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\begin{document}

\title{Molecular evidence of Bartonella spp. in free-living opossums
(Didelphimorphia: Didelphidae) from peri-urban Atlantic Forest fragments
of Brazil}



\author[1]{Amanda Alcantara}%
\author[2]{Helena Thoma}%
\author[1]{Sabrina Campos}%
\author[1]{Rosemeri Teixeira}%
\author[1]{Hans Lima}%
\author[3]{Jeferson Pires}%
\author[4]{Renata Moraes}%
\author[1]{Aline Souza}%
\affil[1]{Universidade Federal Fluminense}%
\affil[2]{Universidade Federal do Rio de Janeiro}%
\affil[3]{Universidade Estacio de Sa - Campus Reboucas-Rio Comprido}%
\affil[4]{Universidade de Vassouras}%


\vspace{-1em}



  
  \date{May 24, 2024}


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\selectlanguage{english}
\begin{abstract}
Many emerging infectious diseases are zoonotic and transmitted by an
arthropod vector; thus, the Global One Health perspective is necessary
for a better understanding of these diseases. Bartonellosis, one of
those emerging infectious diseases, is caused by bacteria of the genus
Bartonella, and can have different clinical manifestations in humans and
animals. Opossums and their ectoparasites have been linked to
human-wildlife conflicts. Knowing this, we aimed to evaluate infection
by Bartonella spp. in free-living opossums seized or rescued from
Atlantic Forest biome of Rio de Janeiro (southeastern Brazil). Whole
blood was obtained from seven Didelphis aurita and eight D. albiventris,
and DNA was assessed by conventional and quantitative polymerase chain
reactions (PCR and qPCR). Positivity was achieved in 40.0\% (PCR) and
46.7\% (qPCR) of the samples, revealing, possibly for the first time,
the presence of Bartonella spp. DNA in opossums from peri-urban areas of
Atlantic Forest biome. Studies should continue, in order to elucidate
synanthropic mammals importance in bartonellosis epidemiology.%
\end{abstract}%



\sloppy


\textbf{Main document}

\textbf{Introduction}

Opossums (\emph{Didelphis} spp.) are neotropical marsupials of the order
Didelphimorphia, family Didelphidae. Among the species that occur in
Brazil, two can be easily found in the great Brazilian biomes, \emph{D.
aurita} (black-eared opossum) and \emph{D. albiventris} (white-eared
opossum) (Al\selectlanguage{ngerman}éssio et al., 2005; Moura et al., 2009; Paglia et al.,
2012). These mammals have opportunistic habits, and can live in wild,
rural and urban environments, maintaining contact with other animals and
human beings (Lucheis et al., 2009; Tarragona et al., 2011). Notably,
white-eared opossum is widely distributed in Brazilian biomes and is
strongly adapted to highly degraded areas, as well as urban forest
fragments (Aléssio et al., 2005).

This type of behavior, called synanthropic, places opossums as a key
group in the connection between wild and domestic epidemiological cycle
of emerging parasites and microorganisms (Forzanari et al., 2011;
Tarragona et al., 2011). For example, opossums and its most common
ectoparasite, \emph{Ctenocephalis felis} flea, have been
epidemiologically linked to flea-borne pathogens transmission and
human-wild life conflicts in residential environments (Krueger et al.,
2016).

Studies support that cat flea (\emph{C. felis} ) is the major competent
vector for \emph{Bartonella henselae} , \emph{B. clarridgeae} , \emph{B.
koehlerae} , and potentially \emph{B. quintana} (Pennisi et al., 2013;
Zangwill, 2013). Those species among others belong to the
\selectlanguage{greek}α-\selectlanguage{english}proteobacteria, are hemotropic, short and pleomorphic Gram-negative
bacteria that cause intra-erythrocytic infections in animals and humans
(Jacomo et al., 2002; Deng et al., 2012). \emph{Bartonella} species
appear to be highly adapted to one or few reservoir hosts, within which
these bacteria have coevolved and thence remain for long periods in
bloodstream (Kaiser et al., 2011; Harms and Dehio, 2012).

Until 1990, only two diseases were linked to \emph{Bartonella} species,
Carri\selectlanguage{ngerman}ón disease (\emph{B. bacilliformis} ) and Trench fever (\emph{B.
quintana} ) (Karem et al., 2000). Since then, many other Bartonellae
have been identified as causative agents of diseases in animals and
humans (Iannino et al., 2018). Importantly, the list of
mammalian-adapted\emph{Bartonella} species continues to grow, as well as
the number of human disease associated with genus \emph{Bartonella}
(Kaiser et al., 2011; Harms and Dehio, 2012; Buffet et al., 2013).

Currently, Cat-scratch disease (CSD) is the most common human disease
caused by \emph{Bartonella} species, including \emph{B. henselae} ,
and\emph{B. clarridgeiae} (Iannino et al., 2018). Clinical manifestation
of CSD depends on patient immune status, and pet cats and dogs often
develop similar manifestations as humans (Chomel et al., 2009;
Breitschwerdt et al., 2010; Beerlage et al., 2012). Typical CSD denotes
a self-limiting illness characterized by fever and lymphadenopathy;
however, atypical manifestations can occur (Jacomo et al., 2002;
Breitschwerdt et al., 2010; Zangwill, 2013).

\emph{Bartonella} genus has already been isolated or detected in
domestic and wild animals worldwide, including bats, birds, canids,
cattle, deer, felids, horses, marine mammals, rodents, sheep and
reptiles (Deng et al., 2012). Little is known about wild reservoirs of
these bacteria in Brazil, but recently, a study reported \emph{B.
henselae} and \emph{B. clarridgeiae} in domestic cats from an Atlantic
Forest area of Rio de Janeiro state (RJ), where humans, domestic,
synanthropic and wild animals can easily interact (Silva et al., 2019).
Opossums are often identified as likely maintainers of infectious agents
with zoonotic potential (Abel et al., 2000; Ruiz-Piñaand Cruz-Reyes,
2002; Fornazari et al., 2011; Pena et al., 2011).

Considering the growing spectrum of animal reservoir hosts
for\emph{Bartonella} species, and given the presence of
\emph{Bartonella}spp. in RJ state, this study aimed to evaluate natural
infections by those bacteria in free-ranging opossums that lived in
peri-urban areas of the Atlantic Forest of RJ, Brazil.

\textbf{Materials and Methods}

This cross-sectional study was approved by Ethics Committee on Animal
Use (CEUA) of Universidade Federal Fluminense (UFF) (8405150718) and
licensed by Biodiversity Information and Authorization System
(SISBIO/IBAMA63464-2).

Adult and young free-living opossums were included in this study.
Animals were seized or rescued by environment alauthorities and samples
were collected at wild life rehabilitation facilities of Universidade
Estácio de Sá (22° 58' 46'' S, 43° 27' 24'' W) (eight \emph{D. aurita} )
and Universidade de Vassouras (22° 24' 14'' S, 43° 39' 45'' W)
(seven\emph{D. albiventris} ), both located in RJ state, Brazil. Animals
were physically restrained and venipuncture was proceeded from ventral
caudal vein, obtaining up to 1.0mL of blood, which was immediately
placed in ethylenediaminetetraacetic acid (EDTA) tubes.

DNA extraction was performed with the Master Pure\textsuperscript{TM}DNA
Purification Kit for Blood Version (Epicentre®, Madison, Wisconsin,
USA). In order to avoid false negative results, DNA samples were tested
for presence of amplifiable DNA using the primer set GAPDH-F
(5'-CCTTCATTGACCTCAACTACAT-3') and GAPDH-R
(5'-CCAAAGTTGTCATGGATGACC-3'), resulting in amplification of a 399 bp
fragment of the housekeeping \emph{gapdh} gene, encoding
glyceraldehyde-3-phosphate dehydrogenase enzyme, which is present in all
mammals (Birkenheuer et al., 2003).

Subsequently, presence of bacterial DNA was tested by conventional and
quantitative polymerase chain reactions (PCR and qPCR) targeting a 365
bp fragment of \emph{Bartonella} spp. citrate synthase (\emph{gltA} )
gene with primers BhCS781p (5' GGGGACCAGCTCATGGTGG 3') and BhCS1137n (5'
AATGCAAAAAGAACAGTAAACA 3'), as previously proposed (Norman et al.,
1995). Positive (purified DNA of \emph{B. henselae} extracted from a
naturally infected cat) and negative (nuclease-free water) controls were
used for each reaction. Amplicons underwent electrophoresis on 1.5\%
agarose gel stained with ethidium bromide.

\textbf{Results and discussion}

Conventional PCR identified \emph{Bartonella} spp. DNA in 40\% of
opossums (6/15). All positive samples belonged to \emph{D.
aurita}species, representing 85.7\% among evaluated samples of this
species (6/7). Using the qPCR assays, we found 46.7\% (7/15) of positive
results. It is important to note that simultaneous positivity in both
PCR and qPCR assays was observed in the same specimens of \emph{D.
aurita} . One animal, of \emph{D. albiventris} species, was negative in
conventional PCR and positive in qPCR.

This is, for the best of author's knowledge, the first report of
natural\emph{Bartonella} spp. infection in opossums in areas of Atlantic
Forest biome at RJ state. Although not expected, the high frequency of
PCR positive animals may be justified, since other studies have
successfully demonstrated presence of \emph{Bartonella} spp. in wild
mammals and ectoparasites in different Brazilian biomes (Favacho et al.,
2015; Sousa et al., 2018). In addition, \emph{C. felis} , the main
vector involved in\emph{Bartonella} spp. transmission among cats, has
been observed in opossums from RJ (Oliveira et al., 2010). It is also
necessary to emphasize the occurrence of \emph{B. henselae} and \emph{B.
vinsonii}subsp. \emph{arupensis} in \emph{C. felis} collected from the
Virginia opossum, \emph{D. virginiana} , from California, USA (Reeves et
al., 2005; Krueger et al., 2016). According to Reeves et al., 2005, the
presence of \emph{B. henselae} in cat fleas collected from opossums,
implicates both \emph{C. felis} and opossums in maintaining this
pathogen.

Recently, Silva et al., 2019 revealed the natural infection
by\emph{B.henselae} and \emph{B. clarridgeiae} in stray cats that
illegally inhabited a preserved area of \selectlanguage{english}\selectlanguage{english}Atlantic Forest in RJ State.
According to the authors, study area harbored a wide variety of fauna
and flora, as well as allowed human visitation and ecotourism. Although
cats are important hosts for \emph{B. henselae} , \emph{B. clarridgeiae}
, and \emph{B. koehlerae} , there are CSD patients that deny the
occurrence of bite, scratch or even contact with cats, making the
possibility of transmission from other animal hosts more and more
probable (Breitschwerdt, 2014).

In this context, roaming animal population, such as that found by Silva
et al., 2019, can be a source of infection for pet cats. Added to this,
we suggest that in epidemiological cycle of bartonellosis, cat flea is
probably the bridge that links wild mammals (such as \emph{D. aurita}and
\emph{D. albiventris} ) to domestic or stray cats and these latter to
humans.

Temperature and relative humidity are relevant factors for successful
reproduction, development, and survival of fleas (Dryden et al., 1994),
and, apparently, cats are more parasitized by fleas during summer and
autumn months (Farkas, 2009). Given that, several places of Atlantic
Forest biome in RJ may provide a favorable scenario for interaction
among \emph{Bartonella} -infected cats, their fleas and susceptible
opossums.

We concluded that Bartonellae have many important
human-animal-environment aspects. For this reason, it is crucial to
adopt the One Health strategy for emerging zoonosis management in stray
animal populations and synanthropic animals, involving collaborative
work of environmentalists, medical and veterinary researchers. The study
continuity is necessary to identify \emph{Bartonella} species involved,
potential risk factors and epidemiological function of synanthropic
mammals (including opossums) as reservoirs in maintenance and
transmission of these bacteria.

\textbf{Acknowledgements}

We are grateful to Pro-Reitoria de Pesquisa, Pos-Graduacao e Inovacao,
Universidade Federal Fluminense (Proppi/UFF) for financial support and
scholarship.

\textbf{Conflict of Interest Statement}

The authors declare no conlict of interest.

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