Passive epidemiological surveillance in wildlife in Costa Rica identifies pathogens of zoonotic and conservation importance.

Epidemiological surveillance systems for pathogens in wild species have been proposed as a preventive measure for epidemic events. These systems can minimize the detrimental effects of an outbreak, but most importantly, passive surveillance systems are the best adapted to countries with limited resources. Therefore, this research aimed to evaluate the technical and infrastructural feasibility of establishing this type of scheme in Costa Rica by implementing a pilot program targeting the detection of pathogens of zoonotic and conservation importance in wildlife. Between 2018 and 2020, 85 carcasses of free-ranging vertebrates were admitted for post-mortem and microbiology analysis. However, we encountered obstacles mainly related to the initial identification of cases and limited local logistics capacity. Nevertheless, this epidemiological surveillance scheme allowed us to estimate the general state of health of the country's wildlife by establishing the causes of death according to pathological findings. For instance, 60% (51/85) of the deaths were not directly associated with an infectious agent. Though in 37.6% (32/85) of these cases an infectious agent associated or not with disease was detected. In 27.1% (23/85) of the cases, death was directly related to infectious agents. Furthermore, 12.9% (11/85), the cause of death was not determined. Likewise, this wildlife health monitoring program allowed the detection of relevant pathogens such as Canine Distemper Virus, Klebsiella pneumoniae, Angiostrongylus spp., Baylisascaris spp., among others. Our research demonstrated that this passive surveillance scheme is cost-effective and feasible in countries with limited resources. This passive surveillance can be adapted to the infrastructure dedicated to monitoring diseases in productive animals according to the scope and objectives of monitoring wildlife specific to each region. The information generated from the experience of the initial establishment of a WHMP is critical to meeting the challenges involved in developing this type of scheme in regions with limited resources and established as hotspots for emerging infectious diseases.

Introduction

Zoonotic diseases directly threaten public health systems, generating costs in medical treatment, outbreak control, and overloading health systems. In addition, it generates significant losses due to the slaughter of livestock and the affectation of other domestic animals [1,2]. Examples of how these diseases can impact public health, animal health, and wildlife have been the recent outbreaks of yellow fever and West Nile virus, which show the need to have the infrastructure and diagnostic capacity to ensure constant surveillance of potentially zoonotic agents [3,4].

Wildlife populations act as reservoirs and can play various roles in the epidemiology of numerous pathogens [5-7]. These roles assign to wildlife the important function of sentinels of the health of ecosystems and allow early detection of environmental alterations and the distribution, re-emergence, or emergence of certain pathogens in a specific region [8,9].

Tropical regions are among the areas of most extraordinary natural diversity with a concomitant high diversity of pathogens and, thus, a high potential for disease emergence [10,11]. Moreover, this risk has increased drastically because of anthropogenic pressures linked to over-exploitation of natural resources and increased land use change, increasing the possibility of contact between wildlife, domestic animals, and humans [12,13].

One of the preventive strategies against the risk of epidemic events promoted by the World Organization for Animal Health (OIE) and the World Health Organization (WHO) is to increase the efforts to establish early detection mechanisms for pathogens, of both zoonotic and conservation importance, via Wildlife Health Monitoring Programs (WHMP) [14-16].

One of the first steps to knowing the health status of the wildlife in a region is monitoring through passive surveillance, which identifies the causes of mortality in a range of species based on their pathological profiles through post-mortem examinations. This approach offers advantages like cost-effectiveness and the ability to carry out convenience samplings, taking advantage of the established infrastructure and diagnostic capacity. Furthermore, when these schemes are set in the long term, it has been shown that they provide the core information for decision-making and the establishment of policies, norms, and strategies, prioritizing disease prevention, even when the sampling is biased and with incomplete geographic coverage [17-20].

In Latin America have been made some significant efforts to improve epidemiological surveillance systems aimed at animal health. Some national programs are installed and functioning perfectly where wild animals are used as sentinels to monitor specific diseases [21,22]. However, there are still no monitoring programs for the general health status of wildlife, making clear the need to optimize and expand the coverage of these schemes [23,24]. For example, according to the U.S. Department of Agriculture, Costa Rica has the infrastructure and maintains adequate surveillance programs to detect and control zoonotic diseases in livestock [25]. However, it does not contemplate local wildlife within its scheme as it should [26].

Several pathogens, such as zoonotic parasites, vector-borne infectious agents, and direct transmission viruses, have been identified in Costa Rican wildlife [27-38]. This evidence reflects the urgency of establishing a permanent WHMP, where aspects such as general health status and monitoring of zoonotic pathogens in wildlife are considered, facilitating knowledge of the ecoepidemiology of these agents at the local level.

Countries with limited resources, such as Costa Rica, face severe financial and logistical restrictions in monitoring the health and circulation of pathogens in wildlife. Nevertheless, in the short term must extend the coverage of this type of program to tropical regions. Therefore, this research aims to evaluate the technical and infrastructural feasibility of establishing this type of scheme in Costa Rica by implementing a pilot program for passive epidemiological surveillance of wildlife. Although we encountered obstacles such as a lack of data collection legislation and a willingness to cooperate among agencies, our research demonstrated the logistical capacity and that it is possible to adapt the established infrastructure to implement this program. Furthermore, this allowed wild animal carcasses to be analyzed, detecting zoonotic pathogens and pathogens of conservation importance.

Material and methods

Statement of ethics

All samples were obtained from dead wildlife (found dead in the field or euthanized after veterinary care in specialized centers). The study was approved by the Ministry of Environment and Energy (MINAE) (wildlife authority) through permits (R-SINAC-PNI: -ACAT-040, ACAHN-18, ACTo-022, ACT-OR-DR-43, ACG-026, ACLAC-039, ACLAP-023, ACOPAC-005, ACC-037), and with the support of the animal health authority, the National Animal Health Service through the office (SENASA-DG-0277-18).

WHMP schema proposal and case definition

For the implementation of a WHMP, a passive epidemiological surveillance scheme was proposed adapting the current country’s technical diagnostic resources and infrastructure. To create a network for detecting dead and diseased wild animals, officials from the wildlife management centers and officials from wildlife authorities reported cases and voluntarily sent specimens. Officials were encouraged to send complete carcasses from free-ranging vertebrates after death due to any associated disease or trauma, both found dead in the field or deceased in management centers. Carcasses of animals that remained more than 48 hours in the management centers before death, received medication, or were frozen for more than a week were excluded from the study. The proposed WHMP scheme is shown in Fig 1.

Fig 1

Pilot WHMP work scheme design proposal.

DVM-MC: Doctor of veterinary medicine of wildlife management centers; MC: Wildlife management centers; PD: Pathology Department of Escuela de Medicina Veterinaria, Universidad Nacional.

Basic information was requested and registered for every sample submission: geographic location, the standard and scientific name of the animal, clinical signs, and any information considered relevant to the case, following the scheme recommended by the OIE for the notification of cases for disease surveillance system in wild animals [16,39]. All carcasses were shipped under refrigerated conditions at 2–8°C.

Pathological analysis

The carcasses received were classified by autolysis degree according to an established scale of one to five [40]. Thus, ranging from a fresh carcass or recently dead animal (grade 1) to advanced decomposition (grade 4) and partial, mummified carcasses or skeletal remains (grade 5). Only carcasses with grades 1 to 3 were included in the study for post-mortem analysis and tissue sampling [41]. Therefore, 96 specimens were received, of which 85 were admitted to the study. Specimens were divided by sex and age according to the development of sexual organs and phenotypic characteristics of the species. Also, they were divided by taxonomic order.

All morphological findings were recorded. In addition, tissue samples were taken for routine histopathological and microbiological analysis as required. Tissue samples for histopathology were processed based on standard routing protocols [41].

Detection of different infectious agents

Virus detection

Molecular methods were used to detect different viral agents. All molecular methods were done in the presence of positive and negative controls. The samples analyzed were fresh tissues collected sterile during post-mortem analysis. In addition, we performed RNA extraction using the commercial kit DNeasy Blood and Tissue (QIAGEN, Venlo, The Netherlands), following the manufacturer’s recommendations. The methods used and the samples collected are specified in Table 1.

Table 1

Molecular techniques for the detection of viral agents and protozoa.

Infectious agent	Target region	Method	Primer	Sequence	Reference protocol	Used material
Canine Distemper Virus (CDV).	N gene	NestedRT-PCR	First round: CDV-1F	5’- ACT GCT CCT GAT ACT GC-3’	Da Budaszewski et al., 2014. [42]	Tissuea
			CDV-2R	5’- TTC AAC ACC RAC YCC C-3’
			Second round:CDV-3F	5’- ACA GRA TTG CYG AGG ACY TRT-3’
			CDV-4R	5’- CAR RAT AAC CAT GTA YGG TGC-3’
Alphaviruses.	nsP4	NestedRT-PCR	First round:--	5’- TTT AAG TTT GGT GCG ATG ATG AAG TC-3’ (500 nM)	Grywna et al., 2010. [43]	Tissuea
				5’- GCA TCT ATG ATA TTG ACT TCC ATG TT-3’ (500 nM)
			Second round:--	5’-GGT GCG ATG ATG AAG TCT GGG ATG T-3’ (200nM)
				5’- CTA TGA TAT TGA CTT CCA TGT TCA TCC A-3’ (100 nM)
				5’-CTA TGA TAT TGA CTT CCA TGT TCA GCC A-3’ (100 nM)
Flaviviruses.	NS5 gene	Semi-nested RT-PCR	First round: MAMD	5’- AAC ATG ATG GGR AAR AGR GAR AA-3’	Scaramozzino et al., 2001. [44]	Tissuea
			cFD2	5’-GTG TCC CAG CCG GCG GTG TCA TCA GC-3’
			Second round: FS 778	5’-AAR GGH AGY MCD GCH ATH TGG T-3’
			cFD2	5’-GTG TCC CAG CCG GCG GTG TCA TCA GC-3’
Avian Influenza virus (AI).	matrix (M) gene	qRT-PCR	M + 25	5’-AGA TGA GTC TTC TAA CCG AGG TCG-3’	Spackman et al., 2002. [45]	Tissue and swab b
			M 124	5’-TGC AAA AAC ATC TTC TTC AAG TCT CTG-3’
			M + 64	5’-FAM-TCA GGC CCC CTC AAA GCC GA-TAMRA-3’
Rabies virus.	Nucleoprotein	RT–PCR	RAB504	5’-TAT ACT CGA ATC ATG AAT GGA GGT CGA CT-3’	Primers: Oliveira et al. 2010. [46]Protocol:Carnieli et al. 2008 [47]	Tissuec
			RAB304	5’-ACG CTT AAC AAC AAR ATC ARA G-3’
Newcastle virus.	Fusion gene, F0	RT-PCR	NCD3	5’-GTC AAC ATA TAC ACC TCA TC-3’	STAUBER, 1995. [48]	Tissue and swab b
			NCD4	5’-GGA GGA TGT TGG CAG CAT T-3’
Toxoplasma gondii.	529bp repetitive segment	PCR	Tox-8	5’-CCC AGC TGC GTC TGT CGG GAT-3’	Homan et al., 2000. [49]Reischl et al., 2003. [50]	FFPEd
			Tox-11	5’-GCG TCG TCT CGT CTA GAT CG-3’
Trypanosoma cruzi.	18S rRNA gene	Nested PCR	First round:SSU4_F	5’-GTG CCA GCA CCC GCG GTA AT-3’	First round primer:Pinto et al., 2015. [51]Second round primer:Noyes et al., 1999. [52]Protocol: Aleman et al., 2017. [53]Murphy & O’Brien, 2007.[54]	FFPEe
			18Sq1R	5’-CCA CCG ACC AAA AGC GGC CA-3’
			Second round:SSU561F	5’-TGG GAT AAC AAA GGA GCA-3’
			SSU561R	5’-CTG AGA CTG TAA CCT CAA AGC-3’
Leishmania spp.	Kinetoplast	PCR	13A	5’- GTG GGG GAG GGG CGT TCT-3’	Medeiros et al. 2002. [55]Sosa-Ochoa et al. 2015. [56]	FFPEf
			13B	5’-ATT TTA CAC CAA CCC CCA GTT-3’

FFPE: Formalin-fixed paraffin-embedded.

a brain and lung.

b Lung and Trachea tissue and cloacal swab.

c hippocampus, cerebellum, and medulla oblongata.

d spleen, lung, and liver.

e heart.

f spleen.

Detection of protozoan parasites`

Confirmation was performed using molecular techniques for pathogen identification when a previous presumptive protozoa presence was established in the histopathological study. All molecular methods were done in the presence of positive and negative controls. Tissue samples previously embedded in paraffin were used for this purpose. The deparaffinization procedure was done using xylol washes following the method recommended to perform DNA extraction from the tissue [57]. We performed DNA extraction using the commercial kit DNeasy Blood and Tissue (QIAGEN, Venlo, The Netherlands) according to the manufacturer’s instructions. The methods used and the samples collected are specified in Table 1.

Bacteriological detection

Tissue samples from animals with inflammatory processes (suppurative or abscesses) were cultured following standard bacteriological procedures. For bacterial isolation, samples were inoculated on non-selective and selective agar media. Significant bacterial growth was identified using the automated VITEK-2 Compact system, software version 8.02 (bioMérieux, Marcy l’Etoile, France). VITEK test cards for Gram-negative [GN], Gram-positive [GP], and anaerobes [ANC] were used for identification according to the manufacturer’s instructions.

Identification of metazoan parasites

All the parasites in the carcasses were collected and washed with physiological saline, preserved in alcohol, acetic acid, and formalin (AFA) solution. No more than one week after collection, they underwent identification to the genus level through morphometric characteristics [58]. Physical and morphometric characteristics were recognized after fixation and clarification with Hoyer’s solution by light microscopy [59-61]. In addition, processed cestodes were stained with dilute Harris’ hematoxylin solution.

Information management, geocoding, and spatial analysis

The information on each case was included in the epidemiological surveillance information system (SIVE) from the animal health authority. Each case was geocoded using the latitude and longitude generated by GPS of the point where the specimen was found by field personnel. When the GPS was unavailable, they were geocoded using the latitude and longitude of the approximate location where they were found, and this was generated by Google Earth Pro v7.3 (2021, Google Inc.). With the georeferenced points of each sample admitted created a map using ArcGIS 10.7 (ERSI), according to territorial division by conservation area: Arenal Huetar Norte Conservation Area (ACAHN); Arenal Tempisque Conservation Area (ACT); Central Conservation Area (ACC); Guanacaste Conservation Area (ACG); La Amistad Caribe Conservation Area (ACLAC); La Amistad Pacífico Conservation Area (ACLAP); Osa Conservation Area (ACOSA); Pacífico Central Conservation Area (ACOPAC); Tempisque Conservation Area (ACT); Tortuguero Conservation Area (ACTo). Additionally, a feedback report was sent to the field staff with the relevant findings per case.

Results

Participation in the WHMP and distribution of cases by age, sex, and taxonomic classification

The notification of cases was made by officials from the wildlife authority, with 24.7% (21/85) of the cases and 75.3% (64/85) by officials from wildlife management centers. Only four management centers reported and sent cases for analysis. The conservation areas with the most significant participation in the WHMP were the same ones where the participating wildlife management centers were located. The geographical location of the management centers, diagnostic laboratory, and cases analyzed is shown in Fig 2. The conservation areas where there was no participation are those located furthest from the diagnostic laboratory and with significant obstacles for shipment, as mentioned in Table 2.

Fig 2

Geocoding of the cases analyzed by conservation area.

The number corresponds to the cases analyzed in each conservation area. Wildlife management centers shown are those that collaborated with the WHMP.

Table 2

Participation in the WHMP of detectors of cases and obstacles found in each conservation area.

Conservation area	Number of cases	Cases detector		Obstacles to sending cases
		Wildlife Officer	MC
ACAHN	3	0	3	Inability to store. Coordination problems with the health agency for the transport of specimens. Few rescue centers motivated to participate.
ACAT	6	0	6	Coordination problems with the wildlife agency to submit specimens.
ACC	27	6	21	No significant obstacles.
ACG	11	7	4	Coordination problems with the health agency for the transport of specimens.
ACLAC	0	0	0	Few rescue centers motivated to participate. Coordination problems with the wildlife agency to submit specimens. Coordination problems with the health agency for the transport of specimens. Distant from the diagnostic laboratory.
ACLAP	0	0	0	There are no rescue centers in the region. Coordination problems with the wildlife agency to submit specimens.
ACOSA	0	0	0	There are no rescue centers in the region. Distant from the diagnostic laboratory.
ACOPAC	23	0	23	Coordination problems with the wildlife agency to submit specimens.
ACT	12	5	7	Few rescue centers in the region. Inability to store
ACTo	3	3	0	There are no rescue centers in the region. Insufficient field staff. Coordination problems with the health agency for the transport of specimens.

ACAHN: Conservation area Arenal Huetar Norte; ACT: Conservation area Arenal Tempisque; ACC: Conservation area Central; ACG: Conservation area Guanacaste; ACLAC: Conservation area La Amistad Caribe; ACLAP: Conservation area La Amistad Pacifico; ACOSA: Conservation area Osa; ACOPAC: Conservation area pacific central; ACT: Conservation area Tempisque; ACTo: Conservation area Tortuguero.

Of the 85 specimens admitted to the study, there was an age distribution of 27.1% (23/85) young animals and 72.9% (62/85) adults. The sex distribution was 56.5% (48/85) males and 43.5% (37/85) females. According to the taxonomic order, we received 29.4% (25/85) Carnivora, 29.4% (25/85) Primate, 12.9% (11/85) Pilosa, 5.9% (5/85) Didelphimorphia, 4.7% (4/85) Rodentia, 4.7% (4/85) Artiodactyla, 2.3% (2/85) Cingulate, 2.3% (2/85) Pelecaniformes, 2.3% (2/85) Accipitriformes, 2.3% (2/85) Anseriformes, 1.2% (1/85) Ciconiiformes, 1.2% (1/85) Piciformes and 1.2% (1/85) Coraciiformes. The geographical distribution of admitted cases by conservation area is shown in Fig 2.

Identification of causes of death according to pathological findings

According to pathological findings, the distribution of the presumptive cause of death corresponded to 60% (51/85) of death not associated with an infectious agent. Of these, 54.1% (46/85) associated with traumatic events (mainly roadkill and electrocution), 2.4% (2/85) with a degenerative disease, and in 3.5% (3/85) of cases, death was presumptively associated with intoxication. Additionally, of individuals with a cause of traumatic death, 37.6% (32/85) concomitantly presented some infectious agent with or without an associated disease (24 with gastrointestinal and pulmonary metazoan parasites, three with bacteria, one with protozoa, and four with multiple microorganisms). In 27.1% (23/85) of cases, death was directly related to infectious agents, ten presented lesions associated with viruses, five with metazoan parasites, two with protozoan parasites, one with bacteria, and five presented lesions associated with multiple etiologies. In 12.9% (11/85) of cases, the cause of death was not determined. The absolute and relative values of the causes of death for each taxonomic group according to the presence of infectious agents are specified in Table 3.

Table 3

Absolute and relative values of the causes of death for each taxonomic group.

Cause of Death / Taxon	DAIA	DNAIA-PD	DNAIA-IAD	DNAIA	UD
Mammals
Carnivora	40% (10/25)	28% (7/25)	16% (4/25)	8% (2/25)	8% (2/25)
Primate	32% (8/25)	36% (9/25)	16% (4/25)	8% (2/25)	8% (2/25)
Pilosa	0% (0/11)	9.1% (1/11)	27.3% (3/11)	45.4% (5/11)	18.2% (2/11)
Didelphimorphia	20% (1/5)	0% (0/5)	60% (3/5)	0% (0/5)	20% (1/5)
Rodentia	25% (1/4)	0% (0/4)	0% (0/4)	25% (1/4)	50% (2/4)
Artiodactyla	25% (1/4)	0% (0/4)	0% (0/4)	75% (3/4)	0% (0/4)
Cingulate	0% (0/2)	0%% (0/2)	0%% (0/2)	100% (2/2)	0%% (0/2)
Birds
Pelecaniformes	100% (2/2)	0% (2/2)	0% (2/2)	0% (2/2)	0% (2/2)
Accipitriformes	0% (0/2)	0% (0/2)	50% (1/2)	50% (1/2)	0% (0/2)
Anseriformes	0% (0/2)	0% (0/2)	0% (0/2)	100% (2/2)	0% (0/2)
Ciconiiformes	0% (0/1)	0% (0/1)	0% (0/1)	0% (0/1)	100% (1/1)
Piciformes	0% (0/1)	0% (0/1)	0% (0/1)	100% (1/1)	0% (0/1)
Coraciiformes	0% (0/1)	0% (0/1)	0% (0/1)	0% (0/1)	100% (1/1)
Total	27.1% (23/85)	20% (17/85)	17.6% (15/85)	22.4% (19/85)	12.9% (11/85)

DAIA: Death associated with an infectious agent; DNAIA-PD: Death not associated with an infectious agent, with a pre-existing infectious disease; DNAIA-IAD: Death not associated with an infectious agent, with infectious agent detection; DNAIA: Death not associated with an infectious agent; UD: Undetermined death.

Infectious agents detected in the WHMP

Ten viruses, seven protozoa, and seven bacteria were identified in mammalian specimens. In 22 cases, these pathogens were involved with lesions or systemic disease, of which 19 were directly associated with the cause of death of mammals. Only Sarcocystis spp. detected in two cases was an incidental finding. Additionally, 38 mammals had metazoan parasites. Multi-parasitosis was observed in 15.3% (13/85) of the cases. Parasites such as Prosthenorchis spp. (n = 15), Angiostrongylus spp. (n = 6), and Cilycospirura spp. (n = 1) were responsible for severe parasitosis with systemic disease. Some of the lesions, such as pyogranulomatous abscessing bronchopneumonia and nodular and sclerosing gastritis associated with infectious agents, are observed in Fig 3 (see legend). In 50.6% (43/85) of the cases, the mammals presented infectious agents with a zoonotic potential, such as Klebsiella pneumoniae, Toxoplasma gondii, Angiostrongylus spp. The etiological agents identified by taxonomic groups and the number of specimens analyzed are specified in Table 4.

Fig 3

Infectious agents in lesions identified in wild animals.

A) Lung (Alouatta palliata-howler monkey). Lymphoplasmacytic pneumonia with the presence of tissue cyst, morphology compatible with Toxoplasma gondii, confirmation by PCR (arrowhead; H&E 600x). B) Lung (Alouatta palliata-howler monkey). Pyogranulomatous abscessing bronchopneumonia with intralesional bacteria Klebsiella pneumonia, confirmation by culture (arrowhead; H&E 200x). C) Brain (Didelphis marsupialis-opossum). Presence of nematode Angiostrongylus spp. identified by morphology (arrowhead; H&E 400x). Inset: Nematode magnification (H&E 200x). D) Lung (Cebus imitator-white-faced monkey). Bronchopneumonia associated to multiple Nematodes, Filariopsis spp. identified by morphology (more cuts of the female in microphotograph) (arrowhead; H&E 40x). E) Stomach (Herpailurus yagouaroundi-jaguarundi). Nodular and sclerosing gastritis associated with multiple Cylicospirura spp. Nematodes identified by morphology (arrowhead; H&E 40x). F) Skin (Sphiggurus mexicanus-porcupine) Pyogranulomatous and eosinophilic dermatitis associated with massive infestation of Sarcoptex spp. (arrowhead; H&E 400x). Inset: Mites magnification (H&E 100x).

Table 4

Number of infectious agents tested and positive in mammals according to etiology.

Mammalian taxonomic groups / infectious agent		Primate	Carnivora	Pilosa	Didelphimorphia	Rodentia	Artiodactyla	Cingulate
Viral	CDV (n = 18)	0	10	0	0	0	0	0
	Alphaviruses (n = 9)	0	0	0	0	0	0	0
	Flaviviruses (n = 9)	0	0	0	0	0	0	0
	Influenza virus (n = 8)	0	0	0	0	0	0	0
	Rabies virus (n = 76)	0	0	0	0	0	0	0
Bacterial	C. perfringens (n = 18)	0	0	0	0	0	1	0
	E. coli (n = 18)	1	0	0	0	0	0	0
	K. pneumoniae (n = 18)	1	0	0	0	0	0	0
	T. pyogenes. (n = 18)	0	0	0	0	1	0	0
	S. aureus (n = 18)	1	1	1	0	0	0	0
	Mycobacterium spp. (n = 18)	0	0	0	0	0	0	0
Protozoan parasites	Toxoplasma gondii (n = 4)	2	0	0	0	0	0	0
	Trypanosoma spp. (n = 14)	0	0	0	3	0	0	0
	Leishmania spp. (n = 8)	0	0	0	0	0	0	0
	Sarcocystis spp. (n = 5)	0	1	0	0	0	1	0
Metazoan parasites 1	Angiostrongylus spp.	0	5	0	1	0	0	0
	Dirofilaria spp.	0	4	0	0	0	0	0
	Dipetalonema spp.	5	0	2	0	0	0	0
	Gnathostoma spp.	0	0	0	1	0	0	0
	Baylisascaris spp.	0	1	0	0	0	0	0
	Ancylostoma spp.	0	1	0	0	0	0	0
	Cylicospirura spp.	0	1	0	0	0	0	0
	Prosthenorchis spp.	10	5	0	0	0	0	0
	Macracanthorhynchus spp.	0	1	0	0	0	0	0
	Spirometra spp.	0	2	0	0	0	0	0

n: Number tested.

1 only zoonotic metazoan parasites are shown.

All birds submitted were evaluated for virus presence (n = 9); two of these were positive for flaviviruses. Additionally, three birds had metazoan parasites. Most of the pathogens identified were directly associated with the cause of the death of birds. Only Procyrnea spp. identified in one case was an incidental finding. In 2.3% (2/85) of the cases, the birds presented infectious agents with zoonotic potential, such as Contracaecum spp. The etiological agents identified in birds and the number of samples analyzed are specified in Table 5.

Table 5

Number of infectious agents tested and positive in birds according to etiology.

Avian taxonomic groups / infectious agent		Pelecaniformes	Accipitriformes	Anseriformes	Ciconiiformes	Piciformes	Coraciiformes
Viral	Alphaviruses (n = 3)	0	0	0	0	0	0
	Flaviviruses (n = 3)	2	0	0	0	0	0
	Influenza virus (n = 9)	0	0	0	0	0	0
	Newcastle virus (n = 9)	0	0	0	0	0	0
Bacterial	C. perfringens (n = 1)	0	0	0	0	0	0
	E. coli (n = 1)	0	0	0	0	0	0
	K. pneumoniae (n = 1)	0	0	0	0	0	0
	Salmonella spp. (n = 1)	0	0	0	0	0	0
	S. aureus (n = 1)	0	0	0	0	0	0
Metazoan parasites 1	Contracaecum spp.	2	0	0	0	0	0

n: Number of tested.

1 only zoonotic metazoan parasites are shown.

Geospatial distribution of detected infectious agents and their accumulation by geographic region

We established the distribution of the most frequently identified infectious agents in the analyzed specimens (Fig 4). First, a wide distribution of zoonotic parasites was evidenced in the country. Then, there was an accumulation in the Central Pacific region of specimens with acanthocephaliasis (12 with Prosthenorchis spp., one with Macracanthorhynchus spp.), and an accumulation of specimens with gastrointestinal nematodes in the great metropolitan area and tourist areas of Guanacaste (six with Angiostrongylus spp., one with Baylisascaris spp., one with Ancylostoma spp.). Additionally, vector-borne diseases occurred exclusively in specimens from coastal regions and altitudes less than 300 meters above sea level (11 with filariae, two with flaviviruses). The CDV in carnivores from various areas of the country did not show a specific distribution pattern (n = 10). The analyzed specimens associated with these infectious agents can be observed in S1 Table.

Fig 4

Geographical distribution of the most frequently identified infectious agents in the referred specimens.

The individuals reported as negative were depicted even though the infectious agent was not detected in the complementary analyzes or no lesions suggestive of the disease were found in the pathological analysis.

Discussion

The WHMP schemes have proven to be a fundamental tool in monitoring pathogens of zoonotic importance [62-64]. These surveillance systems are even more critical in geographical areas where high rates of biodiversity are prominent [14]. For example, Costa Rica is economically dependent on its ecotourism services, and its fauna is one of its most important assets [65]. However, no epidemiological surveillance system is currently directed to wildlife to study outbreaks or other health events.

Furthermore, implementing these types of schemes is essential for a country considered a "hotspot" for the appearance or emergence of new infectious agents; however, we encountered some obstacles when performing this study [11,13]. These obstacles are mainly related to lack of legislation for data collection, willingness to cooperate between agencies, financial disincentives and logistical problems for the storage and transport of carcasses, and difficulties similar to those described by some authors [66,67].

The storage capacity and transport logistics directly impact WHMPs. Urban areas with transportation facilities reported and dispatched more carcasses, in contrast to remote or difficult-to-access regions with less participation. These patterns tally with previous reports indicating that notification of wildlife mortality or morbidity generally depends on the initial detection of cases by the general public. Consequently, detected cases are biased towards events in populated or easily accessible areas [17,68,69]. Nevertheless, this shows that the existing logistics in ACC and ACOPAC (urban areas with the highest number of reported cases) can be used to maintain the WHMP at least on a regional level. Likewise, it is necessary to expand the network of laboratories to include other institutions with pathological diagnosis capacity within the WHMP scheme to reduce reliance on the storage and transportation of carcasses. This measure has been shown to improve coverage in distant regions and increase case reporting [19,20,63].

The lack of guidelines and legislation also limited participation and case detection. This means that the system is maintained by the self-interest of officials and interpersonal relationships of people from different institutions. These findings are consistent with previous evaluations of the veterinary services of Costa Rica [26]. To ensure the long-term sustainability of the WHMP, legislation and regulations are necessary to provide financial support and clarify the specific functions of each institution. This could facilitate coordination and cooperation between institutions to notify and transport specimens [19,20,62,63].

The notification and referral of cases relied heavily on the management centers that provide veterinary care to wild animals. Other studies have proposed these institutions as an indispensable tool within the WHMPs due to the large amount of information they can generate for the system [70,71]. The performance of necropsy by the veterinary doctors of management centers would greatly support the efficiency and sustainability of this WHMP, reducing the demand for transportation (only samples would be transported, not complete carcasses), and it would eliminate freezing; facilitating diagnosis. This could encourage the participation of management centers from distant regions without storage capacity, thus increasing coverage. However, the previous regulations, manuals and procedures for post-mortem analysis and sampling procedures are necessary, as occurs with other surveillance schemes, to avoid affecting the diagnosis since pathological analysis is vital in passive surveillance schemes [17,20,21].

Carnivores and primates were the taxa with higher representation. These data can be associated with the fact that they are medium to large-sized animals, more charismatic, and with a more significant contact of these species with human environments, facilitating the recognition of morbidities and mortalities by the population [69]. Therefore, taxa should be prioritized in the WHMPs, since it allows for optimizing the use of resources. Furthermore, in addition to their easy detection, they are taxa in which various pathogenic agents can circulate [5,7].

In contrast, obtaining viable bird carcasses for post-mortem analysis was challenging due to the advanced degree of autolysis, wasting important transport and storage resources, an obstacle experienced in other studies [72]. Wild birds should be included in surveillance programs for influenza virus and Newcastle disease virus that maintains in the poultry production systems in the country. This would maintain monitoring as is done in other WHMPs without the need for post-mortem analysis, thus avoiding wasted resources [19].

In addition, most cases with a traumatic cause of death presented some pre-existing infectious pathology. Free-living animals are naturally exposed to infectious agents, so it is common to find them in incidental lesions in post-mortem analysis [73-75]. These cases allow the detection of infectious agents in passive surveillance, whether or not they are associated with the cause of death [73,76-78]. Therefore, they must be kept within the cases to be analyzed.

The diagnostic capability allowed the WHMP to detect infectious agents that could affect the health of domestic animals, public health, and the conservation of wild species. For example, our study shows the presence of potentially zoonotic bacterial infectious agents classified as emerging diseases in some regions [79-81]. The most relevant are Klebsiella pneumoniae, Escherichia coli, and Staphylococcus aureus, which were associated with primary disease in some of the analyzed specimens. In addition, these bacteria currently top the list of infectious agents with antibiotic resistance genes, thus showing the importance of monitoring these agents in WHMP schemes [28,82-84].

We also detect vector-borne diseases, which are recognized as agents with epidemic potential in Latin America due to tropical regions’ climatic, health, and socioeconomic conditions that favor their spread [85-87]. We identified primates, carnivores, and birds with infectious agents of vector transmission, for example, Dirofilaria spp., Dipetalonema spp., and flavivirus mainly present by the coast. Most of these cases come from regions already defined as endemic areas for these infectious agents in domestic animals, which reveals a possible transmission by this route and a potential risk for the conservation of the species [88-91].

Detection of these vector-borne pathogens also reveals a potential risk to public health in places with a high rate of tourists visiting Costa Rica. This risk is reinforced by health system reports showing at least three disease cases in humans associated with Dirofilaria immitis and isolated cases of subcutaneous filariasis [92-94]. Furthermore, detecting virus-related mortalities such as West Nile in wild birds (as was possibly our case) allows early alerts. It has been shown that there is a higher risk of exposure for human populations close to the regions where mortalities of wild birds occur [95,96].

The CDV was frequently detected in our study, reflecting the relevance of this virus in the role of spillover towards carnivore species and possibly the implications of a spillback towards susceptible or non-vaccinated domestic canines [97-99]. Endemic CDV outbreaks have been reported anecdotally throughout Costa Rica and America in dog populations. More recently, sporadic outbreaks in wild carnivores of urban and suburban areas have been recorded [97,100]. Unfortunately, Costa Rica does not have official data on the domestic dog population. Therefore, herd immunity data in this population is uncertain, especially for dogs without an owner or in non-urban areas. This poses a risk to wild carnivores, especially in urban areas with susceptible canine populations. Furthermore, the possibility of transmission of this virus to other species beyond carnivores is a hypothesis that has been investigated [101]. Given the high diversity of vertebrates in Costa Rica and the high circulation of CDV detected, this virus should be considered within epidemiological surveillance programs.

Also, this study’s gastrointestinal and pulmonary metazoan parasites are relevant for public health and wildlife conservation programs. For instance, we detect the nematodes Angiostrongylus spp., Baylisascaris spp., Ancylostoma spp., and Cylicospirura spp. in mammalian species located in densely populated areas. In addition, we detected cases with acanthocephalans (Prosthenorchis spp., Macracanthorhynchus spp.) concentrated in the Central Pacific region and parasites transmitted by water or aquatic food such as the cestode Spirometra spp. in the country’s northern region. This result proves a cost-effective tool for the WHMP, which does not require financial resources beyond qualified personnel for the morphological identification of worms and allows the detection of pathogenic agents that primarily impact children [102-104].

This study did not detect rabies virus infections. These findings are supported by previous studies on wild animals in Costa Rica [29]. However, this passive surveillance program allowed expanding coverage in the number of species and geographic regions through constant monitoring of wild species. Human and livestock fatalities have been reported associated with rabies infections, which stresses the relevance of its continuous monitoring of species that can act as reservoirs [35,105]. A similar situation applies to Newcastle and Influenza virus. In our samples, none of the birds showed evidence of disease or associated clinical signs; however, due to the great relevance of these diseases to the country and the risk for national production, it is advisable to establish routine monitoring by the animal health agency under the WHMP scheme [35,106]. Including serological monitoring of tuberculosis and brucellosis of wild species in the scheme would even be advisable. These national epidemiological surveillance programs already include some wild species, and coverage could be expanded [107].

Finally, we could not identify the cause of death in some of the samples analyzed. Although we tested for the main circulating infectious agents in Costa Rica, no conclusive data was obtained. Ranges of 17–22% have been reported in pathological studies in wild species, where the causative agent of the disease cannot be determined, mainly associated with the degree of autolysis and the diagnostic complexity [68,74,76]. These results are consistent with the percentages of an absence of identification of the etiological agent in our samples. Although proving that diagnostic capacity is acceptable, further work is necessary to develop robust diagnostic techniques for wild animal testing. Further efforts and incentives, financed by government authorities, are required for pathogen surveillance in wildlife through the consistent implementation of tools such as new generation metagenomics [108-111].

Although the proposed program is limited to the country’s resources and infrastructure, and it is clear that it is not generally applicable, it is important to start evaluating the implementation of these programs in regions where disease surveillance in wildlife is minimal. For example, this study shows that this passive surveillance scheme is cost-effective and feasible to establish in countries with limited resources. Furthermore, this scheme was possible since we could adapt the infrastructure dedicated to monitoring diseases in productive animals according to the scope and objectives of monitoring wildlife specific to each region. We also showed sufficient diagnostic capacity in the country for detecting infectious agents of zoonotic and conservation importance in wild animals. If this scheme is maintained over time, it will generate data to allow the decision-making to promote the conservation of species, animal health, and public health by knowing the circulation and behavior of these pathogens [68].

This study highlights the need for an inter-institutional and trans-institutional commitment to the sustainability over time of this surveillance scheme. Participant institutions must remain motivated and focused on the benefits beyond the economic part. The feedback to field staff and the frequent reports of the importance of detected pathogens are crucial to maintaining motivation and detection network, as in our case. In addition, the information generated from the experience of the initial establishment of a WHMP is critical to meeting the challenges involved in developing this type of scheme in regions with limited resources and established as hotspots for emerging infectious diseases [13,112]. Although it is necessary to standardize methods and techniques for monitoring pathogens in wildlife, the development of pilot schemes allows sharing experiences with programs already installed and leads to subsequent optimization and standardization studies that will facilitate the exchange of information and expand coverage [112].

Biological data and more representative pathological findings of the analyzed specimens.

(DOCX)

Click here for additional data file.

30 Mar 2022

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Daniel Becker

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. We note that Figure 3 in your submission contain map images which may be copyrighted. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For these reasons, we cannot publish previously copyrighted maps or satellite images created using proprietary data, such as Google software (Google Maps, Street View, and Earth). For more information, see our copyright guidelines: http://journals.plos.org/plosone/s/licenses-and-copyright.

We require you to either (1) present written permission from the copyright holder to publish these figures specifically under the CC BY 4.0 license, or (2) remove the figures from your submission:

1. You may seek permission from the original copyright holder of Figure 3 to publish the content specifically under the CC BY 4.0 license.

We recommend that you contact the original copyright holder with the Content Permission Form (http://journals.plos.org/plosone/s/file?id=7c09/content-permission-form.pdf) and the following text:

“I request permission for the open-access journal PLOS ONE to publish XXX under the Creative Commons Attribution License (CCAL) CC BY 4.0 (http://creativecommons.org/licenses/by/4.0/). Please be aware that this license allows unrestricted use and distribution, even commercially, by third parties. Please reply and provide explicit written permission to publish XXX under a CC BY license and complete the attached form.”

Please upload the completed Content Permission Form or other proof of granted permissions as an "Other" file with your submission.

In the figure caption of the copyrighted figure, please include the following text: “Reprinted from [ref] under a CC BY license, with permission from [name of publisher], original copyright [original copyright year].”

2. If you are unable to obtain permission from the original copyright holder to publish these figures under the CC BY 4.0 license or if the copyright holder’s requirements are incompatible with the CC BY 4.0 license, please either i) remove the figure or ii) supply a replacement figure that complies with the CC BY 4.0 license. Please check copyright information on all replacement figures and update the figure caption with source information. If applicable, please specify in the figure caption text when a figure is similar but not identical to the original image and is therefore for illustrative purposes only.

The following resources for replacing copyrighted map figures may be helpful:

USGS National Map Viewer (public domain): http://viewer.nationalmap.gov/viewer/

The Gateway to Astronaut Photography of Earth (public domain): http://eol.jsc.nasa.gov/sseop/clickmap/

Maps at the CIA (public domain): https://www.cia.gov/library/publications/the-world-factbook/index.html and https://www.cia.gov/library/publications/cia-maps-publications/index.html

NASA Earth Observatory (public domain): http://earthobservatory.nasa.gov/

Landsat: http://landsat.visibleearth.nasa.gov/

USGS EROS (Earth Resources Observatory and Science (EROS) Center) (public domain): http://eros.usgs.gov/#

Natural Earth (public domain): http://www.naturalearthdata.com/

4. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match.

When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #2: N/A

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The study addresses a critical gap in wildlife disease surveillance by implementing a useful and needed tool to survey wild animals passively. They highlight the urgency of this epidemiological surveillance system, with an extensive pathological work paired with a broad literature review on the topic. The authors show the relevance of such approach with significant examples such as the circulation of Canine Distemper Virus, which could potentially be a treat for wildlife and domestic dogs. However, although the authors state that they aim “to evaluate the technical and infrastructural feasibility to establish” a passive surveillance system, they are only demonstrating the feasibility of this approach by describing in detail they pathological findings and discussing the relevance of their results for public health and conservation, without any further analysis or clear evaluation of viability of their program.

The information their research provided is however, of great value, and the authors make a strong case of the need of such passive surveillance in a low-resource country like Costa Rica. They showed they have the infrastructural and expertise to carry this surveillance program, but probably lack the financial and logistical support to make this program permanent. However, they don’t seem to propose a protocol or guidelines on how to make this program available and reproducible in the country (including the sites they identify as under-studied) and other regions. For instance, they could address aspects such as that most of their samples came from wildlife rescue center as possible ways of improving and sustaining their surveillance program. In line 278-280 they even mentioned that they encounter obstacles, but do not mention how those obstacles were overcome. I suggest they proposed clearer steps moving forward, if they truly want to evaluate the implementation of this valuable wildlife surveillance program.

The text has some grammatical errors and redundant sentences, that makes the reading hard to follow. I mention some of these issues below, in addition to other observations.

Lines 50-53. Covid-19 would not make a good example. Although it may have a zoonotic origin, it is considered a human-to-human disease. May be consider another example.

Lines 54-56. These two sentences seem redundant. Consider rephrasing.

Line 59. Consider changing “countries” for “regions”, and eliminating the “(including Costa Rica)”

Line 61. Consider changing “geographic regions” for “countries”

Line 64. Do you mean increased land use change?

Lines 76-77. In that case, since they are post-mortem examinations, it would not be morbidity, right?

Line 77-79. I don’t follow this sentence. What do you mean by “obtaining a sustainable tool that allows understanding the emerging potential of different pathogens.”? Also, consider changing “profitability” to “cost-effective” because the latter has a connotation of lucrative.

Line 88. A disease is not a synonym for pathogen, please change “vector-borne diseases” accordingly.

Lines 89-93 and 96-98. Please rephrase these sentences, as they are unclear and hard to follow.

Lines 99-101. Avoid giving specific results here (e.g., 85 carcasses), just general and main findings.

Line 108. Isn’t the permit R-SINAC-PNI-ACLAC-039 for the Area de Conservacion La Amistad Caribe only?

Table 1. superscript b. Cloacal swab is not a tissue, please change accordingly.

Lines 167-168. Please change “specimens of the birds and 76 (89.5%) of the mammals” to “bird and 76 (89.5%) mammal specimens”

Lines 170-173. This information doesn’t seem to belong in this subsection but rather in either of the following ones.

Lines 178-181. 24 + 3 + 1 + 4 = 32, not 31 as stated below. Please check this. Also, please be specific on what are you referring to with “parasites”.

Lines 184-193. I am confused with this whole paragraph and the data presented below (including in Table 2). I imagine that you have several tissue samples from the same carcass, because you had 85 individuals total, but are now presenting 266 lesions total (199 + 67). Please clarify.

Lines 195-196. 11 + 5 + 4 = 20, it doesn’t add to 21. Please check this.

Lines 199-200. Do you mean that the rest of the tissues with infection lesions were associated with a pathogen? And what is the complementary analysis?

Table 2. I suggest you change “Bird” for “Aves”, to keep the correct taxa name for all the groups sampled.

Lines 241-242. Do you mean in here that all the birds, total of nine, were screened for viruses? From which, only the two that were involved in a mass mortality event were positive for flaviviruses? Please rephrase accordingly.

Line 300. Rephrase to: Regarding mammals, carnivores and primates were the taxa with higher representation

Lines 302-304. It is unclear what do you mean with this sentence. Are you suggesting that human proximity makes transmission of this infectious agents more common, or that the sole proximity facilitates pathogen detection? Also, it could be possible that cases of more charismatic animals are over-represented.

Lines 305-306. Anthropogenic effect it’s a broad term. Can you rephrase and be more specific?

Lines 313-315. What do you mean by human conditions? Also, I am not sure if you are using the term “evolve” correctly here. Do you mean pathogen evolution or how the disease develops?

Lines 331-332. This sentence is very vague. Please rephrase and be more specific.

Line 335. Please change “(CoastLine)” to “by the coast”.

Lines 336-340. What about conservation implications? What if this pathogen detection reveals the transmission of pathogens from domestic animals to wildlife?

Line 349 (and others) Please be consistent with the naming. If you used “CDV” at the beginning after explaining what it stands for, you should keep using the same name. Also, include the genus information at the beginning, where you mentioned the pathogens you are going to be screening for.

Line 353. Please change “studies” to “study”.

Lines 355-356. Can you provide a reference for this estimation?

Line 363. With “productive animal” do you mean livestock?

Line 379. Replace “the Nasua narica species” to “Nasua narica”

Figure 3. Giving that you mentioned that samples are biased toward the wildlife management centers, it would be informative to show in the map the location of the centers that sent the carcasses. If there is no clear purpose for the color coding on the map (I imagine indicating elevation), I suggest removing it, because it makes the figure harder to interpret. Finally, consider using other type of icons to show the pathogens detected that are not 3D.

Figure S1. This map was very noisy and hard to interpret. Consider simplifying as much as possible. May be to order, instead of genus, and removing the elevation color.

Please check your references. Scientific names should be in italics and there are a few mistakes, such as in Line 534, where Panama and Costa Rica are not capitalized.

Reviewer #2: Initially I would like to congratulate Dr. Alfaro-Alarcon for this manuscript, we know that research efforts to develop studies like the one presented here require a network of motivated institutions and people, this can be evidenced in this manuscript. So please feel proud of this manuscript fruit of his continuous work for almost three years. Congratulations.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: PEDRO ENRIQUE NAVAS SUÁREZ

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Submitted filename: PONE-D-21-39637 REV1.docx

Click here for additional data file.

1 Jun 2022

Rebuttal Letter

PONE-D-21-39637

Passive epidemiological surveillance in wildlife in Costa Rica identifies pathogens of zoonotic and conservation importance by Alfaro-Alarcón et al.

PLOS ONE

Dear reviewers:

General comments:

Initially, we would like to thank you for the positive and accurate comments you have made. Undoubtedly, these changes substantially improve the information and scientific relevance of this manuscript. Previously, we believed that we had addressed the aspects associated with the feasibility of implementing a passive epidemiological surveillance system for diseases in wild animals in Costa Rica. However, with these changes, the arguments are more direct with each point addressed and broader in the improvements needed for this system to be sustainable over time and reproducible in other regions, obtaining the important benefits generated by monitoring the health of wild animals, as exemplified in this research.

Academic Editor

Both reviewers note a disconnect between the stated aims of the paper (to evaluate the feasibility of implementing a passive surveillance system) and the actual content of the paper (results of pathological findings and their relevance for conservation/public health). Both reviewers in turn suggest the authors provide substantially more attention to a forward-looking component of their manuscript and propose concrete recommendations to implement such a program and how to make it sustainable/reproducible (one reviewer also suggests that the manuscript would greatly benefit from a flowchart of methodologies).

Response: These changes were accepted. The presentation of the results and the discussion of the arguments were reformulated, with the purpose to be much more prospective and linked to the objective of this study. A flowchart was added on the proposed surveillance system to make this reproducible.

Both reviewers also provide a number of more minor suggestions to improve clarity and precision of the manuscript. Some important clarifying points are also noted about inclusion/exclusion criteria for animals from rescue centers, given the possibility for pathogen exposure within the centers themselves (rather than from the forests).

Response: These changes were accepted. The answer is given at every minor suggestion.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf.

Response: The manuscript was adjusted to the style requirements established by PlosONE according to the indicated templates.

2. We note that Figure 3 in your submission contain map images which may be copyrighted. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For these reasons, we cannot publish previously copyrighted maps or satellite images created using proprietary data, such as Google software (Google Maps, Street View, and Earth). For more information, see our copyright guidelines: http://journals.plos.org/plosone/s/licenses-and-copyright.

We require you to either (1) present written permission from the copyright holder to publish these figures specifically under the CC BY 4.0 license, or (2) remove the figures from your submission:

Response: The maps submitted as figures in this manuscript are not copyrighted. The maps were created by the researchers for this study, using ArcGIS Desktop 10.8.1 software, license type: advanced, paid for by the National University of Costa Rica.

4. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match.

When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section

Response: The correct information has been sent.

Reviewer #1:

The study addresses a critical gap in wildlife disease surveillance by implementing a useful and needed tool to survey wild animals passively. They highlight the urgency of this epidemiological surveillance system, with an extensive pathological work paired with a broad literature review on the topic. The authors show the relevance of such approach with significant examples such as the circulation of Canine Distemper Virus, which could potentially be a treat for wildlife and domestic dogs. However, although the authors state that they aim “to evaluate the technical and infrastructural feasibility to establish” a passive surveillance system, they are only demonstrating the feasibility of this approach by describing in detail they pathological findings and discussing the relevance of their results for public health and conservation, without any further analysis or clear evaluation of viability of their program.

The information their research provided is however, of great value, and the authors make a strong case of the need of such passive surveillance in a low-resource country like Costa Rica. They showed they have the infrastructural and expertise to carry this surveillance program, but probably lack the financial and logistical support to make this program permanent. However, they don’t seem to propose a protocol or guidelines on how to make this program available and reproducible in the country (including the sites they identify as under-studied) and other regions. For instance, they could address aspects such as that most of their samples came from wildlife rescue center as possible ways of improving and sustaining their surveillance program. In line 278-280 they even mentioned that they encounter obstacles, but do not mention how those obstacles were overcome. I suggest they proposed clearer steps moving forward, if they truly want to evaluate the implementation of this valuable wildlife surveillance program.

Response: The presentation about the results and discussion of the arguments was reformulated. In the current manuscript, the obstacles that the country faces for the implementation of a vigilance system in wild animals are developed. At the same time, we included recommendations that could be useful to overcome these difficulties and facilitate permanent implementation.

The text has some grammatical errors and redundant sentences, that makes the reading hard to follow. I mention some of these issues below, in addition to other observations.

Lines 50-53. Covid-19 would not make a good example. Although it may have a zoonotic origin, it is considered a human-to-human disease. May be consider another example.

Response: It is exemplified by covid-19 as a pathogen with possible origin in wild animals that can generate outbreaks. But this change was accepted. The change appears on lines 50-53:

“Examples of how these diseases can impact public health, animal health and wildlife have been the recent outbreaks of yellow fever and West Nile virus, which show the need to have infrastructure and diagnostic capacity to ensure a constant surveillance of potentially zoonotic agents.”

Lines 54-56. These two sentences seem redundant. Consider rephrasing.

Response: This change was accepted. The change appears on line 54: “Wildlife populations act as reservoirs and can play various roles in the epidemiology of numerous pathogens.”

Line 59. Consider changing “countries” for “regions”, and eliminating the “(including Costa Rica)”

Response: This change was accepted. The change appears on line 58.

Line 61. Consider changing “geographic regions” for “countries”

Response: This change was accepted. The change appears on line 60.

Line 64. Do you mean increased land use change?

Response: Yes, the change appears on line 63.

Lines 76-77. In that case, since they are post-mortem examinations, it would not be morbidity, right?

Response: Yes, morbidity was removed. The change appears on line 74.

Line 77-79. I don’t follow this sentence. What do you mean by “obtaining a sustainable tool that allows understanding the emerging potential of different pathogens.”? Also, consider changing “profitability” to “cost-effective” because the latter has a connotation of lucrative.

Response: This sentence was removed.

Line 88. A disease is not a synonym for pathogen, please change “vector-borne diseases” accordingly.

Response: This change was accepted. The change appears on line 87.

Lines 89-93 and 96-98. Please rephrase these sentences, as they are unclear and hard to follow.

Response: These sentences were rephrased. The change appears on lines 88-90:

“This reflects the urgency of establishing a permanent WHMP, where aspects such as general health status and monitoring of zoonotic pathogens in wildlife are considered, facilitating knowledge of the ecoepidemiology of these agents at the local level.”

Lines 99-101. Avoid giving specific results here (e.g., 85 carcasses), just general and main findings.

Response: This change was accepted. The change appears on lines 95-99.

Line 108. Isn’t the permit R-SINAC-PNI-ACLAC-039 for the Area de Conservacion La Amistad Caribe only?

Response: Yes, by mistake we only included one. The change appears on lines 102-106: The study was approved by the Ministry of Environment and Energy (MINAE) (wildlife authority) through permits (R-SINAC-PNI: -ACAT-040, ACAHN-18, ACTo-022, ACT-OR-DR-43, ACG-026, ACLAC-039, ACLAP-023, ACOPAC-005, ACC-037), and with the support of the animal health authority, the National Animal Health Service through the office (SENASA-DG-0277-18).

Table 1. superscript b. Cloacal swab is not a tissue, please change accordingly.

Response: The change appears on the Table 1. “Lung and Trachea tissue and cloacal swab.”

Lines 167-168. Please change “specimens of the birds and 76 (89.5%) of the mammals” to “bird and 76 (89.5%) mammal specimens”.

Response: These sentences were rephrased. The change appears on lines 179-184:

“According to the taxonomic order, we received: 29.4% (25/85) Carnivora, 29.4% (25/85) Primate, 12.9% (11/85) Pilosa, 5.9% (5/85) Didelphimorphia, 4.7% (4/85) Rodentia, 4.7% (4/85) Artiodactyla, 2.3% (2/85) Cingulate, 2.3% (2/85) Pelecaniformes, 2.3% (2/85) Accipitriformes, 2.3% (2/85) Anseriformes, 1.2% (1/85) Ciconiiformes, 1.2% (1/85) Piciformes and 1.2% (1/85) Coraciiformes.”

Lines 170-173. This information doesn’t seem to belong in this subsection but rather in either of the following ones.

Response: This sentence was removed.

Lines 178-181. 24 + 3 + 1 + 4 = 32, not 31 as stated below. Please check this. Also, please be specific on what are you referring to with “parasites”.

Response: These data were reviewed, certainly there are 32. The change appears on lines 196-198: “Of the individuals with a cause of traumatic death, 67.4% (32/85) concomitantly presented some infectious agent with or without associated disease (24 with gastrointestinal and pulmonary parasitic worms, three with bacteria, one with protozoa and four with multiple microorganisms).”

Lines 184-193. I am confused with this whole paragraph and the data presented below (including in Table 2). I imagine that you have several tissue samples from the same carcass, because you had 85 individuals total, but are now presenting 266 lesions total (199 + 67). Please clarify.

Response: Yes, lesions of different tissues were identified in each carcass. However, this paragraph and Table 2 were removed.

Lines 195-196. 11 + 5 + 4 = 20, it doesn’t add to 21. Please check this.

Response: This paragraph was removed.

Lines 199-200. Do you mean that the rest of the tissues with infection lesions were associated with a pathogen? And what is the complementary analysis?

Response: This paragraph was removed.

Table 2. I suggest you change “Bird” for “Aves”, to keep the correct taxa name for all the groups sampled.

Response: Table 2 includes the correct taxonomic name at the order level of the different groups sampled.

Lines 241-242. Do you mean in here that all the birds, total of nine, were screened for viruses? From which, only the two that were involved in a mass mortality event were positive for flaviviruses? Please rephrase accordingly.

Response: This sentence was rephrased. The change appears on line 230:

“All birds submitted were evaluated for virus presence (n=9), two of these were positive for flaviviruses.”

Line 300. Rephrase to: Regarding mammals, carnivores and primates were the taxa with higher representation

Response: The change appears on line 291.

Lines 302-304. It is unclear what do you mean with this sentence. Are you suggesting that human proximity makes transmission of this infectious agents more common, or that the sole proximity facilitates pathogen detection? Also, it could be possible that cases of more charismatic animals are over-represented.

Response: This paragraph was rephrased. The change appears on lines 291-295:

“Carnivores and primates were the taxa with higher representation. These data can be associated with the fact that they are medium to large-sized animals, more charismatic and with a more significant contact of these species with human environments, facilitating the recognition of morbidities and mortalities by the population. These taxa should be prioritized in the WHMPs, since it allows to optimize the use of resources because, in addition to their easy detection, they are taxa in which various pathogenic agents can circulate.”

Lines 305-306. Anthropogenic effect it’s a broad term. Can you rephrase and be more specific?

Response: This paragraph was removed.

Lines 313-315. What do you mean by human conditions? Also, I am not sure if you are using the term “evolve” correctly here. Do you mean pathogen evolution or how the disease develops?

Response: Yes, reference was made to how the disease develops. However, this paragraph was removed.

Lines 331-332. This sentence is very vague. Please rephrase and be more specific.

Response: These sentences were rephrased. The change appears on lines 315-316:

“We also detect vector-borne diseases, which are recognized as agents with epidemic potential in Latin America due to the climatic, health and socioeconomic conditions of tropical regions that favor their spread.”

Line 335. Please change “(CoastLine)” to “by the coast”.

Response: The change appears on line 318.

Lines 336-340. What about conservation implications? What if this pathogen detection reveals the transmission of pathogens from domestic animals to wildlife?

Response: It is a risk to public health and to the conservation of species. The cases were in endemic regions with high transmission of these pathogens in domestic animals. This is mentioned on line 318-320.

Line 349 (and others) Please be consistent with the naming. If you used “CDV” at the beginning after explaining what it stands for, you should keep using the same name. Also, include the genus information at the beginning, where you mentioned the pathogens you are going to be screening for.

Response: The change was accepted. This was corrected throughout the manuscript.

Line 353. Please change “studies” to “study”.

Response: This change was accepted. The change appears on line 327.

Lines 355-356. Can you provide a reference for this estimation?

Response: No, it was corrected on lines 331-332:

“Costa Rica does not have official data on the domestic dog population”.

Line 363. With “productive animal” do you mean livestock?

Response: This change was accepted. The change appears on line 348

Line 379. Replace “the Nasua narica species” to “Nasua narica”

Response: This sentence was removed.

Figure 3. Giving that you mentioned that samples are biased toward the wildlife management centers, it would be informative to show in the map the location of the centers that sent the carcasses. If there is no clear purpose for the color coding on the map (I imagine indicating elevation), I suggest removing it, because it makes the figure harder to interpret. Finally, consider using other type of icons to show the pathogens detected that are not 3D.

Figure S1. This map was very noisy and hard to interpret. Consider simplifying as much as possible. May be to order, instead of genus, and removing the elevation color.

Response: The figures corresponding to the maps were edited. The Color coding shows the different conservation areas; however, this was previously not specified. The location of the participating rescue centers was included.

Please check your references. Scientific names should be in italics and there are a few mistakes, such as in Line 534, where Panama and Costa Rica are not capitalized.

Response: References were checked and corrected.

Reviewer #2:

General comments: Initially I would like to congratulate Dr. Alfaro-Alarcon for this manuscript, we know that research efforts to develop studies like the one presented here require a network of motivated institutions and people, this can be evidenced in this manuscript. So please feel proud of this manuscript fruit of his continuous work for almost three years. Congratulations.

This MS is a bit difficult to evaluate, since it could have two different perspectives:

1. Formulation and implementation of the pilot of a Wildlife Health Monitoring Program (WHMP).

2. A study of causes of death and main pathological processes in various species of vertebrates.

My review is in order to analyze this MS with the postulating of WHMP. I strongly believe that this is a manuscript that with some revisions can be used as an example for the installation of a wildlife health surveillance program. Below I respectfully highlight some points that should be considered by you.

I initially consider that some infectious agents should be considered as "primary" for monitoring. I believe that the rabies virus and the flaviviruses would be good pathogens in mammals and influenza and the New Castle virus in birds. These diseases must be carefully selected since they will be the ones that will generate the demand for a national program by the responsible agency. For the other pathogens, I consider appropriate the use of complementary techniques based on histopathological analysis (CDV, bacterial).

In order to replicate this program, I felt a huge lack of a flowchart in the methodology describing each of the institutions with their respective activities.

Response: “Primary” agents were established for monitoring, but it was not indicated. The flowchart specifies the surveillance scheme for the different infectious agents (microbiological diagnosis based on pathological findings or routine diagnostics), and the role of each participating institution.

I understand that all the necropsies were carried out by the veterinary pathologists of the university involved in this study, but in order to improve the efficiency and effectiveness of this program, they do not believe that it would be better if the necropsies and sample collection were carried out directly in the institution (in this way the freezing of corpses would be avoided, which improves the histopathological evaluation).

Response: This is one of the measures proposed to improve the sustainability of the WHMP. This change appears on lines 281-290.

The carcasses that were found in the "forest" can really provide insight into the pathogens that are circulating in the ecosystem. However, animals that have entered a rescue center may be exposed to pathogens that are stabilized on the installations. Considering this, was there any inclusion criteria for animals from rescue centers, for example, animals that arrived dead or that lasted a maximum of 24 hours in the institution?

Response: Exclusion criteria were not mentioned above. Lines 112-114 included the previously established exclusion criteria:

“Carcasses of animals that remained more than 48 hours in the management centers before death were excluded also those that received medication, and those carcasses that were frozen for more than a week when it was necessary to store them.”

I think that seeking to create a diagnostic algorithm within the wildlife health program that you propose, perhaps a classification of causes of death like this could be better:

• Death associated with an infectious agent.

• Death not associated with an infectious agent, with a pre-existing infectious disease.

• Death not associated with an infectious agent, with a molecular diagnosis of an infectious pathogen.

• Death not associated with an infectious agent.

• Undetermined death.

With these categories you could see which species may be sensitive (which would be ideal for passive surveillance by acting as sentinels) and which are resistant (which would be ideal for serological inquiries) to the selected pathogens. I believe that in order to establish a wildlife health surveillance program, particularly in countries with limited resources, it is important to prioritize efforts, to those pathogens of greatest importance to human health (based on epidemiological data from the Health Agency), as well as for biodiversity conservation (based on wildlife mortality data).

Response: Thanks for the observation. This algorithm was used. This information appears in Table 3.

I confess that when presenting absolute and proportional results I find it interesting to use the following form: X% (n/N). It is not something that they should replicate but if you find it interesting it would be great.

Response: This change was accepted. The data is displayed in this format.

Introduction: Despite being a somewhat extensive introduction (for me), I consider that addressing the points that will be dealt with in the manuscript, I make some specific comments in the minor revision section.

MM: If you accept my comments please describe the WHMP better, I have no objection to the proposed diagnostic methodologies. I am sure that you are quite judicious about it.

Response: To better describe the WHMP, a flowchart has been included detailing the role of each participating institution and the communication of the results obtained.

Results and discussion: As things change in the methodology you will see that the results can be written in another way, I will make two suggestions for tables, and please when you are writing the results do not forget that the focus of the manuscript is not pathology, it is the implementation of the WHMP. Please, be a little more summarized in the pathological aspects (S2 table is enough) and increase the information about the program. I make this same comment for discussion. Remember that discussing whether or not the non-infectious cause of death is frequent, for the purpose of this manuscript, may be secondary. But discussing the limitations, benefits, diagnostic techniques, costs, can be much more interesting.

Tables:

If the authors consider my suggestions, Table 1 should include the absolute and relative values of the causes of death for each of the taxonomic groups, that is:

Cause of Death / Taxon DAIA DNAIA-PD DNAIA-ADT DNAIA UD

Mammals % (n/N) % (n/N) % (n/N) % (n/N) % (n/N)

Carnivores % (n/N) % (n/N) % (n/N) % (n/N) % (n/N)

Birds % (n/N) % (n/N) % (n/N) % (n/N) % (n/N)

Pelecaniformes % (n/N) % (n/N) % (n/N) % (n/N) % (n/N)

DAIA= Death associated with an infectious agent; DNAIA-PD= Death not associated with an infectious agent, with a pre-existing infectious disease; DNAIA-ADT= Death not associated with an infectious agent, with an additional diagnostic tool; DNAIA= Death not associated with an infectious agent; UD= Undetermined death.

Response: These changes were accepted, and due to the new way of presenting the results, there is a new Table 2 with new information. The pathological aspects were considerably reduced, the table 2 and the paragraphs that indicated the results by lesions were removed. The same was done with the discussion. This was a great observation to improve the quality of the manuscript with respect to WHMP.

Table 1 include the pathogens diagnosed with the respective techniques.

Table 3 was included for additional information about the program.

Tables 4 and 5 were already in the manuscript. Agents detected by birds and mammals were divided because it is impossible to include them all in a single table.

S1. Table: please add the scientific name of all specimens.

Response: This change appears in table S1.

Figures:

Please homogenize the formatting of the figures, if you are going to place a scale bar, place them in all of them, otherwise, remove them in all of them. In the case of metazoan parasites, describe if they were identified morphologically and/or molecularly, otherwise if they were by tissue characteristics, mention which guide was used (eg, Gardiner or Chitwood), and describe the order and/or the superfamily, and later if they want they can mention the potential genus (explaining that this genus is postulated by previous accounts in the species or close taxonomic group). I confess that I feel a lack of macroscopic photos. but they are not necessary

Response: This change was accepted, all scale bars were eliminated. Also, after the histopathological description of each microphotograph, the diagnostic technique used is indicated.

Figure 1.

A. To exemplify the cases of CDV in none of the animals were inclusion corpuscles observed? If these were observed, my suggestion is to place a photo of one of them. The caption provided describes the image well.

Response: The purpose of this figure was to show inflammatory-type lesions. However, with the reduction of the pathological component and with the reformulation of the results. This figure was removed.

B. The inflammatory component is not fully observed in the microphotograph, I confess that it was a bit difficult for me to show the granulomatous component (If it is possible to take another microphotograph where it is more evident, it is highly recommended), otherwise, I recommend modifying the legend of this Photo.

Response: With the reduction of the pathological component and with the reformulation of the results. This figure was removed.

C. In this microphotography it is not possible to show the Trypanozoma amastigotes, the myocardial inflammatory process is well described in the legend, my suggestion is to place a microphotograph at a higher magnification (eg 400x or 1000x) only of the amastigote, if you have a field where are inside the myocardial fiber would be great.

Response: With the reduction of the pathological component and with the reformulation of the results. This figure was removed.

D. I strongly recommend taking a photomicrograph that expresses the necrotizing component of hepatitis, otherwise you should change the caption on this photo.

Response: With the reduction of the pathological component and with the reformulation of the results. This figure was removed.

E. In my opinion if you don't have a potential cause for this injury, I recommend removing it.

Response: With the reduction of the pathological component and with the reformulation of the results. This figure was removed.

F. I recommend removing this photo, since initially this is not a finding associated with an infectious component.

Response: With the reduction of the pathological component and with the reformulation of the results. This figure was removed.

Figure 2.

A. Surely you analyzed it, but to exemplify toxoplasmosis I think a microphotograph of a liver with tachyzoites and the lobular inflammatory and necrotizing process would be much more illustrative. If this was not possible, a pity, the photograph and the legend are fine, just a change, according to Dubey in the latest updates on toxoplasmosis, the term that should be used is no longer pseudocyst, now tissue cyst should be used.

Response: This change was accepted, figure 2 now becomes figure 3. The previous microphotograph was changed for a lung microphotograph where the inflammatory process and the presence of the tissue cyst are observed.

B. They should be proud of their histotechnician, those histological cuts are excellent!!!! I only recommend not mentioning the type of inflammatory cell since at this magnification it is not possible to identify them (even zooming in on the image).

Response: This change was accepted.

C. Excellent microphotography, the marvelous lesion!!! (poor primate), just a recommendation, we know that by culture you are sure of the bacteria, but in the histopathological slide my suggestion is to report with intralesional bacteria (Klebsiella pneumoniae by culture), if you want to place an insert I would suggest placing a photo of it histological gram field. Otherwise, I would remove the inset.

Response: This change was accepted. The inset was removed.

D. These lungworms were identified morphologically and/or by molecular techniques, or solely by the characteristics of the worm in the tissue. if it was solely for tissue characteristics please don't use gender, as we know that morphological keys are only suitable up to order or superfamily. It would be interesting to mention that in this cut they have both males and females (many more cuts of the female), since this helps to understand the cycle of this beautiful nematode. As in photo B, due to the magnification, it is not possible to identify the inflammatory cells.

Response: All nematodes were collected and identified by morphological characteristics until the gender level. This information appears in the current methodology on lines 154-158:

“All the parasites present in the carcasses were collected and washed with physiological saline, preserved in alcohol, acetic acid, and formalin (AFA) solution. No more than one week after collection they underwent identification to the genus level through morphometric characteristics. Physical and morphometric characteristics were recognized after fixation and clarification with Hoyer's solution by light microscopy. In addition, processed cestodes were stained with dilute Harris' hematoxylin solution.”

Likewise, this information is detailed in the legend of Figure 3, and the inset was removed.

E. Same previous comment on classification, additionally in that photo the nematodes are not illustrated (in the same way as in the other microphotographs).

Response: This microphotograph was removed

F. This microphotograph is not very illustrative, the different layers of the tissue are not observed, in my opinion this photo could be removed.

Response: This microphotograph was removed

G. Same previous comment on classification, additionally I want to tell you that this microphotograph is very pretty. I suggest that in the legend they mention the fibroplasia component (which is evident) and that it helps the morphological diagnosis of this Nodular and sclerosing gastritis condition.

Response: This change was accepted. The morphological diagnosis was corrected.

H. The photo is very good, but it is nothing more than a photo of a sarcocystis, if it were associated with an inflammatory process, I would recommend including it in the manuscript, otherwise I recommend removing it.

Response: This microphotograph was removed

I. Excellent microphotography, I don't know if this condition is reported in that particular species, congratulations.

Minor comments

Line 28: please add space in “the detection”.

Response: This change was made.

Line 30: please remove a space (there are double spaced) between words “a solid”

Response: This change was made.

Line 47-53: Dear author, I would like to better understand your statement, it represents a burden for society in what sense (economic due to the overload of health systems, due to treatment costs, etc.). In the manuscripts that you are citing there is interesting material for you to formulate a sentence with greater impact. I fully understand that the pandemic caused by SARS-CoV 2 can seduce us to cite it as an example; however there are other diseases that can serve as an example and can generate impacts for both humans and wildlife (e.g., yellow fever, tuberculosis, rabies, influenza, West Nile Virus (interesting example in your specific case :D) etc.). Respectfully, I encouraged you to write a new sentence.

Response: This paragraph was rephrased. The change appears on lines 48-53:

“Zoonotic diseases represent a direct threat to public health systems, generating costs in terms of medical treatment, outbreak control and overloading health systems. In addition, it generates significant losses due to the slaughter of livestock and affectation of other domestic animals. Examples of how these diseases can impact public health, animal health and wildlife have been the recent outbreaks of yellow fever and West Nile virus, which show the need to have infrastructure and diagnostic capacity to ensure a constant surveillance of potentially zoonotic agents.”

Line 83-87: Dear authors, I considered that it would be interesting to add some additional information on the programs currently installed in some Latin American countries where wildlife is considered within the national surveillance programs (eg, non-human primates in yellow fever in Brazil [https://bvsms.saude.gov.br/bvs/publicacoes/guia_vigilancia_epizootias_primatas_entomologia.pdf]; in Argentina, game species are used to monitor some diseases such as trichinellosis [https://www.argentina.gob.ar/sites/default/files/informe_trichinellosis_2010_2019.pdf]; unfortunately those are the only two programs that I know of but with this information you could mention that there are some national programs installed and working perfectly where wild animals are used as sentinels for surveillance of some diseases).

Response: This change was accepted. The change appears on lines 80-82.

Line 139: change histological by histopathological.

Response: This change was accepted. The change appears on line 140.

Line 165: typing error “sender”

Response: This typing error was removed.

Line 166-167: I believe that in order to successfully replicate this study it would be interesting if the authors describe the criteria used to classify the age-range of the specimens.

Response: This information is included in the current methodology. The specimens were classified according to age, considering the development of the sexual organs and the phenotypic characteristics of the species. Because the classification by histological analysis of the teeth is really expensive. However, the juvenile specimens were very young, and it was easy to classify them.

Line 184-193: Does the number of 199 include all the organs evaluated in the pathology?

Response: Yes, all the organs evaluated in the pathology were included. Nevertheless, in the current version, this information was removed.

Final comments: Once again, I consider that this manuscript should be published since it brings a potential benefit to the scientific community that the readers of this prestigious journal will surely take advantage of. Obviously, the manuscript as it is at this moment needs some changes that I have no doubt that Dr. Alfaro-Alarcon and his team can make without any difficulty. I thank you for the opportunity to evaluate this important manuscript and I send you my best energies so that you can send it back with the suggested comments. Again, I remind you that I made this evaluation with great respect and my greatest critical sense so that you have a highly competitive manuscript.

Submitted filename: Rebuttal letter Response to Reviewers.docx

Click here for additional data file.

28 Jun 2022

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Daniel Becker

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: No

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #2: N/A

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors present a tool of passive surveillance that is both cost-effective and essential to monitor wildlife health and potential risk of spillover and spillback events. With their results, they demonstrate that despite the challenges developing countries face, there is a need to stay vigilant, as pathogens like Canine Distemper Virus and zoonotic gastrointestinal parasites were detected in their study. They also show and discuss important research gaps, such as the lack of information from certain areas in the country due to lack of resources and remoteness, and the misrepresentation of birds in their surveillance program.

In their first submission, the authors only presented their results without proposing a feasible and standardized method to implement this tool. In this version, they provide more detail (exemplified in figure 1), but I am afraid that their suggestions are very country specific. If this manuscript is accepted, I suggest that it would be under the study type “Methods, software, databases, and tools”, which should meet the criteria of utility, validation and availability (see https://journals.plos.org/plosone/s/submission-guidelines#loc-methods-software-databases-and-tools), and should be developed more broadly to propose a scheme for other countries with similar challenges.

Although, their epidemiological findings are interesting by themself, they are not the real focus of the paper, but rather are the evidence to stress the need to implement their proposed passive surveillance program in a sustainable manner with the tools that are already available. However, it seems like the authors shift the focus of their paper in presenting their findings. As an example, tables 3, 4 and 5 could be supplemental materials.

Finally, I found the manuscript difficult to follow. Occasionally, the sentences read awkward (e.g., Lines 50-53), had typos (e.g., Line 55) or wrong terms (e.g., “academy” in Lines 312-314), were grammatically incorrect (e.g., Line 80-82 and Line 95-97), or lacked a clear structure (e.g., Lines 108-112). The authors should do some intense editing to their manuscript, with a more concise language, and, if available, consider asking for English-proof reading services.

Reviewer #2: Dear authors, Initially, I want to express my good energies to you, for accepting the comments made and implementing them in an appropriate way in the manuscript. I confess that the manuscript with the modifications made is much clearer and meets the proposed objectives. A few comments below.

1. I recommend the use the following categories: metazoan parasites, protozoan parasites.

2. Please, double check the grammar of the text, mainly in the introduction, in general the English could be a little more fluent. However, I emphasize that this does not affect at all the understanding of the ideas they expose.

Minor comments:

Line 5: delete of (repeated)

Line 197: change pulmonary parasitic worms to lungworm.

Table 4: change parasitic worms to metazoan parasites.

For my part, I have no further comments, I believe that this manuscript will be of great importance to show feasible examples of disease surveillance in wildlife in Latin America. Once again congratulations Dr. Alfaro and coatures.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Pedro Enrique Navas-Suárez

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

11 Aug 2022

Reviewer 1

In their first submission, the authors only presented their results without proposing a feasible and standardized method to implement this tool. In this version, they provide more detail (exemplified in figure 1), but I am afraid that their suggestions are very country specific. If this manuscript is accepted, I suggest that it would be under the study type "Methods, software, databases, and tools", which should meet the criteria of utility, validation and availability (see https://journals.plos.org/plosone/s/submission-guidelines#loc-methods-software-databases-and-tools), and should be developed more broadly to propose a scheme for other countries with similar challenges. Although, their epidemiological findings are interesting by themself, they are not the real focus of the paper, but rather are the evidence to stress the need to implement their proposed passive surveillance program in a sustainable manner with the tools that are already available. However, it seems like the authors shift the focus of their paper in presenting their findings. As an example, tables 3, 4 and 5 could be supplemental materials.

R/ We thank reviewer #1 for the constructive and helpful comments. The reviewers' suggestion to standardize a general passive wildlife surveillance scheme for countries with limited resources is an exciting objective. However, we consider that that idea exceeds the scope and intentions of this research. This manuscript aims to communicate our experience of what could be the establishment of a passive epidemiological surveillance system in the wildlife of Costa Rica as a model. This study is a pilot effort with the intention that the scientific community of other countries can visualize what aspects should be considered when aspiring for a surveillance system for wildlife animals. We believe that the current scope of this study can be helpful to countries pursuing this goal.

We also want the manuscript to illustrate aspects such as the type of agents detected and how to approach their diagnosis based on the available diagnostic capacities. This is essential in countries with low income dedicated to epidemiological surveillance of wildlife. In addition, we want to highlight the importance of linkage among human, animal, and wildlife health authorities and higher education institutions under the one health approach. This is based on the urgent need to raise awareness of the benefits of this type of disease monitoring for the countries and its regional and global impact.

Based on this study's limitations, we have recognized the weaknesses in lines: #357-365. However, the proposed program is limited to Costa Rica's resources and infrastructure and does not apply to every country. Therefore, we emphasize that it is important to start evaluating the implementation of these programs in regions where disease surveillance in wildlife is minimal based on experiences like ours. Furthermore, although it is necessary to standardize methods and techniques for monitoring pathogens in wildlife, the development of pilot schemes allows sharing experiences with other programs already installed.

In tune with the European Wildlife Disease Association and OIE, although efforts have been made to expand and unify surveillance programs dedicated to wildlife, significant variations persist. There are still variations in the scope, scale, and capacity for establishing surveillance schemes in many regions. These limitations are mentioned in lines: #372-375.

We are confident that in future studies, we will be able to compare our scheme with other WHMPs in the region to contribute to building these systems in other regions. In addition, other upcoming experiences would help standardize and optimize monitoring systems in the scientific community. Based on the reasons mentioned above and by recognizing the limitations of our study in the manuscript, we believe that the study should maintain the initially conceived structure and leave for future studies the pertinent approach proposed by reviewer 1.

Finally, I found the manuscript difficult to follow. Occasionally, the sentences read awkward (e.g., Lines 50-53), had typos (e.g., Line 55) or wrong terms (e.g., "academy" in Lines 312-314), were grammatically incorrect (e.g., Line 80-82 and Line 95-97), or lacked a clear structure (e.g., Lines 108-112). The authors should do some intense editing to their manuscript, with a more concise language, and, if available, consider asking for English-proof reading services.

R/We apologize for these mistakes. The English language and grammar were revised throughout the text, and the manuscript was improved to make it more concise and fluent.

Reviewer #2:

We would like to thank reviewer #2 for the positive comments

I recommend the use the following categories: metazoan parasites, protozoan parasites.

This was modified as requested

Please, double check the grammar of the text, mainly in the introduction, in general, the English could be a little more fluent. However, I emphasize that this does not affect at all the understanding of the ideas they expose.

The English language was revised throughout the text.

Minor comments:

Line 5: delete of (repeated)

This was corrected

Line 197: change pulmonary parasitic worms to lungworm.

This was corrected

Table 4: change parasitic worms to metazoan parasites.

This was corrected

Submitted filename: Response to reviewers.pdf

Click here for additional data file.

30 Aug 2022

5 Sep 2022

Departamento de Patología

Escuela Medicina Veterinaria

Universidad Nacional

Dr. Alejandro Alfaro, Ph.D.

alejandro.alfaro.alarcon@una.cr

Heredia, September 5, 2021

To

Academic Editor

Reviewers

PLOS ONE

Dear academic editor, dear reviewers,

We would like to thank you all for your observations which will improve our paper. We hope this article would be published soon, the published information could be of great value for wildlife scientists.

Journal Requirements

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

R/ References were checked:

References #23, #93 and #103 have been replaced with current and relevant references.

References #5, #7, #70 and #73 were removed as they were not relevant.

Reviewer 1

I want to congratulate the authors, not just for the relevance of their paper and the amount of work they did with the implementation of the passive surveillance, but also for the great improvement they made to present their findings, implications and recommendations. I just have a few very minor comments that will be easily corrected.

We would like to thank reviewer #1 for the positive comments.

L34-36: “For instance, 60% (51/85) of the deaths were not associated with an infectious agent. In 67.4% (32/85) of the cases, deaths were not associated with an infectious agent, but an infectious agent was detected.”

These two sentences seem to be repeating the statement, and there seems to be a mistake: 32/85 is 37.6%, not 67.4%. Please check this. Same error in L191.

This was corrected. Line 34-35: “For instance, 60% (51/85) of the deaths were not directly associated with an infectious agent. Though in 37.6% (32/85) of these cases an infectious agent associated or not with disease was detected.”

Line 191: the percentage was corrected.

L37: The acronym “WHMP” is mentioned here, without first stating what does it stand for.

This was corrected.

L84: Consider removing the acronym “LMIC” since it is not used in the rest of the paper, and it is confusing.

This was modified as requested.

Sincerely yours,

Dr. Alejandro Alfaro-Alarcón Ph.D.

Departamento de Patología

Escuela de Medicina Veterinaria

Universidad Nacional

alejandro.alfaro.alarcon@una.cr

Submitted filename: Response to Reviewers.pdf

Click here for additional data file.

11 Sep 2022

Passive epidemiological surveillance in wildlife in Costa Rica identifies pathogens of zoonotic and conservation importance

PONE-D-21-39637R3

Dear Dr. Alfaro-Alarcon,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Daniel Becker

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

14 Sep 2022

PONE-D-21-39637R3

Passive epidemiological surveillance in wildlife in Costa Rica identifies pathogens of zoonotic and conservation importance

Dear Dr. Alfaro-Alarcón:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Daniel Becker

Academic Editor

PLOS ONE