemiological study in humans in Durango City, we found an abattoirs in Durango City. Of these 519 pigs, 3 (0.6%) were juveniles and association of T. gondii infection with the consumption of chorizo (a 516 (99.4%) were adults. The 3 juvenile pigs were pure bred Yorkshire. The red, fresh sausage made mostly of raw pork tissues mixed with raw adult pigs included 485 (94.0%) females and 31 (6.0%) males; 490 (95.0%) chili pepper) (Alvarado-Esquivel, Liesenfeld, Ma´rquez-Conde et al., were Yorkshire and 26 (5.0%) were a mixed breed. All 519 pigs were apparently healthy. Data on these pigs were provided by blood collectors 2010). Information regarding T. gondii infectioninpigsinDurango and veterinarians at the abattoir, but we have no way to determine the State is lacking. Because pork is widely consumed in Mexico, and a validity of these data. These pigs were said to be raised on many properties, number of typical Mexican dishes are made of this meat, we and we could not obtain reliable information with respect to owners or the determined the seroprevalence of T. gondii infection in pigs raised in management of these pigs. These pigs were slaughtered in November 2010 3 geographical regions in Durango State, Mexico as well as in pigs and blood samples were collected directly from the hearts. slaughtered in the largest abattoir in Durango City, Mexico. Serological examination Blood samples were transported to the laboratory on the day of Received 17 January 2011; revised 22 January 2011; accepted 28 January collection. Sera were collected from whole blood by centrifugation and 2011. stored at 220 C until tested. Pig sera were tested for T. gondii antibodies *Laboratory of Animal Pathology, Regional Livestock Union of using 2-fold serial dilutions from 1:25 to 1: 3,200, with the modified Durango, Km 4.5 Carretera Durango-Parral, 34217 Durango, Durango agglutination test (MAT), as described by Dubey and Desmonts (1987). State, Mexico. The antigen was prepared at the Laboratory of Parasitology, National {Abattoir TIF, Regional Livestock Union of Durango, Km 4.5 Carretera Reference Centre on Toxoplasmosis (Reims, ) as described by Durango-Parral, 34217 Durango, Durango State, Mexico. Desmonts and Remington (1980). {Laboratory of Parasitology–Mycology, National Reference Centre on Toxoplasmosis, Biological Resources Centre Toxoplasma, Hospital Statistical analysis Maison Blanche, 45 rue Cognac-Jay, 51092 Reims Cedex, France. }United States Department of Agriculture, Agricultural Research Service, Statistical analysis was performed using Epi Info software v. 3.5.1 Animal and Natural Resources Institute, Animal Parasitic Diseases (Centers for Disease Control and Prevention: http://wwwn.cdc.gov/ Laboratory, Building 1001, Beltsville, Maryland 20705-2350. epiinfo). The Yates’ corrected Chi square test was used for comparison I To whom correspondence should be addressed. of the frequencies among groups. A P-value of ,0.05 was considered DOI: 10.1645/GE-2755.1 statistically significant.

616 ALVARADO-ESQUIVEL ET AL.—TOXOPLASMOSIS IN PIGS IN MEXICO 617

TABLE I. Seroprevalence of T. gondii infection in domestic pigs raised in backyards in Durango, Mexico.

Pigs tested Seroprevalence of T. gondii infection Region Municipality Homes surveyed No. No. %

Valleys .Canatla´n 28 54 17 31.5 .Durango 24 112 17 15.2 .Vicente 27 64 6 9.4 . 4 24 2 8.3 . 17 51 3 5.9 .Pen˜on Blanco 10 20 1 5 .Poanas 14 32 1 3.1 .Nombre de Dios 2 5 0 0 .All 126 362 47 13 Semi-desert .Cuencame 23 73 13 17.8 .Santa Clara 10 20 0 0 .All 33 93 13 14 Mountains .Pueblo Nuevo 19 45 18 40 .San Dimas 13 39 9 23.1 .All 32 84 27 32.1* All . 191 539 87 16.1

* Significantly higher seroprevalence in pigs in the mountainous region than in those in the valley region (P 5 0.00004) and in the semi-desert region (P 5 0.006).

RESULTS study on Mexican pigs in the last 20 yr (Dubey, 2009b). Garcia- Vazquez et al. (1993) tested sera of 1,203 6- to 10-mo-old pigs Overall, antibodies to T. gondii were found in 136 (12.7%)of slaughtered in , Mexico for antibodies to T. gondii the 1,074 pigs with MAT titers of 1:25 in 29, 1:50 in 23, 1:100 in using an in-house ELISA. Toxoplasma gondii antibodies were 18, 1:200 in 22, 1:400 in 12, 1:800 in 8, 1:1,600 in 2, and 1:3,200 or found in 8.9% of pigs; the pigs were said to be raised in 4 higher in 22. A significantly (P 5 0.0008) higher seroprevalence of unspecified Mexican states. It is of interest that pigs slaughtered in T. gondii infection was observed in pigs raised in Durango State the abattoir in Durango had a 9.1% seroprevalence of T. gondii (16.0 ) than in those from Sonora State (9.1 ) (Table II). Pigs % % infection, similar to the study of Garcia-Vazquez et al. (1993), raised in backyards in Durango State (20/89, 22.5 ) had a % although different serological tests (ELISA vs. MAT) were used. significantly higher frequency of high antibody titers (1:3,200 or Recently, Galva´n-Ramirez et al. (2010) examined 48 pork higher) than did pigs from Sonora State (2 of 47: 4.2%; P 5 0.01). samples from Ocotla´n, , Mexico for T. gondii infection, and The seroprevalence of T. gondii infection was significantly (P 5 T. gondii DNA was not detectable by PCR in any of the pork 0.01) higher in mixed (15.7%) than in pure-breed (10.3%) pigs but samples. Homogenate of each pork samples was also digested in similar in males (13.0%) and females (12.6%) (Table II). pepsin and, after washing, digest of each sample was inoculated Of 555 pigs in Group A, 89 (16.0%) were seropositive with into a mouse. The recipient mice were tested for T. gondii MAT titers of 1:25 in 11, 1:50 in 13, 1:100 in 11, 1:200 in 16, 1:400 infection by histopathology and serology by ELISA. Toxoplasma in 9, 1:800 in 7: 1:1,600 in 2, and 1:3,200 or higher in 20. gondii was not detectable in tissues of any of the 48 bioassayed Seroprevalence of T. gondii infection in pigs older than 8 mo was significantly (P 5 0.009) higher than that found in younger pigs. TABLE II. General characteristics of the 1,074 pigs studied and the Pigs up to 8-mo-old had a seroprevalence of 10.9% (24 of 221) seroprevalence of T. gondii infection. with titers of 1:25 in 2, 1:50 in 6, 1:100 in 3, 1:200 in 1, 1:400 in 3, 1:800 in 4, and 1:3,200 or higher in 5. Pigs older than 8 mo had a Seroprevalence of seroprevalence of 19.5% (65 of 334) with titers of 1:25 in 9, 1:50 in Pigs tested T. gondii infection 7, 1:100 in 8, 1:200 in 15, 1:400 in 6, 1:800 in 3, 1:1,600 in 2, and Characteristics No. No. % P value 1:3,200 or higher in 15. Seroprevalence varied with geographic source of pigs (Table I). Pigs raised in backyards in the Age (mo) mountainous region had a higher seroprevalence of T. gondii 4–8 224 25 11.2 0.51 infection (32.1%) than did those raised in the valley region 9–48 850 111 13.1 . (13.0%) or in the semi-desert region (14.0%). Seropositive pigs Gender were found on 44 (23.0%) of the 191 properties sampled. Of 519 Male 200 26 13 0.96 pigs in Group B from the abattoir, 47 (9.1%) were seropositive Female 874 110 12.6 . with MAT titers of 1:25 in 18, 1:50 in 10, 1:100 in 7, 1:200 in 6, Breed 1:400 in 3, 1:800 in 1, and 1:3,200 or higher in 2. Pure 610 63 10.3 0.01 Mixed 464 73 15.7 . DISCUSSION Origin Durango State 555 89 16 0.0008 There is very little information on T. gondii infection in pigs in Sonora State 519 47 9.1 . Mexico. To our knowledge, there has been only 1 seroprevalence 618 THE JOURNAL OF PARASITOLOGY, VOL. 97, NO. 4, AUGUST 2011 mice, but IgG and IgM T. gondii antibodies were found in 1 and T. gondii can persist in virtually all edible tissues of pigs mouse. Thus, 1 of 48 pork samples might have been infected, but during the life of the pig (Dubey, 1988). We are not aware of any definitive evidence of T. gondii infection was lacking. report of toxoplasmosis in humans directly linked to eating Here, we found an overall seroprevalence of T. gondii infection infected pork in Mexico, but clinical toxoplasmosis and blindness of 12.7% in domestic pigs in 2 states of Mexico. Differences in the have been linked to the ingestion of undercooked pork in Korea production facilities, feeding, and environmental characteristics (Choi et al., 1997). probably explain differences in the seroprevalences in pigs within In the present study of pigs raised in backyards in the different regions of the country. Most of the pigs in Group A in mountainous region, 32.1% had a significantly higher seroprev- this study were raised outdoors in backyards at homes located in alence of T. gondii infection than did those raised in the valley rural communities where pigs are frequently free-ranging, eat region (13.0%)(P 5 0.00004) and the semi-desert region (14.0%) waste food, and drink untreated water. In addition, cats are also (P 5 0.006). The reason for these differences is unclear; however, free-ranging, cohabiting in the same house or in neighboring differences in mean daily temperature, annual rainfall amounts, houses. In a study of 123 farms across Illinois, Weigel et al. (1995) and other microclimatic factors may impact survival of oocysts in found that higher T. gondii prevalence in sows was associated with soil, which is the likely source of infection in pigs. access of cats to sows (oocyst exposure) and to fewer sows in the Prevalence of T. gondii varies dramatically among the classes of herd; outdoor housing, per se, was not the determinant factor. In pig surveyed (market pigs vs. sows, indoor pigs from biosecure another study of 47 swine farms in Illinois, 68.3% of 391 cats on housing systems vs. free-range pigs). In the United States, the pigs these farms were seropositive, and viable T. gondii were isolated used for unprocessed pork consumption (feeder pigs, market pigs, from cat feces, soil, and feed given to pigs (Dubey et al., 1995). We finisher pigs [these terms are synonymous]) are mostly raised have no information on cats on these farms in Durango, but indoors in well-managed facilities. Under these conditions, 21.0% of 105 (Alvarado-Esquivel et al., 2007) and 9.3% of 150 prevalence of T. gondii has declined drastically in the last decade (Dubey et al., 2008) domestic cats from Durango City had T. (Dubey et al., 2005; Dubey, 2009b; Hill et al., 2010). Currently, gondii antibodies. ,1.0% of feeder pigs in the United States have antibodies to T. Unlike in the United States, most pigs in Mexico are raised in gondii (see Dubey, 2009b; Davies, 2010); nonetheless, on some small holdings in ‘backyard’ production units; in the present study poorly managed farms up to 90.0% of pigs are infected with T. of pigs raised in Durango, the average number of pigs per property gondii (Dubey et al., 2002, 2008). A comparison is made here with was 3. In the present study, pigs raised in backyards in Durango pigs from the United States because of the close geographic State had a significantly higher frequency of high titers (1:3,200 or settings; however, it is difficult to compare results from the higher) of T. gondii antibodies (22.5%) than did pigs raised on the present study in Mexico with surveys from other countries due to farm from Sonora State (4.3%). This is noteworthy because 8.5% of differences in a variety of conditions. In the United States, most 1,000 pigs from had high levels of antibodies, and viable T. pigs are raised in midwestern states, and there is little information gondii was isolated from 75.0% of pigs with MAT titers of 1:400 or on T. gondii seroprevalence in pigs raised in Arizona or New higher from these sows (Dubey et al., 1995; Dubey, 2009b). These Mexico, which border the Sonora State, Mexico from where results may be related to repeated exposure to T. gondii in the Group B pigs were derived (Dubey, 2009b). Although we do not backyard setting. It is of interest that the magnitude of antibody have reliable information concerning the sources of pigs in Group titers in some South American countries is higher than in the B, from an epidemiologic perspective, what is important is that United States, perhaps related to the probability of repeat these pigs were destined for human consumption in Durango infections (Frenkel and Ruiz, 1981; Dubey, 2009a). State. The MAT test used here has been extensively evaluated for the Our results indicate that T. gondii infections occur widely in diagnosis of latent infection in pigs, using isolation of the parasite pigs produced in Durango State. This finding increases public as a standard (Dubey et al., 1995, Gardner et al., 2010). The health concerns because consumption of chorizo (made of pork) isolation of viable parasite from 37.1% of naturally exposed sows has been associated with T. gondii infection in humans in with a low MAT titer of 1:20 (Dubey et al., 1995) suggests that the Durango (Alvarado-Esquivel, Liesenfeld, Ma´rquez-Conde et al., screening dilution of 1:25 we used is indicative of T. gondii 2010). This is the first study documenting the seroprevalence of T. infection in pigs. Richomme et al. (2009), using the same gondii infection of pigs in Durango, Mexico. These results will serological procedure as used in the present study, isolated viable hopefully contribute to the development of optimal preventive T. gondii from 35.0% (21 of 60) seropositive (MAT, 1:6 or higher) measures against T. gondii infection resulting from consumption of wild pigs from France; their results provide additional data on of infected pork. the validity of the MAT. In the United States, pigs are considered an important source of ACKNOWLEDGMENTS infection for T. gondii infections in humans (Dubey and Jones, The authors thank Heriberto Quin˜ones-Soria, Araceli Dominguez, 2008) and this may be true in Mexico. There were more than Francisco Ontiveros-Vazquez, Jorge Marcos Salazar-Rios, and Israel 9 million pigs in Mexico in the 2007 census and 18.8% of them Ortiz-Dı´az from the Regional Livestock Union of Durango for their valuable support in obtaining data and collecting blood samples from the were raised in Sonora State (http://www.fira.gob.mx:8081/sas/ pigs, and Dr. Dolores Hill for helpful suggestions. docs/InformacionEconomica/Notas_de_Analisis/Principales%20 Resultados%20del%20Censo%202007.pdf). Although there is no LITERATURE CITED current census, the number of pigs in Mexico is increasing. Assuming ALVARADO-ESQUIVEL, C., H. M. CRUZ-MAGALLANES,R.ESQUIVEL-CRUZ,S. a 10.0% prevalence of T. gondii in 9 million pigs, 900,000 infected pig ESTRADA-MARTI´NEZ,M.RIVAS-GONZA´ LEZ,O.LIESENFELD,S.A. carcasses enter the food supply in Mexico on an annual basis. A MARTI´NEZ-GARCI´A,E.RAMI´REZ,A.TORRES-CASTORENA,A.CASTA- single 50-kg market pig would account for over 300 servings of meat, N˜ EDA ET AL. 2008. Seroepidemiology of Toxoplasma gondii infection ALVARADO-ESQUIVEL ET AL.—TOXOPLASMOSIS IN PIGS IN MEXICO 619

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