ABSTRACT
Paratuberculosis (PTB) is a disease that affects cattle (Bos taurus), goats (Capra aegagrus hircus), sheep (Ovis aries), and wild animals, such as white-tailed deer (Odocoileus virginianus), since all ruminants are susceptible. The causal agent is Mycobacterium avium subsp. paratuberculosis (MAP). The disease is chronic, consumptive, and incurable; it causes chronic granulomatous gastroenteritis with lymphangiectasis and lymphangitis leading to a syndrome of malnutrition and eventually to death. Mycobacterium avium subsp. paratuberculosis is transmitted in feces mainly orally; however, it can also be transmitted vertically. Thus, the objective of this study was to determine the seroprevalence of MAP antibodies and its relationship to age and sex of Texas white-tailed deer in the subclinical stage of PTB in Coahuila, Mexico. The entire population (n=99) belonging to the Wildlife Management and Conservation Unit (WMCU) San Juan, Monclova, Coahuila, Mexico was captured. Mycobacterium avium subsp. paratuberculosis was diagnosed using an enzyme-linked immunosorbent assay by serologic test. Seroprevalence variables of adult vs. young females and males vs. females were compared. The treatments were assigned at random. For the analysis of data, the chi-square test was used. Total seroprevalence in an intensive WMCU was 16% (16/99). Total seroprevalence by sex was 5.0% (5/99) for males and 11% (11/99) for females, and total seroprevalence by age was 7% (7/99) for young and 9% (9/99) for adult. Within sex, the seroprevalence in males was 16% (5/31) and 16% (11/68) in females. There were no statistical differences for any of the comparisons. Total seroprevalence of the white-tailed deer population in the WMCU was 16%, and PTB seroprevalence was independent of sex or age of the sampled individuals of this population.
INTRODUCTION
Paratuberculosis (PTB) or Johne disease is a disease that affects cattle (Bos taurus), goats (Capra aegagrus hircus), and sheep (Ovis aries), but it can appear in all ruminants and has been considered a problem for wild fauna, such as free-ranging white tailed deer (Odocoileus virginianus; Sleeman et al. 2009) and Key deer (Quist et al. 2002). In the same way, PTB affects wild non-ruminant species, such as the wild European rabbit (Oryctolagus cuniculus) and carnivores (Chiodini et al. 1984; Carta et al. 2013). Reports of PTB in wild fauna include monogastric species, and so the disease represents an addition risk in regard to propagation of the disease, and it has been hypothesized that Mycobacterium avium subsp. paratuberculosis (MAP) could be involved in Crohn disease in humans. Moreover, the incubation period of MAP has been estimated to be up to 5 yr in cattle and 2 yr in sheep (Whittington et al. 2012). Paratuberculosis is a chronic, consumptive, and incurable disease; it causes severe chronic granulomatous gastroenteritis with lymphangiectasis and associated lymphangitis, whose end consequence is the appearance of malnutrition syndrome with weight loss, chronic or intermittent diarrhea, and eventual death (Chiodini et al. 1984; Whittington et al. 2012). According to Palmer et al. (2019), the most common clinical signs of PTB in deer are chronic diarrhea, weight loss, and a decline in body condition.
In domestic and wild ruminants, horizontal transmission of MAP is oro-fecal (Windsor and Whittington 2010), and vertical transmission through placenta and semen also has been suggested (Khol et al. 2010; Rindi and Garzelli 2014). However, intrauterine transmission is more common in farmed deer than in cattle and sheep (Carta et al. 2013). The presence of MAP in semen (42.9%) and in reproductive tissue (epididymis tissue, 14.3%; Cowper gland, 28.6%; and prostate, 14.3%) has been confirmed in sheep in clinical state (Velázquez-Morales et al. 2019). Moreover, PTB infection has been reported in wild and exotic species, including red deer (Cervus elaphus) and rabbits (Oryctolagus cuniculus). Transmission to exotic species likely occurs through ingestion of grasses contaminated by sheep and cattle (Carta et al. 2013). The animals most susceptible to contracting the infection are mainly animals younger than 6 mo old and lactating offspring infected by contamination of the udder with feces, which can contain MAP, or by intake of forage contaminated by mycobacteria (Chiodini et al. 1984; Windsor and Whittington 2010). When domestic cattle share the area with huemul deer (Hippocamelus bisulcus), an epidemiologic interaction occurs, verified when determining the common infection of mycobacteria; it was suggested that the infection was acquired by consuming contaminated water (Corti et al. 2020).
Scientific information about PTB in deer is scarce (Kopecna et al. 2008). Mandujano (2004) reported that only 13% of the 502 studies were peer-reviewed publications. Of these publications, 74% dealt only on its biology, ecology, and management and 26% on its distribution. In Mexico, there are no studies of the health status of deer populations. In addition, PTB has been a problem on deer farms in many countries (Kopecna et al. 2008). The mean prevalence of MAP recorded in wildlife was 2.41% (Carta et al. 2013), and in North America, the seroprevalence to MAP in white-tailed deer populations ranges from 2% to 50% (Wolf et al. 2008; Palmer et al. 2019).
There are several diagnostic tests for detection of MAP that vary in sensitivity and specificity. In this sense, the enzyme-linked immunosorbent assay (ELISA) is the most frequently used method in prevalence studies because of its relatively low cost and ease of use (Stau et al. 2012). Our objective was to determine the prevalence of antibodies by MAP and its relationship with age and sex of Texas white-tailed deer, in a subclinical state of PTB in Coahuila, Mexico.
MATERIALS AND METHODS
Location and description of the study area
The study area is located in the Wildlife Management and Conservation Unit (WMCU) San Juan, municipality of Monclova, Coahuila, Mexico. The coordinates of the WMCU San Juan centroid are 26°50′9″N, 101°1′50″W, covering an area of 3,237.1 ha (Fig. 1). The orography of the terrain includes areas with altitudes of 400 m with hills that rise to approximately 700 m. Mean annual precipitation is 300 mm to 400 mm, and mean annual temperature is 20 C to 22 C. Climate is warm arid and semiarid (García 2004). The soil types are luvic Xerosols, calcic Xerosols, and calcaric Regosols. Generally, these soils are representative of arid regions and are classified as light-colored soils with organic matter and salts, such as carbonates. The main vegetation covers are scrub and grasslands (INEGI 2014). The vegetation types are desert rosetophile scrub, desert microphyll scrub, and a small portion of natural grassland, which are apt for extensive livestock raising because of its floristic composition (INEGI 1997).
Location of the Wildlife Management and Conservation Unit San Juan Ranch, municipality of Monclova, Coahuila, Mexico, containing the population of Texas white-tailed deer (Odocoileus virginianus) captured for detecting Mycobacterium avium subsp. paratuberculosis with an enzyme-linked immunosorbent assay.
Location of the Wildlife Management and Conservation Unit San Juan Ranch, municipality of Monclova, Coahuila, Mexico, containing the population of Texas white-tailed deer (Odocoileus virginianus) captured for detecting Mycobacterium avium subsp. paratuberculosis with an enzyme-linked immunosorbent assay.
Within the WMCU San Juan, there is no permanent current of water. However, 6 km to the northeast is a creek, and 30 km to the west is the Monclova River (Maderey-Rascón and Torres-Ruata 1990). There are also small reservoirs that supply water for wildlife, and 39 feeding sites for protein supplementation are evenly distributed throughout the WMCU.
Study population
The study was conducted in the San Vicente Reserve on the San Juan Ranch. Ninety-nine deer were captured with drop nets; seven were adult males, 50 adult females, 24 young males, and 18 young females. Females and males were considered young if they were ≤2.5 yr old and adult if they were ≥2.6 yr old. During the sampling period, no individual showed signs of PTB.
Sample preparation
Blood samples (5 mL) were collected by puncture of the jugular. Later the samples were centrifuged (1000 × G for 10 min) to recover the serum in sterile 2 mL collection tubes, which were frozen at –20 C until analysis.
Enzyme-linked immunosorbent assay
Serum samples were processed to calculate seroprevalence at the Centro Nacional de Investigación Disciplinaria en Microbiología Animal of the Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias. Detection of previous infection with MAP was performed with the ELISA test, recorded in domestic sheep samples. Additionally, the ELISA has been validated for cattle, and it is considered a prototype procedure for cervids such as deer (Quist et al. 2002).
The assay was carried out with a MAP protoplasmic antigen (strain 3065), according to the protocol of Martínez-Covarrubias et al. (2012). The protein of the Mapcon PPA-3 antigen Paratuberculosis Protoplasmic Antigen (Allied Monitor Inc®, Fayette, Missouri, USA) was fixed on the wells of the ELISA plates. At the end of the 24 h incubation period, the samples in each well were washed four times with 300 µL of phosphate buffered saline-TWEEN 20 solution (PBST; 0.05%, pH 7.4) and 100 µL of 1% albumin blocking solution (Fluka Biochemika, Buchs, Switzerland). The plates were incubated at 37 C for 1 h, then washed four times with PBST and stored at 4 C wrapped in plastic and aluminum foil until use. On each plate, 100 µL of the control, negative, or positive serums was placed in the corresponding wells. A positive control serum was acquired from Allied Monitor. All the serums were tested in pairs; they were incubated at room temperature for 30 min and washed four times with 300 µL PBST. Conjugated anti-bovine immunoglobulin G conjugated with horseradish peroxidase (100 µL) was then added to each well, which was incubated at room temperature and washed with PBST four times. To each well, 100 µL of the substrate solution ABTS (2,2′-azino-di-[3-ethylbenzthiazoline sulfonic acid]; AMRESCO, Albany, New York, USA) was added and kept in the dark at room temperature and shaken for 30 min. Readings were taken at 650 nm in an eight-channel spectrophotometer (ELx800, Bio-Tek, Winooski, Vermont, USA). The highest values at 0.22 optical densities were considered positive.
Statistical analysis
Groups of young females, young males, adult females, and adult males were assigned to treatments at random and compared. For data analysis, the chi-square test was used with α=0.05 in SPSS Statistics® (IBM Statistical Software, version 24, Chicago, Illinois, USA).
RESULTS
The total seroprevalence of MAP in Texas white-tailed deer in an intensive WMCU was 16% (16/99). The total seroprevalence by sex was 5% (5/99) for males and 11% (11/99) for females, and the total seroprevalence by age was 7% (7/99) for young deer and 9% (9/99) for adult deer. Moreover, seroprevalence of young females was 4% (4/99) compared with 7% (7/99) in adult females. Seroprevalence of young males was 3% (3/99), while in adult males it was 2% (2/99). Within sex, seroprevalence in males was 16% (5/31) and 16% (11/ 68) in females (Table 1).
Paratuberculosis seroprevalence as determined with an enzyme-linked immunosorbent assay in a population of Texas white-tailed deer (Odocoileus virginianus) in Coahuila, Mexico, according to age and sex. There was no statistical difference (P>0.05; χ2) between age groups and sexes in infected groups.

DISCUSSION
The seroprevalence of MAP antibodies was estimated in a population of Texas white-tailed deer in an intensive WMCU. Animals there have been reported to be exposed to MAP but never expressed clinical signs of PTB (Chiodini et al. 1984). Control of the disease in domestic ruminants depends on timely detection and elimination of infected animals, but this may be limited by a lack of adequate diagnosis. For this reason, strategies for detection, monitoring, and control of MAP must be implemented (Jaimes et al. 2008). Serologic, molecular, and culture-based tests can be used to estimate the number of infected individuals to prevent the mycobacterium from disseminating throughout the population or from creating a site that puts other susceptible species at risk. There is little relationship between age of the deer and seroprevalence of MAP antibodies in ruminants. For red deer, it has been reported that the most susceptible animals are 8 to 15 mo old (Mackintosh et al. 2010). However, in our study using the serologic test, there were no statistical differences among the variables studied: young and adult individuals, and males and females. On the contrary, the values of seroprevalence tended to be higher in adult animals than in young animals since MAP has a long incubation period, which varies among species. For example, in cattle the estimated incubation period is 3 to 10 yr, and in sheep and goats it can be shorter because in sheep most of the deaths occur at 3 to 4 yr old (Whittington et al. 2012).
Paratuberculosis is distributed worldwide, and its prevalence varies from 5 to 25% (Martínez Covarrubias et al. 2012). However, in white-tailed deer, Woodbury et al. (2008) reported a prevalence of 13.3%, a value they obtained with Elisa, and Hattel et al. (2004) found only two cases of MAP in postmortem examination of 160 captive white-tailed deer while Wolf et al. (2008) reported a serologic prevalence of 2%, only in adult white-tailed deer. In North America, the seroprevalence to MAP antibodies in populations of white-tailed deer generally ranges from 2% to 50% (Wolf et al. 2008; Palmer et al. 2019). We obtained a seroprevalence of 16%, despite the deer population being well distributed in the study area without aggregation at specific points; the 39 sites of feed supplementation were evenly distributed throughout the WMCU, considerably reducing social interactions of the deer. In intensive production units, risk of infection increases due to factors such as high animal concentration, exposure of offspring to contaminated feces, milk, and water, low nutritional value feed, acidic soils, and stress factors that suppress immunity such as transport, birthing, and lactation. Reports of clinical PTB, in deer and elk (Cervus canadensis) in North America, indicate the presence of factors such as confinement, high population density, association with infected livestock, and stress (Quist et al. 2002), as those that most affect risk of infection.
Average PTB seroprevalence in the entire population of white-tailed deer was 16%, even though no animal had clinical symptoms and the seroprevalence was independent of sex or age of the deer. We recommend diagnostic studies such as bacteriologic culture of feces, as it allows clinical and subclinical detection of infection. This bacteriologic culture test has a specificity of 100% but a sensitivity less than 50% (Jaimes et al. 2008). We recommend the nested PCR test, which has greater sensitivity (67.8%) and specificity of 84% and allows detection of both viable and non-viable bacteria (Martínez-Covarrubias et al. 2012) and allows identification and elimination of animals that may spread or be the cause of establishing PTB in herds (Velázquez-Morales et al. 2019). A deer herd may be monitored at least every 4 yr, since MAP infections in white-tailed deer resemble those of cattle during calfhood and are followed by a prolonged period of incubation about 2 yr (Palmer et al. 2019). Further studies are necessary to clarify the incubation period of MAP in white-tailed deer.
ACKNOWLEDGMENTS
The authors thank the personnel of the WMCU San Juan in Monclova, Coahuila, for their support. We thank the Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias and especially Centro Nacional de Investigación Disciplinaria en Microbiología Animal, under the direction of Marco Antonio Santillán-Flores, for serologic testing.