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    Distribution of Bartonella species in shelter cats by period (months).

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Molecular Survey of Bartonella Species in Shelter Cats in Rio De Janeiro: Clinical, Hematological, and Risk Factors

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  • 1 Departament of Veterinary Medicine and Surgery, Veterinary Institute, Federal Rural University of Rio de Janeiro, Seropédica, Brazil;
  • 2 Departament of Animal Parasitology, Veterinary Institute, Federal Rural University of Rio de Janeiro, Seropédica, Brazil;
  • 3 Oswaldo Cruz Foundation, Rio de Janeiro, Brazil;
  • 4 Oswaldo Cruz Foundation, Campo Grande, Brazil

The present study aimed to detect Bartonella DNA in cats belonging to shelters, and to evaluate risk factors, clinical signs, and hematological abnormalities associated with infection. Complete blood counts and screening for the presence of Bartonella DNA were performed on cats’ ethylenediamine tetraacetic acid anticoagulant–blood samples. Eighty-three cats (39.9%) were positive for Bartonella species. Bartonella DNA was also detected in fleas and in the blood of cats infested by positive flea. Cats that had not been sterilized, had outdoor access, had histories of fights, and had concurrent flea infestation were more likely to be infected by Bartonella species (P < 0.05). Age and sex were not associated with infection. Fifty-one (38.6%) symptomatic cats were positive to Bartonella species (P > 0.05). Clinical conditions most commonly observed were signs of respiratory abnormality and Sporothrix species coinfection (P > 0.05). Regarding hematological changes, eosinophilia was associated with infection (P < 0.05). A high frequency of Bartonella species infection was found in shelter cats and highlights the importance of adequate flea-control programs to prevent infection in cats and consequently in adopters and other animals.

INTRODUCTION

Bartonella species are Gram-negative facultative intracellular and arthropod-transmitted bacteria that infect numerous host species, including humans, rodents, rabbits, felids, canids, and ruminants.1 Cats are likely the main mammalian reservoir hosts for the zoonotic species Bartonella henselae, Bartonella clarridgeiae, and Bartonella koehlerae, agents commonly associated with human disease and transmitted between cats by Ctenocephalides felis fleas.2 Human transmission occurs by means of cat bites and scratches with teeth and nail contaminated with flea feces. In humans, these bacteria are responsible for many clinical syndromes, including cat scratch disease, endocarditis, bacillary angiomatosis, bacillary peliosis, meningitis, and glomerulonephritis.2,3 Some of these can be fatal, especially for immunocompromised patients.2,3

Although Bartonella infection in cats is common, details of the relationship of these bacteria with the cat, including the possible role of Bartonella species in the pathogenesis of feline diseases, are far from clear.1 Most infected cats remain asymptomatic carriers for years. However, studies have suggested a potential association between Bartonella DNA detection and clinical signs in cats, such as endocarditis,4,5 stomatitis,6 pyogranulomatous myocarditis and diaphragmatic myositis,7 fever of unknown cause,8,9 and central nervous system diseases.10 Regarding hematological findings, limited data are available in cats that were naturally or experimentally infected. Nevertheless, some cats have shown transient anemia, persistent eosinophilia, and mature neutrophilia.11,12

Bartonellosis in cats occurs worldwide. However, in Brazil, there are very few studies, especially focusing on detection and molecular characterization of Bartonella species.1316 Considering that bartonellosis can lead to nonspecific signs, studies involving these agents are important, particularly to assess their actual occurrence in veterinary clinical practice.

The aims of this study were to detect Bartonella DNA in blood of potentially naturally infected cats by means of polymerase chain reaction (PCR) diagnosis and to evaluate associated risk factors, clinical signs, and hematological abnormalities in cats from shelters located in Metropolitan Region of Rio de Janeiro, Brazil.

METHODS

Cat samples.

The procedures followed in this study were previously approved by the Ethics Committee for the use of animals at the Universidade Federal Rural do Rio de Janeiro (protocol number 027/2014).

The survey was carried out in shelters that are autonomous and independent organizations that aim to rescue, sterilizate, and promote adoption of abandoned cats located in the Metropolitan Region of Rio de Janeiro, Brazil. Most of the cats probably still live in the shelter and stay available for adoption in events often promoted in fairs in public spaces, playgrounds, and agricultural stores.

Two-hundred eight cats were sampled and divided into groups: group 1, those in the Rio de Janeiro municipality (68 cats in four shelters), and group 2, those in the Belford Roxo, Duque de Caxias, Guapimirim, Itaguaí, Mesquita, Nova Iguaçu, São Gonçalo, and Seropédica municipalities (140 cats in 14 shelters).

Blood samples were aseptically obtained by cephalic phlebotomy, transferred into sterile tubes containing ethylenediamine tetraacetic acid anticoagulant, and maintained at 4°C until hematological analysis (up to 12 hours after blood collection).

Cats were physically examined for ectoparasites presence, which were also collected when possible. All ectoparasites were identified based on the morphological criteria observed with a stereoscopic microscope, according to standard taxonomic keys.17

Tutors or those responsible for the shelters have signed a consent form, allowing the study at the shelter and ensuring to provide accurate and honest answers to the semi-structured questionnaire applied. As so, the following data were recorded for each cat: age (kitten ≤ 1 year versus adult > 1 year), sex (male versus female), outdoor access, sterelity status, evidence of fight history, ectoparasites status (previous and current infestation), ectoparasiticide use, vaccination status, worm therapy, and contact with other animal species. Following clinical evaluation, cats were classified as symptomatic or asymptomatic according to clinical manifestation previously reported on the literature6,18 and other clinical signs observed during feline physical examination.

Hematological analysis.

Complete blood count was measured using the fully automated analyzer Poch-100 iV (Roche, Basel, Switzerland), in accordance with the manufacturer’s recommendations. Differential leukocyte count, hematological abnormalities, and hemoparasites examination were performed manually on Diff Quick–stained thin blood smears, using an optical microscope, with immersion objective lens magnification of ×100 and total plasma protein concentration determined by refractometry.19 After hematological tests were completed, blood samples were stored at −80°C until molecular analyses were undertaken.

Molecular assays.

DNA extraction from 200 μL of whole blood was performed using a ReliaPrepTM Blood gDNA Miniprep System kit (Promega, Madison, WI), in accordance with the manufacturer’s instructions. Flea DNA extraction was performed individually as previously described.20 Concentration and purity were determined using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA). For monitoring contaminant DNA during the process of DNA extraction, sterilized ultrapure water was used as a negative control and processed in parallel with the samples. DNA samples were screened for the presence of the 16S-23S rRNA intergenic spacer (ITS) region and the gltA gene using primers previously reported.21,22 Following amplification, PCR products were subjected to horizontal electrophoresis on 1% agarose gel and stained with GelRed (Biotium, Fremont, CA). The positive control consisted of B. henselae (Houston strain) cultured in HEp-2 cells. All PCR runs were performed with nuclease-free water (Invitrogen, Waltham, MA) as negative control.

Sequence analysis.

A total of 51 positive cat samples were randomly selected, purified using an Illustra GFX PCR Purification Kit (GE Healthcare, Pittsburgh, PA), and submitted to sequencing using an automatic sequencer (ABI3730xl, Applied Biosystem, Waltham, MA). Sense and antisense sequences were analyzed using DNA Sequence Assembler v.4 software and compared with those deposited in the GenBank DNA database using the basic local alignment search tool (BLAST, National Center for Biotechnology Information, available at https://blast.ncbi.nlm.nih.gov).

Statistical analysis.

Association between Bartonella species infection and risk factors, months of blood collection, clinical signs, and hematological abnormalities was evaluated by comparing frequencies with Chi-square and Fisher’s exact test, with a significance level of 5%. Hematological analyses were evaluated with the Lilliefors normality test, followed by analysis of variance or the Mann–Whitney test at the 5% significance level. In addition and in accordance to reference intervals,23 frequencies of the hematological abnormalities eosinophilia (eosinophils count > 0.8 × 103 cells/µL), thrombocytopenia (platelets count < 300 × 103 cells/µL), lymphopenia (lymphocytes count < 1.5 × 103 cells/µL), and hyperproteinemia (total plasma protein concentration > 7.5 g/dL) were correlated with Bartonella positivity. All statistical tests were performed using Bioestat 5.0 statistical software (Instituto de Desenvolvimento Sustentável Mamirauá, Amazonas, Brazil).

RESULTS

Of the 208 shelter cats sampled, 83 (39.9%) were positive for Bartonella species based on ITS (16S-23S) and gltA gene results. The frequencies in groups 1 and 2 were 22.1% and 48.6%, respectively (P < 0.01) (Table 1). Bartonella DNA was also detected in fleas from cats infected by the bacteria. Cats were more frequently infected by Bartonella species during the period of March to June (52.2%) than during December to March (27.7%) (P < 0.05) (Figure 1, Table 2). Among the 51 positive samples selected, sequencing confirmed B. henselae (68.6%, 35/51), B. clarridgeiae (23.5%, 12/51), and B. koehlerae (17.6%, 9/51) infections in cats. Cases in which ITS and gltA sequences from a single same cat corresponded to different feline Bartonella species were considered coinfections. In this way, three (5.9%) and two (3.9%) cats were concurrently infected with B. henselae plus B. clarridgeiae and B. clarridgeiae plus B. koehlerae, respectively. Cat fleas were found to harbor B. henselae and B. clarridgeiae DNA. No B. koehlerae specie was detected in fleas.

Table 1

Distribution of Bartonella species bacteremia in shelters cats located in municipaties at the Metropolitan Region of Rio de Janeiro, Brazil

Bartonella species infection
GroupMunicipalityNo. of catsPositiveNegative%P-value
1Rio de Janeiro68155322.10.0004
2Others140687248.6
Duque de Caxias74357.1
Itaguaí169756.3
Mesquita2091145.0
Nova Iguaçu2011955.0
Seropédica46242252.2
Belford Roxo74357.1
São Gonçalo156940.0
Guapimirim91811.1
Figure 1.
Figure 1.

Distribution of Bartonella species in shelter cats by period (months).

Citation: The American Journal of Tropical Medicine and Hygiene 100, 6; 10.4269/ajtmh.18-0585

Table 2

Statistical analysis of factors associated with Bartonella species bacteremia in cats from shelters located in the Metropolitan Region of Rio de Janeiro state, Brazil

VariableCats (n)Positive (%)P-value (X2)Odds ratioCI (95%)
Age
 Young (≤ 1 year)6828 (41.2)1
 Adult (> 1 year)14055 (39.3)0.91221.080.60–1.95
Sex
 Female11041 (37.3)1
 Male9842 (42.9)0.49711.260.72–2.20
Outdoor access
 No13546 (34.1)1
 Yes7337 (50.7)0.02881.991.11–3.55
Sterilized
 No10149 (48.5)2.02
 Yes10734 (31.8)0.020211.15–3.56
Historic of fights
 No15152 (34.4)1
 Yes5731 (54.4)0.01382.271.22–4.22
Present ectoparasite infestation
 No276 (22.2)1
 Yes18177 (42.5)0.07182.591.00–6.73
Past ectoparasites infestation
 No8427 (32.1)1
 Yes12456 (45.2)0.08241.740.97–3.10
Present tick infestation
 No19678 (39.8)1
 Yes125 (41.7)0.99981.080.33–3.53
Present lice infestation
 Yes248 (33.3)1
 No18475 (40.8)0.63321.380.56–3.38
Present flea infestation
 No10032 (32.0)1
 Yes10851 (47.2)0.03591.91.08–3.35
Past flea infestation
 No286 (21.4)1
 Yes18077 (42.8)0.05252.741.06–7.09
Ectoparasiticide use
 Yes13546 (34.1)1
 No7337 (50.7)0.02881.991.11–3.55
Vaccination
 Yes8928 (31.5)1
 No11955 (46.2)0.04471.871.05–3.33
Worm therapy
 Yes16865 (38.7)1
 No4018 (45.0)0.58051.30.64–2.60
Contact with other species
 No7824 (30.8)1
 Yes13059 (45.4)0.05271.871.03–3.38
Contact with dogs
 No239 (39.1)1
 Yes18573 (40.0)0.88441.040.43–2.52
Contact with opossums
 No19675 (38.3)1
 Yes128 (66.7)0.06853.220.94–11.1
Contact with horse
 No18672 (38.7)1
 Yes2211 (50.0)0.42811.580.65–3.84
Period (months)
 September 22–December 204315 (34.9)
 December 21–March 194713 (27.7)1
 March 20–June 96735 (52.2)0.01542.861.29–6.36
 June 20–September 215120 (39.2)

In addition, cats that were not sterilized, had outdoor access, showed evidence of fight histories, were infested with fleas at the time of collection, and did not receive ectoparasiticide prophylactic management or vaccination protocols were more likely to be infected by Bartonella species (P < 0.05). Young and adult cats of both sexes had a similar chance of becoming infected (P > 0.05).

Cats with past or present ectoparasite infestation or prior flea infestation had greater chances of bacterial infection, although these findings were not statistically significant (P > 0.05). By contrast, tick or lice infestation was not linked to infection (P > 0.05). Cats in contact with other species, especially opossums as reported by the owners, were more likely to be infected, although the correlation was not significant (P > 0.05) (Table 2).

Symptomatic (38.6%) and asymptomatic (42.1%) cats had similar chances of becoming infected (P > 0.05). Bacterial infection was seen most commonly in cats presenting respiratory signs (42.6%) or Sporothrix species infection (39.3%). Less commonly, it was observed in cats with oral (28.6%) or ocular (20%) signs and chronic diarrhea (16.7%) (P > 0.05). Nonspecific clinical signs such as anorexia, lethargy, dehydration, fever, and lymphadenopathy were not associated with infection (P > 0.05) (Table 3). Hemoparasites were not detected at the microscopical evaluation of blood smears. Eosinophilia was associated with infection (P < 0.05), being observed in 64% of Bartonella species positive cats. No other hematological abnormality was associated with infection (P > 0.05) (Tables 4 and 5).

Table 3

Association between Bartonella species bacteremia in asymptomatic and symptomatic cats, clinical signs, and diseases reported in 208 shelter cats from the Metropolitan Region of Rio de Janeiro state, Brazil

Clinical signs or diseasePositiveNegativeTotal%P-value
Asymptomatic32447642.10.7301
Symptomatic518113238.6
 Anorexia011110.00.0036
 Lethargy0770.00.0434
 Reproductive failure0110.01.0000
 Diarrhea2101216.70.1295
 Skin lesions5172222.70.1312
 Ocular signs14520.00.6502
 Oral signs4101428.60.4142
  Stomatitis0110.01.0000
  Gengivitis3101323.10.2515
  Ulcers in the tongue11250.01.0000
 Urinary tract disorders13425.00.6519
 Fever (> 39.2°C)12253732.40.3729
 Dehydration19385733.30.3029
 Sporotrichosis11172839.30.8921
 Respiratory Signs20274742.60.8008
 Hyperthyroidism11250.01.0000
Table 4

Hematological results from 208 shelter cats screened for Bartonella species infection

Bartonella species
ParameterResultMeanDPEPP-valueReference23
RBC (106/µL)Positive8.31.50.20.66455.0–10.0
Negative8.11.60.2
Hematocrit (%)Positive37.76.50.70.309830–45
Negative36.17.00.6
Hemoglobin (g/dL)Positive11.62.00.20.23359.8–15.4
Negative11.12.30.2
MCV (fL)Positive45.54.80.50.419739–55
Negative44.74.10.4
MCHC (%)Positive30.82.10.20.865530–36
Negative30.81.80.2
Platelet (103/µL)Positive37113515.00.0494300–800
Negative33414212.7
TPPC (g/dL)Positive7.90.80.10.01096.0–7.5
Negative8.31.10.1
WBC (103/µL)Positive17.147.550.830.45875.5–19.5
Negative16.708.130.73
Band neutrophils (103/µL)Positive0.250.310.030.68060.0–0.3
Negative0.350.590.05
Segmented neutrophils (103/µL)Positive10.216.770.740.97192.5–12.5
Negative10.236.830.61
Lymphocytes (103/µL)Positive4.963.130.340.21161.5–7.0
Negative4.352.580.23
Monocytes (103/µL)Positive0.450.380.040.25680.0–0.9
Negative0.530.470.04
Eosinophils (103/µL)Positive1.270.880.100.01690.0–0.8
Negative1.141.140.10
Basophils (103/µL)Positive0.010.070.010.96150.0–0.2
Negative0.010.060.01

DP = standard deviation; EP = standard error; SD = standard deviation; SE = standard error; RBC = red blood cell count; MCV = mean corpuscular volume; MCHC = mean corpuscular hemoglobin concentration; TPPC = total plasma protein concentration; WBC = white blood cell count.

Table 5

Hematological results for Bartonella infection in cats from shelters located in the Metropolitan Region of Rio de Janeiro state, Brazil

Bartonella species n (%)
Hematological changePositive (n = 83)Negative (n = 125)P-value
EosinophiliaYes53 (63.9)60 (48.0)0.0352
No30 (36.1)65 (52.0)
ThrombocytopeniaYes20 (24.1)50 (40.0)0.0259
No63 (75.9)75 (60.0)
LymphopeniaYes02 (2.4)14 (11.2)0.0305
No81 (97.6)111 (88.8)
HyperproteinemiaYes52 (62.7)92 (73.6)0.1280
No31 (37.3)33 (26.4)

DISCUSSION

Zoonosis studies have become more important in recent years because the cat population has risen noticeably and adopting domestic animals has become more common. In Brazil, few previous studies have addressed Bartonella infection in cats, including risk factors, and hematological and clinical abnormalities. In the present study, 39.9% of cats tested positive for Bartonella DNA, which is higher than those found in previous reports, including those that address cats from shelters.13,15,2426 Similar results were reported in California, United States.26 Notably, it was found that 97.3% of cats harbored bacterial DNA in a shelter in Volta Redonda municipality, Rio de Janeiro, which is attributable to local conditions.27

Bartonella infection was significantly higher in cats from group 2 when than in those from group 1 (Rio de Janeiro municipality), which may be explained by differences in hygiene conditions and preventive flea control applied in these shelters. Approximately 69% (97/140) of the cats from group 2 shelters were flea-infested, which represents four times the level observed in cats from group 1 (16.1%, 11/68). This finding supports us to establish a correlation between bacteremia and flea infestation. The presence of Bartonella DNA in C. felis fleas collected in bacteremic cats also suggests that these ectoparasites play an important role in the transmission of Bartonella species to cats. Ctenocephalides felis was shown to be a potential vector for Bartonella species, including those for which cats serve as a natural reservoir, B. henselae and B. clarridgeiae.28

Apparently, there is a seasonal trend of this bacterial infection. In the study, we observed that the number of cats infected by the bacteria (52.2%, 35/67) and infested by fleas (55.5%, 60/108) were both highest in the autumn months (March 20–June 19), a finding also observed in previous studies.26,29 A possible explanation is that owners, thinking that cat flea season is over, reduce measures to control flea infestation in their pets. Indeed, autumn months represent an active period for fleas, especially in Rio de Janeiro, where temperatures remain moderate year round and so fleas can proliferate. This pattern of seasonal incidence was also observed in humans infected with Bartonella in France. Authors have attributed such finding to cat flea activity and cat’s behavior of spending more time inside the house during autumn months, and therefore, it increases the risk for transmission to humans.30

Previous or current ectoparasite infestation showed a nonsignificant association with Bartonella species bacteremia in cats, with animals that had fleas at any time in their life being almost three times more likely to be bacteremic than those that had not. Cats harboring fleas at the time of blood collection had significantly increased risk of being infected by Bartonella species. A similar correlation to B. henselae bacteremia was found.31 In addition, vector-control programs were crucial to avoid Bartonella species infection, as cats that did not use ectoparasiticides were significantly more likely to be infected. These findings reinforce the association between Bartonella species bacteremia in cats and past or present flea contact, and highlights C. felis as a primary vector for these bacterial agents. It is noteworthy that prevention of Bartonella infection in cats is best accomplished by preventing exposure to ectoparasites, especially fleas.

Non-sterilized cats and those with outdoor access were significantly more likely to be bacteremic than other cats. Although it was not associated with infection, a study found that non-sterilized cats were twice as likely to be bacteremic than sterilized ones.32 These findings reinforce the importance of fleas in cat infection as non-sterilized cats and those with outdoor access are greater exposed to arthropod vectors.

Interestingly, cats with histories of fight behavior had twice the chance of becoming infected. Experimental intradermal inoculation of Bartonella species has induced bacteremia in cats.33,34 In addition, Bartonella species DNA has been successfully detected from saliva and nails of naturally infected cats.26,35,36 This evidence suggests the possibility that cats have other sources of infection besides direct horizontal transmission via fleas. Previous studies have demonstrated that Bartonella bacteremia is higher in younger animals,24,37 although our results demonstrate that young and adult cats of both sexes have similar chance of becoming infected. Such results are consistent with other studies.32,38

Although the association was not significant, cats that lived in the presence of other animals generally showed almost two times more chance of becoming infected by Bartonella species. Interestingly, the presence of opossums in the neighborhood increased the risk of infection by three times. Opossums (Didelphis species) are synanthropic animals and, therefore, adapted to peridomicile environments. Ctenocephalides felis flea, the main ectoparasite involved in transmission of Bartonella species among cats, has been shown to parasitize opossum species in Brazil, including Rio de Janeiro State.39,40 Along these lines, the presence of these ectoparasites in opossums indicates their proximity to human residences and to domestic animals. This interaction highlights the imminent risk of pathogen transmission between animal species through arthropod vectors and, consequently, to humans, thus posing a public health risk.39 More studies are needed to understand the importance of opossums in epidemiological chain of this disease.

Endoparasites’ presence or absence seems not to be related to Bartonella infection in cats. On the other hand, cats without adequate vaccinations were at significantly higher risk of infection. It is important to note that the shelter cats studied generally live under constantly stressful conditions, including poor nutrition, crowding, inadequate hygiene, and vaccination measures, which may interfere with the cats’ immune status.41 In such scenarios, inadequate or absent vaccinal support predispose the animals to such imunossupressive diseases as retroviruses, which appear to increase the pathogenicity of B. henselae infection in cats.42

No clinical signs were associated with Bartonella species infection because symptomatic and asymptomatic cats have similar chances of becoming infected. This finding is consistent with those of other authors,18 according to whom most naturally infected cats do not show clinical signs and appear to tolerate chronic bacteremia without obvious clinical abnormalities. However, previous studies have suggested Bartonella species are causative agents of clinical condition such as endocarditis, stomatitis, pyogranulomatous myocarditis and diaphragmatic myositis, fever of unknown cause, and central nervous system diseases.410 It should be mentioned that it is difficult to establish causal associations between clinical conditions and such pathogens as Bartonella species, which have high prevalence in the reservoir host population. This high prevalence of asymptomatic bacteremia indicates that there is a long history of coevolution between Bartonella and their cat hosts.43

Many of the symptoms exhibited by the cats of this study are nonspecific and common to other diseases affecting these animals routinely. Therefore, the likelihood that some clinical conditions have multiple causes must also be considered, particularly in cats exposed to arthropod vectors. Thus, a differential diagnosis with other diseases of common occurrence in the feline clinical routine is necessary.12

Regarding laboratory findings, eosinophilia was highly associated with Bartonella species infection in cats, being present in almost 64% of bacteremic cats. In accordance with our results, a study has observed persistent eosinophila in cats experimentally infected with Bartonella species and related this hematological finding with infection chronicity.11 On the other hand, researchers demonstrated that hematological findings in Bartonella spp.–infected cats may be uncommon.44 It is important to note that other clinical conditions may also cause this hematological change, such as infection by intestinal parasites and skin or respiratory abnormalities found in cats from this study. Further studies are needed to establish the correlation between eosinophilia and Bartonella infection.

Concerning the zoonotic aspect of Bartonella infection, we highlight the importance of testing cats before adoption and a rigorous management of flea control in the ambient and cat adopted, especially because of the potential risk to immunocompromised patients. The high occurrence of Bartonella species in cats from shelters at Metropolitan Area of Rio de Janeiro emphasizes the need for local veterinary community, owners, and public health authorities to be alert to the risk of infection. Ectoparasites control measures, especially those designed to prevent flea infestation, should be implemented to minimize the risk of infection by cats, other vertebrate hosts, and humans.

Acknowledgments:

We acknowledge the researchers James R. Welch for revising English language of this manuscript and Maria L. Corrêa and Caio J. B. Coutinho Rodrigues for helping in the blood sample collection.

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Author Notes

Address correspondence to Cristiane D. Baldani, Departament of Veterinary Medicine and Surgery, Veterinary Institute, Federal Rural University of Rio de Janeiro, BR 465, Km 47, Seropédica, Rio de Janeiro CEP 23890-000, Brazil. E-mail: crisbaldani@gmail.com

Financial support: This research was financially supported by Carlos Chagas Filho Foundation for Research Support in the State of Rio de Janeiro (FAPERJ; grant number: E-26/110.386/2014). The American Society of Tropical Medicine and Hygiene (ASTMH) assisted with publication expenses.

Authors’ addresses: Juliana M. Raimundo, Andresa Guimarães, Gleice M. Amaro, Aline T. da Silva, Camila F. M. Botelho, Carlos L. Massard, and Cristiane D. Baldani, Federal Rural University of Rio de Janeiro, Seropédica, Brazil, E-mails: julianam_rj@hotmail.com, andresaguimaraes02@yahoo.com.br, gleicemamaro@outlook.com, aline_tonussi@yahoo.com.br, camilafmbotelho@gmail.com, carlosmassard@ufrrj.br, and crisbaldani@gmail.com. Elba R. S. de Lemos, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil, E-mail: elemos@ioc.fiocruz.br. Alexsandra R. M. Favacho, Oswaldo Cruz Foundation, Campo Grande, Brazil, E-mail: crisbaldani@gmail.com.

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