Borrelia miyamotoi and Candidatus Neoehrlichia mikurensis in Ixodes ricinus Ticks, Romania.

To the Editor: Ixodes spp. ticks are vectors for human and animal pathogens. Ix. ricinus ticks are widely distributed, frequently reported to feed on humans, and the main vector for a large variety of tickborne pathogens (). The effect of ticks and tickborne diseases on public health, animal health and welfare, and animal production appears to be an increasing global problem, which will lead to considerable economic costs ().

Borrelia miyamotoi is a spirochete that belongs to the relapsing fever group and causes symptoms similar to those of other relapsing fever group pathogens and Lyme borreliosis, including erythema migrans−like skin lesions (). The geographic distribution of B. miyamotoi is sporadic; it has been detected in Ixodes spp. ticks in many countries in Europe and in North America and Asia. In Russia, the United States, and recently in the Netherlands, B. miyamotoi was detected in humans and confirmed to cause disease (,). In Romania, pathogens that cause Lyme borreliosis and reptile-associated borreliae were identified in different tick populations (,). However, no information is available on the presence of relapsing fever group borreliae in this country.

Candidatus Neoehrlichia mikurensis and Anaplasma phagocytophilum are obligate, intracellular, tickborne pathogens of the family Anaplasmataceae; both are emerging zoonotic agents. Candidatus N. mikurensis causes monocytotropic ehrlichiosis in canids and humans and granulocytic anaplasmosis in humans and domestic animals (). These 2 pathogens are found throughout Europe in Ix. ricinus ticks (). A. phagocytophilum has been reported in questing Ix. ricinus ticks, dogs, wild boars, hedgehogs, and tortoises in Romania (). Recently, Candidatus N. mikurensis was detected in an Ix. ricinus tick that had bitten a human in Romania (). This recently discovered tickborne agent was shown to be a risk for disease in humans and has been detected in questing Ix. ricinus ticks throughout Europe and in animal tissue samples and human patients ().

Relapsing fever spirochetes and potential public health risks associated with tickborne pathogens are a serious medical problem. Thus, we assessed the presence of B. miyamotoi, A. phagocytophilum, and Candidatus N. mikurensis in questing Ix. ricinus ticks in Romania.

Questing Ix. ricinus ticks were available from previous studies conducted by our research group. A random sampling approach was used as described (). To detect potentially pathogenic bacteria, 468 questing Ix. ricinus ticks were collected from 4 regions from Romania, randomly selected, and analyzed.

Detection of pathogens was performed by using multiplex quantitative PCRs (qPCRs) specific for the flaB and ospA genes of B. miyamotoi, the msp2 gene of A. phagocytophilum, and the groEL gene of Candidatus N. mikurensis. We used IQ Multiplex Powermix (Bio-Rad, Carlsbad, CA, USA) and a final reaction volume of 20 μL (). For detection of A. phagocytophilum and Candidatus N. mikurensis, we also performed multiplex qPCR as described (). For detection of B. miyamotoi, a specific region of the flab gene was targeted by using multiplex qPCR according to a previous described protocol (). For quality control of qPCRs, we included positive and negative controls. Sequences of qPCR products were analyzed and compared with sequences available in GenBank.

B. miyamotoi was detected in 7 ticks: 2 (1.59%) of 126 males, 2 (0.68%) of 296 females, and 3 (6.52%) of 46 nymphs. A. phagocytophilum was detected in 16 ticks: 1 (0.79%) of 126 males, 11 (3.72%) of 296 females, and 4 (8.70%) of 46 nymphs. Candidatus N. mikurensis was detected in 25 ticks: 5 (3.97%) of 126 males, 18 (6.08%) of 296 females, and 2 (4.35%) of 46 nymphs. Overall prevalences were 1.50% for B. miyamotoi, 3.42% for A. phagocytophilum, and 5.34% for Candidatus N. mikurensis. Prevalences of each pathogen in specific varied by locality (Table). No co-infections were detected.

Table

Prevalence of 3 bacterial species in questing Ixodes ricinus in 14 localities, Romania

Locality (county)	No. ticks tested (nymphs, males, females)	No. (%) ticks positive, by bacterial species
		Borrelia miyamotoi	Anaplasma phagocytophilum	Candidatus Neoehrlichia mikurensis
Cugir (Alba)	19 (8, 4, 7)	0	2 (10.53)	1 (5.26)
Vladimirescu (Arad)	17 (0, 5, 12)	0	2 (11.76)	0
Bicaci (Bihor)	23 (12, 5, 6)	0	4 (17.4)	0
Bistrița (Bistriţa-Năsăud)	30 (0, 10, 20)	0	0	0
Poiana Mărului (Brașov)	66 (0,10, 56)	2 (3.03)	0	0
Vultureni (Cluj)	44 (3, 10, 31)	1 (2.27)	3 (6.82)	2 (4.55)
Micești (Cluj)	62 (0, 15, 47)	1 (1.62)	0	2 (3.23)
Reșița (Caraș-Severin)	21 (0, 10, 11)	0	0	0
Corund (Harghita)	59 (7, 17, 37)	0	1 (1.7)	4 (6.78)
Bistra (Maramureș)	26 (1, 10, 15)	0	0	0
Icland (Mureș)	37 (6, 5, 26)	2 (5.41)	4 (10.81)	8 (21.62)
Mediaș (Sibiu)	12 (2, 4, 6)	1 (8.33)	0	2 (16.67)
Rătești (Satu Mare)	22 (7, 10, 15)	0	0	1 (4.55)
Lugoj (Timiș)	30 (0, 11, 19)	0	0	5 (16.67)
Total	468 (46, 126, 296)	7 (1.5)	16 (3.42)	25 (5.34)

We analyzed flab, msp2, and groEL gene sequences obtained by qPCR. These sequences showed 99%–100% identities with gene sequences of B. miyamotoi (GenBank accession no. KJ847050), A. phagocytophilum (accession no. KP164415), and Candidatus N. mikurensis (accession no. FJ966365).

In Romania, the density of Ix. ricinus ticks is high and their host diversity is extensive (). However, data for effects of tickborne pathogens on public health are scarce in this country. In this study, we detected B. miyamotoi, A. phagocytophilum, and Candidatus N. mikurensis in questing Ix. ricinus ticks in Romania, which confirms the emerging trend of these pathogens in Europe. Because of the scarcity of information on human infections with these pathogens in Romania, serologic and molecular investigations and their implementation are needed for diagnosis, which might help in assessing the effect of these pathogens on public health.