Detection of anti-Neospora spp. antibodies associated with different risk factors in horses from Mexico

Padilla-Díaz, Kenia Jasher; Medina-Esparza, Leticia; Cruz-Vázquez, Carlos; Vitela-Mendoza, Irene; Gómez-Leyva, Juan F.; Quezada-Tristán, Teódulo; Padilla-Díaz, Kenia Jasher; Medina-Esparza, Leticia; Cruz-Vázquez, Carlos; Vitela-Mendoza, Irene; Gómez-Leyva, Juan F.; Quezada-Tristán, Teódulo

doi:10.22319/rmcp.v12i1.5267

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Revista mexicana de ciencias pecuarias

On-line version ISSN 2448-6698Print version ISSN 2007-1124

Rev. mex. de cienc. pecuarias vol.12 n.1 Mérida Jan./Mar. 2021 Epub Sep 20, 2021

https://doi.org/10.22319/rmcp.v12i1.5267

Articles

Detection of anti-Neospora spp. antibodies associated with different risk factors in horses from Mexico

Kenia Jasher Padilla-Díaz^a

Leticia Medina-Esparza^a^*

Carlos Cruz-Vázquez^a

Irene Vitela-Mendoza^a

Juan F. Gómez-Leyva^b

Teódulo Quezada-Tristán^c

^{^a} Instituto Tecnológico El Llano Aguascalientes, Km 18 Carretera Ags-SLP., Municipio de El Llano, Ags., 20330, Aguascalientes, México.

^{^b} Universidad Autónoma de Aguascalientes. Centro de Ciencias Agropecuarias, Aguascalientes, México.

^{^C} Instituto Tecnológico de Tlajomulco, Tlajomulco de Zúñiga, Jalisco, México.

Abstract

Neospora spp. is a protozoan parasite that causes abortions and diseases in the Central Nervous System (CNS) of several domestic and wild animal species. In horses, this parasite causes abortions, neonatal mortality, and CNS diseases. The Neospora species identified in horses is different from Neospora caninum and is called Neospora hughesi. This study aimed to detect the presence of anti-Neospora spp. antibodies associated with different risk factors in horses from Mexico. Risk factors were identified by surveying each stable and individual animal from four different regions (Center, North, West, and South). A total of 684 serum samples were obtained from horses in the different regions, 52.3 % (358) males and 47.7 % (326) females. Samples were subjected to an indirect immunofluorescence (IIF) assay; results were analyzed to estimate the association between seropositivity and risk factors. The seroprevalence of Neospora spp. was 2.34 %. The positive cases were mainly found in three of the four regions included in this study and were significantly associated with anti-Neospora spp. antibodies. The coexistence of the horses with other animals obtained an OR value of 2.34 (95% CI : 0.28 - 19.0; P<0.04). This study concludes that Neospora spp. is present in horses from Mexico.

Key words Neospora hughesi; Neospora spp.; Horses; Risk factors

Resumen

Neospora spp. es un parásito protozoario causante de abortos y enfermedad del Sistema Nervioso Central (SNC) en diversas especies domésticas y silvestres. En equinos, se le ha involucrado como causa de aborto, mortalidad neonatal y enfermedades del SNC. La especie identificada en equinos es distinta a Neospora caninum y se denomina Neospora hughesi. El objetivo del presente, fue detectar la presencia de anticuerpos anti-Neospora spp., asociados a factores de riesgo en caballos de México. Se realizó una encuesta de cada cuadra y animales individuales de cuatro regiones del país (Centro, Norte, Occidente y Sur) para identificación de los factores de riesgo. Se obtuvo un total de 684 muestras de sueros de caballos de las diferentes regiones, 52.3 % (358) machos y 47.7 % (326) hembras. Los sueros fueron conservados a -20 °C. El diagnóstico se realizó mediante la técnica de inmunofluorescencia indirecta (IFI), los resultados de este fueron analizados para estimar la asociación entre la seropositividad y los factores de riesgo. La seroprevalencia a Neospora spp. fue de 2.34 % en la población estudiada; los casos positivos se encontraron principalmente en tres de las cuatro regiones incluidas, que presentaron una relación significativa a la presencia de anticuerpos anti-Neospora spp.; la convivencia de los caballos con otros animales obtuvo un valor de OR de 2.34 (IC 95%: 0.28 - 19.0; P<0.04). Se concluye que, Neospora spp., está presente en los caballos de México.

Palabras clave Neospora hughesi; Neospora spp.; Equinos; Caballos; Factores de riesgo

Introduction

The first case of neosporosis reported in horses was that of a late-term fetus and placenta infected with Neospora caninum¹. The second case corresponded to a one-month-old foal with congenital blindness and neurological disorders². Molecular, antigenic, and structural studies have shown that the species found in horses with neurological problems does not correspond to the one reported, identifying it as Neospora hughesi³. N. caninum and N. hughesi are obligate intracellular protozoa. These species belong to the phylum Apicomplexa, class Sporozoea, order Eucoccidiida, and family Sarcocystidae⁴. In horses, N. caninum has been associated with abortions and reproductive problems⁵^-⁸, and N. hughesi with neurological diseases⁹^-¹¹ and Sarcosystis neuronae, the causal agent of the equine protozoal myeloencephalitis (EPM). EPM is a severe neurological disease that produces significant losses in equine production; this has been mainly reported in the United States¹².

The definitive hosts of N. caninum are domestic dogs¹³, coyotes¹⁴, Australian dingoes¹⁵, and gray wolves¹⁶. Additionally, several domestic and wild animal species have been identified as intermediate hosts¹⁷. However, the definitive hosts of N. hughesi are still unknown, and horses are considered the only potential intermediate host¹⁸. Infection can occur after ingesting sporulated oocysts in contaminated feed or water. Vertical transmission is currently considered only as an alternative route¹⁹^,²⁰.

The serological techniques used to detect anti-Neospora spp. antibodies are indirect immunofluorescence (IIF), enzyme-linked immunoassay (ELISA), Neospora agglutination test (NAT), and Western blot (WB). Additionally, N. caninum tachyzoite antigens are used to evaluate seropositivity for Neospora spp. due to the cross-reaction of this parasite²¹; DNA-based techniques are used to differentiate between N. hughesi and N. caninum²²^). This study aimed to detect the presence of anti-Neospora spp. antibodies associated with different risk factors in horses from Mexico.

Material and methods

Area of study

This study was carried out in the Central, North, West, and South regions of Mexico (Figure 1). Regions were selected according to their geographic and climatic characteristics.

Figure 1 Geographic division of Mexico by study region

Study design

A transversal epidemiological study was conducted from October 2016 to October 2017. This study consisted of two stages: field and laboratory. During the field stage, 5 mL of venous blood were collected in Vacutainer^® tubes without anticoagulant (BD Vacutainer^®) by puncturing the jugular vein of apparently healthy horses. A total of 684 samples were collected; 75 corresponded to the Central region (10.96 %), 54 to the North region (7.89 %), 298 to the West region (43.57 %), and 257 to the South region (37.57 %).

All horses were subjected to a physical exam to determine their health status. Additionally, a survey was applied to the owners to collect information about the general characteristics of the animals (breed, age, sex, and reproductive status) and the specific management of the stable (use of horses, feed, housing, water source, and contact with other animals). Blood samples were transported under refrigeration at 4 °C. In the laboratory, samples were centrifuged at 3,500 rpm for 10 min to separate the serum, which was then transferred to 1.5-mL microtubes and stored at -20 °C until further analysis.

Serologic test

The IIF assay was performed using a commercial kit to detect the circulating IgG antibodies against Neospora spp. This kit (SLD-IFA-NC) employs slides antigenated with N. caninum tachyzoites (strain NC-1) and an anti-equine IgG-fluorescein isothiocyanate conjugate (FITC-Conjugate VMRD). Serum samples were diluted 1:25 in phosphate-buffered saline (PBS). Positive and negative controls were used as standards. The assay was performed following the manufacturer's instructions. All sera that fluoresced at the initial dilution were considered positive and were further diluted to the titer endpoint. The highest serum dilution showing fluorescence was considered the endpoint titer.

Data analysis

The data obtained through the surveys (independent variables) and the presence of anti-Neospora spp. antibodies (dependent variable) were analyzed using the statistical software STATA, version 10. The seroprevalence distribution was obtained with the independent variables, and the association between the seroprevalence of N. caninum in horses and other animals was obtained using a logistic regression model (Chi-square), considering P<0.05. Finally, the odds ratios (OR) between the positive results, the negative results, and the relationship with the confidence interval were calculated.

Results

The presence of anti-Neospora spp. antibodies was detected in serum samples obtained from horses using the IIF technique (N. caninum NC-1). Serum samples with titers at a 1:50 dilution were considered positive. The overall seroprevalence was 2.34 % (16/684).

The reproductive status variable was analyzed based on the total number of animals seropositive for Neospora spp. A total of 50 % (8/16) of the animals had anti-Neospora spp. antibodies at different dilutions, greater at the cut-off point (1:50). Table 1 shows that in entire males (4/16) the maximum dilution was 1:200; however, pregnant females (3/16) reached dilutions of 1:200, two of them reached 1:400.

Table 1 Percentage of anti-Neospora spp. antibody titers according to serial dilutions using IIF technique

Animals	Titers
Animals	1:50	1:100	1:200	1:400
Castrated	18.75 (3/16)	-	-	-
Entire	25.00 (4/16)	18.75 (3/16)	6.25 (1/16)	-
Filly	6.25 (1/16)	-	-	-
Mare	12.50 (2/16)	-	-	-
Pregnant	18.75 (3/16)	18.75 (3/16)	18.75 (3/16)	12.50 (2/16)
Empty	6.25 (1/16)	-	-	-
Recent foaling mare	12.50 (2/16)	12.50 (2/16)	-	-
Total	100.0 (16/16)	50.00 (8/16)	25.00 (4/16)	12.50 (2/16)

The survey variables included in the study were analyzed to identify possible risk factors associated with the presence of anti-Neospora spp. antibodies. Table 2 shows the results obtained from this analysis; the highest number of positive cases (62.5 %) was observed in the horses from the West region, 6.25 % corresponded to the Central region, and 31.25 % to the South. There were no positive cases in the North region. This variable had no statistical significance (P<0.05). Of the positive animals, 43.75 % were male and 56.25 % female. The variable of coexistence of horses with other animals obtained an OR of 2.34 (95% CI: 0.28 - 19.0; P=0.04). The use of horses and housing variables had a P-value <0.20. After analysis with logistic regression, the housing variable had an OR of 3.12. The age, breed, reproductive status, and water source variables were not statistically significant (P>0.05).

Table 2 Distribution of Neospora spp. in positive and negative sera and associated factors of horses from Mexico

Variable	Samples		Neospora spp.		P-value	OR (95% CI)
Variable	N	%	+	-	P-value	OR (95% CI)
Region:
Central North West South	75 54 298 257	10.96 7.89 43.57 37.57	1 (6.25) 0 (0.00) 10 (62.50) 5 (31.25)	74 54 288 252	0.36	0.79 (0.49-1.29)
Sex:
Male Female	358 326	52.34 47.66	7 (43.75) 9 (56.25)	351 317	0.49	0.45 (0.03-6.18)
Age:
Young (7-24 m) Adult (25-48 m) Old (>48 m)	68 375 241	9.94 54.82 35.23	1 (6.25) 10 (62.50) 5 (31.25)	67 365 236	0.21	1.09 (0.47-2.51)
Breed:
Creole Pure	262 422	38.30 61.70	6 (37.50) 10 (62.50)	256 412	0.68	1.09 (0.22-5.40)
Reproductive status:
Entire Castrated Filly Mare Pregnant Empty Recent foaling mare	293 65 26 121 53 115 11	42.84 9.50 3.80 17.69 7.75 16.81 1.61	4 (25.00) 3 (18.75) v1 (6.25) 2 (6.25) 3 (18.75) 1 (6.25) 2 (12.50)	289 62 25 119 50 114 9	0.13	0.69 (0.32-1.49)
Use of horses:
Racing Not racing	329 355	48.10 51.90	6 (37.50) 10(62.50)	323 345	0.10	0.25 (0.04-1.35)
Feed:
Forage Grazing Mixed	325 269 90	47.51 39.33 13.16	9 (56.25) 4 (25.00) 3 (18.75)	316 265 87	0.06	1.14 (0.27-4.76)
Housing:
Stable Paddock	340 344	49.71 50.29	8 (50.00) 8 (50.00)	332 336	0.20	3.12 (0.49-21)
Coexistence with other animals:
Yes No	598 86	87.43 12.57	15 (93.75) 1 (6.25)	583 85	0.04	2.34 (0.28-19)
Water sources:
Open pit Water bank	341 343	49.85 50.15	8 (50.00) 8 (50.00)	333 335	0.78	0.86 (0.31-2.41)
Total	684	100.00	16 (100.00)	668	-	-

Discussion

The seroprevalence reports of neosporosis in horses vary worldwide²³^-²⁶. This study, reported a seroprevalence for anti-Neospora spp. antibodies of 2.34 %. Other studies have showed similar results. In Brazil, a study reported a seroprevalence of 2.5 % and 4.1 % of Neospora spp.⁶^,¹⁸; 3.5 % in Costa Rica; 3.7 % in Nineveh, Iraq; and 3% in Durango, Mexico, for the presence of anti-N. hughesi antibodies²⁷^-²⁹. However, some studies have reported higher values for anti-Neospora caninum antibodies; 34 % in the United States, 20 % in Iran, 48.27 % in Brazil, and 12 % in Peru²³^-²⁶. The detection of antibodies against Neospora spp. contributes to the epidemiological information of this disease in Mexico and horses, representing an important production system in livestock farms. The seroprevalence of Neospora spp. in females and males was 2.7 % and 2.0 %, respectively. Studies in Brazil reported a seroprevalence of 4.3 % in females and 3.7 % in males⁶, while in Israel, a study reported 10.9 % in females and 13.0% in males⁸. These results coincide with those reported in Brazil, which despite not being statistically significant, it should be noted that both females and males have a similar risk of infection.

The horses in this study were from different regions in Mexico. The West and South regions had the highest percentage of horses with anti-Neospora spp. antibodies compared to the Central and North regions. Similar studies in Brazil reported differences in the coastal (5.6 %) and mountain (2.6 %) regions⁶. In Jordan, the presence of Neospora spp. in horses varies across regions³⁰. This suggests that the geographic localization and the climate of each region influence the presence of Neospora spp. in horses.

The OR in this study was 1.1 for the variables of age, breed, and feed. There were no statistical differences. These results are similar to those reported in Israel⁸, Brazil²⁵, Jordan³⁰, and Italy³¹ and suggest that the increase of these variables, although not statistically different, augments the probability of infection by Neospora spp. As for the housing variable, the OR obtained in this study was 3.12, similar to that reported in Israel⁸, which means that housing is an important risk factor that increases the probability of infection with Neospora spp. The variable of coexistence with other animals was associated with the presence of Neospora spp., the OR= 2.34 (P<0.05), which coincides with several studies in Brazil that analyzed the same variable with an OR= 1.35 (P<0.05)⁵^,⁶^,²⁵. These results indicate that coexistence with other animals increases the risk of infection with Neospora spp.

Conclusions and implications

In this study, was detected anti-Neospora spp. antibodies in horses from different regions in Mexico; this demonstrates the presence of Neosporosis in equine farms. None of the positive cases showed characteristic clinical signs of the disease. The coexistence with other animals was the risk factor with the greatest association to seropositivity. However, it is necessary to perform an analysis considering the different categories of each variable. Further studies are required to identify the definitive host of Neospora spp. and its transmission mechanisms to horses. Additionally, it is essential to identify the Neospora species that affects horses.

Acknowledgments

The authors would like to thank the owners of the animals for facilitating this study.

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Received: February 19, 2019; Accepted: March 12, 2020

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