First steps towards building a participatory surveillance network of marsh deer morbidity and mortality
The participatory surveillance network was made up of researchers, field partners (veterinarians, park rangers, livestock and timber producers, and local community), and decision makers in the two largest populations of marsh deer in Argentina located in Ibera Wetlands (IW) and Lower Delta (LD). In IW, the network included personnel from national and provincial protected areas, NGOs, and private veterinarians. In LD, the activities were integrated into the work programme and management structure of the “Marsh Deer Scientific Technical Committee” (CCP) and included samplings during live captures carried out by "Pantano Project”.
The work was gradually articulated in both study areas. Training classes and workshops led to an increased knowledge and improved sampling and biosecurity skills of local partners. During 2014 and 2015, local partners received practical training in the field. In 2016, together with the National Parks Administration, we organized a workshop for the local community in IW. Participants exchanged information on marsh deer and discussed issues, concerns, and approaches in wildlife health, with the purpose of expanding and strengthening the network. The theoretical-practical workshop was attended by 60 persons, including representatives from multiple public, private, and academic institutions and the local community. A similar workshop, held during 2016 in LD, was attended by 15 local partners (Fig. 1A).
Mortality events
With the support of Provincial Wildlife Agencies and the National Parks Administration, reports of mortality events of marsh deer within the framework of the network took place in May 2014 and June 2015 (Fig. 1A). A total of 27 and 100 carcasses of marsh deer were reported in the Mburucuyá National Park (western IW) and in Cambyretá Area - Iberá National Park (north-eastern IW), respectively. Although most of the dead animals were registered by park rangers during field monitoring in remote areas, a few marsh deer with apparent signs of weakness were observed in the days prior to their death. Necropsies of three marsh deer were performed by our group at each site; the remaining individuals could not be evaluated due to advanced autolysis. During the winter of 2016, trained field partners registered only seven dead animals in IW. A complete necropsy was performed by local partners (veterinarians and park rangers) on two of them. In LD, an extraordinary flood between April and August 2016 coincided with wildlife mortality due to disease and starvation, but some individuals died because they were hit by vehicles, attacked by dogs, or hunted due to increased visibility of animals grouped in the few remaining high and dry areas [28]. Several cases of dead, injured, and hunted marsh deer (n=233) were reported to the CCP [28]. Complete necropsies were performed on nine animals, two of which were found dead with visible signs of disease, four had been sighted sick in the days prior to their death, while the remaining three had been killed by poachers.
In both areas, local partners also reported dead deer that had been hunted, hit by vehicles o killed by dogs outside mortality events; they were sampled opportunistically.
Marsh deer survey
A total of 44 marsh deer, mostly adult males, were analysed between May 2014 and April 2017. Thirty-five dead animals were sampled in both areas, 19 of which were clinically sick individuals sampled during mortality events (Fig. 2). Live marsh deer (n=9) and the other 16 dead animals, including 10 hunted deer, one road-kill, three dog-kills and two visibly sick individuals, were sampled outside of mortality events. Some deer sampled during and outside mortality events (n=17) had been seen alive before their death (Table 1, Fig. 1A).
Half of the studied individuals (n=22) had a good body condition (score 3). The other half showed a regular (score 2) or a poor body condition (score 1), and most of them were sampled during mortality events (Table 1). All marsh deer with body score 1 showed cachexia and skin laceration. Submandibular oedema and diarrhoea were frequent in individuals with regular or poor body condition score. During winter, five females were pregnant at time of death, in correspondence with the reproductive season. Five adult or juvenile males had lost their antlers (Table 1). The temporal distribution of sampling, body scores, and cause of death (if applicable) of marsh deer are detailed in Fig. 1B.
Table 1. Overview of general information about sampled marsh deer (Blastocerus dichotomus) in Argentina.* includes 9 live marsh deer.
|
|
Marsh deer sampled
|
|
|
During Mortality events
|
Outside Mortality events
|
Total
|
Area
|
Ibera Wetlands
|
11
|
3
|
14
|
|
Lower Delta
|
8
|
22*
|
30
|
Age class
|
Adult
|
12
|
13
|
25
|
|
Yearling
|
6
|
12
|
18
|
|
Fawn
|
1
|
0
|
1
|
Sex
|
Male
|
13
|
14
|
27
|
|
Female
|
6
|
11
|
17
|
Body Condition Score
|
Score 1
|
10
|
1
|
11
|
|
Score 2
|
9
|
2
|
11
|
|
Score 3
|
0
|
22
|
22
|
Submandibular oedema
|
Presence
|
8
|
1
|
9
|
Cachexia
|
Presence
|
10
|
2
|
12
|
Bone fractures
|
Presence
|
0
|
9
|
9
|
Skin laceration
|
Myiasis, alopecia, erosions
|
11
|
16
|
27
|
Diarrhoea
|
Presence
|
9
|
1
|
10
|
Ticks load
|
Zero - Low
|
1
|
17
|
18
|
|
Medium
|
6
|
4
|
10
|
|
High
|
8
|
1
|
9
|
|
Not evaluated
|
4
|
3
|
7
|
Pregnancy (n=17, only females)
|
Presence
|
3
|
2
|
5
|
Antler drop (n=26, only adult and yearling males)
|
Presence
|
4
|
1
|
5
|
Haematology and biochemistry values
Haematological and serum biochemistry parameters of marsh deer were determined in animals sampled alive and with good body condition. In addition to the nine live animals, we included three individuals that had been rescued with gunshot wounds and were sampled while alive, and then died (Table 2). Almost all of the mean haematology values were within the range previously reported for free-living marsh deer in Brazil [29]. Significant differences with the values reported by Szabó et al. [29] were observed in the packed cell volume for both sexes; red blood cell count for both sexes; mean cell haemoglobin concentration in females; and total protein in both sexes [29] (Supplementary material, Additional files 1, 2).
Table 2. Haematological and serum biochemistry parameters for marsh deer (Blastocerus dichotomus) in Argentina. Sample sizes differed between parameters because insufficient blood could be obtained from some animals.
|
|
|
Parameter
|
n
|
Median
|
Range
|
|
Packed cell volume (%)
|
12
|
32
|
20-39
|
|
Red blood cell count (106/µl)
|
12
|
6.22
|
1.09-10.54
|
|
White blood cell count (103/µl)
|
12
|
6.20
|
4.05-9.82
|
|
Haemoglobin (g/dL)
|
5
|
10.9
|
8.16-15.7
|
|
Mean cell volume (fl)
|
4
|
41.43
|
32.25-43.2
|
|
Mean cell haemoglobin (%)
|
4
|
15.68
|
13.15-18.5
|
|
Mean cell haemoglobin concentration (g/dL)
|
4
|
40.92
|
33.03-44.5
|
|
|
|
|
|
|
Total protein (g/dL)
|
12
|
6.7
|
5.8-7.2
|
|
Albumin (g/dL)
|
12
|
3.16
|
2.88-3.56
|
|
Blood urea nitrogen (mg/dL)
|
12
|
39.03
|
27.81-78.70
|
|
Creatinine (mg/dL)
|
12
|
1.33
|
1.00-2.27
|
|
Aspartate aminotransferase (IU/L)
|
12
|
87.44
|
23.80-420.02
|
|
Alanine transferase (IU/L)
|
12
|
20.58
|
5.74-81.01
|
|
Alkaline phosphatase (IU/L)
|
12
|
362.53
|
90.40-1104.60
|
|
Creatine phosphokinase (IU/L)
|
7
|
203.90
|
43.60-815.10
|
|
|
|
|
|
|
Total calcium (mg/dL)
|
11
|
7,06
|
1.55-8.63
|
|
Phosphorus (mg/dL)
|
9
|
6.29
|
3.07-8.11
|
|
Magnesium (mg/dL)
|
11
|
1.94
|
0.75-4.20
|
|
Serological analyses
The results of the serological analyses are shown in Table 3. Two marsh deer from LD showed evidence of exposure to Leptospira interrogans serovar pyrogenes. One of them, sampled alive during a CCP rescue, had a good body condition and a L. pyrogenes pyrogenes titre of 1/200; the other one, sampled during a mortality event, had a poor body condition and a titre of 1/100. One hunted marsh deer from IW with regular body condition showed evidence of exposure to brucellosis in all four serological techniques (Table 3).
Table 3. Serological tests and methods used, and results of tests performed in marsh deer (Blastocerus dichotomus) pathogens in Argentina.
Pathogen
|
Test procedure (positive titre)
|
Number positive/number tested
|
Bluetongue virus
|
AGID (1:4)
|
0/29
|
Infectious bovine rhinotracheitis virus
|
ELISA
|
0/29
|
Bovine viral diarrhoea virus
|
ELISA
|
0/29
|
Brucellosis
|
BPA /ROSEBEN /2ME (1:100) / SAT (1:100)
|
1/29
|
Foot-and-mouth disease virus
|
VIAA
|
0/29
|
Johnes’ disease (Mycobacterium avium subsp. paratuberculosis)
|
AGID
|
0/29
|
Leptospira interrogans (11 serovars) a
|
MAT (1:50)
|
2/29
|
Bovine leucosis
|
AGID
|
0/29
|
Q Fever b
|
Indirect multi-species ELISA
|
0/29
|
Chlamydial abortion c
|
Indirect multi-species ELISA
|
0/29
|
Vesicular Stomatitis Virus Indiana and New Jersey Serotype
|
ELISA-LP (liquid phase)
|
0/29
|
References: AGID: Agar Gel Immunodiffusion; ELISA: Enzyme-linked immunodiffusion assay; BPA: buffered plate antigen test; ROSEBEN: rose bengal test; 2ME: 2-mercaptoethanol test; SAT: tube agglutination test; VIAA: Virus infection-associated antigen; MAT: Microagglutination test. Tests performed at National Service of Agri-Food Health and Quality (SENASA).
a Leptospira interrogans serovars ballum, castellonis, canicola, grippotyphosa, icterohaemorrhagiae, copenhageni, pomona, pyrogenes, sejroe, wolffi, tarassovi.
b ID Screen® Q Fever Indirect ELISA Multi-species kit (ID.vet, France).
c ID Screen® Chlamydophila abortus Indirect ELISA Multi-species kit (ID.vet, France).
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 from individuals in LD were identified as A. triste. Tick loads were estimated in 37 marsh deer, including live and dead animals sampled up to eight hours post-mortem. High tick loads were found in nine individuals (24.3%), eight of which were from IW and showed regular or poor body condition. In LD, most marsh deer (74%) had a low tick load (Table 1).
The molecular detection of vector-borne agents (VBA) was performed in 40 marsh deer (Table 4). Regarding piroplasmid parasites, Babesia sp. was not detected in any deer, whereas T. cervi was found in 21 individuals, 11 of them with regular or poor body condition score. Ehrlichia chaffeensis was found in five marsh deer, four of which had a poor body condition score. In both areas, different Anaplasma species occurred in marsh deer with good, regular, and poor body condition scores. The identified species of the family Anaplasmataceae included E. chaffeensis, A. platys, A. odocoilei, A. marginale, and Candidatus A. boleense. 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 of the examined individuals.
A total of 20 individuals in both areas had co-infections with different VBAs during and outside mortality events. Co-infections involved two VBAs in 13, and three VBAs in six marsh deer. Only one animal with poor body condition sampled during a mortality event in LD was co-infected with four VBAs.
Table 4. Results for PCR identification of pathogens in marsh deer (Blastocerus dichotomus) samples from Iberá Wetlands (n=14) and Lower Delta (n=26) populations, by body condition score.
|
Body Score Condition 1 n positive (%)
|
|
Body Score Condition 2 n positive (%)
|
|
Body Score Condition 3 n positive (%)
|
|
Total n positive (%)
|
|
|
IW
|
LD
|
|
IW
|
LD
|
|
IW
|
LD
|
|
IW (n=14)
|
LD (n=26)
|
Babesia sp.
|
0
|
0
|
|
0
|
0
|
|
0
|
0
|
|
0
|
0
|
Theileria cervi
|
4 (28.6%)
|
2 (7.7%)
|
|
4 (28.6%)
|
1 (3.8%)
|
|
0
|
10 (38.5%)
|
|
8 (57.1%)
|
13 (50.0%)
|
|
|
|
|
|
|
|
|
|
|
|
|
Ehrlichia chaffeensis
|
3 (21.4%)
|
1 (3.8%)
|
|
0
|
0
|
|
0
|
1 (3.8%)
|
|
3 (21.4%)
|
2 (7.7%)
|
Anaplasma platys
|
0
|
0
|
|
1 (7.1%)
|
0
|
|
0
|
1 (3.8%)
|
|
1 (7.1%)
|
1 (3.8%)
|
Anaplasma odocoilei
|
0
|
1 (3.8%)
|
|
1 (7.1%)
|
1 (3.8%)
|
|
0
|
5 (19.2%)
|
|
1 (7.1%)
|
7 (26.9%)
|
Candidatus A. boleense
|
0
|
0
|
|
0
|
0
|
|
0
|
1 (3.8%)
|
|
0
|
1 (3.8%)
|
Anaplasma marginale
|
0
|
2 (7.7%)
|
|
0
|
0
|
|
0
|
4 (15.4%)
|
|
0
|
6 (23.1%)
|
|
|
|
|
|
|
|
|
|
|
|
|
Trypanosoma theileri
|
0
|
2 (7.7%)
|
|
1( 7.1%)
|
1 (3.8%)
|
|
0
|
4 (15.4%)
|
|
1 (7.1%)
|
7 (26.9%)
|
Trypanosoma evansi
|
1 (7.1%)
|
0
|
|
0
|
0
|
|
0
|
2 (7.7%)
|
|
1 (7.1%)
|
2 (7.7%)
|
|
|
|
|
|
|
|
|
|
|
|
|
Rickettsia sp.
|
0
|
0
|
|
0
|
0
|
|
0
|
0
|
|
0
|
0
|
Quantitative and qualitative analyses of faeces
Faeces from 43 marsh deer were analysed for parasites. More than half of all faecal samples (28/43, 65.1%) had less than 100 parasite eggs per gram (EPG), of which 60.7% (17/28) were from animals with a good body condition. Of the remaining 34.9% (15/43), one marsh deer with poor body condition score had an EPG value of 1880, and two with regular body condition score had EPG values of 826 and 827, respectively. The remaining 12 individuals had EPG values between 100 and 330 (Fig. 3A).
Thirty-one out of 43 animals (72.1%) had less than 10 oocysts per gram (OPG) of faeces. Eleven deer had OPG values between 11 and 100. The highest value (OPG=790) was found in a dead marsh deer during a mortality event. This animal also had a poor body condition, high tick load, and EPG=156, including Trichostrongylus spp., Haemonchus spp., Ostertagia, and Paramphistomum spp. (Fig. 3B).
Results of the qualitative faecal analyses are shown in Table 5. Trichostrongylina [30], Strongyloides spp., Capillaria spp., and Paramphistomum spp. eggs were found. A high frequency of infection (79%) was detected for Trichostrongylina eggs. Larval culture of 18 faecal samples evidenced the occurrence of Trichostrongylus spp. and Strongyloides spp., and third-stage larvae morphologically compatible with the genera Haemonchus, Ostertagia, Oesophagostomum, and Cooperia. Nine marsh deer were positive for Haemonchus sp.; seven of them had a regular or poor body condition and died during mortality events, whereas the three marsh deer positive for Ostertagia spp., also sampled during mortality events, had a poor body condition.
Adult parasites of Paramphistomum cervi were identified macroscopically in the rumen of marsh deer during necropsies, and their eggs detected in faeces (Table 5). Six of the seven animals positive for this parasite were from IW; five of them had died during mortality events and showed a poor or regular body condition.
Table 5. Qualitative analysis of the gastrointestinal parasites identified in faecal samples of marsh deer (Blastocerus dichotomus) in Argentina.
Parasite
|
Sugar Flotation
|
|
Larval culture
|
Number of samples with eggs or oocysts/Total (%)
|
|
Number of samples with third stage larvae/Total (%)
|
Trichostrongylina eggs*
|
34/43 (79%)
|
|
-
|
|
|
|
|
Trichostrongylus spp.
|
-
|
|
12/18 (67%)
|
Haemonchus spp.
|
-
|
|
9/18 (50%)
|
Ostertagia spp.
|
-
|
|
3/18 (17%)
|
Cooperia spp.
|
-
|
|
1/18 (6%)
|
Oesophagostomum spp.
|
-
|
|
1/18 (6%)
|
Strongyloides spp.
|
13/43 (30%)
|
|
5/18 (28%)
|
Capillaria spp.
|
1/43 (2%)
|
|
-
|
Paramphistomum sp.
|
7/43 (16%)
|
|
-
|
|
|
|
|
Eimeriidae
|
22/43 (51%)
|
|
-
|
* Ellipsoidal shape, double membrane, smooth surface, medium size (85 µm), with blastomeres according to the different stages.
|
Gross and histopathological findings
Complete necropsies were performed on 23 marsh deer (14 males and 9 females); 12 of them were adults, 10 juveniles, and one was a fawn. Eight (35%) deer had a good, five (21.5%) a regular, and 10 (43.5%) a poor body condition score. Table 6 summarizes the main microscopic findings in dead animals with score 1 and 2 sampled during mortality events vs. animals with score 3 that died of traumatic causes. One marsh deer with score 2 and sampled outside a mortality event was excluded from Table 6. This animal was reported dead in a field in LD. The deer had inflammation and myiasis in the metacarpophalangeal joint of the left forelimb and a fracture in the second phalanx of the same forelimb.
Table 6. Microscopic findings in the main organs examined in dead marsh deer (Blastocerus dichotomus) from Argentina.
Microscopic findings
|
Marsh deer (body condition score 1-2) found dead during mortality event
|
Marsh deer (body condition score 3) dead by trauma
|
Number of positive by histopathology /Total
|
Heart
|
Inflammation
|
2/13
|
1/8
|
Haemorrhages
|
1/13
|
0/8
|
Necrosis
|
1/13
|
0/8
|
Lungs
|
Congestion
|
3/13
|
4/8
|
Pneumonia
|
4/13
|
3/8
|
Oedema
|
2/13
|
3/8
|
Haemorrhages
|
2/13
|
3/8
|
Abomasum
|
Inflammation
|
4/10
|
4/7
|
Oedema
|
3/10
|
0/7
|
Liver
|
Inflammation
|
7/14
|
5/8
|
Hepatocellular steatosis
|
3/14
|
0/8
|
Congestion
|
3/14
|
1/8
|
Necrosis
|
1/14
|
0/8
|
Kidneys
|
Inflammation
|
5/13
|
2/8
|
Necrosis
|
2/13
|
0/8
|
Spleen
|
Hemosiderosis
|
4/12
|
2/8
|
Lymphoid hyperplasia
|
2/12
|
2/8
|
Lymphoid necrosis
|
1/12
|
0/8
|
Congestion
|
2/12
|
0/8
|
Brain
|
Congestion
|
1/9
|
1/4
|
Inflammation
|
0/9
|
1/4
|
Eight of the necropsied marsh deer died of traumatic causes. Three animals had received gunshot wounds to the thorax and were diagnosed with oedema, congestion, and pulmonary haemorrhages (Table 6). Three individuals had been wounded in other parts of the body, and two animals had been attacked by dogs, showing haemorrhages and non-specific lesions in different tissues.
In addition to the necropsied individuals that died of traumatic causes, histopathological analysis allowed us to determine the cause of death of seven animals (two from IW and five from LD) that had poor body condition and died during mortality events. The severe leucocytosis in most organs and multiple histopathological lesions (multifocal hepatic necrosis, lymphoid necrosis of the spleen, and interstitial pneumonia) (Table 6) detected in one marsh deer from IW were compatible with septicaemia. This animal also showed cachexia, submandibular oedema, skin laceration, high tick load, OPG=790, and was co-infected with T. cervi, E. chaffeensis, Trichostrongylus spp., Haemonchus spp, Ostertagia spp., and Paramphistomum sp. The other marsh deer from IW had a severe acute cortical nephrosis; it also showed high tick load and was co-infected with T. cervi and T. theileri.
One deer from LD had an acute multifocal myocardial necrosis (Table 6). In addition, it showed oedema in multiple organs, a mild nephritis, abomasitis, and EPG=1880. Cachexia, submandibular oedema, skin laceration, and high tick load were also found, and the deer was co-infected with T. cervi, T. theileri, Trichostrongylus spp., and Ostertagia spp.
Fibrinous bronchopneumonia may have been the cause of death of one deer sampled in 2016 during the mortality event in LD. This animal was co-infected with T. cervi, T. theileri, and Trichostrongylus spp. Another marsh deer sampled in LD died of nephrotoxic nephrosis (Table 6). Clinical conditions and tissue lesions related to malnutrition were detected in this animal and in two other marsh deer sampled in the same area during the mortality event. All three animals were males, showed cachexia, and two of them had lost their antlers days before their death (the third one was a fawn). One deer was co-infected with T. cervi, A. odocoilei, A. marginale, and T. theileri, and had a hepatocellular atrophy. The fawn had a multifocal pustular rumenitis, atrophy of the thymus, absence of fat reserves, and hepatic lipidosis, and was infected with E. chaffeensis.
In the other eight animals, the lesions were non-specific and did not allow us to establish the cause of death. One deer died with a regular body condition score outside the mortality events, while the other eight individuals showed poor or regular body condition and died during mortality events. Six marsh deer showed cachexia, four of which also had submandibular oedema. Five had high, and four intermediate tick loads.
Cysts of Sarcocystis sp. were detected in the cardiac muscle of six animals; in two of them, cysts were also found in skeletal muscle. Embryonated eggs and larvae of Metastrongylus nematodes were found in seven marsh deer (three juveniles and four adults), whereas adult nematodes were observed only in one adult deer. Adult forms of F. hepatica were present in the liver of five animals from IW (22%). Unidentified scattered pyriform microorganisms were observed in erythrocytes in brain vessels of one of these animals, a male with poor body condition.
Skin lesions from two marsh deer were examined. A juvenile male presented a chronic, locally extensive, ulcerative dermatitis with associated panniculitis on 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.
Data analysis
The MCA showed that lower body condition (score=1), higher tick loads (tick load =1), infection with E. chaffeensis, and presence of harmful gastrointestinal parasites (Ostertagia sp., Paramphistomum sp., and Haemonchus sp.) were correlated (Fig. 4). Low body scores and high parasite loads were correlated with the sampling period from May to August (winter season). In the opposite way, the MCA showed that a higher body score was correlated with the September-December sampling period, and with the absence of harmful parasites. These associations were not statistically confirmed.