Seroprevalence of Leptospirosis,Brucellosis,and Q Fever in a Wild Red Deer ( Cervus elaphus ) Population Kept in a Fenced Reserve in Absence of Contact with Livestock

Abstract

Wildlife health is of interest for public and animal health because wild animals have been identified as important sentinels for the surveillance for zoonotic pathogens. This work investigated Brucella spp., Coxiella burnetii, and Leptospira spp. infection seroprevalence in a free-ranging red deer population. The study was conducted in a fenced reserve with controlled hunting activity in central Spain with animals that did not have any contact with livestock. Sampling was performed at two time points before and 5 years after the implementation of new management measures, including a reduction in the red deer population in the reserve. In addition, the presence of Leptospira DNA was tested in placental and fetal samples from seropositive pregnant animals. Antibodies against Brucella and Coxiella were not detected in any sample. The seroprevalence of Leptospira was 9.4% (13/137) in the first sampling for serovars Canicola and Panama. Five years later, the prevalence rose to 38.5% (97/252) with Pomona, the only serovar detected. Animals older than 2 years (50%; 70/140) were more likely to be Pomona seropositive than animals ≤2 years old (25.2%; 27/107; p < 0.001). Leptospira DNA was not detected in any sample tested. In conclusion, wild red deer in this area without contact with livestock seem not to play an important role in Brucella spp. and C. burnetii maintenance. The high seroprevalence of Leptospira spp. serogroup Pomona could indicate a risk for people with narrow contact with these animals, but the carrier status was not assessed. Consequently, it is unknown if red deer would represent a risk for human infection. Considering that wild boar could be the source of infection to red deer, the role of wild boar in the spread of leptospirosis and the risk for human infection should be investigated.

Introduction

Wildlife health is of interest in terms of conservation and because wild animals are important sentinels for the surveillance for zoonotic pathogens (Rabinowitz et al. 2006, Halliday et al. 2007). In Spain, the red deer (Cervus elaphus) has notably increased its population size (Acevedo et al. 2008). This finding is most likely due to the abandonment of fields, the advance of forests, and the implementation of hunting management practices (Acevedo et al. 2005). The current range of the red deer extends throughout Spain with higher densities in the southwest, where it is one of the most culturally and economically important hunting species (Carranza 2004). Exposure of hunters to wildlife during game carcass dressing has been proposed as a potential zoonotic risk (Bengis et al. 2004). In this context, brucellosis, Q fever, and leptospirosis are important zoonotic bacterial diseases. Brucellosis is caused by bacteria of the genus Brucella (Bengis et al. 2004), Brucella abortus and B. melitensis being the species most regularly transmitted between wild and domestic ungulates (Bengis et al. 2004, Van Campen and Rhyan 2010). Q fever is a zoonosis with a worldwide distribution that is caused by the Gram-negative intracellular bacterium Coxiella burnetii (González-Barrio et al. 2015). A high prevalence of this pathogen in deer and the deer-human contact may represent an important risk factor for human infection, especially in areas where a significant percentage of animals have been exposed to the bacterium (Kirchgessner et al. 2013). Leptospirosis is a worldwide zoonotic infection that occurs in domestic and wild mammals (Bharti et al. 2003) and is considered one of the most important reemerging human health hazards by the OIE (Bengis et al. 2004). Among wildlife species, rodents and wild boars are considered to be important reservoirs for leptospirosis (Bharti et al. 2003). Finally, these diseases are also important causes of reproductive failure in domestic ruminants (Bengis et al. 2004, Gortazar et al. 2007, García-Ispierto et al. 2014). As a result of this situation, surveillance studies that assess the role that red deer may play in the maintenance of different zoonotic pathogens in Spain could have important contributions to the prevention and control programs. The aim of this work was to determine the seroprevalence of Brucella spp., C. burnetii, and Leptospira spp. infections in a wild red deer population kept in a fenced reserve with controlled hunting activity. The animals did not have any contact with livestock for several decades. The samplings were performed at two different time points before and 5 years after the implementation of several management measures. The influence of different risk factors (age, gender, and pregnancy) was also evaluated. Finally, the presence of Leptospira DNA was investigated in placental and fetal collected samples from seropositive pregnant animals.

Materials and Methods

Sampling area

The samplings were performed in the Quintos de Mora reserve, which is a fenced hunting area of 6864 ha located in the province of Toledo (Central Spain) that extends on a plain 800 meters above sea level (m.a.s.l.) and a mountainous area that reaches 1200 m.a.s.l. The climate is Mediterranean with rather cold winters (average temperature of the coldest month between 0°C and 18°C) and hot (average temperature in the hottest month above 22°C) and dry summers (“Csa” following the climate classification of Köppen-Geiger) (Iberian climate Atlas, AEMET). The most representative mammals are the hunting species red deer, wild boar (Sus scrofa), fallow deer (Dama dama), and roe deer (Capreolus capreolus). Livestock has not been present in Quintos de Mora for at least five decades.

Experimental design

The seroprevalence of Brucella spp., C. burnetii, and Leptospira spp. was studied in red deer samples collected in 2007 and 2012 during the hunting season (from January to March). Changes in the management system of the Quintos de Mora reserve were implemented during this 5-year period. The new management system included a reduction in the red deer population, which decreased from 2500 to 1700 animals through controlled selective hunting. The number of roe deer and fallow deer present in “Quintos de Mora” is below 100 animals and the census has not varied over time. In addition, traditional food supplementation done in fixed troughs during the dry season was replaced by opening fenced grazing areas to guarantee enough food supply.

Animals were hunted and blood samples were opportunistically collected from the culled animals. A total of 137 and 252 samples were collected in the first and second samplings, respectively. The number of animals sampled was representative, corresponding to 5.4% (137/2500) and 14.8% (252/1700) of the red deer population from the area, respectively. Blood was collected by heart puncture in plastic tubes and sent to the laboratory at 4°C. In the laboratory, serum samples were collected after centrifugation and stored at −80°C before analysis. Brucella spp.-, C. burnetii-, and Leptospira spp.-specific antibodies were detected using the Rose Bengal Test, ELISA, and Microscope Agglutination Test (MAT), respectively, as described below. The ages of the free-ranging deer were determined using canine morphological and metric variables (D'Errico and Vanhaeren 2002), and the animals were classified into the following age groups: animals ≤2 and >2 years. Gender and pregnancy data were annotated when possible. In pregnant animals, a sample was collected from the placenta and the fetal brain, lung, kidney, and liver tissues, or the whole fetus (depending on the fetus size). All samples were stored at −20°C before analysis.

This study did not involve the purposeful killing of animals. All samples originated from legally hunted dead wildlife.

Rose Bengal Plate agglutination test

To detect antibodies against smooth Brucella spp., serum samples were analyzed using the Rose Bengal Test according to the OIE recommendations (OIE 2015).

ELISA

The LSIVet™ Ruminant Q Fever commercial test (LSI, Lissieu, France) was employed for the detection of specific antibodies against C. burnetii. The manufacturer's instructions were followed for the testing and interpretation of results. C. burnetii ELISA was validated using 23 positive and 23 negative reference red deer sera from a previous study (González-Barrio et al. 2015), and observed a perfect concordance (Cohen's kappa coefficient = 1) (data not shown).

Microagglutination test

The test was performed at the “Laboratorio Central de Veterinaria” (Algete, Madrid, Spain), which is the Spanish reference laboratory for leptospirosis, using the following live strains of Leptospira: L. borgpetersenii serovars Australis, Ballum, Bataviae, Castellonis, Djasiman, Hardjo, Javanica, and Tarassovi; L. interrogans serovars Autumnalis, Bratislava, Canicola, Copenhageni, and Pomona; L. kirschneri serovars Cynopteri and Grippotyphosa; L. noguchi serovars Louisiana and Panama; and L. weilii serovar Sarmin. All strains were originally supplied by the Veterinary Research Laboratories (Stormont, Belfast, United Kingdom) with the exception of the strains of serovars Castellonis, Djasiman, and Panama, which came from the National Laboratories Services (Ames, IA).

The strains were cultured in a liquid medium with Tween 80/40-bovine serum albumin (Ellis and Thiermann 1986), incubated at 29°C, and used after 4–8 days. The concentration of the antigens used for the test was adjusted to a transmittance of 60–70% measured at a 400 nm wavelength. The sera were initially examined at a 1:30 dilution against each of the 18 serovars. Sera with some degree of agglutination activity at the 1:30 dilution were retested against each serovar to which they reacted using 1:10, 1:30, 1:100, 1:300, 1:1000, and 1:30,000 dilutions. Any serum sample for which approximately ≥50% of the leptospires were agglutinated at a 1:100 dilution was classified as positive. The final titer was the highest dilution of the serum at which ≥50% or more of the leptospires were agglutinated. A reference antiserum (National Veterinary Services Laboratories) and positive and negative controls were included in each assay.

Leptospira real-time polymerase chain reaction

A total of 22 pregnant females with a positive MAT Leptospira spp. result were selected in the second sampling. Placental and fetal samples were thawed. Total DNA was extracted separately from the placental and pooled fetal tissues (using the BioSprint 96 DNA Blood + Buffer ATL Kit (Qiagen GmbH, Germany) following the manufacturer's instructions. Real-time PCR was performed according to the previously published protocol (Stoddard 2013); negative and positive extraction controls were included in each assay.

Data analysis

Apparent prevalence rates according to gender, pregnancy status, and age were organized in contingency tables and the data were analyzed using the chi-squared or Fisher exact test (Table 1). Risk factors were also studied by estimating the odds ratios (OR). Age between samplings was compared by Student's t-test. p Values <0.05 were considered statistically significant.

Table 1.

Risk Factors Related to Seropositivity Against Leptospira spp. in Red Deer

			Leptospira spp. seroprevalence
Factor	Sampling	Category	No. positive/no. tested (%)	95% CI	OR (95% CI); p-value
Age^a
	First sampling	≤2 years	7/54 (12.9)	4.1–21.8	0.61 (0.19–1.93); p = 0.58
		>2 years	6/72 (8.3)	2.0–14.6
	Second sampling	≤2 years	27/107 (25.2)	17.3–33.2	2.96 (1.71–5.12); p = 0.0001
		>2 years	70/140 (50)	41.9–57.9
Sex^b
	First sampling	Male	7/66 (10.6)	3.3–17.9	0.95 (0.3–3.02); p = 0.83
		Female	6/61 (9.8)	2.5–17.2
	Second sampling	Male	36/98 (36.7)	27.4–46.0	1.13 (0.67–1.9); p = 0.74
		Female	61/154 (39.6)	32.1–47.0
Pregnancy status^c
	First sampling	Pregnant	0/5 (0)	0.0–45.0	0.29 (0.01–5.56); p = 0.5
		Nonpregnant	6/49 (12.2)	3.2–21.0
	Second sampling	Pregnant	35/86 (40.7	30.6–50.8	1.25 (0.63–2.4); p = 0.47
		Nonpregnant	22/62 (35.5)	23.7–47.2

Age data were not recorded for 11 and 5 animals in the first and second sampling, respectively.

Sex data were not recorded for 10 animals in the first and second sampling.

Pregnancy status data were not recorded for 83 and 104 animals in the first and second sampling, respectively.

CI, confidence interval; OR, odds ratio.

Results

Prevalence and risk factor analysis

All samples were seronegative for Brucella spp. and C. burnetii in both samplings. The results showed that 9.4% of the samples (13/137; 95% confidence interval [CI]: 4.7–14.2) had antibodies against Leptospira spp. in the first sampling, whereas the apparent prevalence rate 5 years later rose to 38.5% (97/252; 95% CI: 32.9–44.0) (OR = 5.9; CI: 3.19–11.16; χ² = 35.39; p < 0.0001). Most animals had antibodies against only one serovar. During the first sampling, 1.4% (2/137; 95% CI: 0.0–3.4) and 6.5% (9/137; 95% CI: 2.5–10.6) of the samples were only positive for serovars Canicola and Panama, respectively, and only two samples were positive for both serovars. Only serovar Pomona was found during the second sampling.

During the first sampling, no significant differences (p > 0.05) were observed between age, gender, and pregnancy status (Table 1). However, in the second sampling, animals >2 years old (50%) were more likely to be Pomona seropositive than animals ≤2 years old (25.2%). No significant difference in the mean age was found between samplings (p > 0.05; Student's t-test).

The antibody titers (Table 2) against serovar Canicola detected during the first sampling were 1:100 and 1:1000. In addition, most animals had a 1:100 titer against serovar Panama. Interestingly, the two samples showing coinfections had MAT titers of 1:100 and 1:1000 against serovars Canicola and Panama, respectively. In contrast, most animals had antibody titers of 1:300, 1:100, and 1:1000 against serovar Pomona in the second sampling.

Table 2.

Distribution of Specific Antibody Titers for the Different Leptospira spp. Serovars Studied in Wild Ruminants During the Two Samplings

Serovar	1:100	1:300	1:1000
Canicola	50% (2/4)^a	0	50% (2/4)^a
Panama	90.9% (10/11)^a	0	9% (1/11)^a
Pomona	33% (32/97)^b	46.4% (45/97)^b	20.6% (20/97)^b

Data corresponding to the first sampling (2007).

Data corresponding to the second sampling (2012).

Finally, Leptospira DNA was not detected in any of the placental and fetal samples from pregnant animals seropositive for Leptospira spp.

Discussion

Brucella spp., C. burnetti, and Leptospira spp. are important worldwide zoonotic pathogens; however, the role wildlife plays in maintaining these diseases outside of their target domestic animal or human populations is uncertain (Godfroid 2002, Arricau-Bouvery and Rodolakis 2005, Kirchgessner et al. 2013). In our study, antibodies against Brucella spp. were not detected in the sampled red deer population. This finding suggests that red deer seems not to be a suitable host for smooth Brucella species. Our results are in agreement with previous studies performed in red deer and other wild ruminants in different regions of Spain (Boadella et al. 2010, Muñoz et al. 2010, Serrano et al. 2011). Red deer also tested negative for Brucella in studies from the Czech Republic (Hubálek et al. 1993) and Central Italian Alps (Gaffuri et al. 2006). The sporadic cases reported in red deer were primarily associated with animals living with infected livestock (Hars et al. 2003). C. burnetii antibodies were not detected in our study; therefore, red deer could not play an important role in the sylvatic cycle of C. burnetii in this area. Previous Q-fever studies in wildlife in Spain showed a seroprevalence in wild red deer of 9.5% and 0% in the northern and southern areas, respectively (Ruiz-Fons et al. 2008). A seroprevalence of 3.8% in unmanaged red deer was found near the area where our hunting reserve was located (González-Barrio et al. 2015). Other studies performed in wild ruminants in Europe have shown an anti-C. burnetii antibody prevalence of 25% in red deer from the Czech Republic (Hubálek et al. 1993). These differences may depend on the living environments and higher contact with domestic ruminants.

The most remarkable results corresponded to the presence of Leptospira infections. However, the circulating serovars and the seroprevalence rates varied temporally. The first noticeable finding was a significant increase in the overall prevalence rate in the second sampling that rose from 9.4% to 38.5%. In agreement with the low prevalence rate found in the first sampling, a prevalence of 6.3% deer was reported in the central Italian Alps (Andreoli et al. 2014). In Spain, low seroprevalence rates were found in red deer from the southern (4.6%, Arenas et al. 1991) and northern (2.6%, Espí et al. 2010) areas. Although it is difficult to compare studies due to the different cutoff values employed (usually 1:320), in our study, 27.2% of the samples had titers equal to or higher than 1:300.

Antibodies to serogroups Canicola (1.4%) and Panama (6.5%) were detected in the first sampling. Most of the positive red deer from the first sampling harbored antibodies against serovar Panama, whose origin is probably associated with carnivores and rodents. In France, a prevalence of 18% against this serovar has been reported in European polecat (Mustela putorius) (Moinet et al. 2010), this mustelid species is present in “Quintos de Mora” (Arija 2010). In the past, Pomona and Icterohaemorrhagiae were the most prevalent serovars described in red deer from southern Spain (León-Vizcaíno et al. 1994). In northern Spain, Pomona (1.6%), Bratislava (1.1%), Grippotyphosa (0.7%), Muenchen (2.6%), and Panama (1.2%) were found in red deer (Espí et al. 2010). These differences might be due to ecological and environmental factors that determine the geographic distribution of the maintenance hosts for each serovar. During the second sampling, the overall leptospirosis seroprevalence significantly increased. Only antibodies against the Pomona serogroup were detected, suggesting variability in exposure over time. Variations in prevalence could also reflect variations in the interaction between hosts and higher environmental contamination. Contact with some epidemiologically relevant species could have increased the risk of exposure of red deer to this pathogen. In the case of the Pomona serogroup, the most likely source of infection could be different rodent species and wild boar. Leptospira kirschneri serovar Mozdok (serovar belonging to serogroup Pomona) has been isolated from small rodents trapped in the vicinity of “Quintos de Mora” (Arent et al. 2017). Regarding the role of wild boar, it is known that there is an increase of wild boar populations across Europe. Mild winters, reforestation, intensification of crop production, supplementary feeding, and compensatory population responses of wild boar to hunting pressure might explain this population growth (Acevedo et al. 2007). Although the number of wild boars could not be determined in our study, hunting numbers could be used as indicator of animal numbers. In this study, a total of 35 and 211 wild boars were culled during the first and second sampling season, respectively, suggesting a higher number of wild boars. Moreover, in “Quintos de Mora,” the new management system included a reduction in the red deer population and the growth of natural vegetation available for animals during the dry season to guarantee enough food supply. These measures could have influenced the spread of Pomona, increasing aggregation, for example at feeding sites, since red deer and wild boar could share the same feeding areas (open fenced grazing areas) and the habit of walling in water pools in summer. Moreover, adult males mark their territory during the mating season and spread the infection. This contact and behavior most likely favors the transmission of the infection through contaminated pasture, mud, and water (Johnson et al. 2004, Barasona 2015). In addition, Pomona has been also retrieved in cattle and pigs near to “Quintos de Mora” area, being the most likely source of infection in wild boars (Arent et al. 2017). A high Pomona prevalence (38.1%) was detected in a fallow deer population in northern Spain together with high titers in wild boars after 5 years without positive reactors. Simultaneously, high titers (>1:1280) were detected in several European wild boars in the region (Espí et al. 2010). Unfortunately, boar samples were not collected in our study. Because the wild boar population in Spain shows a tendency to grow, we can infer that wild boars may play an important role in leptospirosis transmission. Further studies on leptospirosis and the isolation of the infective agent are indicated in wild boars in this area. Accordingly, integrative management measures should be designed according to the wildlife populations present in the habitat (e.g., reduction of the current wild boar population).

When the risk factors associated with Pomona infection were studied, red deer older than 2 years showed a significantly higher seropositivity rate than young animals in the second sampling. This result was in agreement with other studies in domestic (Barwick et al. 1997, Ward et al. 2002, Balakrishnan et al. 2011) and wild animals (Vale-Gonçalves et al. 2015) because the possibility of contact with Leptospira increases over the animal lifespan. In our study, Leptospira DNA was not detected in placental and fetal tissues from seropositive pregnant animals. The most plausible explanation is that the pathogen was not located in these tissues or the antibody titers are due to past exposure to strains of the Pomona serogroup. Then, the infection was eliminated in a short period of time because red deer are probably an accidental host for serogroup Pomona (Ayanegui-Alcerreca et al. 2007). However, the lack of Leptospira DNA detection could be also due to the low pathogen numbers in placental and fetal tissues, which made it undetectable by the technique employed. Connections between red deer hind abortions and leptospirosis infections need to be clarified in the future.

Conclusions

In conclusion, free wild red deer without contact with domestic animals seem not to play an important role in Brucella spp. and C. burnetii maintenance in the studied area. A high seroprevalence of Leptospira spp. serogroup Pomona was detected, but the carrier status was not assessed in this study. Consequently, it is unknown if red deer would represent a risk for human infection. On the other hand, considering that wild boar could be the source of infection to red deer, the role of wild boar in the spread of leptospirosis and the risk for human infection should be investigated.

Footnotes

Acknowledgments

This study was financed by the SALUVET research group's own resources. Authors thank Consejería de Agricultura, Medio Ambiente y Desarrollo Rural de Castilla la Mancha, for the institutional authorization to collect the samples. Authors also thank Daniel Gutierrez-Expósito (Saluvet group), Jose María Blasco, Agustín Gutiérrez-Sierra, Carlos Rodríguez-Vigal, Angel Moreno Gómez, and staff of Quintos de Mora for their collaboration in the samplings.

Author Disclosure Statement

No competing financial interests exist.

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