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 . 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 , 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 .
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 . 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 . 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 . Antibodies to L. interrogans were found in related species, such as Mazama gouazoubira  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 .
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 .
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 . 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 . 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 [34–36]. 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  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 [13–15], 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 . 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 .
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  and high loads of trematodes, including P. cervi, were detected in Parana River, Brazil . Both agents have high prevalence in IW, favoured by the adequate temperatures and the simultaneous presence in Limmaea sp. and known definitive hosts (e.g. domestic sheep). In IW, the infection rate in Limmaea sp. increases until the end of summer and autumn, and the clinical signs of disease appear between 2 and 4 months later . Most of the analysed marsh deer had died during extraordinary floods after an extended warm season. The floods increase the habitat for Limmaea sp., favouring the production of metacercariae and increasing the risk of infection. In marsh deer, these factors are combined with the habit of feeding in swampy environments.
This is the first record of Fasciola hepatica in marsh deer. The occurrence of both species of trematodes, often simultaneously, in deer with poor and regular body condition, and co-infected with other agents, suggest that trematodes could contribute to the clinical signs observed in sick/dead deer during mortality episodes. Given the ecological characteristics of B. dichotomus, the detection of liver lesions in some infected individuals, and the high prevalence of Fa. hepatica in the area, future studies on the role of the species in the maintenance of the infection in the area are essential.
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 .
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 . 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 , 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 ; 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  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 , whereas azotaemia and hyperphosphatemia may be related to mild nephritis . 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 co-infections with viral agents .
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 directly related to death. Many of the reported results open the doors for future research on the association of infectious agents with pathological lesions in marsh deer, spillovers of agents to and from populations of domestic ruminants, and the role of marsh deer in the transmission cycles of certain diseases. Given that health information on marsh deer is extremely scarce in the Neotropical region  where the species is native, our work contributes with abundant baseline information about marsh deer in Argentina and may serve as the basis for further scientific investigation.