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Hemoplasmas Are Endemic and Cause Asymptomatic Infection
in the Endangered Darwin’s Fox (Lycalopex fulvipes)
Sophia Di Cataldo,
a
Ezequiel Hidalgo-Hermoso,
b
Irene Sacristán,
a
Aitor Cevidanes,
a
Constanza Napolitano,
c,d
Claudia V. Hernández,
e
Fernando Esperón,
f
Darío Moreira-Arce,
g
Javier Cabello,
h
Ananda Müller,
i,j
Javier Millán
k,l,m
a
Programa de Doctorado en Medicina de la Conservación, Facultad de Ciencias de la Vida, Universidad Andrés Bello, Santiago, Chile
b
Conservation and Research Department, Parque Zoológico Buin Zoo, Buin, Chile
c
Departamento de Ciencias Biológicas y Biodiversidad, Universidad de Los Lagos, Osorno, Chile
d
Instituto de Ecología y Biodiversidad, Santiago, Chile
e
Laboratorio de Diagnóstico Veterinario, HCV, Escuela de Medicina Veterinaria, Universidad Andrés Bello, Lo Pinto, Chile
f
Centro de Investigación en Sanidad Animal, Valdeolmos, Spain
g
Laboratorio de Estudios del Antropoceno, Facultad de Ciencias Forestales, Universidad de Concepción, Concepción, Chile
h
Facultad de Medicina Veterinaria, Universidad San Sebastián, Puerto Montt, Chile
i
Department of Biomedical Sciences, Ross University School of Veterinary Medicine, Basseterre, Saint Kitts and Nevis
j
Instituto de Ciencias Clínicas Veterinarias, Facultad de Ciencias Veterinarias, Universidad Austral de Chile, Valdivia, Chile
k
Facultad de Ciencias de la Vida, Universidad Andrés Bello, Santiago, Chile
l
Instituto Agroalimentario de Aragón-IA2, Universidad de Zaragoza-CITA, Zaragoza, Spain
m
Fundación Agencia Aragonesa para la Investigación y el Desarrollo, Zaragoza, Spain
ABSTRACT Mycoplasma haemocanis is prevalent in the endangered Darwin’s fox
(Lycalopex fulvipes) in its main stronghold, Chiloé Island (Chile). The origin of the in-
fection, its dynamics, its presence in other fox populations and the potential conse-
quences for fox health remain unexplored. For 8 years, hemoplasmal DNA was
screened and characterized in blood from 82 foxes in Chiloé and two other fox pop-
ulations and in 250 free-ranging dogs from Chiloé. The prevalence of M. haemocanis
in foxes was constant during the study years, and coinfection with “Candidatus My-
coplasma haematoparvum” was confirmed in 30% of the foxes. Both hemoplasma
species were detected in the two mainland fox populations and in Chiloé dogs. M.
haemocanis was significantly more prevalent and more genetically diverse in foxes
than in dogs. Two of the seven M. haemocanis haplotypes identified were shared
between these species. Network analyses did not show genetic structure by species
(foxes versus dogs), geographic (island versus mainland populations), or temporal
(years of study) factors. The probability of infection with M. haemocanis increased
with fox age but was not associated with sex, season, or degree of anthropization of
individual fox habitats. Some foxes recaptured years apart were infected with the
same haplotype in both events, and no hematological alterations were associated
with hemoplasma infection, suggesting tolerance to the infection. Altogether, our re-
sults indicate that M. haemocanis is enzootic in the Darwin’s fox and that intraspe-
cific transmission is predominant. Nevertheless, such a prevalent pathogen in a
threatened species represents a concern that must be considered in conservation
actions.
IMPORTANCE Mycoplasma haemocanis is enzootic in Darwin’s foxes. There is a
higher M. haemocanis genetic diversity and prevalence in foxes than in sympatric
dogs, although haplotypes are shared between the two carnivore species. There is
an apparent tolerance of Darwin’s foxes to Mycoplasma haemocanis.
KEYWORDS Canidae, Lycalopex,Mollicutes, risk factors, South America
Citation Di Cataldo S, Hidalgo-Hermoso E,
Sacristán I, Cevidanes A, Napolitano C,
Hernández CV, Esperón F, Moreira-Arce D,
Cabello J, Müller A, Millán J. 2020.
Hemoplasmas are endemic and cause
asymptomatic infection in the endangered
Darwin’s fox (Lycalopex fulvipes). Appl Environ
Microbiol 86:e00779-20. https://doi.org/10
.1128/AEM.00779-20.
Editor Edward G. Dudley, The Pennsylvania
State University
Copyright © 2020 American Society for
Microbiology. All Rights Reserved.
Address correspondence to Sophia Di Cataldo,
sophidica@hotmail.com.
Received 2 April 2020
Accepted 7 April 2020
Accepted manuscript posted online 10
April 2020
Published
PUBLIC AND ENVIRONMENTAL
HEALTH MICROBIOLOGY
crossm
June 2020 Volume 86 Issue 12 e00779-20 aem.asm.org 1Applied and Environmental Microbiology
2 June 2020
The Darwin’s fox (Lycalopex fulvipes) is an endangered carnivore native to Chile. Its
distribution range includes three metapopulations: one on Chiloé Island, with an
estimated population of 412 mature individuals, and two isolated mainland popula-
tions (Nahuelbuta Park and Valdivian Coastal Range) with about 227 mature individuals
in total (1). The presence of dogs in and around parks comprises one of the major
threats to the conservation of this carnivore (2). The presence of free-ranging dogs in
the entire Darwin’s fox distribution range has been documented (3), and the popula-
tions in the continent are completely surrounded by human-dominated lands. Rural
dogs in Chile are usually allowed to range freely (4, 5) and often lack any kind of
prophylactic treatment or veterinary care (6). The naive behavior of the Darwin’s fox can
predispose it to interactions with dogs, ending in physical attacks and potential
pathogen transmission (7).
Hemotropic mycoplasmas (also called hemoplasmas) are small bacteria that attach
to the surface of red blood cells of mammals (8). These species are widely distributed
and can infect humans (9), domestic animals (including dogs and cats) (10), and wildlife
(including wild carnivores) (11, 12). Three species of mycoplasmas have been described
in canids: Mycoplasma haemocanis,“Candidatus Mycoplasma haematoparvum,” and
“Candidatus Mycoplasma turicensis” (10, 13–15). Hemoplasma infection in dogs can
cause acute and chronic hemolytic anemia, with results ranging from asymptomatic
infection or slight lethargy to death; M. haemocanis is the most pathogenic species (16).
Thus far, no clinical signs have been reported in hemoplasma-infected wild canids, and
the pathological and epidemiological relevance for wildlife remains unknown. The
transmission route of hemoplasmas is still under debate. Some species infecting dogs
and cats are believed to be vector borne, but direct and/or vertical transmission has
been demonstrated for others (17, 18). M. haemocanis has recently been proved to be
transmitted vertically (19) in a dog, although it was classically considered to be
transmitted by the brown dog tick, Rhipicephalus sanguineus (20). Nevertheless, since
there are no canine ticks on Chiloé Island, other ways of transmission (i.e., direct
transmission) must be operating.
During a molecular disease survey, Cabello et al. (21) reported an unexpected high
prevalence of hemoplasma DNA among 30 free-living Darwin’s foxes captured in Chiloé
in the period from 2009 to 2012. Sequencing showed that 80% of the sequences
obtained corresponded to M. haemocanis, another to M. haemofelis, and one to an
as-yet-uncharacterized Mycoplasma sp., which was later found to be shared with
domestic cats and the wild cat guigna (Leopardus guigna) in Chile (22). All the foxes
studied by Cabello et al. (21) were apparently healthy and were negative for almost
every other of the nine vector-borne pathogen groups for which these animals were
tested. Pathogen transmission is hindered in small populations of solitary species
such as the Darwin’s fox because of their low rate of interspecific contact (23).
Therefore, the high prevalence of infection reported by Cabello et al. (21) would
support the hypothesis that hemoplasmas more likely persist in foxes based on an
interspecific (i.e., dog-to-fox) rather than an intraspecific (fox-to-fox) transmission
pathway. Considering that poorly managed free-ranging dogs are abundant in rural
parts of Chile (24), including those areas sustaining Darwin’s fox populations, a role
of the domestic dog as a reservoir and source of infection for the fox is a likely
scenario.
Different epidemiological questions regarding hemoplasma infection in the Dar-
win’s fox remain. Whether Darwin’s foxes in Chiloé and mainland populations are
reservoir or spillover hosts for M. haemocanis is still unknown. It has been observed in
wild felids that the domestic cat would act as a source host of hemoplasma (25, 26).
However, proving a role as reservoir for a pathogen is complex. In order to disentangle
the origin of hemoplasma infection in the Darwin’s fox, we took different approaches.
If these bacteria are being transmitted from dogs to foxes, we predicted that shared
sequences would be found in both hosts, with greater hemoplasma genetic diversity in
dogs. We also expected a higher prevalence of infection in foxes living closer to human
settlements. We also aimed to provide some insights into risk factors and effects of
Di Cataldo et al. Applied and Environmental Microbiology
June 2020 Volume 86 Issue 12 e00779-20 aem.asm.org 2
hemoplasma infection in the Darwin’s fox and to determine whether the pathogen is
present in other Darwin’s fox populations. The ultimate goal of our study was to
determine if hemoplasma infection should be considered a disease threat for the last
Darwin’s foxes.
RESULTS
Molecular detection and characterization. The overall observed prevalence of
Mycoplasma DNA for the whole study period was 56.6% (95% confidence interval
[CI] ⫽46.0% to 67.3%; 47 foxes positive). All the newly obtained 16S rRNA sequences
showed between 99.7 and 100% identity with published sequences of M. haemoca-
nis/M. haemofelis (Table 1). Sequencing of a portion of the RNase P gene (accession no.
CP003199) indicated 100% identity with M. haemocanis sequences in all cases. Species-
specific primers confirmed coinfection with “Ca. Mycoplasma haematoparvum” in nine
of the 30 M. haemocanis-positive foxes for the period from 2013 to 2017 (30.0%, 95%
CI ⫽13.6% to 46.4%). In the foxes from the mainland, M. haemocanis DNA was found
in two individuals from Nahuelbuta, one of which was coinfected with “Ca. Mycoplasma
haematoparvum,” and in one from Valdivia (Fig. 1). Six of the recaptured foxes were
positive for M. haemocanis (two of which were coinfected with “Ca. Mycoplasma
haematoparvum”), and one was negative at both capture events. Another fox, which
was positive for M. haemocanis at both capture events, did not present coinfection with
“Ca. Mycoplasma haematoparvum” at the first sampling but 2 years later was coin-
fected with this species (Table 2).
Sixty dogs were positive for Mycoplasma sp. DNA (24.0%; 95% CI ⫽18.7% to 29.3%)
across Chiloé Island (Fig. 1). Of the fifty-three readable sequences, 53.2% corresponded
to M. haemocanis/M. haemofelis and 46.8% to “Ca. Mycoplasma haematoparvum.” M.
haemocanis prevalence in foxes was significantly higher than in dogs (
2
⫽15.908, P⬍
0.001), whereas “Ca. Mycoplasma haematoparvum” prevalence was not significantly
different between species (
2
⫽2.641, P⬎0.1). We did not analyze dogs for potential
M. haemocanis/“Ca. Mycoplasma haematoparvum” coinfections, but even if all dogs
positive for “Ca. Mycoplasma haematoparvum” were coinfected with M. haemocanis,
prevalence of M. haemocanis in foxes would still be significantly higher than in dogs
(
2
⫽28.9, P⬍0.001).
Only one of the foxes surveyed in the period from 2009 to 2017 period was
parasitized by a tick (a larvae of Ixodes sigelos), a typical species of pudu (Pudu puda),
an endemic deer from Chile, and none hosted fleas. No ticks were recovered from dogs,
and 57 individuals (25.2%; 95% CI ⫽19.6% to 30.8%) hosted fleas.
Genetic analysis. Sequencing of nearly 900 bp the 16S rRNA gene from 25 foxes
and 12 dogs revealed the presence of seven different nucleotide sequence types (ntST),
with 99.9% identity among them and between 99.7% and 99.9% identity with other M.
haemocanis sequences (Fig. 2). Two of the seven ntST were shared between foxes and
dogs (Table 1). ntST-1 was the most frequent; it was detected in 29 individuals (19 foxes
and 10 dogs). The other ntST shared between species (ntST-4) was detected in one dog
and one fox. ntST-5 was detected in two foxes, whereas the other four ntST were found
in only a single individual, either a fox (n⫽3) or a dog (n⫽1). Five of the recaptured
TABLE 1 Nucleotide sequence types detected in hemoplasmas in blood samples from Darwin’s foxes and rural sympatric dogs and their
closest GenBank sequences
ntST Animal(s) in which detected (n)
Closest
sequence Species
Identity
(%) Host Country
1 Dog from Chiloé (10), fox from Chiloé (17),
fox from Nahuelbuta (2)
EF416566 Mycoplasma haemocanis 100 Canis lupus familiaris Switzerland
2 Fox from Chiloé (1) AF197337 Mycoplasma haemocanis 99.92 Canis lupus familiaris USA
3 Fox from Chiloé (1) GQ129116 Mycoplasma haemocanis 99.92 Canis lupus familiaris Italy
4 Fox from Chiloé (1), dog from Chiloé (1) GQ129116 Mycoplasma haemocanis 99.84 Canis lupus familiaris Italy
5 Fox from Chiloé (2) DQ825458 Mycoplasma haemofelis 99.92 Lynx lynx Switzerland
6 Fox from Chiloé (1) MK064162 Mycoplasma haemocanis 99.77 Pulex irritans on Lycalopex culpaeus Argentina
7 Dog from Chiloé (1) KY117659 Mycoplasma haemocanis 99.73 Canis lupus familiaris Chile
Hemoplasmas in the Endangered Darwin’s Fox Applied and Environmental Microbiology
June 2020 Volume 86 Issue 12 e00779-20 aem.asm.org 3
foxes presented ntST-1 in both capture events, while the other two presented a
different ntST (Table 2). All these sequences were placed in the M. haemocanis/M.
haemofelis clade in the phylogenetic tree (Fig. 2).
The haplotype diversity (Hd) in foxes was 0.427 (standard deviation [SD] ⫽0.122),
the nucleotide diversity (Pi) was 0.00116 (SD ⫽0.00040), and the average number of
nucleotide differences (k) was 0.547. In dogs, Hd was 0.345 (SD ⫽0.172), Pi was 0.00108
(SD ⫽0.00057), and k was 0.727. The network analysis showed no genetic structure
between dogs and foxes (F
ST
⫽0.02919, P⬎0.05; nearest-neighbor statistic
[Snn] ⫽0.51243, P⬎0.05) (Fig. 3) and no geographic structure between the island and
mainland populations (F
ST
⫽0.030079, P⬎0.5; Snn ⫽0.83647, P⬎0.5) (Fig. 3). The
most prevalent ntST was present through the entire sampling period, and in concor-
dance, no genetic structure was detected among years (F
ST
⫽0.03809, P⬎0.05;
Snn ⫽0.0790, P⬎0.05) (Fig. 3).
Risk factor analysis. Models indicated that prevalence of infection of M. haemo-
canis was significantly higher in adult foxes than in juveniles (62.3% versus 20.0%; Z
value ⫽⫺2.247, P⬍0.05) (Table 3). No other risk factor was related to the probability
of M. haemocanis infection (Fig. 4). Prevalence of M. haemocanis-“Ca. Mycoplasma
FIG 1 Map of the study areas, showing hemoplasma infection status in the surveyed Darwin’s foxes and rural dogs. Dogs from Inio, a small fishing village in
the south of Chiloé, are shown pooled in a pie chart.
Di Cataldo et al. Applied and Environmental Microbiology
June 2020 Volume 86 Issue 12 e00779-20 aem.asm.org 4
haematoparvum” coinfections did not differ depending on fox age (
2
⫽0.099, P⬎
0.5).
Prevalence was also higher in adult dogs than in juveniles (
2
⫽63.2, P⬍0.01), and
no sex-related differences were found (
2
⫽1.7, P⬎0.05).
Hematological analysis. No significant differences were found in the hematological
and biochemical variables evaluated depending on the sex or age of the animals, so the
samples were pooled for further comparisons. None of the studied variables differed
between the infection pattern groups that were compared.
TABLE 2 Hemotropic mycoplasma infection status in Darwin’s foxes recaptured during the study period and Mycoplasma haemocanis
nucleotide sequence type detected in each capture event
Fox Yr Site Season Age
M. haemocanis
status
M. haemocanis
ntST
“Candidatus M. haematoparvum”
status
1 2014 Chiloé Spring Adult ⫹4⫺
2016 Chiloé Winter Adult ⫹1⫹
2 2013 Nahuelbuta Spring Adult ⫺⫺⫺
2016 Nahuelbuta Summer Adult ⫺⫺⫺
3 2014 Chiloé Spring Juvenile ⫹1⫺
2016 Chiloé Winter Adult ⫹1⫺
4 2015 Chiloé Fall Adult ⫹1⫹
2017 Chiloé Fall Adult ⫹1⫹
5 2016 Chiloé Fall Adult ⫹3⫺
2017 Chiloé Fall Adult ⫹1⫺
6 2014 Chiloé Winter Adult ⫹1⫺
2015 Chiloé Fall Adult ⫹1⫺
7 2013 Chiloé Fall Adult ⫹1⫹
2014 Chiloé Spring Adult ⫹1⫹
8 2014 Chiloé Winter Adult ⫹1⫺
2015 Chiloé Fall Adult ⫹1⫺
Mycoplasma haemocanis. Canis familiaris. Nigeria. /KP715857.1
Mycoplasma haemocanis. Canis familiaris. Italy. /GQ129119.1
ntST5
ntST7
ntST2
ntST1
Mycoplasma haemocanis. Nyctereutes procyonoides viverrinus. Japan. /AB848714.1
ntST3
ntST4
Mycoplasma haemofelis. Felis catus. Brazil. /EU930823.1
ntST6
Mycoplasma haemofelis. Felis catus. Brazil. /KM275243.1
Mycoplasma haemofelis. Felis catus. Brazil. /KM275241.1
Candidatus Mycoplasma turicensis. Prionailurus bengalensis iriomotensis. Japan. /AB697739.1
Candidatus Mycoplasma turicensis. Felis catus. Switzerland. /DQ157150.1
Candidatus Mycoplasma turicensis. Panthera leo. Tanzania. /DQ825454.1
Candidatus Mycoplasma turicensis. Felis catus. South Africa. /DQ464424.1
Candidatus Mycoplasma haemominutum. Felis catus. USA. /KF743738.1
Candidatus Mycoplasma haemominutum. Canis familiaris. China. /AM691834.1
Candidatus Mycoplasma haemominutum. Felis catus. Thailand. /EU285281.1
Candidatus Mycoplasma haemominutum. Canis familiaris. USA. /AY297712.1
Candidatus Mycoplasma haematoparvum. Canis familiaris. Italy. /MH094850.1
Mycoplasma pneumoniae NR041751
94
99
70
91
70
72
99
0.05
FIG 2 Maximum-likelihood tree of the 16S rRNA gene (893 bp) of Mycoplasma haemocanis for Darwin’s foxes and domestic dogs. A Mycoplasma pneumoniae
sequence was used as an outgroup. Bootstrap values of ⱖ70 are given at the nodes of the tree. Diamonds mark the nucleotide sequence types (ntST) from
our study.
Hemoplasmas in the Endangered Darwin’s Fox Applied and Environmental Microbiology
June 2020 Volume 86 Issue 12 e00779-20 aem.asm.org 5
DISCUSSION
Our survey revealed that M. haemocanis was present in the Darwin’s fox samples
through the entire study period, indicating a constant exposure of the species to M.
haemocanis. Moreover, the prevalence was higher in the endangered Darwin’s fox than
in dogs. Although the observed prevalence in dogs is in the range of that found in a
previous study in Chile (15) and even higher than the rates of infection detected
elsewhere (20, 27), the higher prevalence in foxes may be explained by a greater risk
of exposure and/or different susceptibility to the infection.
The sharing of the most frequent ntST of the 16S rRNA gene of M. haemocanis in
dogs and foxes suggests cross-infection between these species. Whether dogs are the
main source of infection for Darwin’s foxes cannot be proved with our data. However,
considering that dogs markedly outnumber foxes and that dogs are moved large
distances by their owners (24), it could be assumed that dogs were the origin of M.
haemocanis infection for the fox. This is also supported by the fact that ntST-1 was
found in foxes from both Chiloé Island and Nahuelbuta, which can be explained only
by the movement of dogs between the continent and Chiloé. However, since this
pathogen was introduced in the population, fox-to-fox transmission seems to be
frequent now. This is supported by the higher prevalence in foxes than in dogs, the
higher haplotype diversity detected in foxes, and the fact that the most prevalent ntST
was found in foxes during all the studied years. This last observation would be better
explained by intraspecific transmission than by periodic spillovers from dogs. Persistent
intraspecific transmission of hemoplasmas following spillover from domestic animals
has been also observed in felids (22, 26).
FIG 3 Median joining network of the 16S gene (893 bp) of Mycoplasma haemocanis in rural dogs and Darwin’s foxes. Each circle in the network corresponds
to a different nucleotide sequence type (ntST), the sizes of the circles correspond to ntST frequencies, the colors of the circles correspond to the two host species
(Darwin’s foxes and rural dogs) (A), two geographic sampling sites (Chiloé Island and Nahuelbuta) (B), and years of sampling (C). The networks in panels B and
C were performed with fox samples only.
TABLE 3 Best model representing multivariate relationships between predictor variables and detection of Mycoplasma haemocanis in
Darwin’s foxes, using logistic regression analysis
Variable Estimate ⴞSE Z value
a
AIC Deviance df
Hosmer-Lemeshow
test Pvalue
Intercept 0.32 ⫾0.58 0.56 76.134 68.134 59
Age juvenile ⫺2.07 ⫾0.92 ⫺2.24*0.8
No. of houses ⫺0.17 ⫾0.07 ⫺2.30*
Distance to nearest house 0.02 ⫾0.02 1.41
a
*,P⫽0.01.
Di Cataldo et al. Applied and Environmental Microbiology
June 2020 Volume 86 Issue 12 e00779-20 aem.asm.org 6
The higher prevalence observed in adult foxes compared to juveniles coincides with
studies of free-ranging dogs (9) and cats (28) and with the data of this study in rural
dogs of Chiloé. This indicates that both foxes and dogs face an increasing possibility
of exposure to the pathogen with age. This, together with the apparent lack of
seasonality and interannual differences in the prevalence indicates that M. haemo-
canis infection behaves enzootically in the Darwin’s fox. The data from the recap-
tured individuals further confirmed the widespread nature of the infection in foxes
and that foxes can be infected more than once, as proven by the finding of foxes
reinfected by a different haplotype. Host sex does not appear to be a risk factor for
infection with M. haemocanis in foxes, which concurs with previous studies in
domestic dogs (27,29).
The observed prevalence of “Ca. Mycoplasma haematoparvum” in dogs in our study
is in the range of that reported in Argentina (30) and higher than that reported in the
above-metioned city dogs in Chile (15). Unfortunately, we were unable to characterize
“Ca. Mycoplasma haematoparvum” further to confirm whether this species is also
shared by both hosts.
The lack of effect of landscape features on hemoplasma prevalence in the Darwin’s
fox is in disagreement with a previous study on feline hemoplasmas in a threatened
sympatric felid, the guigna (22). Our results likely reflect a heterogeneity of the risk of
transmission of a multihost pathogen that may be using more than a single method
of transmission (horizontal, vertical, and/or vector borne) and/or more than a single
arthropod vector.
Infections with hemoplasmas appear to occur as chronic conditions in domestic
species (27,29). This seems to be the case in the Darwin’s fox, as indicated by the
high prevalence observed in the absence of clinical signs and the high proportion
of foxes that were found to be infected by the same ntST in both capture events,
which had up to 3 years of difference between them (although the possibility of
periodic reinfections with the same ntST cannot be ruled out). The chronicity could
explain the absence of hematological alterations associated with the presence of
the pathogen. The possible tolerance of the Darwin’s fox to M. haemocanis is
FIG 4 Prevalence of Mycoplasma haemocanis depending on different intrinsic and extrinsic variables in Darwin’s foxes. (A) Age groups; (B) sex groups; (C)
seasons; (D) study years.
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June 2020 Volume 86 Issue 12 e00779-20 aem.asm.org 7
supported by the genetic variability of the pathogen in the fox population, with up
to six different ntST detected in 25 individuals, which according to Kutzer and
Armitage (31) may reflect some degree of tolerance to the infection. Kutzer and
Armitage (31) also proposed that tolerance to a pathogen will confer a fitness
advantage to the host, which could fluctuate by pathogen load and intrinsic factors
of the host, reasons that may explain why we did not detect any risk factor
associated with the infection other than age. Nevertheless, it is worth noting that
when the apparent tolerance is lost, subclinical infections can lead to a reduction
in host fitness, reproduction, survival, or dispersal, any of which can be detrimental
for the population of an endangered species such as the Darwin’s fox (32).
Previous studies identified coinfection with other agents as a risk factor for becom-
ing infected with hemoplasmas (27, 33), and it is known that the presence of other
agents can aggravate the pathogenicity of a primary agent. For example, distemper
outbreaks in African lions were exacerbated by concomitant Babesia spp. (34). We did
not investigate coinfections with other vector-borne pathogens because our prelimi-
nary survey indicated absence of all the other main canine vector-borne pathogens
(21). Moreover, a recent study showed that the same foxes studied here were not
exposed to canine distemper virus (CDV) (35). Therefore, it appears that coinfection is
not an important driver of hemoplasma infection for this species. Rynkiewicz et al. (36)
proposed that a host that has a tolerance response to a pathogen will have higher
fitness due to the avoidance of the energetic cost of clearing an infection, which seems
to be the case for the fox according to the hematological analyses. Nevertheless,
asymptomatic infection could revert, triggered by other concomitant infectious or
noninfectious causes.
Despite the small sample size, we confirmed the presence of both canine
hemotropic mycoplasmas in foxes from both mainland metapopulations. This could
be relevant during health assessment protocols before potential fox translocations
(37). In these two mainland populations, two other sympatric anthropophilic and
more-abundant fox species, the Andean fox (Lycalopex culpaeus) and the gray fox
(L. griseus), could add a potential bridge between dogs and Darwin’s foxes, increas-
ing the complexity of interspecific transmission of this multihost pathogen.
Infectious diseases can pose a threat to the survival of endangered species, espe-
cially those such as the Darwin’s fox, which are losing their natural habitat and
encountering dogs more frequently, increasing the opportunities for pathogen trans-
mission (23, 38). Therefore, information about the health of their populations and the
evaluation of risk factors for infection are imperative.
Conclusion. We showed that the Darwin’s fox can sustain a constant prevalence
of infection with a relatively high genetic diversity of M. haemocanis and with
apparent lack of associated pathology. Nevertheless, although hemotropic myco-
plasmas may seem asymptomatic, the constant circulation of this disease agent in
the reduced Darwin’s fox population is a concern. The apparent tolerance of foxes
to hemoplasma infection could be disrupted due to other factors that could be
favored by poorly managed sympatric dogs. This disease risk is enhanced by
the rapid habitat loss and degradation that the Darwin’s fox is currently suffering
(39).
MATERIALS AND METHODS
Study areas and field techniques. The samples included in this study were obtained between 2009
and 2017. The samples from 2009 to 2012 corresponded to the 30 fox blood samples from Chiloé Island
included in the previous survey by Cabello et al. (21). From 2013 to 2017, we collected 52 additional
fox samples in the three populations of Darwin’s fox: the Nahuelbuta area (n⫽5) (37°45=S, 73°00=W),
comprising Nahuelbuta National Park and surrounding native forests; the Valdivian Coastal Range
(n⫽5) (40°07=S, 73°33=W); and Chiloé Island (n⫽42) (42°S, 74°W) (Fig. 1). Throughout the years of
our study, seven foxes in Chiloé and one in Nahuelbuta were recaptured once. One individual was
juvenile at the initial sampling and adult when recaptured, while all the others were adults in both
capture events.
Foxes were captured using Tomahawk traps baited with chicken or canned fish. Traps were activated
at dusk and checked the next morning at dawn. Foxes were anesthetized with a combination of 1 mg/kg
Di Cataldo et al. Applied and Environmental Microbiology
June 2020 Volume 86 Issue 12 e00779-20 aem.asm.org 8
xylazine (xylazine 2%; Centrovet, Chile) plus 10 mg/kg ketamine (Ketostop; DragPharma, Chile) or with
0.04 mg/kg dexmedetomidine (Dexdormitor; Zoetis, Chile) plus 5 mg/kg ketamine. The latter was
reversed with 0.4 mg/kg atipamezole (Antisedan; Zoetis, Chile). A veterinarian performed an external
clinical evaluation of the anesthetized animals. Blood was obtained from the cephalic, saphenous, or
jugular vein. Foxes were classified as juveniles (less than 1 year) or adults (older than 1 year) based on
tooth eruption.
Between the years 2015 and 2018, 250 rural dogs were sampled across Chiloé (Fig. 1). Blood was
collected individually after the consent of the owner. Whole blood from dogs and foxes was placed in
EDTA tubes. When possible, whole blood was sent by courier to our laboratory for hematological
analyses. Otherwise, it was stored at –20°C. For serum biochemistry analysis, sera of foxes were extracted
and stored at –20°C when possible. Dogs were classified as juveniles (less than 1 year) or adults (older
than 1 year) based on tooth eruption.
All captures were made with the permission of the Servicio Agricola y Ganadero of the Chilean
Government under permits 1262/2009, 2263/2010, 206/2012, 3155/2013, 1492/2014, 3363/2015, 3035/
2016, 2288/2016, and 5029/2017. The study was approved by the authorities on bioethics of Universidad
Andres Bello (permit no. 08/2016).
Molecular detection and characterization. DNA was isolated from the 302 fox and dog blood
samples using a DNeasy blood and tissue kit (Qiagen) according to the manufacturer’s instructions. All
the newly obtained samples from foxes and dogs were initially screened for Mycoplasma spp. by a
conventional PCR targeting a 391-bp fragment of the 16S rRNA gene (40). Amplicons were selected and
assigned to a nucleotide sequence type (ntST). Since M. haemocanis and M. haemofelis are undistin-
guishable based on the characterization of the 16S rRNA gene alone, we confirmed that all positive
samples corresponded to M. haemocanis by a conventional PCR targeting a 175-bp fragment of the
RNase P gene. All these PCR protocols were as described by Millán et al. (40). The complete 16S rRNA
gene (⬃1,400 bp) was then characterized in all the M. haemocanis-positive foxes and in two positive dogs
per hemoplasma ntST (Table 4). In order to detect if foxes had so-far-undetected coinfections with “Ca.
Mycoplasma haematoparvum,” all the sequences corresponding to M. haemocanis/M. haemofelis from
2013 to 2017 were screened through a “Ca. Mycoplasma haematoparvum”-specific protocol targeting a
112-bp fragment of the 16S rRNA gene as described by Martínez-Díaz et al. (41). Positive controls were
obtained from clinical samples of M. haemocanis and “Ca. Mycoplasma haematoparvum” from previously
sequenced dog blood samples, and ultrapure water was used as a negative PCR control. Two percent
agarose gel electrophoresis was performed, and PCR products were visualized under an UV transillumi-
nator. All positive samples were sequenced by Macrogen, and the sequences obtained were compared
with sequences deposited in the GenBank database (NCBI).
The sequences of the 16S rRNA genes obtained from foxes and dogs were aligned using ClustalW
executed in Geneious Prime 2019.2.1 (Biomatters Ltd.). To determine genetic relationships between the
sequences from wild and domestic canids, we constructed a maximum-likelihood phylogenetic tree
using MEGA 7.0.26 (42) and median joining networks using PopART (43). A network containing the
sequences from dogs and Darwin’s foxes from this study was used to infer genetic relationships among
hemoplasma species. A second network was developed to infer relationships according to the sampling
site (Chiloé versus Nahuelbuta only, since the sequence from Valdivia was not readable) using only the
fox sequences. Finally, a network considering the year of fox sampling was used to infer the dynamics
of infection in the Darwin’s fox. The genetic structure was estimated using the pairwise Phi
ST
test
implemented in Arlequin (44) (level of significance assessed with 1,000 permutations) and the nearest-
neighbor statistic Snn (45) executed in DnaSP.5 (46). An analysis of the nucleotide polymorphisms of 16S
rRNA sequences obtained was performed using the software DnaSP.5 (46) in order to determine the
genetic differentiation of hemoplasmas among hosts.
Hematology and serum chemistry analyses. The harsh climatic conditions in southern Chile and
distant locations of many study sites prevented the proper preservation of some of the blood and serum
samples until laboratory analyses. Therefore, hematological and biochemical variables were obtained for
31 of the foxes during the study period. Seven hematological variables (hematocrit, red blood cell,
platelet and total leukocyte counts, hemoglobin concentration, mean corpuscular volume, and mean
corpuscular hemoglobin concentration) were calculated using a HumaCount 80 cell counter (Human
GmbH, Germany). Relative leukocyte differentiation was estimated by microscopic observation after
Diff-Quick staining. Fourteen biochemical variables were analyzed using a BA400 Analyzer (BioSystem SA,
Barcelona, Spain). Measurement units and variables included are listed in Table 5.
Data analysis. M. haemocanis presence/absence was binary coded and examined with a set of
models of possible intrinsic and extrinsic variables affecting animal exposure to hemoplasmas. We
included generalized linear models considering age, sex, and their interaction in relation to M. haemo-
canis infection. A chi-square test was used to determine differences in infection depending on these
factors in dogs and to determine differences in prevalence between foxes and dogs.
Spatial analyses were performed only in Chiloé to test the effect of extrinsic variables on the M.
haemocanis presence/absence, due to insufficient sample sizes in the other study areas. In order to
analyze if foxes’ exposure to hemoplasmas depends on spillover events from dogs more than on
intraspecific transmission (47), we used a model considering landscape anthropization in the fox home
range area. We created a buffer zone around the fox capture site based on individual fox locations and
the home range size described for the species (3.06 km
2
for males and 2.72 km
2
for females) (48).
Independent variables were as follows: presence/absence of houses in the buffer area, total number of
houses in the buffer area, distance of the capture site to the nearest house, land use, and proportion of
vegetation cover in the buffer area (49). To determine if pathogen exposure showed temporal variation
Hemoplasmas in the Endangered Darwin’s Fox Applied and Environmental Microbiology
June 2020 Volume 86 Issue 12 e00779-20 aem.asm.org 9
TABLE 4 Genes targeted and primers used for PCR screening and characterization of hemotropic mycoplasmas in Darwin’s foxes and rural dogs in Chile
Target Primer sequences Primer names
Fragment
length (bp) Purpose Reference
Mycoplasma sp. 16S rRNA 5=-ATGTTGCTTAATTCGATAATACACGAAA-3=(forward),
5=-ACRGGATTACTAGTGATTCCAACTTCAA-3=(reverse)
Mycop16S rRNA-F, Mycop16S rRNA-R 384 Screening 40
Mycoplasma sp. 16S rRNA (seminested) Characterization This study
Template 5=-AGAGTTTGATCCTGGCTCAG-3=(forward),
5=-TACCTTGTTACGACTTAACT-3=(reverse)
HemoF1, HemoR2 1,428
First 5=-ATATTCCTACGGGAAGCAGC-3=(forward),
5=-TACCTTGTTACGACTTAACT-3=(reverse)
HemoF2, HemoR2 1,107
Second 5=-GCCCATATTCCTACGGGAAGCAGCAGT-3=(forward),
5=-GTTTGACGGGCGGTGTGTACAAGACC-3=(reverse)
HemMycop16S-322s, HemMycop16S-1420as 1,029
Third 5=-GYATGCMTAAYACATGCAAGTCGARCG-3=(forward),
5=-CTCCACCACTTGTTCAGGTCCCCGTC-3=(reverse)
HemMycop16S-41s, HemMyco16S-938as 870
M. haemocanis/M. haemofelis RNase P 5=-CCTGCGATGGTCGTAATGTTG-3=(forward),
5=-GAGGRGTTTACCGCGTTTCAC-3=(reverse)
RNAseP-F, RNAseP-R 175 Characterization 40
“Ca. Mycoplasma haematoparvum” 16S
rRNA
5=-GGAATCACTAGTAATCCYGTGTCAGCTATAT-3=(forward),
5=-AATTAAATACGGTTTCAACTAGTACGTTTCTTT-3=
(reverse)
Mycoplasma Species-F, Candidatus
Mycoplasma haematoparvum-R
112 Characterization 41
Di Cataldo et al. Applied and Environmental Microbiology
June 2020 Volume 86 Issue 12 e00779-20 aem.asm.org 10
(e.g., epizootic or seasonal), a third model, including the variables season and year, was developed (50).
Finally, a full model considering all the mentioned variables and a null model were developed. All
variables were compared with presence/absence of M. haemocanis using generalized linear models.
Variables were initially analyzed with univariate models, and then the variables with significant Pvalues
were included in multivariate models. The Akaike information criterion (AIC) was used for model
selection, and its fit was assessed using the Hosmer-Lemeshow goodness-of-fit test (51).
Hematological and biochemical variables were compared for each of the following fox infection
statuses: hemoplasma-positive versus -negative foxes, M. haemocanis-infected foxes versus M.
haemocanis- and “Ca. Mycoplasma haematoparvum”-coinfected foxes, and M. haemocanis- and “Ca.
Mycoplasma haematoparvum”-coinfected foxes versus negative foxes. We used the Shapiro-Wilk nor-
mality test to determine if the data were distributed normally. The two-sample Mann-Whitney U test was
performed to determine differences among the variables. Following the “Guidelines for the Determina-
tion of Reference Intervals in Veterinary Species” of the American Society for Veterinary Clinical Pathology
(available at https://cdn.ymaws.com/www.asvcp.org/resource/resmgr/QALS/Other_Publications/RI_
Guidelines_For_ASVCP_webs.pdf), the median, standard deviation, and range values were provided for
each parameter. All data analyses were performed using R software version 3.4.1 (52).
Data availability. The new sequences obtained in the present study were submitted to GenBank
with accession numbers MN164349 to MN164353.
ACKNOWLEDGMENTS
This study was funded by Morris Animal Foundation grant D16Z0-825, FONDECYT-
Regular 1161593, CONICYT-FONDECYT Iniciación 11150934 (C.N.), CONICYT-FONDECYT
Iniciación 11181180 (D.M.-A.), Morris Animal Foundation (MAF) Fellowship Training
Award D15ZO-413 (C.N.), National Geographic Society Conservation Trust C309-15
(C.N.), Mohamed bin Zayed Species Conservation Fund 152510351 (C.N.), and CONICYT
PAI Convocatoria Nacional Subvención a Instalación en la Academia Convocatoria Año
2019 Folio 77190064 (C.N.).
We thank Catherine Chirgwin, Alan Bannister, and the Tantauco Foundation, Coop-
erativa de Pescadores Mar Adentro, Corporación Nacional Forestal (CONAF), Parque
Ahuenco, Parque Tablaruca, Forestal Arauco. We thank Carla Barría for laboratory
assistance and Daniel González for identifying the tick.
TABLE 5 Hematological and serum chemistry values in Darwin’s foxes from Chiloé Island
Variable (unit)
a
Sample size Median value SD Range
WBC (
l) 31 15,850 5,020 5,960–29,600
RBC (
l) 31 5,870,000 957,455 3,310,000–7,880,000
HB (g/liter) 31 13.9 2.14 7.3–18.7
HTO (%) 31 42.3 7 23.9–59.0
MCV (fl) 25 72 23.31 16.4–90.0
MCH (fl) 31 23.6 0.97 21.8–26.3
MCHC (g/liter) 31 32.3 1.7 27–35.5
PLT (
l) 31 304,000 109,250 141,000–580,000
N(
l) 25 12,648 3,845 4,410–18,480
L(
l) 30 1,738 1,717 752–9,400
M(
l) 30 510 1,167 138–6,150
E(
l) 26 150 681 0–3,071
Ca (mmol/liter) 26 2.46 0.39 1.35–2.94
P (mmol/liter) 28 1.95 3.50 0.1–11.3
BUN (mmol/liter) 28 9.11 5.02 4.82–27.4
Crea (mmol/liter) 28 0.07 0.3 0.04–0.18
Bil (
mol/liter) 21 3.93 2.57 1.71–13.68
Gluc (mmol/liter) 28 2.6 1.69 0.06–6.16
Chol (mmol/liter) 28 5 1.11 2.92–8.02
ALP (IU/liter) 22 40 76.59 3–227
ALT (IU/liter) 26 77 33.40 30–152
AST (IU/liter) 26 83 53.46 21–225
GGT (IU/liter) 24 2 4 1–21
Prot (g/liter) 25 76 11 45–96
Glob (g/liter) 23 40 73 32–58
Alb (g/liter) 28 30 66 17.2–47
a
WBC, white blood cells, RBC, red blood cells; HB, hemoglobin; HTO, hematocrit; MCV, mean corpuscular
volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; PLT,
platelet count; N, neutrophils; L, lymphocytes; M, monocytes; E, eosinophils; Ca, calcium; P, phosphorus;
BUN, blood urea nitrogen; Crea, creatinine; Bil, bilirubin; Gluc, glucose; Chol, cholesterol; ALP, alkakine
phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; GGT, gamma-glutamyl
transpeptidase; Prot, total proteins; Glob, globulins; Alb, albumin.
Hemoplasmas in the Endangered Darwin’s Fox Applied and Environmental Microbiology
June 2020 Volume 86 Issue 12 e00779-20 aem.asm.org 11
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