Apparently high Leptospira antibody seropositivity in donkeys for slaughter in three municipalities in Durango,Mexico

Abstract

We determined the seropositive percentage and the determinants of Leptospira exposure in domestic donkeys presented for slaughter in the State of Durango, Mexico. We examined 194 donkeys in 4 gathering premises. Sera of donkeys were analyzed for anti-Leptospira IgG antibodies using a microscopic agglutination assay with a panel of 7 Leptospira antigens. The association between Leptospira seropositivity and general characteristics of donkeys was assessed by bivariate and multivariate analyses. Antibodies against Leptospira (for at least 1 of the 7 Leptospira serovars examined) were found in 151 of 194 (77.8%) donkeys. Seropositive donkeys were found in all regions, municipalities, and premises examined. The predominant serovar was L. interrogans Icterohaemorrhagiae (n = 117; 60.3%), followed by L. interrogans Sejroe (n = 96; 49.5%), L. interrogans Canicola (n = 10; 5.2%), L. kirschneri Grippotyphosa (n = 6; 3.1%), L. borgpetersenii Ballum (n = 5; 2.6 %), L. biflexa Semaranga (n = 3; 1.5%), and L. noguchii Panama (n = 2; 1%). Leptospira seropositivity was associated with gathering premises (OR = 2.64; 95% CI: 1.27–5.46; p = 0.009) and municipalities (OR = 0.11; 95% CI: 0.01–0.78; p = 0.02). Our results demonstrate an apparently high seropositive percentage of Leptospira infection in the donkeys studied in Mexico.

Keywords

Donkeys Mexico seroepidemiology

Leptospires are spirochete bacteria that can infect domestic animals and wildlife, as well as humans, and cause leptospirosis.^18,20 Leptospirosis is a worldwide zoonosis, especially in countries with tropical and subtropical climates.¹⁶ Human infections with Leptospira are acquired from animals or from an environment contaminated by Leptospira.²¹ Several pathogenic species of Leptospira can cause a wide range of clinical manifestations in humans, varying from mild flu-like illness to severe disease with multi-organ system complications.⁸ In horses, most Leptospira infections are asymptomatic;^14,17 however, clinical manifestations, including reproductive failure and recurrent uveitis, have been reported.^10,17 Detection of Leptospira infection relies on serology.¹⁵ The microscopic agglutination test (MAT) is widely used to detect antibodies against Leptospira serovars in horses.^14,15,17 Predominant Leptospira serovars in horses include Icterohaemorrhagiae in Latin America,¹⁵ and Bratislava in South Africa¹⁷ and Ethiopia.¹⁹

Very little is known about infection with Leptospira in donkeys. In a study of domestic donkeys in northwest Morocco, researchers found 20% seroprevalence of Leptospira infection; the most prevalent serovars were L. borgpetersenii Javanica and L. interrogans Australis.⁷ The seropositive percentage of Leptospira infection and serovars in donkeys in Mexico is largely unknown. In a search for information about prevalence of Leptospira infection in horses in the region, no articles were found. It is unclear whether Leptospira exposure exists among donkeys in Mexico, and information about factors associated with Leptospira infection in donkeys in Mexico is lacking. Therefore, we determined the frequency of anti-Leptospira IgG antibodies and Leptospira serovars in domestic donkeys for slaughter in the northern Mexican state of Durango. As well, we investigated the association between Leptospira seropositive percentage and the general characteristics of donkeys and their environment.

We performed a serosurvey using residual archival sera from a Toxoplasma gondii study in domestic donkeys for slaughter in Durango State, Mexico.¹ Our project was approved by The Bioethics and Animal Welfare Commission (BAWC) of the Veterinary Medicine and Animal Husbandry School (Facultad de Medicina Veterinaria y Zootecnia) of the University of Veracruz (Universidad Veracruzana), Mexico. Donkeys had been sampled in 4 equid gathering premises (trade centers) in Durango, Mexico. Donkeys in these premises came from 3 municipalities (Durango, Mezquital, and San Dimas), and had been gathered for shipment to abattoirs in other states of Mexico. General data on donkeys was obtained with the aid of a questionnaire. In total, 194 mixed-breed donkeys were studied. Donkeys were 0.2–12 y old, 136 (70.1%) were females and 58 (29.9%) were males. Clinical status of the donkeys was based on physical examination by a veterinarian (JA Zamarripa-Barboza). No laboratory tests were performed, other than those for Leptospira antibodies. Of the 194 donkeys surveyed, 156 were apparently healthy, 37 had cutaneous sores, and 1 was malnourished. According to the donkeys’ owners, feeding of donkeys was based on pasture. Also, the owners informed us that donkeys were raised in either the valley region (n = 54) or the mountainous region (n = 140) of Durango State. Donkeys were not raised for meat, but rather were cull animals. Dealers bought the donkeys with no concern about the history of the animals.

Donkey sera were analyzed for anti-Leptospira IgG antibodies using 2-fold serial dilutions from 1:25 with a MAT.⁹ MAT was carried out on microplates, using dilutions with phosphate-buffered saline (pH 7.2) in a final volume of 50 µL. Equal volumes of live Leptospira suspensions grown in EMJH (Ellinghausen–McCullough–Johnson–Harris) medium from 5–6 d were used as antigens. The antigens were standardized by diluting the cultures to obtain ~200 cells per microscopic field, using a 40× objective in dark-field microscopy. A cutoff titer of ⩾1:100 was used for seropositivity. This cutoff has been used in other studies in equids.¹⁵ Heat-inactivation of sera to enhance MAT titers was performed, as reported previously.¹³ Positive controls from animals other than donkeys were included in the MAT. We did not include a positive control from donkeys because we were unable to obtain a serum sample from a donkey with demonstrated clinical leptospirosis in Mexico. A panel of 7 leptospiral antigens was used: L. biflexa Semaranga, L. noguchii Panama, L. borgpetersenii Ballum, L. kirschneri Grippotyphosa, L. interrogans Canicola, L. interrogans Sejroe, L. interrogans Icterohaemorrhagiae. Selection of these 7 leptospiral antigens was based on the availability of Leptospira variants in our laboratory.

Statistical analysis was performed (Excel 2010, Microsoft, Redmond, WA; SPSS v.20.0, IBM, Armonk, NY). Sample size was calculated using a population size of 900 donkeys (a broad estimation of donkeys), a 95% confidence level, and a reference seropositive percentage⁷ of 20% as the expected frequency of exposure. The result of this calculation was 193 donkeys. The association between Leptospira seropositivity and the general characteristics of donkeys was assessed by bivariate analysis using the Pearson chi-squared test and by regression analysis using the Enter method. Only variables of donkeys with a p ⩽ 0.05 obtained by bivariate analysis were further analyzed by logistic regression. Odds ratio (OR) and 95% confidence interval (CI) were calculated, and a p ⩽ 0.05 was considered statistically significant.

Antibodies against Leptospira (for at least 1 of the 7 Leptospira serovars examined) were found in 151 of 194 (77.8%) domestic donkeys studied. The seropositive rate of Leptospira infection did not vary with age or sex of the donkeys (Table 1). Seropositive donkeys were found from all regions, municipalities, and premises examined (Fig. 1). Titers were found to all 7 Leptospira serovars examined. Serum samples of Leptospira-positive donkeys showed reactivity to 1–4 Leptospira serovars. Seropositivity to 1, 2, 3, and 4 Leptospira serovars were found in 79, 58, 12, and 2 donkeys, respectively. The predominant serovar was L. interrogans Icterohaemorrhagiae (n = 117; 60.3%) followed by L. interrogans Sejroe (n = 96; 49.5%), L. interrogans Canicola (n = 10; 5.2%), L. kirschneri Grippotyphosa (n = 6; 3.1%), L. borgpetersenii Ballum (n = 5; 2.6 %), L. biflexa Semaranga (n = 3; 1.5%), and L. noguchii Panama (n = 2; 1%). Positive titers varied from 1:100 to 1:400 among Leptospira serovars (Table 2).

Table 1.

Correlation of general data of the 194 donkeys studied, and seropositive percentage to Leptospira.

Characteristic	Donkeys tested (n)	Seropositivity to Leptospira		p value
Characteristic	Donkeys tested (n)	n	%	p value
Age (y)
<1	10	8	80	0.5
1–5	113	91	80.5
>5	71	52	73.2
Sex
Male	58	48	82.8	0.28
Female	136	103	75.7
Health status
Cutaneous sores or malnourished	38	22	57.9	0.001
Healthy	156	129	82.7
Region
Mountains	140	104	74.3	0.05
Valleys	54	47	87
Municipality
Durango	54	47	87	0.02
Mezquital	135	102	75.6
San Dimas	5	2	40
Premises
1	154	116	75.3	0.01
2	5	2	40
3	30	28	93.3
4	5	5	100

Figure 1.

Map of Mexico that shows the location of the State of Durango, and the frequency of Leptospira seroreactivity in donkeys in the 3 municipalities of Durango State surveyed.

Table 2.

Titers to Leptospira serovars found in donkeys in Durango, Mexico.

Leptospira serovars	No. of seropositive donkeys and their titers			Total
Leptospira serovars	1:100	1:200	1:400	Total
L. interrogans Icterohaemorrhagiae	40	26	51	117
L. interrogans Sejroe	22	14	60	96
L. interrogans Canicola	1	1	8	10
L. kirschneri Grippotyphosa	1	3	2	6
L. borgpetersenii Ballum	2	1	2	5
L. biflexa Semaranga	2	1	0	3
L. noguchii Panama	0	1	1	2

Donkeys from the valley region had a higher (87.0%) seropositive rate to Leptospira than donkeys from the mountainous region (74.3%; p = 0.05). Bivariate analysis also showed that seropositive rate to Leptospira varied significantly (p = 0.02) among municipalities, being the highest (87.0%) rate in donkeys originating from Durango municipality (Table 1). Logistic regression analysis of variables with p ⩽ 0.05 obtained by bivariate analysis showed that Leptospira exposure was only associated with gathering premises (OR = 2.64; 95% CI: 1.27–5.46; p = 0.009) and municipalities (OR = 0.11; 95% CI: 0.01–0.78; p = 0.02). With respect to clinical status, apparently healthy donkeys had a higher (82.7%) seropositive rate to Leptospira than the group of donkeys with cutaneous sores or that were malnourished (57.9%; p = 0.001).

It is not clear why the seropositive percentage of Leptospira infection that we found in donkeys in Durango, Mexico (77.8%) is higher than reported in donkeys in other countries (20% in northwest Morocco;⁷ 41.2% in Azerbaijan, Iran¹²). It is possible that the differences in environmental contamination with Leptospira among the countries might have contributed to a higher seropositive percentage of Leptospira infection in donkeys in Mexico than in donkeys elsewhere. We looked for factors associated with Leptospira infection of donkeys in Durango, Mexico. Logistic regression analysis showed that Leptospira infection was associated both with gathering premises and with municipalities. Donkeys studied were gathered in 4 premises for shipment to abattoirs. These premises may have gathered donkeys from nearby municipalities. This finding suggests that environmental contamination with Leptospira differs among municipalities or premises. Research on water contamination and infection with Leptospira in animals in municipalities in Durango State is needed. The seropositive percentage of Leptospira infection was higher in apparently healthy donkeys than in the group of donkeys with cutaneous sores or that were malnourished. This finding agrees with the observation that most infections with Leptospira in horses are asymptomatic.^14,17

The predominant serovar found was L. interrogans Icterohaemorrhagiae (60.3%) followed by L. interrogans Sejroe (49.5%) and L. interrogans Canicola (5.2%). In a study in northwest Morocco, the most prevalent serovars in donkeys were Javanica and Australis.⁷ In equids (horses, donkeys, and mules) in Azerbaijan, Iran, the predominant serovars were L. interrogans Pomona (39%), followed by Grippotyphosa (33%), Icterohaemorrhagiae (15%), and Canicola (11%).¹² On the other hand, the predominant serovar (Icterohaemorrhagiae) found in donkeys in our study was also predominant in a 2017 study of horses in Latin America.¹⁵

Very little is known about leptospirosis in humans in Durango. Seroprevalence of Leptospira infection varies among population groups in the region, being higher in patients with liver diseases (22.7%),⁴ meat workers (17.7%),⁵ and in the rural general population (15.6%)² than in gardeners (6%)⁶ or in waste pickers (4.4%).³ On the other hand, leptospirosis in donkeys is of epidemiologic importance because shedding of Leptospira by infected animals, if it occurs, might contribute to exposure of other animals and humans. In fact, L. kirschneri has been isolated from urine of a mare post-abortion in Brazil.¹¹

Our survey has some limitations. All donkeys surveyed were being sent for slaughter; it is possible that these donkeys received less health care from their owners than work donkeys. In addition, donkeys studied were raised in only one Mexican state. Few donkeys were included in some categories. Therefore, further research to determine the seropositive percentage of Leptospira infection in donkeys used for purposes other than slaughtering and raised in Mexican states other than Durango should be conducted.

Footnotes

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

This study was financially supported by Juárez University of Durango State, Durango, Mexico, and State University of Veracruz (Universidad Veracruzana), Veracruz, Mexico.

ORCID iD

Cosme Alvarado-Esquivel

References

Alvarado-Esquivel

, et al. Prevalence of Toxoplasma gondii antibodies in domestic donkeys (Equus asinus) in Durango, Mexico slaughtered for human consumption. BMC Vet Res 2015;11:6.

Alvarado-Esquivel

, et al. Seroepidemiology of Leptospira exposure in general population in rural Durango, Mexico. Biomed Res Int 2015;2015:460578.

Alvarado-Esquivel

, et al. Leptospira exposure and waste pickers: a case-control seroprevalence study in Durango, Mexico. J Clin Med Res 2015;7:637–640.

Alvarado-Esquivel

, et al. Leptospira exposure and patients with liver diseases: a case-control seroprevalence study. Int J Biomed Sci 2016;12:48–52.

Alvarado-Esquivel

, et al. High seroprevalence of Leptospira exposure in meat workers in northern Mexico: a case-control study. J Clin Med Res 2016;8:231–236.

Alvarado-Esquivel

, et al. Leptospira exposure and gardeners: a case-control seroprevalence study. J Clin Med Res 2016;8:25–28.

Benkirane

, et al. Preliminary investigations on the distribution of Leptospira serovars in domestic animals in north-west Morocco. Transbound Emerg Dis 2016;63:e178–e184.

Evangelista

Coburn

Leptospira as an emerging pathogen: a review of its biology, pathogenesis and host immune responses. Future Microbiol 2010;5:1413–1425.

Faine

, et al. Leptospira and Leptospirosis. 2nd ed. Melbourne, Australia: MediSci Press, 1999.

10.

Hamond

, et al. Presence of leptospires on genital tract of mares with reproductive problems. Vet Microbiol 2015;179:264–269.

11.

Hamond

, et al. Molecular characterization and serology of Leptospira kirschneri (serogroup Grippotyphosa) isolated from urine of a mare post-abortion in Brazil. Zoonoses Public Health 2016;63:191–195.

12.

Hassanpour

Safarmashaei

Seroprevalence of leptospiral infection in horses, donkeys and mules in East Azerbaijan province. Afr J Microbiol Res 2012;6:4384–4387.

13.

Knudtson

Fetters

The effect of heat-inactivation on agglutinating antibody titers to Leptospira interrogans. J Vet Diagn Invest 1990;2:149–150.

14.

Pikalo

, et al. Vorkommen von Antikorpern gegen Leptospiren bei Pferden im mitteldeutschen Raum [Occurrance of antibodies against Leptospira in horses in Middle Germany]. Berl Munch Tierarztl Wochenschr 2016;129:202–208. German.

15.

Pinto

, et al. A systematic review of leptospirosis on dogs, pigs, and horses in Latin America. Trop Anim Health Prod 2017;49:231–238.

16.

Saito

, et al. [Leptospirosis now—the centennial of the discovery of Weil’s disease pathogen]. Nihon Saikingaku Zasshi 2014;69:589–600. Japanese.

17.

Simbizi

, et al. A study of leptospirosis in South African horses and associated risk factors. Prev Vet Med 2016;134:6–15.

18.

Torres-Castro

, et al. Revision actual de la epidemiologia de la leptospirosis [Current review of the epidemiology of leptospirosis]. Rev Med Inst Mex Seguro Soc 2016;54:620–625. Spanish.

19.

Tsegay

, et al. Circulating serovars of Leptospira in cart horses of central and southern Ethiopia and associated risk factors. Prev Vet Med 2016;125:106–115.

20.

Vieira

, et al. A systematic review of leptospirosis on wild animals in Latin America. Trop Anim Health Prod 2018;50:229–238.

21.

Wasiński

Leptospiroza—aktualne problemy [Leptospirosis—current problems]. Przegl Epidemiol 2011;65:471–476. Polish.