The Occurrence of Spotted Fever Rickettsioses and Other Tick-Borne Infections in Forest Workers in Poland

Abstract

The presence of antibodies to Rickettsia conorii, R. helvetica, R. felis, R. slovaca, R. sibirica, and R. massiliae in sera of 129 forest workers from northeastern and southern Poland was assayed by indirect immunofluorescence. Previous environmental studies revealed presence of spotted fever group (SFG) rickettsiae in ticks collected from these areas. Additionally, the workers were examinated for the presence of antibodies specific to other tick-borne bacteria: Anaplasma phagocytophilum, Bartonella spp., and B. burgdorferi. The results of the studies have shown the presence of specific SFG rickettsiae antibodies in 14.7% of tested forest workers, among them 78.9% had species-specific antibodies to R. massiliae. Contrary to previous detection R. helvetica and R. slovaca in ticks collected in the environment of the examined area, no species-specific antibodies to these species were detected in studied workers. Antibodies to B. burgdorferi (44%) were found in forest workers more often than antibodies to other tested pathogens. B. burgdorferi was also the main component of coinfections. The most frequent confirmed serologically coinfections were simultaneous infections with B. burgdorferi and Bartonella spp. found in 10% of tested individuals. So far, SFG rickettsiae infections have not been diagnosed in Poland; however, the presence of the bacteria in ticks and presence of specific antibodies in humans exposed to arthropods show the need for monitoring the situation. The list of tick-borne pathogens is increasing, but knowledge about the possibility of humans acquiring multipathogens infections after tick bite still needs evaluation.

Introduction

I t has been known since 1909 that ticks are involved in transmission of various infectious diseases to humans (Swanson et al. 2006). Nowadays, tick-borne infectious diseases have become an important component of the differential diagnoses of febrile illnesses occurring between spring and late fall. Knowledge of their etiological agents is crucial for proper diagnosis and consecutive treatment.

Tick-borne rickettsioses, the oldest known tick-borne diseases, are caused by rickettsiae of the spotted fever group (SFG). Rickettsia conorii subsp. conorii was thought for many years to be the only tick-borne rickettsial disease occurring in Europe (Brouqui et al. 2007). Emerging human SFG rickettsiae recently described in Southern and Eastern Europe include Rickettsia conorii subsp. israelensis, Rickettsia conorii subsp. caspia, Rickettsia aeschlimannii, Rickettsia slovaca, Rickettsia sibirica subsp. mongolotimonae, Rickettsia massiliae, and Rickettsia raoultii (Parola et al. 2005, Brouqui et al. 2007). Moreover, species transmitted by other vectors than ticks, Rickettsia felis and Rickettsia akari, have been also recognized as human pathogens (Parola et al. 2005, Brouqui et al. 2007).

Until recently serologic surveys of rickettsioses were not conducted in Poland despite detection of the bacteria in ticks collected on Polish territory. The presence of rickettsiae and rickettsioses were recognized in neighboring countries (Parola et al. 2005, Brouqui et al. 2007, Doobler et al. 2009). SFG rickettsiae were detected in 3.5% of Dermacentor reticulatus ticks collected in 2003/2004 in northeastern Poland. They were found to belong to R. massiliae genogroup (Stanczak 2006a). Later studies conducted in Poland revealed the presence of Rickettsia helvetica in Ixodes ricinus (Stanczak 2006b, Chmielewski et al. 2009) and Rickettsia slovaca in D. reticulatus (Chmielewski et al. 2009), as well as Rickettsia raoultii, in both of the above mentioned ticks' species present in Poland (Chmielewski et al. 2009). Both R. slovaca and R. helvetica have already been identified as agents of human rickettsioses in Europe. However, no clinical cases were reported, and we have no data about clinical and epidemiological characteristic of the rickettsial infections in humans in Poland.

It is well known that I. ricinus ticks serve both as vectors as well as reservoirs for R. helvetica; this species of rickettsia has been isolated in many European countries and for many years they were considered a nonpathogenic rickettsia. R. helvetica has been incompletely described as human pathogen; few so far described cases are documented only serologically. It has been implicated in fatal perimyocarditis, febrile illness, and sarcoidosis (Brouqui et al. 2007).

The group of tick-borne diseases is increasing; the recent data demonstrate not only that Bartonella spp. is able to inhabit ticks, but also that the bacteria of this genus are transmitted efficiently transstadialy in I. ricinus ticks (Cotte et al. 2008, Podsiadly et al. 2009).

The aim of the study was to determine the prevalence of antibodies to SFG rickettsiae in inhabitants of the areas of Poland where Rickettsia spp. were previously detected in ticks. Simultaneously, seroprevalence to other bacterial tick-borne infections caused by Anaplasma phagocytophilum, Bartonella spp., and Borelia burgdorferi was estimated.

Materials and Methods

Studied groups

One hundred and twenty-nine forest workers were tested, including 91 from northeastern (Białowieza Primeval forest) and 38 from southern (Radomsko forest) Poland. Blood samples were collected during 2 months: November 2008 and February 2009. All participants were at high-risk exposure to tick bites due to their profession and living in tick-infested forest areas; 20% were female and 80% were male. They did not show any clinical symptoms at the time of the studies. Consent for the study was obtained from all participants.

Serological studies

For detection IgG Rickettsia spp. antibodies, microimmunofluorescence (MIF), the method accepted as a reference for serodiagnosis of rickettsioses, was used. A two-stage procedure was applied for Rickettsia spp. antibody evaluation. The first stage was detection and differentiation of the presence of antibodies specific to typhus and SFGs. Inactivated R. typhi and R. rickettsii antigens were used (Rickettsia IFA IgG; Focus Diagnostic—sensitivity 100% and specificity 100%). Control sera were delivered by the manufacturer. The screening was performed at a titer of 1:16. All serum samples negative in this titer were regarded as negative to both group of rickettsiae, whereas serum samples with titer ≥16 for SFG rickettsiae were characterized for the presence of antibodies to six SFG rickettsial species (Rickettsia Screen IFA IgG Antibody Kit; Fuller Laboratories). Purified, acetone-fixed antigens of R. conorii, R. helvetica, R. felis, R. slovaca, R. sibirica, and R. massiliae used as an individual substrate on the same slide were applied as diagnostics antigens. According to Unite des Rickettsies in Marseilles (Brouqui et al. 2004) IgG titer ≥64 to a given rickettsial species without cross-reactions with other SFG species was considered indicative of the infection by this specific Rickettsia species. Patients' positive sera were tested in serial dilution to determine their endpoint titer. In both applied serological kits, sera reactive and nonreactive against SFG rickettsia delivered by the manufacturer were used as controls. The intensity of specific fluorescence was evaluated subjectively with scores from 1 to 4, and the antibody titer was defined by the major dilution with a score 2.

Levels of A. phagocytophilum and Bartonella spp. IgG antibodies were determined by indirect immunofluorescence assay (Anaplasma phagocytophilum IFA IgG Kit—sensitivity 100% and specificity 100%, Bartonella IFA IgG; Focus Diagnostics—sensitivity 95% and specificity 97%). Natural antigens were used: human isolate of A. phagocytophilum HE-1 strain in HL 60 cells (Human promyeloblast) and Vero cells infected with B. henselae and B. quintana strains, respectively. Titer of IgG antibodies ≥64 against either of the pathogens was considered positive. Positive and negative controls were delivered by the manufacturer.

B. burgdorferi IgM and IgG antibodies were tested with ELISA test (Borrelia IgG ELISA Recombinant Antigen—sensitivity 98.6% and specificity 84.7%; Borrelia IgM ELISA Recombinant Antigen—sensitivity 98.6% and specificity 100%; Biomedica GmbH). The following recombinant antigens were applied: OspC of B. burgdorferi sensu stricto, B. garini, p18 B. afzelii, p100 B. afzeli, and VLsE fusion protein of different genospecies. Positive results were confirmed by Western blot method (Anti-Borrelia-EUROLINE-WB IgM, Anti-Borrelia-EUROLINE-WB IgG; Euroimmun). Results were interpreted as seropositive according to criteria of the German Society of Hygiene and Microbiology (Guidelines for the microbiological diagnosis of Lyme borreliosis. Available at www.dghm.org/med/index.html?cname=MiQ).

Molecular studies

The positive samples were further tested using polymerase chain reaction and gene sequencing. DNA was extracted from sera with QIAamp Mini Kit (Qiagen). The presence of Rickettsia spp., A. phagocytophilum, Bartonella spp, and B. burgdorferi DNA was examined with primers specific for gene fragments listed in Table 1.

Table 1.

Primers Used for Polymerase Chain Reaction Amplification and Sequencing

Pathogen	Primers	Fragment gene (size, bp)	Nucleotide sequences	Reference
Rickettsia spp.	RpCS.409d	Citrate synthase gene	5′-CCTATGGCTATTATGCTTGC	Roux et al. (1997)
	RpCS.1258n	(850)	5′-ATTGCAAAAAGTACAGTGAACA
	Rr190-70	Outer membrane protein A	5′-ATGGCGAATATTTCTCCAAAA	Roux et al. (1996)
	Rr190-701	(632)	5′-GTTCCGTTAATGGCAGCATCT	Roux et al. (1996)
	Rr17.61p	17-kDa genus-common antigen	5′-GCTCTTGCA ACT TCT ATGTT	Webb et al. (1990)
	Rr17.492n	(434)	5′-CATTGTTCGTCAGGTTGGCG	Webb et al. (1990)
B. burgdorferi	L2	16S rRNA (600)	5′-GGTCAAGACTGACGCTGAGT	Chmielewski et al. (2003)
	P1		5′ -TCGCTTTGTACAGGCCATTG
Bartonella spp.	BhCS.781p	citrate synthase gene (400)	5′-GGGGACCAGCTCATGGTGG	Podsiadly et al. (2007)
	BhCS.1137n		5′-AATGCAAAAAGAACAGTAAACA
A. phagocytophilum	EHR 521	16S rRNA (247)	5′-TGT AGG TTC GGT AAG TTA AAG	Levin et al. (1999)
	EHR 747		5′-GCA CTC ATC GTT TAC AGC GTG

Results

All 129 analyzed serum samples were seronegative to R. typhi. Antibodies to SFG rickettsiae were detected in 19 (14.7%) of the forest workers (Tables 2 and 3). The highest detected titer reached 128. Species-specific antibodies to R. massiliae were found in 15 (79%) of the 19 seropositive individuals. In the remaining 4 rickettsia positive sera, cross-reactions were observed. In the sera of two workers, the titers of antibodies for R. slovaca or R. massiliae were one serial dilution higher than for other species. In other two seropositive persons, the first showed low reactivity to three rickettsial species and the second one had antibodies to R. conorii, R. slovaca, and R. massiliae in titer of 128 (Table 2).

Table 2.

Positive Immunofluorescence Assay Results for Antigens to Six Spotted Fever Group Rickettsia in Forest Workers in Poland ^a
–c

No. of seropositives	R. conori	R. helvetica	R. felis	R. slovaca	R. siberica	R. massiliae
12	0	0	0	0	0	64
1	0	0	32	0	0	64
1	32	0	0	0	0	64
1	32	32	0	32	0	64
1	32	0	64	64	32	64
1	64	64	64	128	0	32
1	64	32	32	64	0	128
1	128	32	64	128	0	128

Thirty-six individuals were seronegative in the first screening test and were not tested for the presence of six SFG rickettsial antigens; 93 sera positive in first stage were tested for the presence of antibodies to six SFG Rickettsia.

Thirty-four of analyzed individuals were seronegative to all six tested SFG rickettsial antigens.

Forty individuals were seropositive to selected SFG rickettsial antigens in titer 32, and they are excluded from the table.

SFG, spotted fever group.

Table 3.

Prevalence of Antibodies Against Tick-Borne Bacteria in Forest Workers

Region	n	A. phagocytophilum	B. burgdorferi	Bartonella spp.	Rickettsia spp.
Bialowieza forest	91	18 (20%)	36 (40%)	26 (29%)	10 (11%)
Radomsko forest	38	2 (5%)	8 (21%)	12 (32%)	9 (24%)
Total	129	20 (15.5%)	44 (34%)	38 (29.5%)	19 (14.7%)

Specific A. phagocytophilum antibodies were detected in 15.5% (20/129) of the studied individuals. The highest titer reached 256. Thirty-four percent (44/129) of forest workers were seropositive to B. burgdorferi. Bartonella spp. antibodies were found in about 30% (38/129) of the individuals; the levels of antibodies ranged from 64 to 256 (Table 3).

Infection with two different bacterial agents was detected serologically in 2% to 10% of the studied individuals depending on the bacterial species. The concomittent occurrence of antibodies to B. burgdorferi and Bartonella spp. was most frequently (10%) detected. B. burgdorferi and A. phagocytophilum, as well as Bartonella spp. and A. phagocytophilum, antibodies were found simultaneously in 4.7% of the individuals, whereas in 1.6% of workers antibodies to both A. phagocytophilum and Rickettsia spp. were present. Approximately 9% and 7% of workers seropositive for Rickettsia spp. showed antibodies to Bartonella spp. and B. burgdorferi, respectively. Infections with three different bacterial agents were recognized in <2% of the forest workers.

Fragments of genes specific for Rickettsia spp. were not present in the sera of any of the Rickettsia spp. seropositive patients. Similarly, DNA of B. burgdorferi and A. phagocytophilum was not detected in any of these bacteria seropositive serum samples.

In case of Bartonella spp. three samples were positive both serologically (titer of 1:64 or more) and by polymerase chain reaction/sequencing, but the majority (n = 35) were positive only serologically. Sequences of the three positive DNA extracts were identified as uncultured Bartonella sp. (accession nos. HM1 16784, HM1 16785, and HM1 16786) deposited in GenBank.

Discussion

The results of the present study have shown the presence of specific SFG rickettsiae antibodies in 14.7% of forest workers, among them 78.9% had species-specific antibodies to R. massiliae. None of the tested workers had antibodies specific for R. typhi. Obtained data clearly show the presence of antibodies against members of the SFG of rickettsiae in sera of persons living in the regions not considered so far as areas of geographic distribution of this group of bacteria. However, no subject showed any symptoms, which did not suggest active infection.

R. massiliae has been recently recognized as a human pathogen (Vitale et al. 2006). The bacteria have been isolated from Rhipicephalus spp. ticks collected in various countries in Europe and also in the Central African Republic (Parola et al. 2005). Rhipicephalus spp. ticks do not constitute a permanent fauna in Poland. There are reports that the ticks are sporadically carried to Poland by birds, and they have also been found on animals such as dogs traveling with their owners to the south of Europe (Siuda et al. 2006, Nowak 2010). In our study the relatively higher seropositivity to R. massiliae than to other SFG species may suggest that these bacteria could spread in Poland through other vectors than Rhipicephalus spp. including typical for this geographical region tick species. Marquez et al. (2003) found R. massiliae genogroup in D. marginatus ticks in Spain. In other studies R. massiliae was detected in 1.7% of I. ricinus ticks in Eastern Bavaria (Dobler and Wolfel 2009). In our studies on the prevalence of Rickettsia spp. in ticks, we found R. massiliae neither in D. reticulatus nor in I. ricinus, which are most common ticks in Poland (Chmielewski et al. 2003).

R. helvetica and R. slovaca have been detected in ticks in Northern and Eastern Europe, including various area of Poland (Chmielewski et al. 2003, Stanczak 2006a, 2006b). In our previous study, R. slovaca and R. helvetica were detected in 2% and 4.5% of ticks, respectively (Chmielewski et al. 2003). So far, TIBOLA or R. helvetica infections were not reported in Poland. The results of our studies did not show the presence of species-specific antibodies in the studied risk groups either.

Human anaplasmosis is known to occur in regions of North America and Europe inhabited by Ixodes spp. In Poland, various studies have shown the presence of A. phagocytophilum in ∼14% ticks (Stanczak et al. 2004, Tomasiewicz et al. 2004). The high prevalence of A. phagocytophilum in the environment correlates with high seropositivity reaching nearly 16% in exposed individuals. Likewise, antibodies specific to A. phagocytophilum, Bartonella anibodies, were detected relatively often in forest workers; however, they were present at low level. High seroprevalence to Bartonella spp. in low titers might be explained by the results of molecular studies. Molecular analysis of the obtained amplicons revealed around 90% similarities with originated from environment Bartonella species like B. koehlerae detected in dogs (GenBank number FJ832088), uncultured Bartonella spp. from arthropods in Peru (GU 583842.1), and B. clarridgeiae detected in ectoparasites in Taiwan (GU 056189). It is supposed that in serological examination cross-reactivity to nonhuman pathogens, environmental Bartonella strains, was probably detected. The route of transmission of Bartonella from the environment to humans is an open question, but such a finding suggests circulating bartonela in nature.

Still, Lyme borreliosis has been recognized as the most frequent tick-borne disease in the Northern Hemisphere. The results of the present studies are concordant with this thesis, as antibodies to B. burgdorferi were found in forest workers more often than antibodies to other tested pathogens. B. burgdorferi was also the main component of coinfections.

The simultaneous occurrence of two pathogens has been reported in 1%–10% ticks (Swanson et al. 2006). This correlates with serological evidence of dual infections with tick-borne pathogens in humans, as infections with two pathogens were found in 10% of workers.

The frequent prevalence of dual infections of Bartonella spp. and B. burgdorferi suggests the possibility of a shared vector–ticks, especially since both B. burgdorferi and Bartonella inhabit wild rodents, reservoirs of both bacteria.

The results of many studies indicate the frequent occurrence of B. burgdorferi and A. phagocytophilum coinfections in humans, ranging from 3% to 26% (Swanson et al. 2006). According to our results, simultaneous infections with B. burgdorferi and A. phagocytophilum were not the most frequent. In our studies we included testing of the prevalence to Rickettsia spp. and Bartonella spp. antibodies in the studied subjects. Taking into consideration these two pathogens as agents of coinfections could have influenced the results.

The list of tick-borne pathogens is increasing, but knowledge about the possibility of humans acquiring multipathogens infections after tick bite still needs evaluation. An important issue is also the recognition of the geographical range and position of new and old species of tick-borne bacteria as well as identification of diseases transmitted by ticks. That specially concerns rickettsia, knowledge of their distribution is crucial for recognition of rickettsiae-induced diseases in the areas where the bacteria were not consider in differential diagnosis of influenza-like or unclear rheumatoid clinical symptoms. Results of our study can efficiently contribute to the understanding of Rickettsia spp. distribution in Europe.

Footnotes

Acknowledgments

We are grateful to all persons who helped with collecting samples from forest workers, especially to Wlodzimierz Pawlowski from Bialowieża. This work was partially supported by the Ministry of Science and Higher Education (MNiSW) Project N404 072 32/2198 and N303 047 31/1487.

Disclosure Statement

No competing financial interests exist.

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No. of seropositives	R. conori	R. helvetica	R. felis	R. slovaca	R. siberica	R. massiliae
12	0	0	0	0	0	64
1	0	0	32	0	0	64
1	32	0	0	0	0	64
1	32	32	0	32	0	64
1	32	0	64	64	32	64
1	64	64	64	128	0	32
1	64	32	32	64	0	128
1	128	32	64	128	0	128

No. of seropositives	R. conori	R. helvetica	R. felis	R. slovaca	R. siberica	R. massiliae
12	0	0	0	0	0	64
1	0	0	32	0	0	64
1	32	0	0	0	0	64
1	32	32	0	32	0	64
1	32	0	64	64	32	64
1	64	64	64	128	0	32
1	64	32	32	64	0	128
1	128	32	64	128	0	128

No. of seropositives	R. conori	R. helvetica	R. felis	R. slovaca	R. siberica	R. massiliae
12	0	0	0	0	0	64
1	0	0	32	0	0	64
1	32	0	0	0	0	64
1	32	32	0	32	0	64
1	32	0	64	64	32	64
1	64	64	64	128	0	32
1	64	32	32	64	0	128
1	128	32	64	128	0	128