pmc951249620657J Vector EcolJ Vector EcolJournal of vector ecology : journal of the Society for Vector Ecology1081-17101948-7134266119681094936310.1111/jvec.12171HHSPA1972360ArticleCoexistence of Bartonella henselae and B. clarridgeiae in populations of cats and their fleas in GuatemalaBaiYing1RizzoMaria Fernanda1AlvarezDanilo2MoranDavid2PeruskiLeonard F.3KosoyMichael1Bacterial Disease Branch, Division of Vector-Borne Disease, Centers for Disease Control and Prevention, Fort Collins, Colorado, U.S.A.Centro de Estudios en Salud, Universidad del Valle de Guatemala, Guatemala City, GuatemalaCenters for Disease Control and Prevention, Central American Regional Office, Guatemala City, Guatemala bby5@cdc.gov 13320241220151932024402327332

Cats and their fleas collected in Guatemala were investigated for the presence of Bartonella infections. Bartonella bacteria were cultured from 8.2% (13/159) of cats, and all cultures were identified as B. henselae. Molecular analysis allowed detection of Bartonella DNA in 33.8% (48/142) of cats and in 22.4% (34/152) of cat fleas using gltA, nuoG, and 16S–23S internal transcribed spacer targets. Two Bartonella species, B. henselae and B. clarridgeiae, were identified in cats and cat fleas by molecular analysis, with B. henselae being more common than B. clarridgeiae in the cats (68.1%; 32/47 vs 31.9%; 15/47). The nuoG was found to be less sensitive for detecting B. clarridgeiae compared with other molecular targets and could detect only two of the 15 B. clarridgeiae-infected cats. No significant differences were observed for prevalence between male and female cats and between different age groups. No evident association was observed between the presence of Bartonella species in cats and in their fleas.

Catscat fleasBartonellaB. henselaeB. clarridgeiaeGuatemalaINTRODUCTION

At least three Bartonella species, B. henselae, B. clarridgeiae, and B. koehlerae, are associated with cats. With a worldwide distribution, B. henselae is the most common of the three species with a considerable variation in prevalence observed across different regions (Chomel et al. 1995, 2002, Bergmans et al. 1996, Branley et al. 1996, Maruyama et al. 2001). B. clarridgeiae is also reported throughout most temperate regions of the world (Heller et al. 1997, Chomel et al. 1999, Marston et al. 1999, Maruyama et al. 2000), while B. koehlerae is less common compared with the other two species (Droz et al. 1999). Bartonella infections are more likely in younger cats (<1 year old) (Chomel et al. 1995). Two main genotypes of B. henselae (Houston I and Marseille) have been identified based on 16S rRNA gene sequences (Bergmans et al. 1996, La Scola et al. 2002). The respective prevalence of these two genotypes varies considerably among cat populations from different geographical areas. B. henselae Marseille is the dominant type in cats from western U.S.A., Australia, and most of Europe, whereas Houston I represents the majority of B. henselae isolates in cats from the eastern U.S.A. and East Asia (Boulouis et al. 2005). Epidemiological evidence and experimental studies have shown that the cat flea (Ctenocephalides felis) plays a major role in the transmission of B. henselae and B. clarridgeiae among cats (Chomel et al. 1996). Cats infected with B. henselae and other Bartonella species are typically asymptomatic with a persistent bacteremia lasting from several months to years (Koehler et al. 1994, Abbott et al. 1997). B. henselae is responsible for various human infectious diseases, including vasoproliferative illness (bacillary angiomatosis), hepatosplenic granulomatosis, peliosis hepatitis, fever, central nervous disorders, and, most commonly, cat scratch disease (CSD) (Welch et al. 1992, Branley et al. 1996). Recently, B. henselae has been identified as the causative agent of infective endocarditis in Thailand (Pachirat et al. 2011, Watt et al. 2014). B. clarridgeiae also has been reported as a causative agent of cat scratch disease (Kordick et al. 1997, Margileth and Baehren 1998), as well as other diseases (Sander et al. 2000).

In Guatemala, Bartonella infections are prevalent in cattle and bats (Bai et al. 2011, 2013). However, cats and their fleas have not been assessed for Bartonella infections in this country. Considering the ubiquity of cats, their association with humans, and the distribution of Bartonella species, it is important to estimate the status of Bartonella infections in local populations of cats and cat fleas in Guatemala. This in turn can provide information for estimating the risk of acquiring cat-originated Bartonella species by people. The present study aimed to identify Bartonella species using both blood culture and molecular detection in cats and their fleas, and determine its prevalence.

MATERIALS AND METHODSSample collection

Cats from pet clinics or neutering and spaying campaigns conducted in seven sites within Guatemala were recruited to the study during January, 2013 and August, 2013. Cat fleas were collected in 70% alcohol; cats were recorded for gender, age, weight, clinical symptoms, and flea infestation status. Collected blood was stored at −70° C until processing.

Isolation of <italic toggle="yes">Bartonella</italic> bacteria from cat blood

Cat blood was thawed at 4° C and re-suspended 1:4 in brain heart infusion broth supplemented with 5% amphotericin B (1μg/ml) for the purpose of reducing fungal contaminants. Then 100 μl diluted blood (25 μl whole blood) was plated on heart infusion agar containing 10% rabbit blood and incubated in an aerobic atmosphere with 5% carbon dioxide at 35° C for up to four weeks. Bacterial growth was monitored at the end of each week. Bacterial colonies were presumptively identified as Bartonella based on colony morphology. Subcultures of Bartonella colonies from the original agar plate were streaked onto secondary agar plates and incubated at the same conditions until sufficient growth was observed. Pure cultures were harvested in 10% glycerol.

Confirmation and multi-locus sequence typing (MLST) of <italic toggle="yes">Bartonella</italic> isolates

Crude genomic DNA was prepared by heating a heavy suspension of pure culture for 10 min at 95° C followed by centrifugation of the lysed cells for 1 min at 3,000 rpm. The supernatant was then transferred to a clean centrifuge tube to be used as the template DNA. All isolates obtained from the blood were first verified as Bartonella species by amplifying and sequencing a specific region in the gltA, and then further characterized by six additional targets, including ftsZ, nuoG, ribC, rpoB, ssrA, and 16S–23S internal transcribed spacer (ITS), using primers that have been previously applied (Bai et al. 2013). All positive PCR products were purified using Qiagen QIAquick PCR Purification Kit (Qiagen, MD) and sequenced in both directions using an Applied Biosystems Model 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA). The obtained sequences were aligned by each locus and compared among the isolates and with other known Bartonella species using the Lasergene software package (DNASTAR, Madison, WI). Based on the allelic profile, each unique combination for the isolates was designated as a sequence type (ST) and sequences for the seven loci were concatenated.

Molecular detection and identification of <italic toggle="yes">Bartonella</italic> species in cat blood and cat fleas

Cat blood DNA was extracted using the Qiagen QIAamp kit following the blood protocol. To determine what targets to apply, a pilot study on 48 cat samples from the present study was first conducted using nested gltA and the other PCR targets applied to characterization of the Bartonella isolates. The pilot study indicated that nested gltA, conventional nuoG, and ITS PCRs were more sensitive than the other targets (data not shown). The nested gltA was performed using the primer for the isolates characterization as the outer primer, and then Bhcs.781p and Bhcs.1137n (Norman et al. 1995) as the inner primers. For flea DNA preparation, individual fleas were first triturated using a bead beater protocol (Halos et al. 2004) and then processed following the Qiagen tissue protocol. Flea DNA was tested for ITS and gltA (using the same primers as used for isolates characterization). All positive cat blood and fleas were subject to sequencing as described above for Bartonella species identification.

RESULTSCats and fleas

In total, blood was collected from 160 cats, consisting of 84 females and 64 males (12 cats were missing gender information). Cat ages varied from one month to seven years old, with 65 cats of <1 year old, 66 cats of 1 to 4 years old, five cats >4 years old (24 cats had no age information). Flea infestation was observed in 71 cats, from which 152 fleas were collected with ranges of 1 to 12 fleas per cat. All fleas were subsequently identified as cat fleas (Ctenocephalides felis). Seventy-seven cats were free from flea infestations, and 12 cats had no flea infestation information.

<italic toggle="yes">Bartonella</italic> culturing and MLST characterization of the isolates

Of the 160 blood samples, 159 were cultured for Bartonella. Bartonella-like bacteria were observed on agar inoculated with 13 (8.2%) samples after one to two weeks post-inoculation. Bacteremia levels varied from 40 to 1,480 CFU per milliliter of blood. PCR amplification of gltA confirmed all 13 isolates as Bartonella species. The gltA sequences showed that all of these isolates belonged to B. henselae Houston type I. The sequences were close to each other (99.7% similarity) and were identical by the gltA PCR assay to two previously identified variants [GenBank: AJ439406 and NC005956]. Characterization of the isolates with the other six targets (ftsZ, nuoG, ribC, rpoB, ssrA, and ITS) further confirmed that all of these isolates are B. henselae Houston type I, with identification of four ftsZ variants, three nuoG variants, two ribC variants, four rpoB variants, and four ITS variants. All isolates were invariant by ssrA and identical to a previously described variant [GenBank:JN029785]. The isolates were of five sequence types based on the MLST allelic profile (Table 1), with divergence of 0.1 to 0.4% among all STs. Novel variants of each target were submitted to GenBank with the following GenBank accession numbers: KP822810 to KP822812 (ftsZ), KP822813 (nuoG), KP822814 to KP822815 (ribC), KP822816 to KP822819 (rpoB), and KP822820 to KP822821 (ITS).

Molecular detection and identification of <italic toggle="yes">Bartonella</italic> species in cat blood

Molecular detection using nested gltA, nuoG, and ITS was applied to 142 of the 160 blood samples based on sample availability for Bartonella infection. A total of 48 (33.8%) were positive for Bartonella DNA for at least one of the three tested targets, showing a significant higher detection rate when compared to culturing (χ2=24.3, p<0.001). Of the 48 positive samples, 27 were positive for all three targets; 15 samples were positive for either two of the three targets; and six samples were positive for a single target. By target, Bartonella species was detected in 44 (31.0%), 44 (31.0%), and 29 (20.4%) by nested gltA, ITS and nuoG, respectively. Of the 13 culture positive samples, blood DNA was available for ten samples. All three targets were positive for Bartonella species in nine of the ten samples, but none of the targets was amplified in one sample which presented a bacteremia of 40 CFU. For all positive samples, there is no statistical difference with respect to either gender or age (p > 0.05).

Sequences were obtained for 47 of the 48 Bartonella-positive samples by one or more targets. Two Bartonella species, B. henselae and B. clarridgeiae, were identified among the sequences, with 32 (68.1%) of them as B. henselae and 15 (31.9%) as B. clarridgeiae. For the 32 B. henselae infected samples, 21 were confirmed by all three targets, seven were confirmed by two targets (including two by ITS and nested gltA, three by nuoG and ITS, and two by nuoG and nested gltA), and four were confirmed by a single target (including two by nested gltA and two by ITS). By target alone, B. henselae was confirmed in 28, 27, and 26 samples by ITS, nested gltA, and nuoG, respectively. Genetic variants identified in cat blood were the same as those in cultures by each of the three targets. For the 15 B. clarridgeiae-infected samples, two samples were confirmed by all three targets, 11 were confirmed by both ITS and nested gltA, and two were confirmed only by one target, either nested gltA or ITS. In fact, the two samples confirmed by all three targets were the only samples that were amplified by nuoG among the 15 B. clarridgeiae samples. The sequences of all B. clarridgeiae positive samples were invariant for each target, and all were previously described with GenBank accession numbers as KC331017, KC331014, and FN645454 for gltA, ITS, and nuoG, respectively.

Molecular detection and identification of <italic toggle="yes">Bartonella</italic> species in cat fleas

Molecular detection of Bartonella infection using gltA and ITS was applied to the 152 fleas collected from 71 cats. Thirty-four fleas (22.4%) collected from 19 cats were positive for Bartonella by at least one of the tested targets. Among the positive fleas, 24 fleas were positive by both gltA and ITS, nine fleas were positive by ITS, and one flea was positive by gltA alone.

Sequences were obtained from 32 fleas, either gltA or ITS or both. Sequencing analysis demonstrated the fleas were infected with the same two Bartonella species, B. henselae and B. clarridgeiae, as found in cats, with 18 fleas (from seven cats) infected with B. henselae and 14 fleas (from 11 cats) infected with B. clarridgeiae. Of the 18 fleas with B. henselae, 11 fleas were confirmed by both gltA and ITS, six by ITS, and one by gltA. The sequences for B. henselae were of the same two variants for gltA and three of the four ITS variants, which were identified in cats. Of the 14 fleas with B. clarridgeiae, nine fleas were confirmed by both gltA and ITS; the other five fleas were confirmed by ITS alone. All ITS sequences and gltA sequences for B. clarridgeiae were identical to those identified in cats.

Relationships of <italic toggle="yes">Bartonella</italic> infection between cats and flea infestations

Flea infestation information was recorded in 132 cats, with 65 cats infested and 67 cats not infested. Bartonella was detected in 40% (26/65) of cats infested with fleas, and in 30.0% (20/67) of cats not infested with fleas. Bartonella infection in cats did not show any significant correlation to flea infestation status (χ2=0.98, p=0.32).

Of the 48 cats that had Bartonella infection in the study, 22 of them were flea-infested; the other 26 were free from fleas. Fleas from 8 of the 22 flea-infested cats were Bartonella-positive, but the rest (14 cats) were Bartonella-negative. Of the 19 cats that had positive fleas (see previous section), blood specimens were available for 13 cats and for Bartonella testing. Eight of them were positive and the rest of the cats were negative. Six cats were infected with the same Bartonella species as their fleas, with B. henselae in four cats and their fleas, and B. clarridgeiae in two cats and their fleas; two cats were infected with B. henselae but their fleas were infected with B. clarridgeiae.

DISCUSSION

Using both culturing and molecular detection by PCR directly in blood, we report the presence of Bartonella infections in cats and their fleas from Guatemala. Similar to reports from other regions (Chomel et al. 1995, 1999, 2002, Bergmans et al. 1996, Branley et al. 1996, Heller et al. 1997, Marston et al. 1999, Maruyama et al. 2000, 2001), Bartonella infections were prevalent in cats in this country. Nevertheless, the prevalence estimated by molecular detection (33.8%) was significantly higher than by culturing (8.2%). It is not surprising that a molecular approach is more sensitive than culturing, but the molecular approach does not provide evidence of viable bacteria in animal samples. In all 13 culture-positive cats, bacteremia levels were quite low (40 to 1,480 CFU per milliliter). The observation of low concentrations of Bartonella bacteria in cat blood can explain the overall low success of culture. Alternatively, the growth requirement for the bacteria may not be met by the media. Prevalence of infection between male and female cats, as well as in different age groups, showed no significant differences between the groups compared.

Two Bartonella species, B. henselae and B. clarridgeiae, were identified in the cats and their fleas, with B. henselae more common than B. clarridgeiae. Interestingly, all cultures obtained from cats exclusively were of B. henselae. It is unknown why no B. clarridgeiae was cultured from any cats. Possibly a very low bacteremia level caused by this particular species limited its detection by culturing. However, B. clarridgeiae may possess some special biological characteristics or requirements that affect the growth of the bacterium on the agar that prevented culture. Results from a recent study by Zhu et al. (2014) suggests that Bartonella species forming a phylogenetic group (lineage-3), to which B. clarridgeiae belongs, lack the gpsA and other metabolically related genes that are important in the phospholipid pathway. Other studies reported that the Bartonella bacteria in lineage-3 are difficult to isolate and culture in artificial medium (e.g., blood agar, BACTEC) compared to Bartonella of other lineages (Podsiadly et al. 2007) but are readily detected by PCR (Zhu et al. 2014). Noticeably, PCR using nuoG, ITS, and nested gltA showed sensitivities to these three targets and were comparable in detecting B. henselae in cats; however, nuoG was less sensitive in detecting B. clarridgeiae compared to the other two targets.

Using the MLST platform, we further demonstrated that all B. henselae isolates obtained in the cats belonged to the Houston type I group, suggesting that it is the major genotype in Guatemala. As the sample size, as well as the investigated area, is relatively small, further studies are required to support this assumption. Although all identified genotypes belong to the same type, our MLST analysis allowed us to distinguish five sequence types among the isolates. The genetic differences demonstrated by MLST may help to identify a link between human cases and their cat sources.

Although cat fleas were frequently infected with both B. henselae and B. clarridgeiae, we could not demonstrate an association between occurrences of Bartonella in fleas and their cat hosts. A Bartonella-infected cat may or may not be infested with fleas and, if infested, the fleas could be either positive or negative for Bartonella infection. On the other hand, fleas collected from positive cats were not always positive. Similar observations were reported in some other studies (Morway et al. 2008, Tsai et al. 2011, Gutiérrez et al. 2014). These observations are challenging considering the well-documented role of fleas in transmitting Bartonella bacteria among cats (Chomel et al. 1996). It is possible that Bartonella infections may persist in both cats and fleas as observed in rodents (Kosoy et al. 2004, Bai et al. 2011). After infecting their hosts, Bartonella bacteria may cause a persistent bacteremia in cats at an undetectable level. The bacteremia level may cyclically fluctuate, occasionally reaching detectable levels of bacteremia. In such a scenario, it would be hard to notice any evident correlation of the infection in cats and their fleas. Also, we cannot exclude alternative modes of transmission, such as cat bites and scratches, which might contribute to the lack of correlation between prevalence of Bartonella infection and infestation of fleas in cats. Bartonella henselae is responsible for most CSD cases in America and across the world. It also causes other clinical manifestations. While most cats are asymptomatic after becoming infected with B. henselae, they serve as reservoirs of the agent and transmit the infection to humans. Data on prevalence of CSD in Guatemala are limited, but people commonly come into contact with cats and are potentially at risk for cat-borne diseases, including CSD. The presence of B. clarridgeiae in cats and cat fleas suggests the need to include this agent when testing clinical samples from human cases suspected for CSD along with B. henselae.

Acknowledgments

This study was supported by the U.S. CDC Global Disease Detection program. We thank the staff from local pet clinics for helping to collect samples. The protocol for collecting blood samples from cats was approved by the Animal Care and Use Committee of the Universidad del Valle de Guatemala (UVG) (approval number I-2013-8).

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Allelic profiles and sequence types (ST) for the 13 Bartonella isolates obtained from cats in Guatemala.

IsolateftsZgltAnuoGribCrpoBssrAITSST
B406831122211ST1
B406841122211ST1
B408851122211ST1
B409151122211ST1
B409161122211ST1
B408882231312ST2
B408872231312ST2
B405753122113ST3
B409173122113ST3
B409183122113ST3
B409144212411ST4
B405774212414ST5
B409194212414ST5