Seroepidemiology of Leptospira Infection in Backyard Pigs in Durango State, Mexico.

PURPOSE: This study aimed to determine the seroprevalence and correlates of Leptospira IgG antibodies in backyard pigs in the northern Mexican state of Durango. We performed a cross-sectional study of 305 backyard pigs. Anti-Leptospira IgG antibodies were detected using microscopic agglutination assay (MAT) with a panel of 12 Leptospira antigens.
RESULTS: Overall, antibodies against Leptospira (any of the 12 Leptospira serovars examined) were found in 186 (61.0%) of the 305 pigs studied. Seropositive pigs were found on 80 (70.2%) of the 114 properties surveyed. The predominant serovar was Leptospira interrogans Pomona (n = 55); followed by Leptospira noguchii Lousiana and Leptospira santarosai Tarassovi (n = 53 each); L. interrogans Bataviae (n = 47); Leptospira biflexa Semaranga and L. interrogans Hebdomadis (n = 36 each); L. interrogans Pyrogenes (n = 30); L. interrogans Djasiman (n = 20); Leptospira borgpetersenii Ballum (n = 11); L. noguchii Panama and L. interrogans Canicola (n = 5 each); and L. borgpetersenii Mini (n = 2). Logistic regression showed that seropositivity was associated with low (<1000 m above sea level) altitude (odds ratio [OR] = 3.24; 95% confidence interval [CI]: 2.01-5.20; P < 0.001).
CONCLUSIONS: This is the first report of Leptospira exposure in backyard pigs in Mexico and of an association between Leptospira exposure in pigs and altitude. Backyard pigs represent a high-risk group for Leptospira exposure.

Introduction

Leptospiras are thin helical bacteria classified into at least 12 pathogenic and 4 saprophytic species [1]. Pathogenic Leptospiras are the etiological agents of leptospirosis, an emerging zoonotic disease that affects humans and animals worldwide [2]. Leptospirosis may affect many species including domestic and wild animals [3, 4]. In humans, most characteristic early clinical signs include fever of sudden onset, severe general malaise, muscular pain, and conjunctival congestion [5]. Whereas jaundice and hemorrhage are clinical features present in patients with the severe form of leptospirosis [5]. In animals, the acute phase of the Leptospira infection is mostly sub-clinical, but chronic infections cause reproductive wastage and great economic losses [6]. More than 300 serovars have been identified among Leptospiras [7]. Pigs are generally considered the reservoir host species for Pomona, Bratislava, and Tarrasovi serovars [8]. Leptospirosis is transmitted by direct or indirect contact with an infected animal, and infection with Leptospira in pigs may lead to considerable losses due to abortion, stillbirths, weakly piglets, or infertility [8]. The seroprevalence of Leptospira infection in pigs varies substantially among countries. For instance, a relatively low (5%–13%) seroprevalence has been reported in pigs in Trinidad [9], Thailand [10], and feral swine in the USA [11]. Whereas intermedium (20.2%–25%) seroprevalence was found in pigs in Portugal [12], a high (55.9%) seroprevalence was reported in pigs in Córdoba, Colombia [13].

The epidemiology of Leptospira infection in pigs in Mexico has been poorly studied. Examination of Leptospira infection in pigs is important because contact with Leptospira-infected pigs might be a risk for Leptospira infection in humans. Epidemiological studies about Leptospira infection in pigs are important to get useful information for an optimal planning of preventive measures against this infection and thus to improve the population health. In a previous study, Vado-Solís et al. found a 25% seroprevalence of Leptospira infection in pigs in the southern Mexican state of Yucatan [14]. However, the seroprevalence of Leptospira infection in pigs in the northern Mexican state of Durango is unknown. Therefore, we sought to determine the seroprevalence of anti-Leptospira IgG antibodies and Leptospira serovars in pigs in Durango, Mexico. In addition, seroprevalence association with the general characteristics of pigs and their environment was also investigated.

Materials and Methods

Study Design and Pigs Surveyed

A cross sectional study using residual sera from a Toxoplasma gondii serosurvey in pigs in Durango State, Mexico [15] was performed. Inclusion criterium of pigs was backyard pigs raised in Durango State. Exclusion criterium was pigs with insufficient serum sample or missing information about their general characteristics. In total, 305 randomly selected backyard pigs were included in the study. Pigs were raised in eight municipalities in three geographical regions of Durango State, Mexico (Table 1). Pigs were apparently healthy and raised in 114 homes. A median of three pigs (range, 1 to 6) per property were surveyed. Of the 305 pigs, 117 (38.4%) were juveniles (4- to 8-month-old) and 188 (61.6%) were adults (9-month-old to 4-year-old); 204 (66.9%) were females and 101 (33.1%) were males. The great majority (n = 269) of pigs were mixed-breed, 35 were Yorkshire, and one was Landrace. Environment characteristics of the municipalities surveyed including climate, mean annual temperature and rainfall, and altitude (meters above sea level) were obtained.

Table 1.

Seroprevalence of Leptospira infection and environment correlates in backyard pigs in Durango, Mexico

Regiona	Municipalityb	Climatec	Meters above sea leveld	Mean annual temperature (°C)	Mean annual rainfall (mm)	Homes surveyed	Homes with positive pigs		Pigs tested no.	Seropositivity to Leptospira
							No.	%		No.	%
Valleys	Durango	Semi-arid	1885	16.9	529	15	14	93.3	67	44	65.7
	Vicente Guerrero	Subhumid	1950	17.4	500–600	15	10	66.7	23	16	69.6
	Coneto de Comonfort	Dry	2000	15.3	450	17	17	100	51	45	88.2
	Peñon Blanco	Subhumid	1800	18	450	9	4	44.4	20	8	40
	All					56	45	80.3	161	113	70.2
Semi-desert	Cuencame	Semi-arid	1580	21.1	243.7	16	11	68.8	45	24	53.3
	Santa Clara	Dry	2050	14.9	300–600	10	4	40	20	4	20
	All					26	15	57.7	65	28	43.1
Mountains	Pueblo Nuevo	Temperate	2560	18	1300	20	9	45	45	17	37.8
	San Dimas	Warm subhumid	520	23.7	1050	12	11	91.7	34	28	82.4
	All					32	20	62.5	79	45	57
All						114	80	70.2	305	186	61

Statistically significant (P = 0.001) difference among regions.

Statistically significant (P < 0.001) difference among municipalities.

Statistically significant (P = 0.001) difference among climates.

Statistically significant (P < 0.001) difference among altitudes.

Detection of Anti-Leptospira Igg Antibodies

Serum samples of pigs were analyzed for anti-Leptospira IgG antibodies using 2-fold serial dilutions from 1:25 with the microscopic agglutination assay (MAT). A titer of 1:100 was used as cut-off for seropositivity in MAT. A panel of 12 leptospiral antigens was used: Leptospira biflexa Semaranga, Leptospira noguchii Panama, Leptospira interrogans Djasiman, L. noguchii Lousiana, Leptospira borgpetersenii Ballum, Leptospira santarosai Tarassovi, L. interrogans Pyrogenes, L. interrogans Bataviae, L. interrogans Pomona, L. interrogans Hebdomadis, L. interrogans Canicola, and L. borgpetersenii Mini.

Statistical Analysis

Results were analyzed with the aid of the software Microsoft Excel 2010, Epi Info 7 (Centers for Disease Control and Prevention: http://wwwn.cdc.gov/epiinfo/), and SPSS 15.0 (SPSS Inc., Chicago, Illinois). We assessed the association of Leptospira seropositivity and the general and environment characteristics of pigs with the Pearson's χ2 test or the two-tailed Fisher's exact test (when values were small). Leptospira seroprevalence was correlated with two groups of mean annual temperatures of the municipalities surveyed: <20 °C, and ≥20 °C. In addition, Leptospira seroprevalence was correlated with three groups of altitudes: <1000, 1000–2000, and >2000 m above sea level. We also assessed the association between Leptospira seroprevalence and two groups of mean annual rainfalls: ≤600 mm and >600 mm). Variables with a P value of ≤0.05 obtained by bivariate analysis were further analyzed by multivariate analysis. Odds ratio (OR) and 95% confidence interval (CI) were calculated by stepwise regression analysis using the backwards elimination method. A P value of <0.05 was considered statistically significant.

Ethics

The Bioethics and Animal Welfare Commission (BAWC) of the Veterinary Medicine and Animal Husbandry School (Facultad de Medicina Veterinaria y Zootecnia) of the State University of Veracruz (Universidad Veracruzana) approved this project. Owners of pigs provided verbal consent to study their animals. Blood sampling in pigs was performed according to guidelines of the BAWC.

Results

Overall, antibodies against Leptospira (any of the 12 Leptospira serovars examined) were found in 186 (61.0%) of the 305 backyard pigs studied. The seroprevalence of Leptospira infection did not vary with age, sex, or breed of the pigs. Table 2 shows a correlation between Leptospira seroprevalence and general characteristics of pigs. Seropositive pigs were found on 80 (70.2%) of the 114 properties surveyed. Positive anti-Leptospira titers to all 12 Leptospira serovars examined were found. Sera of Leptospira-positive pigs showed reactivity to one or up to 6 Leptospira serovars. Seroreactivities to 1, 2, 3, and 4 Leptospira serovars were found in 79, 59, 39, and 7 pigs, respectively, whereas seroreactivities to 5 and 6 serovars were found in one pig each. The predominant serovar was L. interrogans Pomona (n = 55), followed by L. noguchii Lousiana and L. santarosai Tarassovi (n = 53 each); L. interrogans Bataviae (n = 47); L. biflexa Semaranga and L. interrogans Hebdomadis (n = 36 each); L. interrogans Pyrogenes (n = 30); L. interrogans Djasiman (n = 20); L. borgpetersenii Ballum (n = 11); L. noguchii Panama and L. interrogans Canicola (n = 5 each); and L. borgpetersenii Mini (n = 2).

Table 2.

General characteristics of the 305 pigs surveyed and seroprevalence of Leptospira infection

Characteristics	Pigs tested no.	Seroprevalence of Leptospira infection		P value
		No.	%
Age
4 to 8 months	117	72	61.5	0.87
9 months to 4 years	188	114	60.6
Gender
Male	101	66	65.3	0.27
Female	204	120	58.8
Breed
Pure	36	23	63.9	0.70
Mixed	269	163	60.6

The seroprevalence of Leptospira infection varied significantly (P = 0.001) among geographical regions, being the highest (70.2%) in pigs raised in the Valleys region (Table 1). In addition, the seroprevalence of Leptospira infection varied significantly (P < 0.001) among municipalities, being the highest (88.2%) in pigs raised in Coneto de Comonfort municipality. The seroprevalence also varied significantly (P = 0.001) among climates, being the highest (82.4%) in a warm sub-humid climate. Seroprevalence did not vary (P = 0.39) among mean annual rainfall groups: 62.4% in ≤600 mm and 57.0% in >600 mm. Seroprevalence (59.3%) in places with mean annual temperatures of <20 °C was similar (P = 0.30) to that (65.8%) found in places with mean annual temperatures ≥20 °C. Seroprevalence decreased significantly (P < 0.001) with altitude (meters above sea level): 82.4% in <1000, 66.5% between 1000 and 2000, and 32.3% in >2000.

Logistic regression analysis of variables likely (P < 0.05) associated with Leptospira infection obtained by bivariate analysis showed that seropositivity was associated with low altitude (<1000 m above sea level; OR = 3.24; 95% CI: 2.01–5.20; P < 0.001), whereas other variables including region, municipality, and climate were not associated with seropositivity by logistic regression.

Discussion

The seroepidemiology of Leptospira infection in pigs in Mexico has been scantly studied. Few studies about the seroprevalence of Leptospira infection in pigs in Mexico have been reported. Cisneros-Puebla et al. studied 1970 swine from a number of Mexican farms and found a 39.8% seroprevalence of Leptospira infection, and the most frequent serovars were Bratislava (22.5%), Icterohaemorrhagiae strain Palo Alto (14.5%), and Portland-vere strain Sinaloa ACR (13.8%) [16]. In another study, Vado-Solis et al. examined 353 pigs from farms and ranches of the southern Mexican State of Veracruz and found a 25% seroprevalence of Leptospira infection, and the most frequent serovar was Bratislava (51%) followed by icterohemorrhagiae (12%) and Panama (10%) [14]. However, there is no report about the seroepidemiology of Leptospira infection in pigs in the northern Mexican state of Durango. Therefore, this study was aimed to determine the seroprevalence and correlates of Leptospira infection in pigs in Durango, Mexico. We found that more than half (61%) of the pigs examined had anti-Leptospira antibodies. In fact, the seroprevalence found in our study is the highest reported so far. The seroprevalence found is higher than seroprevalences (25%–39.8%) of Leptospira infection reported in pigs in two previous Mexican studies [14, 16]. In an international context, the seroprevalence found in our study is higher than seroprevalences reported in pigs in Trinidad (5%) [9], (8.17%) Vietnam [17], Thailand (11.3%) [10], Portugal (20.2%) [12], and Colombia (55.9%) [13]. Studies of Leptospira infection in feral swine in the USA have found seroprevalences ranging from 13.0% to 48.9% [11, 18, 19]. In a study in Poland, 10.4% of 377 wild boars examined were found seropositive for Leptospira [20]. In contrast, in a study in New South Wales, researchers found antibodies to L. interrogans serovar Pomona in 53% of 239 feral pigs surveyed [21]. Furthermore, a high seroreactivity rate (48.6%) to Leptospira species was found in feral pigs in Queensland, Australia [22]. It is not clear why pigs in our study had a higher seroprevalence than pigs in other studies. The same MAT was used in all studies. However, differences in the serovars tested and in environment or raising characteristics might have influenced the seroprevalences among the studies. We studied backyard pigs, whereas farm or feral pigs were examined in the other studies. The three predominant serovars in pigs in our study were L. interrogans Pomona, L. noguchii Lousiana, and L. santarosai Tarassovi, whereas the serovars Bratislava, Icterohaemorrhagiae, and Portland or Panama were predominant in pigs in other Mexican studies [14, 16]. The difference in predominant serovars can be explained by differences in the serovars tested among the studies. To the best of our knowledge, there is only one previous report about the seroepidemiology of Leptospira infection in backyard pigs. Anampa et al. found an 82.1% seroprevalence of Leptospira infection in 133 backyard pigs slaughtered in two abattoirs in Lima, Peru [23].

We searched for the association between Leptospira seropositivity and environment characteristics of pigs. Multivariate analysis showed that Leptospira seropositivity was associated with decreasing altitude. Pigs raised in places with low altitude (<1000 m above sea level) had the highest seroprevalence rate. To the best of our knowledge, there is no previous report about this association. It is not clear why pigs raised in places with low altitude had a higher seroprevalence of Leptospira infection that those raised in places with higher altitude. It is likely that places with low altitude have a more suitable environment for Leptospira growing than places with higher altitudes. Leptospira survives in moist environments [24]. It is possible that contact with contaminated water source in low altitude places may favor Leptospira transmission.

The present survey has some limitations. All pigs examined were raised in backyards; therefore, further studies to determine the seroprevalence of Leptospira infection in farm pigs raised in Durango State are needed. All pigs tested were apparently healthy, and seroprevalence might vary in ill pigs. It is unknown whether maternal antibodies might have influenced the seroprevalence of Leptospira infection in very young pigs. We did not include the testing of some Leptospira serovars; however, inclusion of more serovars would increase the seroprevalence.

Our results suggest that Leptospira infection is very common in backyard pigs in the northern Mexican state of Durango. Remarkably, all municipalities and the majority (70.2%) of the houses sampled had seropositive pigs. Further research to determine risk factors associated with Leptospira exposure in pigs in northern Mexico should be conducted. It is recommended to avoid contact with Leptospira-infected backyard pigs, their urine, or contaminated environment to prevent Leptospira infection in humans. Wearing protective clothing and covering skin lesions when handling backyard pigs, as well as disinfecting their contaminated areas, are measures to prevent Leptospira transmission.

Conclusions

This is the first report of Leptospira exposure in backyard pigs in Mexico and of an association between Leptospira exposure in pigs and altitude. Results of the present study suggest that backyard pigs represent a high-risk group for Leptospira exposure. Results may be useful for the planning of optimal measures against Leptospira infection in pigs.