Baseline studies on the health of marsh deer ( Blastocerus dichotomus ) populations from Argentina: laying the grounds for an improved interpretation of mortality episodes CURRENT STATUS:

Background: The comprehensive approach to diseases in broad ecological contexts appears as a new challenge for global health and draws on baseline studies to detect changes in the occurrence of pathogens over time. Marsh deer ( Blastocerus dichotomus ) mortality episodes were described in Argentina and several pathogens associated with environmental and eco-epidemiological factors were indicated as risk factors. To date, the lack of basic health information on these populations has hampered interpretation of findings. This study aimed to provide baseline data on health parameters in marsh deer populations from Argentina. Results: Between May 2014 and December 2016 we determined health parameters in 44 marsh deer with different body condition scores, and studied the pathological lesions in dead animals. Marsh deer with poor body condition had a high burden of the ticks Amblyomma triste and Rhipicephalus microplus . Vector borne agents (VBAs), such as Theileria cervi. Trypanosoma theileri, Trypanosoma evansi, Ehrlichia chaffeensis, Anaplasma platys, Anaplasma odocoilei, Anaplasma marginal e, and Candidatus Anaplasma boolense were also found. Haemonchus spp, Ostertagia spp., Trichostrongylus spp. showed the highest infection prevalence. A Multiple Correspondence Analysis suggested a possible association of i) lower body score condition with high tick loads and infection with VBAs and, ii) the impoverished body score with high loads of nemathelminths and well-known harmful gastrointestinal parasites. Conclusions: Our results contribute with knowledge on the life history and health aspects of marsh deer in the study areas. We provide data on the prevalent infectious and parasitic agents in their populations. A range of haematology and serum chemistryvalues, and the occurrence of Fasciola hepatica and Leptospira interrogans serovar pyrogenes were reported for the first time in wild marsh deer from Argentina. The histopathological findings in succumbed animals allowed us to recognize injuries associated or not with their body condition. Our results are the first step in the creation of a baseline on marsh deer health in Argentina. In the future, these data added to new contributions could help improve the interpretation of the findings during mortality events.

Wildlife populations are increasingly threatened by habitat loss and degradation, invasive species, environmental pollution, climate change, and emergent diseases; all of these factors are driven by unsustainable natural resource exploitation by humans [1,2]. Particularly, disease-related mass mortalities have led to the extinction of several species [2,3]. The identification of infectious agents in wild species along with pathogen surveillance in their populations contributes with knowledge about ecosystem health while providing valuable information on the eco-epidemiology of the transmission of infectious diseases.
The marsh deer (Blastocerus dichotomus), the largest South American deer, is listed as Vulnerable by the Red List of mammals from Argentina [4] and the International Union for Conservation of Nature (IUCN) [5]. Habitat loss and fragmentation due to agricultural development and construction of hydroelectric dams are the major threats to marsh [5,6] with poaching, dog attacks and diseases being also recognized [4][5][6] but scarcely studied.
In Argentina, marsh deer mortality episodes have been described in Ibera Wetlands [17,18]. The episodes involved several pathogens associated with environmental and eco-epidemiological factors [17]. The health status of wild marsh deer populations has never been evaluated to date in the area.
A few marsh deer individuals have been studied in Corrientes since the 90 s. Paramphistomum sp.
were found to be infected with R. microplus, Demodex sp., Chorioptes sp. and A. tigrinum [18]. During a mortality episode of marsh deer in winter of 2007, our research group identified high parasite burdens of H. contortus in association with adverse climatic conditions [17]. Recently, our group documented high loads of the ticks A. triste and R. microplus in succumbed marsh deer, and the occurrence of E. chaffeensis in marsh deer and ticks [19].
Accurate data about health parameters in wildlife species is needed to detect changes in the occurrence of pathogens over time. Basic physiological and histological data, haematological values and disease parameters are important in assessing the general health of individuals or populations, and can be used to evaluate the progress of diseases [20]. This study aimed to provide baseline data on health parameters of marsh deer from Argentina, thus complementing the existing literature on the specie in neighbouring countries and improving the understanding of mortality events. In the present study, we collected and analysed a wide range of samples of live and dead marsh deer for 36 months in the two major populations of Argentina.

Results
A total of 44 marsh deer (14 in Iberá Wetlands (IW) and 30 in Lower Delta (LD)), mostly adults and males (Table 1) were analysed between May 2014 and December 2016. A total of 35 dead animals were evaluated; the causes of death are detailed in Table 1. Live marsh deer were only sampled in LD (n=9).
Half of the studied individuals (n=22) were apparently healthy and had a good body condition (score 3). The other half showed a regular body condition (score 2) (25%, 95% Confidence Interval (CI), 15-39%) and a poor body condition (score 1) (25%, 95% CI, 15-39%) ( Table 1); these animals had been sampled during stress situations, such as environmental changes, increased animal density in small areas, and increased competition for resources. All marsh deer with body score 1 showed cachexia and several clinical signs of disease. On visual examination, clinical signs frequently included notable weakness, pale mucous membranes, loss of antlers, drooping of head and ears, rough dull coat, decreased muscle tone, severe emaciation, dehydration, loss of coordination and inability to stand. Submandibular oedema and cachexia were frequent in marsh deer with regular and poor body condition score. Animals with good body condition often exhibited bone fractures caused by dog attacks or road collisions. All marsh deer with poor body condition showed skin laceration. At the time of sampling, five of 17 females were pregnant (all of them were dead) and five of 26 adult or juvenile males had lost their antlers (Table 1).

Haematology and biochemistry tests
Haematological and serum biochemistry parameters of marsh deer were only evaluated in live animals (Table 2a). All the results correspond to animals with good body condition (n=12). Almost all of the mean haematology values were within the ranges expected for the species; significant differences were observed for Packed cell volume (PCV) for both sexes (wild) and for females in captivity; for red blood cell count (RCB) for both sexes (wild); for mean cell haemoglobin concentration (MCHC) in females (captivity and wild) and for total protein in both sexes (wild and captivity) [21,22] (Table 2b). Ten parameters of serum chemistry were tested in marsh deer (Table   2a); the values were within the ranges described for taxonomically related species [21].

Serological tests
The results of the serological tests are shown in Table 3. Two marsh deer from LD showed evidence of exposure to Leptospira interrogans serovar pyrogenes (CP_1065: L. Pyrogenes pyrogenes titre 1/200; CP_D1: L. pyrogenes pyrogenes titre 1/100). One marsh deer from IW showed evidence of exposure to brucellosis by buffered plate antigen test (BPA) and Rose Bengal. 2-mercaptoethanol test (2ME) and the tube agglutination test (SAT) titters were 1:100.
Identification of ticks and diagnosis of tick-borne agents All marsh deer from IW were parasitized with the ticks Rhipicephalus microplus and/or Amblyomma triste, and all ticks collected in marsh deer from LD were identified as A. triste. Tick loads were estimated in 37 marsh deer sampled immediately after death. High tick loads were found in 9 marsh deer (24.3%, 95% CI, 13-40%), 8 of which were from IW. In LD, most marsh deer (74%, 95% CI, 54-87%) had a low tick load (Table 1). In both areas, high and medium tick loads were associated with a regular/poor body condition score, whereas individuals with low tick loads were associated with a good body condition score (X 2 ; p=0.0001).
The molecular detection of VBA was performed in 40 marsh deer ( Ehrlichia chaffeensis was found in three marsh deer from IW and two from LD. In both areas, different species of Anaplasma occurred in marsh deer with good, regular and poor body condition scores.
Trypanosoma theileri and T. evansi occurred in marsh deer with good, regular and poor body condition scores in both areas. Rickettsia sp. was not found in any marsh deer individual.
Quantitative and qualitative analysis of faeces values were low (less than 60) in 93% (95% CI, 81-98%) of the analysed samples, regardless of marsh deer body condition score ( Table 5).
Results of the qualitative faecal analysis are shown in Table 6. Parasitic elements morphologically compatible with trichostrongylid eggs type, Strongyloides spp., Capillaria spp. and Paramphistomum spp. eggs were found. A high prevalence of infection (79%, 95% CI, 65-89%) was detected for the trichostrongylid eggs type (Table 6). Culture was conducted in 18 fecal samples; results showed the occurrence of Trichostrongylus spp. and Strongyloides spp., and morphologically compatible thirdstage larvae from the genera Haemonchus spp., Ostertagia spp., Oesophagostomum spp. and Adult parasites of Paramphistomum cervi were identified macroscopically in the rumen of marsh deer during necropsies and the eggs were detected in faeces ( Table 6). Six of the seven positive marsh deer (86%, 95% CI, 49-97%) were from IW, and five of them had died during mortality episodes and showed a poor or regular body condition.
Cysts of Sarcocystis sp. were detected in the cardiac muscle of 27% (95% CI, 13-48%) animals; in two of them, the cysts were also found in skeletal muscle. Metastrongyle nematodes (embryonated eggs and larvae) were found in 7 marsh deer (3 juveniles and 4 adults) (32%, 95% CI, 16-53%) and adult nematodes were observed only in one adult deer. Adult forms of Fasciola hepatica were found in the liver of five marsh deer from IW (22%, CI: 10-42%). Scattered pyriform microorganisms were found in erythrocytes in brain vessels of one of these animals (a male with poor body condition) (CP_MR1).
Skin lesions from 2 marsh deer were examined. A juvenile male presented a chronic, locally extensive, ulcerative dermatitis with associated panniculitis in the neck. The other lesion was a malignant melanoma in the upper right eyelid of an adult male, characterized by fusiform, anaplastic melanocytes, many of them with intracytoplasmic melanin granules.
We were able to determine the cause of death in 6 animals (1 of IW and 5 of LD) that had regular or poor body condition (Table 8). In the other animals, the lesions were nonspecific and did not allow us to establish the cause of death. In IW, the lesions detected in one marsh deer (CP_MR1) were compatible with septicaemia. In LD we detected a myocardial necrosis of possible toxic origin related to the presence of cardiotoxic agents in CP_D2. The fibrinous bronchopneumonia could have been the cause of death of CP_G1 during an extraordinary flood in the area. In CP_S2 marsh deer the cause of death was a nephrosis of possible toxic origin. Clinical conditions and tissue lesions related to malnutrition were also detected in CP_S2 and in two other marsh deer (CP_S1 and CP_I1). The three animals were sampled in the same field in LD during the extraordinary flood of 2016, when food was significantly scarce.

Data analysis
A two-dimension MCA solution was considered from the Multiple Correspondence Analyses. The MCA i, corresponding to the VBA variables, showed that infection of E. chaffeensis, high tick load and the body condition score=1 ( Figure 1) presented a similar behaviour. Infection with T. evansi and A. marginale were in the same area of the graphic ( Figure 1). The MCA ii, which included the presence of nemathelminthes species in faeces, showed that high and medium load of oocysts, presence of Ostertagia sp., Paramphistomum sp., and Haemonchus sp., and the body condition score=1 were closely related ( Figure 2). The MCA iii, including exposure to infectious agents, was not performed due to their low prevalence.

Discussion
In this study we describe the health conditions of 44 marsh deer of the two largest populations in Argentina. Our results contribute with knowledge on the life history of marsh deer and may be contributed as baseline information on health aspects of the species in the study areas. We provide data on the range of normal blood parameters, the prevalent infectious and parasitic agents in their populations, and the lesions found in marsh deer tissues in apparently healthy and unhealthy individuals. Our results are the first step in the creation of a baseline on marsh deer health in Argentina. In the future, these data added to new contributions could help improve the interpretation of the findings during mortality events.
The two performed MCA showed the possible association i) of lower body score condition with high tick loads and infection with VBAs and, ii) the impoverished body score with high loads of nemathelminthes and well-known harmful gastrointestinal parasites. These associations were not statistically confirmed; further studies including a higher sample size are needed to understand the cascade of events that trigger mortality events.
The haematological data obtained in this work are the first values reported for free-ranging marsh deer in Argentina, and the range of values of serum chemistry are, to our knowledge, the first reported for the species in the world. The range of haematological values agrees with data described by other authors for Brazilian populations of marsh deer [21,22].
Exposure to infectious agents in the analysed marsh deer was low. None of the studied marsh deer showed exposure to BTV, IBRV, BVDV, foot-and-mouth disease, Johne's disease, bovine leukosis, Q fever, chlamydial abortion, or VSV. Published information about these infectious agents in marsh deer is scarce. Some reports describe a high prevalence of antibodies against herpesvirus-1 in marsh deer, although they do not distinguish that virus from cervid herpesvirus-2 due to their antigenic similarity [23]. In Brazil, exposure of marsh deer to epizootic haemorrhagic disease virus (EHDV) (an orbivirus related to bluetongue) was detected with a high seroprevalence (74%) with typical lesions [24], whereas bluetongue virus was reported only in captive Mazama sp. In Argentina, serum samples from 14 free-ranging Ozotocerus bezoarticus celer were negative to Johnes' disease, BVDV, EHDV and BTV [25].
Unlike expected, the antibody findings for Leptospira interrogans were very low in the marsh deer, although they were sampled during and after a flood event. In the central region of Argentina, where LD is included, the highest number of cases of leptospirosis in humans was recorded between 2015 and 2016 which doubled the number of cases reported in 2013-2014 [26]. In the area, only two marsh deer were seropositive to serovar pyrogenes (a pathogenic serovar of Leptospira), with titters of between 1:100 and 1:200; one marsh deer was found dead without lesions and the other was found alive, apparently healthy, with no alterations in the blood test. A single positive titre of 100 can be interpreted as a residual background titre, whereas titters between 100 and 200 can be important in non-vaccinated animals [27]. Although the information on leptospirosis in marsh deer is extremely scarce, the study of the pathogen acquires special relevance in severe flood scenarios, since its incidence is strongly associated with rainfall and wet and hot weather [28]. Antibodies to L.
interrogans were found in related species, such as Mazama gouazoubira [29] and O. bezoarticus (serovars hardjo, mini, wolffi and pomona) [25,30], the latter two being found in Argentina. This study documents the first record of antibodies to L. interrogans serovar pyrogenes in the southernmost marsh deer population in Argentina. The effects of L. interrogans serovar pyrogenes were studied histopathologically in hamsters and were found to cause degenerative, haemorrhagic and necrotic lesions in heart, spleen, kidneys, lung and muscle [31].
One hunted marsh deer from Corrientes showed evidence of exposure to brucellosis, with relatively high titres (1: 100, BPA, Rose Bengal, 2-ME and SAT). No previous serological analyses in marsh deer in Argentina and Brazil reported evidence of Brucella infection [27,32]. In our study area, bovine brucellosis has been detected in some herds, and marsh deer could have become infected in environments shared with livestock, as previously suggested in Brazil [33].
The high burden of the ticks A. triste and R. microplus was an important finding in marsh deer with poor body condition, in which skin lesions contributed to an impoverished general condition. This was especially evident in IW, where R. microplus was the most frequent tick. Rhipicephalus microplus mainly infested cattle and is endemic to northwestern Argentina, although its geographical distribution does not include LD. In agreement with the findings in Brazil [7,9] the VBAs found in the analysed marsh deer were T. cervi. T. theileri, T. evansi, E. chaffeensis, A. platys, A. odocoilei and A.

marginale.
Ehrlichia chaffeensis, which causes a zoonotic disease, has been recently described in marsh deer in Argentina [19]. In the present study, the occurrence of E. chaffeensis in IW was positively associated with poor body condition score. Positive deer, except for the fawn animal, had medium or high tick load. Although it is not possible to attribute the origin of the lesions found in the kidney of a positive deer (i.e. glomerulonephritis secondary to immune complex deposition), to E. chaffeensis, future immunohistochemical studies might confirm the possible association.
In this study we also found T. cervi associated with poor and regular body condition scores. T. cervi has been historically considered of low pathogenicity probably because of a long evolutionary relationship between parasite and host [34]. Theileria cervi is often asymptomatic in naturally infected cervids, except for animals with high parasite load, concurrent disease, malnourishment, immunosuppression, in areas of high deer population densities, or in stressful situations [34][35][36].
Histologically, records of haemosiderosis in tissues of positive marsh deer suggest a possible relationship between the lesions and the agent [37,38]. In this study, of a total of 11 positive deer with poor and regular body condition, 91% was evaluated during stress conditions driven by environmental changes, whereas all of them (100%) were co-infected with other infectious or parasitic agents: 90.9% had medium or high tick loads, 72.7% had more than one infection with a VBA, and 72.7% had medium or high loads of gastrointestinal parasites. In addition, scattered pyriform microorganisms were found in erythrocytes in the brain of one positive deer. Specific studies in tissues of T. cervi-positive deer are essential, especially in populations under stress conditions.
Trypanosoma theileri, T. evansi, A. platys, A. odocoilei, A. marginale, and Candidatus A. boolense were found in marsh deer from both areas, regardless of their body condition. Candidatus A. boolense was first identified in different life stages (eggs, larvae, pupae and adults) from mosquitoes in China [39] and this is the first report in marsh deer in Argentina.
Regarding gastrointestinal parasites, trichostrongylid eggs, including Haemonchus spp, Ostertagia spp., and Trichostrongylus spp., showed the highest infection prevalence in the studied marsh deer. In Argentina and Brazil, helminthic diseases are an important cause of morbidity in marsh deer [13][14][15], and H. contortus was found to be one of the most pathogenic agents involved in mortality events [16,17,40]. Ostertagia sp., which was found in this work, was also described in marsh deer in Brazil [14,15,41]. Ostertagia sp. cause abomasal epithelial hyperplasia and an imbalance in the protein digestion process. During hypobiosis stage, it can cause petechiae and ecchymotic haemorrhages in abomasal mucosa [42]. The three deer positive to Ostertagia spp. showed co-infection with Tricostrongylus spp. and two of them with Haemonchus spp. This record is significant due to the high pathogenicity of these agents in domestic livestock and wildlife [43].
Paramphistomum cervi and Fasciola hepatica were detected in more than 70% of the necropsied marsh deer from IW. The occurrence of P. cervi was previously described in marsh deer in Corrientes The histopathological findings in succumbed animals allowed us to recognize different injuries associated or not with their body condition. In road-killed or hunted (by dogs or humans) marsh deer, most of the lesions found were agonal such as congestion, oedema and pulmonary haemorrhages; or incidental findings, such as inflammatory reactions in liver, muscle, lung and abomasum frequently associated with parasitic agents such as Fasciola sp., Sarcocystis sp, Metastrongyle and Trichostrongyloidea nematodes, respectively, in agreement with the findings described by Navas-Suarez and collaborators [45].
Lesions in the respiratory system were frequent in both healthy and sick animals. Pneumonia was described as one of the most frequent inflammatory processes in cervids [45]. In this study, pneumonia was mild, except in a juvenile marsh deer with a locally extended fibrinous bronchopneumonia. As described by Navas-Suarez and collaborators [45], our results show that lung congestion was the most common hemodynamic disorder in the marsh deer, followed by oedema and haemorrhage.
Hepatic inflammations were frequent lesions in the examined deer, although, in most cases, they were not significant or were incidental findings. Lesions along the digestive tract were detected with high frequency in marsh deer with poor body score. Abomasitis was the most frequent lesion in the abomasum, generally associated with the occurrence of Haemonchus sp. and Ostertagia sp, with some animals showing depressions in the abomasal mucosa. A high percentage of intestine samples showed some degree of autolysis, which hindered bacterial culture and isolation, or a proper histophatological study. Some animals evaluated had clinical signs of diarrhoea. Campylobacter spp.
was isolated from faeces of marsh deer in Brazil [46]; the main clinical sign caused by this bacterium in most species is diarrhoea, usually self-limited. Other enteric infections by Salmonella spp. and Yersinia spp. usually affect cervids [27] but have never been reported for marsh deer.
Only six animals showed lesions that allowed us to determine the cause of death. In IW, the severe leucocytosis in most organs and multiple histopathological lesions detected in CP_MR1 were indicative of a septicaemia. Moreover, some of the lesions were suggestive of anaemia, possibly caused by the high tick load detected and the simultaneous occurrence of Haemonchus sp. in digestive tract and T. cervi in the brain. In LD, we detected a myocardial necrosis of possible toxic origin in CP_D2. Oedema found in multiple organs of CP_D2 may be associated with heart failure, hypoalbuminemia and gastrointestinal parasites. The blood AST levels may be increased after liver or heart damage [47], whereas azotaemia and hyperphosphatemia may be related to mild nephritis [48]. We were not able to isolate the causal agent of fibrinous bronchopneumonia in CP_G1 because of the advanced autolysis at the time of sampling and of logistical limitations; however, we suggest that the clinical conditions could have been caused by bacteria such as Mannhaemia hemolytica, Histophilus sommnys or Pasteurella multocida, which are frequently detected in stress situations or during coinfections with viral agents [45].
Clinical conditions and tissue lesions detected in CP_S2, CP_S1 and CP_I1 were related to malnutrition.
The absence of fat reserves and hepatic lipidosis indicated a negative energy balance, and rumenitis in CP_I1 was possibly the result of dysbacteriosis. Under food scarcity conditions, animals frequently feed on toxic plants, which could have caused nephrosis and death in CP_S2.
In this study we focused on the agents most commonly mentioned as causing disease in marsh deer and related species, and on those infectious and parasitic agents prevalent in the two study areas.
The cause of death of animals with poor or regular body condition was determined only in a low proportion of marsh deer, whereas the remaining animals showed different lesions that would not be  [24,51]. The animals were maintained on thermal padded surfaces and oxygenated through mask (5-8 l/min). Vital signs were continuously measured using a multi-parameter medical monitor (Veterinary Monitor, GT9003C, Fridimex S.A.). The anaesthesia was reversed using Naloxone Hydrochloride 0.05 mg/kg (Naloxona Denver Farma ®) and Yohimbine (Yohimbine Vet ®, Richmond Vet Pharma) 0.2 mg/kg. Live and dead deer were assigned to age classes (fawn, yearling, and mature adult) [52] and body condition was determined (scores 1, 2, 3) using the Body Condition Score Chart, with modifications [53]. Data about reproductive status, characteristics of the antlers, evidence of submandibular oedema, cachexia, and bone fractures were recorded. All animals were sexed and inspected for skin laceration and presence of ticks by visual inspection and palpation. Ticks were collected using acarological tweezers and stored in tubes containing 70º alcohol. External tick load was estimated and categorized into three levels according to the abundance and distribution of ticks on the deer body surface: category 3 (high load) corresponds to more than 50 ticks distributed in one or more parts of the body, category 2 (medium load) corresponds to 30-50 ticks, while category 1 (low / null load) corresponds to a tick load between 0 and less than 30 ticks. In dead deer, tick load was estimated only in deer found immediately after death. Blood samples were collected by jugular vein puncture (10-15 ml, live individuals) or cardiac puncture (15-20 ml, dead individuals) and stored at 4 ºC with EDTA (1ml) and at -20°C and -80°C (1ml, each). The remaining blood was centrifuged and serum aliquots were stored at -80°C. A faecal sample was collected directly from the rectum of the marsh deer and stored without air in nylon bags at 4 ºC.
During necropsies, tissues were macroscopically evaluated and two samples of selected organs (heart, lungs, abomasum, liver, kidneys, intestine, lymph nodes, spleen and brain) were collected, including part of normal tissue and part of injured tissue, if present. Tissue samples were conserved in 10% buffered formalin solution (BFS) and frozen at -80°C.
Laboratory diagnosis. The aliquot of blood collected in EDTA tubes was used for haematology using manual methods and an automated analyser (Reflotron Plus, Roche, Mannheim, Germany).
Chemical analyses were processed on an automated analyser (Metrolab 2100, Wiener lab, Rosario, Argentina) and concentration of total protein was determined using a portable refractometer (REF 302, Arcano, China). The blood parameters were compared with previously published data on captive and wild marsh deer from Brazil [21,22].

Serological diagnoses of bluetongue virus (BTV), infectious bovine rhinotracheitis virus (IBRV), bovine
viral diarrhoea virus (BVDV), brucellosis, foot-and-mouth disease virus, Johne's disease, Leptospira interrogans, bovine leucosis, Q Fever, chlamydial abortion, and vesicular stomatitis virus (VSV) were performed according to the procedures described by the World Organization for Animal Health [54] in the National Service of Agri-Food Health and Quality (SENASA).
DNA was extracted from blood by phenol/chloroform method followed by a standard ethanol precipitation [56]. Blood samples were screened using PCR protocols targeting a fragment of the 16SrRNA gene for the Anaplasmataceae family [57,58], a fragment of the internal transcribed spacer 23S-5S of Rickettsia sp. [59] and a fragment of the 18SrRNA gene for Trypanosoma sp. [60] and Babesia/Theileria [61]. For positive samples, both strands of the amplified fragment were sequenced with a Big Dye Terminator v3.1 kit from Applied Biosystems and analysed on an ABI 3130XL genetic analyser from the same supplier (Genomic Unit, Consorcio Argentino de Tecnología Genómica (CATG), Instituto de Biotecnología, CICVyA, INTA). Raw files from each gene target were processed using the Vector NTI Advanced 10 program (Invitrogen). Both chromatograms were used for assembling a consensus sequence. The final file in FASTA format was used for further sequence analysis.
Parasitic elements in faeces were identified, counted and expressed as the EPG and OPG, using a modified Wisconsin technique. Each marsh deer was assigned to a category according to EPG and OPG values. Infective larvae were cultured. Taxonomic identification of parasitic elements was performed according to the literature [62][63][64] using a magnifying glass (10-40X, Carl-Zeiss SV11, Germany).
Tissue samples fixed in 10% BFS were processed using conventional histopathological protocols.
Then, samples were embedded in paraffin wax and 5 μm sections were obtained, which were stained with haematoxylin and eosin. Microscopic lesions found in each tissue were identified and categorized according to severity using a five-degree scale (mild, mild-moderate, moderate, moderate-severe, severe).

Data analysis.
Given the high number of health variables measured, in order to perform exploratory analysis and visually identify the possible associations between body condition and exposure to the different agents, Multiple Correspondence Analysis (MCA) was performed [65]. The variables were divided into three groups: i. exposure to infectious agents, ii. tick load and vector-born agents (VBA), and iii. EPG, OPG and infection with nemathelminthes species. Only agents with more than one positive individual were included. Proportions and 95% confidence intervals (95% CI) were estimated using Wilson's formula implemented in Epitools [66]. The Chi-square test was performed in Epitools [66] to observe the association between tick loads and body condition; categories of tick loads (high and medium) and score (regular and poor) were grouped for this test. The t-test was also implemented in Epitools to compare the means of the haematological values obtained from our work with the means of two references. A significance level of P≤0.05 was used (2-tailed

Consent for publication: Not applicable
Availability of data and material: The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests:
The authors declare that they have no competing interests.

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