Department of Zoonoses, Institute of Rural Health, Lublin, Poland, 2

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1 Vol. 40, no. 1 Journal of Vector Ecology 75 Bartonella henselae in eastern Poland: the relationship between tick infection rates and the serological response of individuals occupationally exposed to tick bites Violetta Zając 1, Angelina Wójcik-Fatla 1, Jacek Dutkiewicz 1, and Jolanta Szymańska 2 1 Department of Zoonoses, Institute of Rural Health, Lublin, Poland, violetta.zajac@poczta.fm 2 Department of Paedodontics, Medical University of Lublin, Lublin, Poland Received 30 July 2014; Accepted 17 September 2014 ABSTRACT: To explore the potential role of Ixodes ricinus as the presumed vector of Bartonella henselae in eastern Poland, ticks collected in various geographic locations were examined for the presence of B. henselae, and the results were matched against the prevalence of anti-b. henselae antibodies in individuals occupationally exposed to tick bites. The presence of Bartonella DNA was investigated by PCR in a total of 1,603 unfed Ixodes ricinus ticks. The presence of IgG antibodies against B. henselae was investigated in serum samples from 332 people occupationally exposed to tick bites (94 farmers and 238 forestry workers). The total prevalence of B. henselae in ticks was 1.7%; the infection rates in males (3.1%) and females (2.7%) were nearly ten times greater than in nymphs (0.3%). The prevalence of seropositive results in the risk group (30.4%), farmers (27.7%) and forestry workers (31.5%), was significantly greater compared to the control group (8.9%). The results showed a weak positive correlation between the degree of infection of ticks and humans living in the same geographic region. The lack of a direct relationship indicates that exposure to tick bites is only one of the factors contributing to the significant preponderance of a seropositive response to B. henselae in the forestry workers and farmers over the control group. Other factors must be considered, such as contact with cats, which are popular domestic animals in Polish villages, and exposure to cat fleas. Journal of Vector Ecology 40 (1): Keyword Index: Bartonella henselae, ticks, occupationally exposed persons, farmers, forestry workers. INTRODUCTION Bartonella henselae is a zoonotic, alpha Proteobacterium that causes cat scratch disease (CSD), usually appearing as tender lymphadenopathy (Klotz et al. 2011) and is increasingly associated with several other syndromes, particularly ocular infections and endocarditis (Cotté et al. 2008). Cats are the main reservoir of B. henselae, and these bacteria are transmitted to cats by cat fleas. However, new potential vectors are suspected of transmitting B. henselae, in particular ixodid ticks (Cotté et al. 2008, Dietrich et al. 2010, Chomel and Kasten 2010, Mosbacher et al. 2011). Numerous authors have detected the presence of the pathogen in Ixodes ricinus (Dietrich et al. 2010, Mietze et al. 2011, Sanogo et al. 2003, Corrain et al. 2012, Reye et al. 2013) and also in Ixodes persulcatus (Morozova et al. 2004), Ixodes scapularis (Eskow et al. 2001), Ixodes pacificus (Holden et al. 2006), Dermacentor reticulatus (Reye et al. 2013), and Rhipicephalus sanguineus (Wikswo et al. 2007, Tsai et al. 2011). Furthermore, there are described human infections by B. henselae associated with tick bites (Eskow et al. 2001, Morozova et al. 2005, Angelakis et al. 2010, Maggi et al. 2013), and Cotté et al. (2008) experimentally proved the possibility of the transmission of B. henselae by Ixodes ricinus ticks. Nonetheless, the testing of Ixodes ricinus for the presence of B. henselae was negative in several studies (Tijsse- Klasen et al. 2011, Sréter-Lancz et al. 2006), and some researchers are of the opinion that transmission of the infection from ticks to humans has not yet been established (Telford and Wormser 2010). To explore the potential role of Ixodes ricinus as a presumed vector of B. henselae in eastern Poland, ticks of this species collected in seven various geographic locations were examined for the presence of Bartonella henselae. In parallel, individuals inhabiting these locations and occupationally exposed to tick bites (forestry and agricultural workers) were examined serologically for the presence of antibodies against B. henselae. The results of the tick examination were matched against the serological results, aiming to establish a relationship between the degree of infection of ticks and humans living in the same geographic areas. MATERIALS AND METHODS Collection of ticks A total of 1,603 unfed Ixodes ricinus ticks (403 females, 450 males, and 750 nymphs) was collected between April and September of the years in seven localities situated in the Lublin province (eastern Poland): two rural communities (Jabłonna and Piotrowice) and five forest inspectorates (Puławy, Świdnik, Zwierzyniec, Sobibór, and Poleski National Park). Ticks were collected by dragging a woolen flag over the lower vegetation at the peripheral and inner parts of deciduous and mixed forests. Collected ticks were placed in glass tubes with 70% ethanol for further investigation. DNA extraction Bacterial DNA was extracted from ticks after removal from alcohol by boiling in 0.7 M ammonium hydroxide, according to Rijpkema et al. (1996) and stored at -20º C. Single adult ticks were prepared separately while nymphs were prepared in pools of five specimens. Minimum infection rate in nymphs was calculated according to Kahl et al. (1989). The concentration of DNA in the extracts was determined with the NanoDrop ND1000 spectrophotometer. The DNA concentrations ranged from ng/µl for females, from ng/µl for males, and from 18-

2 76 Journal of Vector Ecology June ng/µl for nymphs. Detection of Bartonella spp. DNA by PCR The fragment of the gene glta encoding citrate synthase was used as a genetic marker to detect Bartonella spp. DNA by Polymerase Chain Reaction (PCR) (Norman et al. 1995, Li et al. 2007). The following pair of primers was used: BhCS.781p: 5 GGG GAC CAG CTC ATG GTG G 3 and BhCS.1137n: 5 AAT GCA AAA AGA ACA GTA AAC A 3. Each PCR reaction was carried out in a 25 μl reaction volume which contained the following mix of reagents: 0.6 U Taq DNA polymerase (Qiagen, U.S.A.), 1 PCR buffer containing 1.5 mm MgCl 2, 2.5 µl 2 mm dntp (Fermentas, Vilnius, Lithuania), 1 μl 10 μm each of primer (Eurogentec, Seraing, Belgium), 5 μl of DNA, and nuclease-free water (Applied Biosystems, U.S.A.). As a positive control, DNA extracted from the antigen of B. henselae and B. quintana fixed on the slide used for detection of anti-bartonella antibodies (Focus Diagnostics, U.S.A.) was used, while nuclease-free water was used as a negative control. The amplification was carried out in a C1000 Thermal Cycler (BioRad, U.S.A.) in consecutive stages: preliminary denaturation 5 min at 95 C; 35 cycles, each consisting of 20 s denaturation at 95 C, 20 s annealing at 51 C, and 2 min elongation at 72 C; final elongation 10 min at 72 C. The size of the amplified DNA fragment was 380 base pairs (bp). Products of amplification were identified in 2% agarose gel after electrophoresis in standard conditions and staining with ethidium bromide solution (2 μg/ml). DNA sequencing and sequence comparison DNA sequencing was performed with an ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Inc., Foster City, CA, U.S.A.) using Abi Prism Big Dye Terminator v Cycle Sequencing Kits and Big Dye XTerminator Purification Kit (Applied Biosystems). The results were compared with the sequences in the GenBank database using the BLAST software at the National Center for Biotechnology Information (Bethesda, MD, U.S.A.). Serologic response to Bartonella henselae in individuals occupationally exposed to tick bites Blood serum samples were acquired from 332 people occupationally exposed to tick bites (219 men and 113 women, mean age ± years) employed in Lublin province, i.e., 94 farmers (29 men and 65 women, mean age ± years) from the villages of Jabłonna and Piotrowice and 238 forestry workers (190 men and 48 women, mean age ± years) employed at the inspectorates Puławy, Świdnik, Zwierzyniec, and Sobibór and at the Polesie National Park. The study areas were the same as those where ticks were previously collected for the determination of the B. henselae infection rate. The control group consisted of 56 people (20 men and 36 women, mean age ± 10.12) whose occupation did not involve exposure to tick bites. The blood samples were collected in The separated blood serum samples were stored at 4 C until the tests were carried out. Bartonellosis had not been previously diagnosed in any of the tested individuals, and all the examined farmers and forestry workers reported numerous tick bites. The presence of specific IgG antibodies against B. henselae and B. quintana was detected with the indirect fluorescent antibody test (IFA) using commercial kits (Bartonella IFA IgG, Focus Diagnostics, Cypress, CA, U.S.A.) according to the manufacturer s instructions. Vero cells infected with B. henselae and B. quintana were used as antigens. The samples that were positive at a titer of 64 were tested again at higher dilutions; a positive result at a titer of 256 or higher may indicate an active infection. The sensitivity and specificity of the test are 95% and 97%, respectively (Regnery et al. 1992, Peter et al. 1994). The relationship between the degree of Ixodes ricinus infection by B. henselae and serologic response to B. henselae among people employed in the same area and occupationally exposed to tick bites was examined by matching the results presenting the rate of B. henselae infection in Ixodes ricinus ticks against the results showing the serological response of the people occupationally exposed to tick bites in the same area. The degree of the correlation was assessed by Pearson s test. Statistical analysis Student s t- test, the χ 2 test, and Pearson s test for correlation were applied in the analyses using the STATISTICA v. 6.0 package (Statsoft, Tulsa, OK, U.S.A.). RESULTS Infection rate of Ixodes ricinus ticks with Bartonella henselae As observed in Table 1, the total prevalence of B. henselae in the examined Ixodes ricinus ticks was low, amounting to 1.7%. The variability of the infection rate depending on the developmental stage was statistically significant (χ 2 =17.293; df=2; p=0.0002); the infection rates in males and females (3.1% and 2.7%, respectively) were nearly ten times greater than in nymphs (0.3%), and the differences were statistically significant (p= and p=0.0003, respectively). The prevalence of B. henselae in male and female ticks was significantly dependent on the geographic location (p<0.001), whereas such a relationship was not observed for nymphs. For the total collected ticks, the infection rate varied between %, depending on the locality (χ 2 =17.293; df=2; p=0.0002). An analysis of the nucleotide sequence of all obtained amplificates showed 100% identity with the sequence of a fragment of the gene encoding citrate synthase in Bartonella henselae, strain Houston-1, in GenBank under the accession number BX Accordingly, all the Bartonella sequences detected in the ticks were assumed to belong to Bartonella henselae. Serological response to Bartonella henselae in people occupationally exposed to tick bites With the use of an indirect fluorescent antibody test, only antibodies against B. henselae were found; antibodies against B. quintana were not. The serological response to Bartonella henselae depending on employment and geographic locality is presented in Table 2. The prevalence of seropositive results in individuals occupationally exposed to tick bites (farmers and forestry workers) was significantly greater compared to the control group not exposed to tick bites (30.4% vs 8.9%, p=0.0009). No significant difference was found between the serologic response in farmers compared to forestry workers (27.7% vs 31.5%, p=0.498). The place of work (geographic locality) did not significantly affect the

3 Vol. 40, no. 1 Journal of Vector Ecology 77 Table 1. Infection of Ixodes ricinus ticks with B. henselae depending on the developmental stage and geographic locality. Ixodes ricinus Locality No. infected/no. examined (%) Nymphs Males*** Females*** Total*** Jabłonna 1/220 (0.5) 2/20 (10.0) 1/13 (7.7) 4/253 (1.6) Piotrowice 1/75 (1.3) 2/71 (2.8) 2/68 (2.9) 5/214 (2.3) Forest inspectorate in Sobibór 0/75 (0.0) 2/77 (2.6) 1/63 (1.6) 3/215 (1.4) Polesie National Park 0/20 (0.0) 1/52 (1.9) 1/36 (2.8) 2/108 (1.9) Forest inspectorate in Świdnik 0/120 (0.0) 7/101 (6.9) 4/102 (3.9) 11/323 (3.4) Forest inspectorate in Puławy 0/130 (0.0) 0/67 (0.0) 0/48 (0.0) 0/245 (0.0) Forest inspectorate in Zwierzyniec 0/110 (0.0) 0/62 (0.0) 2/73 (2.7) 2/245 (0.8) ***χ 2, p< Total*** 2/750 (0.3) 14/450 (3.1) 11/403 (2.7) 27/1,603 (1.7) Table 2. Anti-B. henselae antibody prevalence according to foresters and farmers place of work (geographic locality). Locality Total Positive serologic results n (%) Jabłonna (35.6) Piotrowice (20.4) Total farmers (27.7) Forest inspectorate in Sobibór (32.8) Polesie National Park (38.2) Forest inspectorate in Świdnik (36.6) Forest inspectorate in Puławy (26.0) Forest inspectorate in Zwierzyniec (26.9) Total forestry workers (31.5) Total occupationally exposed to tick bites (30.4) People not occupationally exposed to tick bites (controls) 56 5 (8.9) test results (χ 2 = 6.854; df = 7; p = 0.444), and the percentages of seropositive results in the exposed group according to the place of work varied from %. In the occupationally exposed group, the percentages of seropositive reactions to B. henselae were similar in men and women (Table 3), though the difference was not statistically significant (p=0.389). In the control group, specific antibodies were found more frequently in women than in men, but this difference was also not significant (p=0.446). Among the forestry workers and farmers, the highest percentage of people with positive serological results was found in the youngest age group, below 19 (40.0%). Indeed, the percentage of seropositive patients decreased with age, but the differences were not statistically significant (χ 2 = 2.082; df = 7; p = 0.954). In the group of people occupationally exposed to tick bites, a positive result was most frequently obtained at a titer of 64 (Table 4). Positive results at a dilution of 1:256 or higher, indicating a possible recent infection, were found in only 2.7% of the studied population. The frequency of high titer results ( 128) was similar in the farmers and forestry workers (p=0.516) and did not show a significant relationship with the gender or age of the tested individuals (p=0.722 and p=0.943, respectively).

4 78 Journal of Vector Ecology June 2015 Table 3. The prevalence of anti-b. henselae antibodies depending on the gender and age of the tested individuals. Characteristic Occupationally exposed group Control group Total Positive serologic results n (%) Total Positive serologic results n (%) Gender female (27.4) 36 4 (11.1) male (32.0) 20 1 (5.0) Age (in years) < (40.0) 0 0 (0) (30.6) 34 5 (14.7) (30.9) 22 0 (0) (29.5) 0 0 (0) (0) 0 0 (0) Total (30.4) 56 5 (8.9) Table 4. Specific anti-b. henselae IgG antibody titers in the tested groups. Serological results B. henselae titers Occupationally exposed group Farmers n (%) Forestry workers n (%) Total n (%) Control group n (%) (22.3) 66 (27.7) 87 (26.2) 5 (8.9) (2.2) 3 (1.3) 5 (1.5) 0 (0) (3.2) 6 (2.5) 9 (2.7) 0 (0) Total positive 26 (27.7) 75 (31.5) 101 (30.4) 5 (8.9) Relationship between the degree of infection of Ixodes ricinus with B. henselae and serologic response to B. henselae in people occupationally exposed to tick bites The prevalence of B. henselae in unfed ticks collected in seven geographic localities was positively correlated with the frequency of seropositive reactions in the people working in the same localities and occupationally exposed to tick bites (r=0.367, Figure 1). This relationship, however, was not statistically significant (p=0.419). Although the maximal difference between the tick infection rate in the Puławy and Świdnik areas (0,0 vs 3.4%) was accompanied by a marked rise in a serologic response exceeding 10% (26.0 vs 36.6%), in some other cases, the relations between these two variables were not consistent. DISCUSSION The total prevalence of B. henselae in the examined Ixodes ricinus ticks (1.7%) was slightly below the median values reported from various European countries. The prevalence is similar to the value of 1.48% reported by Sanogo et al. (2003) for ticks collected from humans in Belluno province, Italy, and greater compared to the values reported from Luxembourg (0.3%) (Reye et al. 2010), Belarus (0.7%) (Reye et al. 2013), Italy (all negative) (Trotta et al. 2012), Hungary (all negative) (Sreter-Lancz et al. 2006), Sweden (all negative) (La Scola et al. 2004), and the Netherlands (all negative) (Tijsse-Klasenet et al. 2011). In contrast, our results are lower compared to earlier Polish studies ( %) (Sytykiewicz et al. 2012, Podsiadły et al. 2007), to data reported from Germany ( %) (Dietrich et al. 2010, Mietze et al., 2011, Janecek et al. 2012), to data from France ( %) (Halos et al. 2005, Dietrich et al. 2010), to results from Portugal (0-32.3%) (Dietrich et al. 2010), and to the results obtained by Corrain et al. (2012) in Imola, Italy (38.3%). It is noteworthy that our study shows a significantly greater prevalence of B. henselae in adult ticks compared to nymphs. These results differ from the data reported by Dietrich et al. (2010), who found that 15 times more nymphs (31.9%) from France, Germany, and Portugal contained B. henselae DNA than did adults. These differences could be most likely explained by different geoclimatologic conditions. The serologic response of occupationally tick bite-exposed people to the antigen of B. henselae was significantly greater compared to the response of people not exposed to tick bites (30.4% vs 8.9%). High titer results were found in 11.5% of the seropositive farmers employed in the Lublin macroregion, which may indicate an active infection. Very similar results were also obtained in the study conducted among forestry workers in south and northeast Poland (Podsiadły et al. 2011). The seroprevalence in farmers and forestry workers in the present study was greater compared to the data for farmers in

5 Vol. 40, no. 1 Journal of Vector Ecology 79 Figure 1. Correlation between infection rates of Ixodes ricinus ticks with B. henselae (IR_TICKS) and the serologic response (SEROPOS) to B. henselae antigen in people occupationally exposed to tick bites. China (9.6%, %) (Zhang et al. 2008, Zhang et al. 2011) and Brazil (13.7%) (da Costa et al. 2005) and similar to the response rate found in a rural population in Colombia (30%) (Buelvas et al. 2008). In addition to agricultural and forestry workers, other groups are also at risk of infection with B. henselae, namely veterinarians and cat owners. In a Polish study, a very high serologic response to B. henselae was found in these groups, 45% and 53.3%, respectively (Chmielewski et al. 2007). In other countries, the response rate was lower, being % in veterinarians (Noah et al. 1997, Ferrés et al. 2006, Chang et al. 2006) and 28.9% in cat owners (Blanco Ramos et al. 1998). A very high incidence of seropositive results (65%) was reported by Juncker-Voss et al. (2004) for the occupational group of workers from a zoological garden. As reference groups, theoretically showing a low response rate, mainly blood donors or groups defined as healthy populations have been investigated. Of these, blood donors from various countries responded to B. henselae with a frequency of 1.2%-11.4% (Blanco Ramos et al. 1998, Mc Gill et al. 2005, Yilmaz et al. 2009, Mansueto et al. 2012). It is noteworthy, that Yilmaz et al. (2009) found a significant relationship between a history of tick bites and a seropositive reaction in an investigated blood donor group. A number of authors have examined the antibody response to B. henselae in healthy populations (Tea et al. 2003, Pons et al. 2008, Lamas et al. 2008, Kamoi et al. 2009, Liu et al. 2012), and most of them have noted a seroprevalence ranging from %, except Kamoi in Japan (2009), Sander in Germany (1998) and Cimolai in Canada (2000) (25.3%, 30% and 36.8%, respectively). Comparing the reviewed results with those obtained in the present study, it could be stated that the earlier results reported for groups of blood donors largely correspond to those obtained for our control group, whereas the response rates reported for healthy populations are mostly greater than the control group but usually lower than in the group occupationally exposed to tick bites. Matching of the results presenting the tick infection rate against the results of the serologic survey (Figure 1) showed a weak positive correlation that did not attain the level of statistical significance. In spite of the lack of a direct significant relationship between the infection rate of B. henselae in ticks and exposed people, the role of I. ricinus in the transmission of Bartonella cannot be excluded. Cotté et al. (2008) recently showed the transovarial transmission of B. henselae in I. ricinus and the transmission of bacteria from ticks to blood through an artificial membrane, which indicates that the tick is a competent vector for B. henselae. Other indirect data, such as the detection of Bartonella sp. DNA in ticks, identification of B. henselae in patients without contact with cats but after tick bites, and coinfection of Bartonella with other tick-borne pathogens (Angelakis et al. 2010), suggest that ticks could be potential Bartonella vectors. However, Telford and Wormser (2010) indicated that although ticks harbor many microbial agents, this does not necessarily imply that ticks might transmit these agents during blood feeding or that they are pathogenic. Indeed, these authors emphasize that in almost all studies, the presence of Bartonella DNA in field-collected ticks was not confirmed by culture of the bacteria. Additionally,

6 80 Journal of Vector Ecology June 2015 they indicated that Cotté et al. (2008) used very large doses of bacteria for tick feeding, a situation that does not realistically occur in cats, and that the B. henselae strain Houston-1 used in the study of Cotté et al. may not represent strains found in nature. Additionally, medical literature searches did not confirm the tickborne transmission of Bartonella species and the simultaneous existence of Lyme disease and Bartonella infection (Lantos and Wormser 2014). Thus, B. henselae transmission to humans and animals by ticks is still controversial, and experimental evidence in an animal model is needed. Cat fleas are well-established vectors of B. henselae among cats, and bites or scratches by infected cats are associated with the development of cat scratch disease in humans. Cats are very common in Polish villages, and the seroprevalence of B. henselae in Polish cats (86%) is one of the highest in Europe (Podsiadły et al. 2003). Among 180 farmers interviewed in another study of ours, 162 (90%) declared that cats and dogs are present on their farms and that only half of them deworm their pets (data not published). Close contact with home pets increases the risk of scratches or bites and of potential infection with B. henselae. The lack of a direct relationship between tick and human infection found in the present work appears to indicate that exposure to tick bites is only one of the factors contributing to the significant preponderance of a seropositive response to B. henselae in the group of forestry workers and farmers over the control group. Thus, other factors must be considered to explain the common occurrence of B. henselae infection among Polish farmers and foresters. Among these factors, the most important are likely contact with cats, which are popular domestic animals in Polish villages, and exposure to cat fleas. 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