Accepted Manuscript

Filarial nematodes with zoonotic potential in ring-tailed coatis

(Nasua nasua Linnaeus, 1766, Carnivora: Procyonidae) and
domestic dogs from Iguaçu National Park, Brazil

Marcela Figuerêdo Duarte Moraes, Marina Xavier da Silva, Paulo

Cesar Magalhães-Matos, Ana Cláudia Alexandre de Albuquerque,
José Hairton Tebaldi, Luis Antônio Mathias, Estevam G. Lux
Hoppe

PII: S2405-9390(16)30061-2
DOI: doi: 10.1016/j.vprsr.2017.01.003
Reference: VPRSR 60
To appear in: Veterinary Parasitology: Regional Studies and Reports
Received date: 22 April 2016
Revised date: 6 January 2017
Accepted date: 14 January 2017

Please cite this article as: Marcela Figuerêdo Duarte Moraes, Marina Xavier da Silva,
Paulo Cesar Magalhães-Matos, Ana Cláudia Alexandre de Albuquerque, José Hairton
Tebaldi, Luis Antônio Mathias, Estevam G. Lux Hoppe , Filarial nematodes with zoonotic
potential in ring-tailed coatis (Nasua nasua Linnaeus, 1766, Carnivora: Procyonidae) and
domestic dogs from Iguaçu National Park, Brazil. The address for the corresponding
author was captured as affiliation for all authors. Please check if appropriate. Vprsr(2017),
doi: 10.1016/j.vprsr.2017.01.003

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 ACCEPTED MANUSCRIPT

Filarial nematodes with zoonotic potential in ring-tailed coatis (Nasua nasua Linnaeus,
1766, Carnivora: Procyonidae) and domestic dogs from Iguaçu National Park, Brazil

Marcela Figuerêdo Duarte Moraes, Marina Xavier da Silva, Paulo Cesar Magalhães-Matos, Ana
Cláudia Alexandre de Albuquerque, José Hairton Tebaldi, Luis Antônio Mathias, Estevam G.
Lux Hoppe

Marcela Figuerêdo Duarte Moraes

Universidade Estadual Paulista - Unesp, Faculdade de Ciências Agrárias e Veterinárias,
Departamento de Medicina Veterinária Preventiva e Reprodução Animal. Via Prof. Paulo
Donato Castellane km 05, CEP:14884-900, Jaboticabal, SP, Brasil

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Marina Xavier da Silva
Projeto Carnívoros do Iguaçu. Parque Nacional do Iguaçu, BR 469, km 22,5, CEP: 85570-970,

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Foz do Iguaçu, PR, Brasil
Paulo Cesar Magalhães Matos

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Universidade Federal Rural do Rio de Janeiro, Instituto de Veterinária, Departamento de
Ciências Veterinárias. BR – 465, km 07, CEP:23.890-000, Seropédica, RJ, Brasil.
Ana Cláudia Alexandre de Albuquerque
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Universidade Estadual Paulista - Unesp, Faculdade de Ciências Agrárias e Veterinárias,
Departamento de Medicina Veterinária Preventiva e Reprodução Animal. Via Prof. Paulo
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Donato Castellane km 05, CEP:14884-900, Jaboticabal, SP, Brasil

José Hairton Tebaldi
Universidade Estadual Paulista - Unesp, Faculdade de Ciências Agrárias e Veterinárias,
Departamento de Medicina Veterinária Preventiva e Reprodução Animal. Via Prof. Paulo
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Donato Castellane km 05, CEP:14884-900, Jaboticabal, SP, Brasil

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Luis Antônio Mathias

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Universidade Estadual Paulista - Unesp, Faculdade de Ciências Agrárias e Veterinárias,

Departamento de Medicina Veterinária Preventiva e Reprodução Animal. Via Prof. Paulo
Donato Castellane km 05, CEP:14884-900, Jaboticabal, SP, Brasil
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Estevam G. Lux Hoppe

Universidade Estadual Paulista - Unesp, Faculdade de Ciências Agrárias e Veterinárias,
Departamento de Medicina Veterinária Preventiva e Reprodução Animal. Via Prof. Paulo
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Donato Castellane km 05, CEP:14884-900, Jaboticabal, SP, Brasil

Tel: +55 (16) 32097338 / Fax: +55 (16) 32097338
e-mail: hoppe@fcav.unesp.br; e.hoppe@gmail.com

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Abstract: Iguaçu National Park, which contains the largest remnant of Atlantic Forest in

southern Brazil and Iguaçu Falls as one of its main visiting points, is a year-round attraction for

thousands of domestic and international tourists. Ring-tailed coatis are generalist, opportunistic

carnivores that benefit from human association. These animals are the most abundant

carnivores in this park, where they come into close contact with tourists and the resident

population around the park. Moreover, as the park is surrounded by human dwellings, free

roaming domestic dogs are frequently present, favoring the exchange of pathogens with wild

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animals. Wild carnivores are known to be infected with several pathogens, from viruses to

arthropods, some of them passed on by domestic carnivores. Among the nematodes that infect

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wild carnivores, those of the Onchocercidae family are of concern due to their zoonotic

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potential. The objective of this research was to assess the prevalence of filarial nematodes in

coatis and dogs in Iguaçu National Park, as well as the potential impact of infection on host
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health and body condition. To this end, 75 coatis and 50 adult dogs were captured in the

aforementioned area. Seven species of filarioids, Dirofilaria immitis, Dirofilaria repens,

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Acanthocheilonema reconditum, Brugia sp., Mansonella sp. and two undetermined species,

confirmed by histochemical tests, were diagnosed in the area. Knott’s concentration test in

coatis and dogs showed a prevalence of 81.6% and 16.0%, respectively. Seven microfilarial
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morphotypes were diagnosed in the coatis and two morphotypes were found in the dogs. A
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specific immunoassay test for Dirofilaria immitis revealed the presence of D. immitis infection in
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1.33% of the coatis and 22% of the domestic dogs. The parasitic infection had negligible effects
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on the body condition of both dog and coati hosts, but an increase was found in eosinophil

counts in coatis with filarial infection, as well as decreases in hematocrit and hemoglobin in the
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infected domestic dogs. These findings represent new locality and host records for all the

filarioids diagnosed in the area of this study, with some of the diagnosed parasites, given their

zoonotic potential, representing a health risk for park visitors and the local population.

Keywords: Histochemistry, Knott’s test, Filariasis, host-parasite interactions, Atlantic rainforest.

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1. INTRODUCTION

Iguaçu National Park contains the largest remnant of Atlantic Forest in southern Brazil.

Along with the Iguaçu National Park in Argentina, these areas form a green corridor between

the two countries that protects endangered species, such as the southernmost population of

jaguars in Brazil (Instituto Brasileiro de Desenvolvimento Florestal, Brasília, 2000). Furthermore,

Iguaçu Falls are located in this park, attracting thousands of visitors from around the world.

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When people enter the park, they become vulnerable to possible contact with vectors and

pathogens associated with the local wildlife, since most of these visitors spend an average of

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four days in areas contiguous to the park, increasing the risk of contact with possible vectors of

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these parasites (Secretaria Municipal de Turismo de Foz do Iguaçu, Foz do Iguaçu, 2012).

Among the carnivores that inhabit Iguaçu National Park, the ring-tailed coati (Nasua
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nasua Linnaeus, 1766) is the most abundant and most seen by tourists because of their diurnal,

gregarious and scansorial habits (Trovati, 2004). These carnivores belong to the Procyonidae
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family and, as an opportunistic species, they often invade farms or houses in search of food,

favoring contact with domestic animals and humans, with whom they live sympatrically (Alves-

Costa et al., 2004).

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Of the several pathogens associated with wild carnivores (Anderson, 2000), four
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species of the family Onchocercidae have been reported to parasitize wild carnivores in Brazil
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(Vieira et al., 2008). Among these, only Dirofilaria immitis – a common parasite of dogs and an
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important emerging zoonotic disease, is relatively known, while the other species are

occasionally reported in taxonomic papers.

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Domestic dogs are the most abundant and widely distributed carnivores, acting as

carriers of several diseases, many of them of relevance to wildlife conservation and public

health (Cleaveland et al., 2006). Also, the probability of transmission of zoonotic diseases is

greater in areas where humans, domestic animals and wildlife are in close contact, even in the

absence of direct contact, since some pathogens can be transmitted by vectors (Osofsky et al.,

2005).

In view of the above, we assessed the diversity and prevalence of filarial parasites in

ring-tailed coatis and domestic dogs in Iguaçu National Park, an area with a strong interface

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between humans, domestic animals, and wildlife, and evaluated the influence of the parasitic

infection on the health of the hosts.

2. MATERIAL AND METHODS

2.1 Study Area

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The research was conducted at Iguaçu National Park, located in the southwest region of

the state of Paraná, Brazil (Figure 1), between the parallels 25°05' and 25°40' S and the

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meridians 54°30' and 54°40' W (Instituto Brasileiro de Desenvolvimento Florestal, Brasília,

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2000). The park contains 185,000 hectares of semi-deciduous forest.

The park is strongly influenced by anthropogenic action, since its surroundings have
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been transformed into agricultural landscapes for grain production, and to a lesser extent, for

dairy farming to meet the growing demand for food. The absence of a buffer zone between the
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park and farms favors the presence of wildlife outside the park and domestic animals, especially

dogs, inside the park. In addition, tourism has a negative impact on fauna because human

activity leads to behavioral changes on wildlife, with a greater presence of opportunistic species
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such as coatis in affected areas (Silva, 2014).

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2.2 Sample collection

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Between July 2014 and March 2015, 75 ring-tailed coatis were captured using baited
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Tomahawk traps and nets. The captured animals were chemically restrained with tiletamine and

zolazepam (5mg/kg, IM) for biometric and clinical examination. Each animal was individually

identified with a numbered ear tag. Blood samples were collected by jugular venipuncture and

fecal samples were collected from the rectum. The animals’ age was estimated as described by

Oliviers et al. (2010). Fifty free roaming dogs were sampled between July and December 2014,

on the 26 farms immediately adjacent to the park, with the owners’ permission. These animals

were physically restrained for blood sampling and clinical examination. Three free-roaming dogs

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were captured with a leash pole inside the park. The sampled domestic dogs were privately

owned dogs that were allowed to roam free close to and inside the national park.

2.3 Evaluation of body condition

The body mass index (BMI) was calculated based on the morphometric data obtained

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from the captured animals (Guimarães, 2009), in order to evaluate the effect of parasitism on

the body condition of the host.

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2.4 Parasitology diagnostics

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Knott’s concentration technique modified by Newton and Wright (1956) was employed
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to detect blood microfilaria. Preparations of the collected material were stained with May-

Grünwald-Giemsa (MGG) for later morphometric identification. Commercial immunodiagnostic

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kits (Witness Dirofilaria, Zoetis®), in combination with the aforementioned method, were used

for the detection of D. immitis antigens in serum samples from coatis and dogs.
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In addition to these tests, a histochemistry technique based on Peribánez et al. (2001)

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was used for microfilarial identification. This involved air-drying buffy coat smears of the positive
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animals and submerging them for 30 seconds in a citrate-acetone solution for fixation. After

drying at room temperature, the smears were stored at -20ºC until they were stained. The
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samples were immersed in May Grunwald-Giemsa (MGG) stain for one hour in a dark incubator

at 37°C. Subsequently, the slides were rinsed for three minutes in deionized water, immersed
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for five minutes in acid hematoxylin solution, and washed again for three minutes in deionized

water. The slides were dried at room temperature and kept away from light until they were read

in a light microscope.

The microfilariae were taxonomically identified from the combined MGG-stained

samples, histochemical test, and morphological and morphometric data.

2.5 Hematological and biochemical evaluation

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A complete blood count (CBC) was made on an aliquot of 3mL of blood in K3-EDTA.

The biochemical levels were determined from aliquots of 4 mL of blood collected in plain

vacuum tubes. Total protein concentration (TP, g/dL), albumin (Alb, g/dL), urea (NPN, mg/dL),

creatinine (CREA, mg/dl), and the enzymes glutamate oxaloacetate transaminase (GOT, U/L),

pyruvic glutamic transaminase (SGPT, U/L) and alkaline phosphatase (ALP, U/L) were
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measured using commercial kits (LABTEST Diagnóstica, Belo Horizonte, MG, Brazil). The
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readings were taken in a LabQuest automatic biochemical analyzer (LABTEST Diagnóstica,

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Belo Horizonte, MG). The globulin concentration was determined based on the difference

between the total protein and albumin levels (Eckersall, 2008).

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2.6 Data Interpretation and statistical analysis
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The animals were divided into two groups: "infected" and "uninfected,” considering the

uninfected group as the negative control in the health tests. To ascertain whether filarial
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infection interfered in the hematological and biochemical parameters, the normal distribution of

data was assessed using the Anderson-Darling test. Whenever the data was heteroscedastic,

logarithmic or radical transformation was employed to induce homoscedasticity (Sampaio,

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2010). When the data distribution was normal, the F test was applied for a comparison of
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variance, and in the case of equal variances, the t-test was employed for comparisons. If the
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data remained heteroscedastic even after transformation, the infected and uninfected groups

were compared using Wilcoxon’s nonparametric test (Siqueira and Tibúrcio, 2011). Linear and
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logistic regression analyses were also performed, as were all the statistical analyses. The R
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software was used for the calculations, with P adjusted to 0.05.

2.7 Ethical aspects

All the procedures adopted in this study were approved by the Comissão de Ética no

Uso de Animais FCAV/UNESP (under Protocol no. 07553/14) and are in accordance with

international standards. The captures were authorized by Sistema de Autorização e Informação

em Biodiversidade – SISBIO (under Protocol no. 38006-2).

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3. RESULTS

The capture efforts represented a total of 3,440 hours and resulted in the capture of 75

ring-tailed coatis: 39.8% (30/75) adults, 42.8% (32/75) subadults, and 17.4% (13/75) pups. In

addition, 50 adult domestic dogs were evaluated on visits to the homes surrounding the park.

These dogs were in poor body condition, with frequent presence of Rhipicephalus sanguineus

ticks and Ctenocephalides sp. fleas. Only 54% of these dogs were vaccinated against rabies,

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but 46% of them had been given at least one dose of anthelmintic medication during their life.

Seven species of filarial nematodes were identified in the captured animals (Figures 2 to

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6). The coatis were infected by seven nematode species: Dirofilaria repens, D. immitis,

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Acanthocheilonema reconditum, Mansonella sp., Brugia sp., and two undetermined species

(Figure 7). The dogs were parasitized by D. immitis and one undetermined species (Figure 8).
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Table 1 lists the prevalence rates of infection by each species of microfilariae in wild coatis and

dogs and Table 2 offers morphometric data and host-range of the observed microfilariae species
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in comparation to literature data. The histochemical tests were negative for domestic dogs due

to the lower microfilaremia levels.

Among the parasitized coatis, 56.6% had monospecific infections, 23.3% were
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coinfected with two species and 16.6% with more than two species of filarial nematodes. In
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domestic dogs, covert D. immitis infection was observed in 6% of the evaluated animals. No
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differences were observed between the prevalence of infection in adult and subadult coatis (p =
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0.5029), males and females (p = 0.3334), and pregnant and non-pregnant females (p = 0.5651).

Also, no effects of parasitism were observed in the body condition and biochemical tests of the
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evaluated coatis.

Of all the factors evaluated in the linear regression analysis, these pathogens only

showed an association with total protein and albumin (p=0.0340 and p=0.0153, respectively). As

for filarial infections, there was no interaction with any of the evaluated parameters, including

the host body condition. However, when age, filarial infection and eosinophil absolute counts

were considered in the same model, a strong association was noted between adults and

eosinophilia, which showed 11.6-fold higher values than among young infected animals

(p=0.0007). The subadults also showed this interaction, but only when a significance level of

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10% was taken in account, as the p value was 0.052. In this case, the subadults showed a 9.6-

fold higher chance of exhibiting eosinophilia than the young animals. In dogs affected with

filarioids, the only changes observed in the tests were lower hematocrit (p = 0.0430) and

hemoglobin (p = 0.0103) levels in comparison to those of negative animals.

4. DISCUSSION

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According to official data (Ministério da Saúde, Brasília, 2015), there are no recent

reports of the presence of human filarial infection in the state of Paraná. However, canine

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infections by these parasites are described in the coastal area of this state (Reifur, 2000), with

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prevalence rates ranging from 15 to 20%. The dogs evaluated in this study showed prevalence

rates of 5.47% for D. immitis, 22.6% for A. reconditum, and 4.68% for a third undetermined
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species. Because the two identified species are known to cause disease in humans, knowledge

about the epidemiology of filarial nematodes in domestic and wild animals is required, given
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their zoonotic potential and risk to domestic animal health (Rani et al., 2010).

It is important to note that all filarioids have indirect life cycles and that their vectors are

widely distributed in South America (Cerqueira, 1959; Cavallazzi et al., 2002; Garcez, 2006).
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The vectors of Dirofilaria and Brugia are Aedes, Culex and Anopheles mosquitoes, while fleas,
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ticks and lice are vectors of Acanthocheilonema. The vectors of Mansonella are Simulium and
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Cerqueirellum blackflies (Cerqueira, 1959; Cavallazzi et al., 2002; Simón et al., 2007; Garcez,
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2006).

The probable vectors of these parasites are well documented on both the Brazilian and
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Argentinian sides of Iguaçu National Park. Some of these species, even those that are not

synanthropic, were captured in peridomiciliary areas inside the park during feeding, suggesting

that human action could have led to behavioral changes in the insects (Guimarães et al., 2003;

Gil-Azevedo et al., 2005). The presence of two species of the genus Simulium, S. incrustatum

and S. subpallidum, has been reported on the Brazilian side, and of S. acarayense and S.

paraguayense on the Argentinian side (Gil-Azevedo et al., 2005). As for mosquitoes, 10

species of the subfamily Anophelinae, 17 species of the subfamily Culicinae, and 16 species of

the tribe Sabethini have been recorded in the area (Guimarães et al., 2003). Thus, the

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ecological conditions of Iguaçu National Park are favorable for maintaining potential vectors of

filariasis, and there is even a record of non-synanthropic species feeding inside buildings,

indicating the potential for transmission of diseases to humans.

The covert filarial infection observed in the dogs of this study is characterized by the

presence of adult parasites without microfilaremia, often related to monosexual infections, low

parasitic intensities or early infection (Nogami et al., 2000; Guilarte et al., 2011). Due to its

nature, this type of infection may be detected only in serological tests. The combined use of

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morphological and histochemical techniques is essential for the proper identification of

microfilariae, especially in wildlife, since the possibility of obtaining adult filarial worms from

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necropsies is rare, as is the chance of discovering novel undescribed species.

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The difference in prevalence rates obtained in this study may be due to the higher

sensitivity of the modified Knott test, as this technique requires the use of a larger blood volume
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(1 mL) than the histochemical methods (10 μL). The occurrence of D. immitis in dogs and coatis

may suggest that the parasite circulates between both host populations, creating an associated
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risk to human health. In addition, all canine cases of D. immitis in the surrounding areas are

autochthonous, since the owners stated that their dogs never traveled to other parts of the

country. It is worth noting that domestic dogs are considered reservoirs of D. immitis (McCall et
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al., 2008), which may suggest a possible spillover of this helminth between the two host
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populations under study.

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Dogs are one of the definitive hosts of D. repens and D. immitis, which were diagnosed
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in this study and are related to human disease (Labarthe and Guerrero, 2005). Cases of

infection by Dirofilaria may be more common than presently known in the country, given that
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many human cases are diagnosed by chance (Rodrigues-Silva et al., 1995; Dantas-Torres et

al., 2014). In addition, there may be other unknown filarial species parasitizing humans in Brazil,

as was observed in a case of intraocular dirofilariasis, in which the species was not determined

(Otranto et al., 2011). It is important to note that Dirofilaria infection was not previously

diagnosed in the studied area, implying that the distribution of dirofilariasis is, in fact, more

common than the literature suggests. With regard to D. repens, the increasing number of cases

indicates that this disease is an emerging zoonosis, contradicting some earlier notions that

classified human dirofilariasis as casual or incidental (Rodrigues-Silva et al., 1995; Pampiglione

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et al., 2001).

Another filarial nematode found in coatis, A. reconditum, is also a parasite of domestic

dogs, affecting subcutaneous tissues, perirenal fat, and the peritoneal cavity, and it is

occasionally linked to disease in humans (Magnis et al., 2013). The prevalence of infection by

this nematode in domestic dogs is still underestimated in Brazil, with only a few studies

reporting prevalence rates ranging from 0.6% to 22.6%, with the highest descriptors of infection

obtained in a study conducted in the coastal region of the same state where this study was

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conducted (Reifur et al., 2004).

The coatis evaluated here showed a high prevalence of Mansonella, 65.3%. Three

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species of this genus are known to be of zoonotic importance. One of the zoonotic species, M.

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streptocerca, occurs in Africa, and the other two, M. perstans and M. ozzardi, have been

recorded in South and Central America. Mansonella perstans was recently introduced in
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Northern Brazil by the migration of workers between states, while M. ozzardi is probably

autochthonous (Klei and Rajan, 2002). The adults of this genus are found in body cavities, with
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M. ozzardi preferentially located in visceral adipose tissue and M. perstans in the peritoneal

cavity and occasionally in the pericardial sac (Bogitsh et al., 2005). Considering the zoonotic

importance of some species of this genus, active monitoring of wildlife in the region is advisable,
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with emphasis on specific identification by molecular methods, in addition to morphological

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studies.
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The observation of Brugia infection in the animals of this study is remarkable, as these
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parasites had not previously been diagnosed in Brazil or in Paraguay and Argentina, both

countries with borders along Iguaçu National Park. A well known agent of lymphatic filariasis in
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humans, this genus of the family Onchocercidae was considered limited to Africa and some

parts of southern Asia, until the description of three American species. Two of them, Brugia

lepori and Brugia beaveri, were described parasitizing rabbits and raccoons, respectively, in the

USA, while Brugia guyanensis was discovered parasitizing coatis in Guyana (Orihel and

Beaver, 1989; Eberhard et al., 1993; Elenitoba-Johnson et al., 1996). In the USA, the frequency

of human infection with B. lepori was higher in rural or suburban areas, where the prevalence of

rabbits infected by this parasite ranged from 40% to 60% (Richardson and Krause, 2003). Given

that the frequency of infection by these species in the coatis under study in Iguaçu National

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Park was lower than in the American case, i.e., 8%, human cases should not be as frequent,

but there could be a risk to human health if the nematodes found here have a zoonotic potential.

Some host traits, such as age, sex and reproductive status, may influence parasitism,

affecting the prevalence and other descriptors of infection (Rossin et al., 2010). From the

hematological standpoint, one of the main alterations in infected animals is eosinophilia

(Ovington and Behm, 1997). However, the magnitude of the eosinophilic response is influenced

by the parasite species, since helminths that cause major tissue damage or have marked tissue

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migrations during their life cycles lead to more pronounced eosinophilia than parasites that are

limited to the gastrointestinal lumen (Amato Neto, 1970). Considering that filarial infections are

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essentially chronic and involve several immunological processes due to repeated exposure to

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the agent during inoculation by the vector, the penetration of the infective larvae, maintenance

of adult worms of considerable longevity, continuous release and migration of microfilariae, and
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significant increases in eosinophil counts are expected (Klion and Nutman, 2004).

Although this type of eosinophilic response is beneficial and responsible for mediating
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protective immunity against helminths, it can undoubtedly induce pathological reactions in the

host through the release of cationic or pro-inflammatory enzymes by activated cells. This

cellular mechanism ends up causing severe tissue damage due to the occurrence of
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eosinophilic infiltration, with consequent deleterious effects on the health and life expectancy of
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the long-term host (Couissinier, 2006).

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Dogs infected with D. immitis often present normal or slightly elevated liver enzyme
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activity, and low values of RBC, hemoglobin and hematocrit, characterizing normochromic

normocytic anemia (Ettinger and Feldman, 2003; Niwetpathomwat et al., 2007; Sevimli et al.,
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2007; Ranjbar-Bahadori et al., 2010). However, these hematological changes are nonspecific

and should not be taken alone to characterize filarial infection in animals.

Parasites of domestic animals were widespread throughout human migrations. Several

of these parasites adapted to their new environments and established stable ecological

relations (Klei and Rajan, 2002). Recently, due to environmental degradation and disruption of

natural ecosystems, these ecological relations have changed, leading to the emergence of

diseases, with humans acting as new or accidental hosts for these pathogens (Woolhouse et

al., 2001; Altizer et al., 2011). Therefore, the presence of zoonotic parasites both in wild and

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domestic animals in Iguaçu National Park indicates that, in addition to the current risk to human

health, environmental changes could lead to changes in the observed epidemiological patterns,

which could result in impacts on wildlife, domestic animals and human health.

Conclusion

This study describes the diversity of Onchocercidae parasites in wild coatis and

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domestic dogs in the area of Iguaçu National Park. Due to the zoonotic potential of these filarial

nematodes, active monitoring of human cases and wildlife health surveillance should be

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implemented in the region. The fact that the park is visited year-round by tourists from several

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parts of the world reinforces the need for active surveillance.

Acknowledgments
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We are grateful to the Fundação de Amparo à Pesquisa do Estado de São Paulo –
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FAPESP for its financial support (Process no. 2014/08180-0), and to the Carnívoros do Iguaçu

Project for its logistical support. The authors also wish to thank the anonymous reviewers for
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their helpful comments.

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Table 1. Prevalence of microfilariae identified by modified Knott’s, histochemical and serological

tests in wild ring-tailed coatis and domestic dogs from Iguaçu National Park

Table 2. Morphometric and host-range of the observed microfilariae species in comparation to

literature data.

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Figure 1. Geographic location of Iguaçu National Park, state of Paraná, Southern Brazil.

Figure 2. Microfilaria of Nasua nasua in Iguaçu National Park. Dirofilaria immitis: A. Knott Test:
coalesced cell bodies, curved tail and free headspace; B. D. immitis histochemical patterns:
activity in the excretory pore and anal pore (Chalifoux and Hunt, 1971); C. Schematic model of
acid phosphatase activity patterns for Dirofilaria immitis microfilariae. 40x magnification.

Figure 3. Microfilaria of Nasua nasua in Iguaçu National Park. Dirofilaria repens: A. Knott Test:
coalesced cell bodies at the front end, straight and abruptly tapered tail and without colors; B.
D. repens histochemical patterns: activity only in the anal pore (Balbo and Abate, 1972;
Radhika, 2005); C. Schematic model of acid phosphatase activity patterns for Dirofilaria repens
microfilariae. 40x magnification.

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Figure 4. Microfilaria of Nasua nasua in Iguaçu National Park. Acanthocheilonema reconditum:
A. Knott Test: robust and well defined cell bodies throughout the body, reducing to a row of

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three or four nuclei in the tail; B. Histochemical patterns for A. reconditum uniform or evenly
marking the body with less intense areas in the cranial to the excretory pore or with areas
marked strongly between the excretory pore and anal pore. (Chalifoux and Hunt, 1971; Vanveen

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and Blotklamp, 1978; Schrey and Trautvetter, 1978; Peribáñez et al., 2001); C. Schematic
model of acid phosphatase activity patterns for Acanthocheilonema reconditum microfilariae.
40x magnification.
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Figure 5. Microfilaria of Nasua nasua in Iguaçu National Park. Mansonella sp.: A. Knott Test:
microfilaria thin, spaced cell bodies spreading from the leading edge to the tail; B.
Histochemical patterns for Mansonella: studies on species of Mansonella (Tetrapetalonema) in
primates showed patterns of red blotches throughout the body (Chalifoux et al., 1973); C.
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Schematic model of acid phosphatase activity patterns for Mansonella microfilariae. 40x
magnification.

Figure 6. Microfilaria of Nasua nasua in Iguaçu National Park. Brugia sp.: A. Knott Test: sheath
left in the anterior and posterior end, the tail nuclei form a single row containing six to seven
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nuclei. B. Histochemical patterns for Brugia: There is no standard marking at all. Some authors
suggest that microfilariae of the genus Brugia may have either histochemically uniform labeling
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in the whole body or present only weak labeling on the surface of the microfilarial body
(Redington, 1975). C. Schematic model of acid phosphatase activity patterns for Brugia
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microfilariae. 40x magnification.

Figure 7. Unidentified microfilariae of Nasua nasua in Iguaçu National Park. A. Pattern 1:

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histochemical activity patterns in nerve ring and excretory pore; B. Pattern 2: histochemical
activity patterns at the anterior end, excretory pore and anal pore. This pattern was described by
Chalifoux (1973) parasitizing primates of the New World, but was also not identified.
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Figure 8. Microfilariae of domestic dogs in Iguaçu National Park. A. Morphotype 1: coalesced

cell bodies B. Morphotype 2: coalesced cell bodies, curved tail with hook-shaped nuclei and
free headspace. Fast Panoptic staining, 40x magnification.

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Table 1. Prevalence of microfilariae identified by modified Knott’s, histochemical and serological

tests in wild ring-tailed coatis and domestic dogs from Iguaçu National Park

FILARID SPECIES KNOTT HISTOCHEMICAL SEROLOGY

Dogs Coatis Dogs Coatis

Coatis Dogs

Dirofilaria immitis 10.6 16.0 1.33 0 1.33 22.0

(%)
33.2 0

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Dirofilaria repens 10.6 0 NA NA
(%) 12.0 0

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Acanthocheilonema 65.3 0 10.6 0 NA NA
reconditum (%)
8.0 0

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Mansonella sp. (%) 26.7 0 NA NA
0 10.0
Brugia sp. (%) 0 0 8.0 0 NA NA
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Not identified 1 (%) 5.3 0 NA NA

Not identified 2 (%) 4.0 0 NA NA

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* NA – Not applicable
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Table 2. Morphometric and host-range of the observed microfilariae species in

comparation to literature data.

1
FILARID TL Sheat A.P N. E.P Widt Histochemi Host Referenc
2 3 4
SPECIES h . R . h cal es
patterns
Dirofilaria 32 Abse - - - 7– A.P. Canis Tarello
repens 5– nt - - - 8.3 familiaris (2011)
37 Abse 41 45- 75 6– A.P. Canis Bucklar
5 nt – 66 – 8.0 familiaris et al.
26 Abse 64 93 4,9 Nasua (1998)

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0– nt – nasua Present
30 6,4 study
8
23

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8–
30
0

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Dirofilaria 20 Abse - - - 5– E.P. e A.P. Canis Bucklar
immitis 5- nt - - - 6.5 familiaris et al.
28 Abse - - - - Canis (1998)
3 nt - - - 5.0 familiaris Kelly
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28 Abse - - - 5.4- Mustela (1979)
6- nt 53 - 44- 69 - 6.5 E.P. e A.P. nigripes Wisely et
34 Abse 55 51 87 - Canis al. (2008)
9 nt 39 - 22- 69- 4,1- familiaris Fuellebor
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16 Abse 65 29 80 5,2 Canis n (1912)

5- nt 4,2- familiaris Newton
18 Abse 4,9 Nasua & Wright
5 nt nasua (1956)
23 Abse Canis Present
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3- nt familiaris study
27 Present
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0 study
30
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7-
32
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0-
25
8
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23

5-

25

Dirofilaria 23 Abse - - - 2.0- - Lynx Orihel &

striata 0- nt 3.0 rufus Ash
24 (1964)
0
Dirofilaria tenuis 22 Abse - - - 2.0- - Procyon Orihel &
0- nt 3.0 lotor Beaver
25 (1965)
0

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Dirofilaria 29 Abse 165 47- 66- 5.0 - P. Eberhard

cancrivora 9- nt - 61 85 cancrivor (1978)
31 205 us
0
Acanthocheilon 21 - - - - Canis Whiteley
ema reconditum 3- - - - - 4– Whole familiaris (1988)
24 - - - - 5.0 body Canis Kelly
0 - 36 - 19- 38 - 4.3- - familiaris (1979)
25 41 26 51 4.8 Whole Canis Newton
8- 5,4 body familiaris & Wright
29 – Nasua (1956)
2 6,1 nasua Present
26 study

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9-
28
3
19

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9-
24
2

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Acanthocheilon 18 - - - - 3.0- - Mephitis Webster
ema mephitis 6- 3.8 mephitis &
21 Beaureg
8 ard
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(1964)
A. procyonis 13 Abse 115 34- 48- 3.9- - Procyon Smith
3- nt - 40 56 4.9 lotor (1980)
15 136
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6
Brugia 21 Prese 86. 25. 31. 4– - Nasua Orihel
guyanensis 3- nt 4 5 6 5.0 nasua (1964)
23
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Brugia beaveri 28 Prese 43 67- 96- 4.5- - Procyon Ash &

Brugia sp. 5- nt – 76 108 6.0 Body lotor Little
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32 Prese 77 30- 62- 5,3 surface Nasua (1964)

5 nt 35 34 75 – nasua Present
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14 – 6,4 study
9- 43
22
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3
Mansonella 29 - - - - 2.5 - Procyon Prince
llewellyni 0 ± Abse 41 25- 44 3,2 All body lotor (1962)
Mansonella sp. 5 nt – 42 – – Nasua Present
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Not identified 22 Abse 61 - 65 4,3 nasua study

1- nt 55 - 77 - 4,0 Canis Present
25 61 86 familiaris study
0
25
2-
25
8
1 2 3 4
TL: Total length, P.A.: Anal pore, N.R: Nervous ring, E.P.: Excretory pore * All

morphometrics data in µm.

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Graphical abstract

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HIGHLIGHTS

 Filarial nematodes are identified in wild coatis and domestic dogs in PARNA
Iguaçu
 Some filarioids identified in coatis had origin in domestic dogs
 All filarioid species found have zoonotic importance.
 Monitoring human and animal health is recommended in the studied area

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