Bartonella in Norway rats ( Rattus norvegicus ) from the urban slum environment in Brazil

: Bartonella are rodent-borne bacteria that cause varied human etiologies. Studies on synanthropic rodents are rare, causing gaps in epidemiological knowledge. We tested bloodclot samples from 79 rats from an urban slum in Salvador, Brazil through PCR targeting gltA gene. Nine samples (11.4%) were positive: six had 100% identity with Bartonella sp. isolate JF429580 and 99.5% with B. queenslandensis strain AUST/NH8 ; three were 100% identical to isolate JF429532 and 99.7% to B. tribocorum . This is the second report on urban rat Bartonella indicating bacterial circulation at detectable rates. Its presence in rats from vulnerable human settlements demands public health attention.


INTRODUCTION
Infections by bacteria from the genus Bartonella represent an array of neglected health issues that can range from asymptomatic chronic infections, to serious, and potentially fatal manifestations (Lins et al. 2019, Chomel et al. 2003, Rolain et al. 2004).Mammals are the primary hosts, with emphasis on rodents, both wild and synanthropic (Gonçalves et al. 2016, Gonçalves-Oliveira et al. 2020).Human infections are associated to close proximity to the hosts and their fleas, and unsanitary conditions.At least 18 genotypes of Bartonella are associated to human infections (Lins et al. 2019, Frank et al. 2018).
Synanthropic rats (genus Rattus) are key hosts of zoonotic pathogens in poor urban settlements, where environmental conditions are prone to rodent infestation, and human populations are forced to coexistence closely, contributing to rat-borne spillover transmissions (Costa et al. 2014b).Rodents have recently been identified as carriers of Bartonella spp. in sylvatic and urban contexts (Costa et al. 2014a, Gonçalves et al. 2016).This context poses rats as key subjects for One Health research, allowing to understand pathogen circulation, predict disease incidence, and inform public health policies (Costa et al. 2014b(Costa et al. , 2021)).
However, in spite of its public health relevance and evidence of circulation in wild and urban fauna, the ecoepidemiology of Bartonella is still understudied and underestimated (Lins et al. 2019).Brazil suffers with particular lack of studies (Silva et al. 2019), especially evaluating non-human hosts and their potential as sources of infection (Braga et al. 2012).The present study reports the presence and molecular characterization of Bartonella in rats captured in poor, urban communities in Salvador, Bahia, Brazil.

MATERIALS AND METHODS
The present study was conducted in 2012 as a pilot to test a systematic rodent sampling methodology (see Panti-May et al. (2016)) at Pau da Lima, a poor urban community in the city of Salvador, Bahia, Brazil.This area is characterized by inadequate and/or absent coverage of urban sanitation services (trash collection, sewage and drainage systems), and is known for high rodent infestation and high incidence of rodent-associated zoonosis; the area is subdivided in three valleys, and this study was conducted in Valley 1 (for a full characterization, see Panti-May et al. (2016)).One hundred and eight sampling points were semi-randomized within the study area in the peridomicile (both around households and within yards of the houses), with two live traps per point for four nights.Captured animals were taxonomically identified by trained biologists during necropsy.Procedures for tissue sampling and Bartonella spp.detection was performed by PCR, with material extracted from blood clot samples following the methodology of Kosoy et al. (1997), briefly the samples were extracted using the QIAamp Tissue Kit (Qiagen Inc., California, USA).Amplification was performed using primers targeting the gltA gene, 0.5 µM of each primer, 10 µL of the sample in a mix of 50 mM KC1, 10 mM Tris-HC1, 1.5 mM MgC12, 0.001% gelatin, 0.1% Brij-35, 200 µM of each deoxynucleotide triphosphate, and 0.2 U of thermostable Ampli-Taq DNA polymerase (Perkin-Elmer-Cetus, Connecticut, USA).Incubation happened at 95°C for two minutes, amplification occurred for 40 cycles at: 95°C (1 minute), 50°C (1 minute), and 72°C (1 minute).All amplifications were verified by an agarose (2%) in Tris-borate-EDTA buffer (0.1 M Tris, 0.09 M of boric acid, 0.001 M of EDTA) electrophoresis.Purification was performed using Wizard PCR Preps (Promega, Wisconsin, USA) for sequencing.Sequencing was performed using a Cetus 9600 thermocycler (Perkin-Elemer-Cetus), where the PCR results were sequenced in both directions with PRISM dye-terminator cycle sequencing kit (Applied Biosystems Inc.), following all manufacturers' specifications.Resolution was obtained using an ABI-Prism autosequencer (Applied Biosystems, Inc.) by polyacrylamide gel (4%) electrophoresis, with the reaction performed at 51°C and constant 1500V.All activities were conducted with approval from the Ethics Committee on Use of Animals of the Oswaldo Cruz Foundation, Salvador, Bahia, Brazil (protocol 003/2012), and the Yale University's Institutional Animal Care and Use Committee (IACUC), New Haven, Connecticut (protocol number 2012-11498).

RESULTS
A total of 117 Brown rats (Rattus norvegicus) were captured, but only 79 had blood clots were collected and used for Bartonella detection by PCR.Nine samples were positive (Table I): six had 100% identity with Bartonella sp.isolate JF429580 (Gundi et al. 2012) and 99.5% identity with B. queenslandensis strain AUST/NH8 (3 mature females, one immature female, one mature male, and one male of unknown age); while the remaining three samples were 100% identical to Bartonella sp.isolate JF429532 (Gundi et al. 2012) and 99.7% to B. tribocorum (all females, 2 immatures and 1 mature).Overall positivity rate for the samples tested was 11.4%.The lack of studies on the subject in Brazil, although not totally unexpected given Bartonellosis' status as neglected infections (Lins et al. 2019), is cause for concern given the absence of basic epidemiological data for the country on an infection that is underreported Silva et al. 2019, Lins et al. 2019).The problem of rodent-borne diseases mainly affects the most vulnerable of society, that are susceptible to disease transmission due to the environmental conditions of poor urban areas.These populations disproportionately carry a higher burden of disease, which is one mechanism that leads to the poverty trap.(Garchitorena et al. 2017, Costa et al. 2014b, 2017).This is particularly true for Bartonella infections, commonly more prevalent in marginalized or underserved populations (Chamberlin et al. 2002).The present results can be considered a sign for concern regarding a neglected group of potentially pathogenic bacteria circulating in a synanthropic reservoir already implicated in the endemic transmission of zoonotic pathogens in Salvador (Costa et al. 2014a, b, Felzemburgh et al. 2014).However, further studies are necessary to properly characterize the situation of Bartonella circulation in order to inform an action plan.

Table I .
Results for the PCR tests performed on rat blood samples to detect Bartonella sp., with results indicating whether the gltA gene was positively detected and posterior sequence (Seq) identity.

Table I .
Continuation.

Table I .
Continuation.