Renate Ranka

Vectors of tick-borne diseases and epidemiological situation in Latvia in 1993-2002.

During the period of 1993-2002 an increase and the remarkable changes in the incidence of tick-borne encephalitis (TBE) and Lyme borreliosis (LB) as well as annual activities of vector species were noticed. The highest increase of TBE morbidity in Latvia has been observed in 1994 and 1995, and less expressed also in 1998 which was followed by a significant decrease during subsequent years. Whereas the highest peak of LB morbidity has been noticed in 1998 with only a minor decrease during subsequent years. Two epidemiologically significant Ixodes tick species are common in Latvia. Ixodes ricinus L. spread in the western and central part of Latvia; but rarely and in small numbers also in the eastern part. Ixodes persulcatus P. Sch. seems to dominate only in the eastern part of the country. The changes of seasonal and annual activities of I. ricinus were observed in two types of monitoring sites--sylvatic and peridomestic, located in the central part of Latvia. Observations of I. persulcatus were made in the eastern districts. Comparing geographically the Tick-borne diseases (TBD) incidence data for three tick distribution regions (I. ricinus dominated, I. persulcatus dominated and mixed regions), it was observed that the trend of annual changes in LB incidence during the last decade is almost of the same kind for all regions. Whereas TBE incidence extremes in 1994 and 1995 were observed almost only in I. ricinus dominated region. The annual field-collected adult tick infection rate with TBE virus (TBEV) from 1993 to 2002 for I. ricinus adults varied between 1.7% and 26.6% and for I. persulcatus--between 0% and 37.3%. The infection level in ticks removed from humans was much higher and from 1998 to 2002 surpassed 30%. TBEV investigations of removed adults and nymphs in 2002 discovered a high TBEV prevalence also in I. ricinus nymphs (43%). Typing of TBE virus isolated from ticks and patient serum samples in collaboration with German and Swedish virologists revealed that all three TBEV subtypes were co-circulating in Latvia. The mean annual tick infection rate with Borrelia burgdorferi s.l. analysed by means of a nested polymerase chain reaction of OspA gene fragment during the period of 1999-2001 was 18%-38% for I. ricinus adults and 25%-51% for I. persulcatus adults. Typing of Borrelia-positive DNA samples indicated that B. afzelii was dominant in Borrelia-positive I. ricinus (64.9%) and I. persulcatus (81.2%) followed by B. garinii (I. ricinus: 24.3%; in I. persulcatus: 14.5%). Investigations of Ehrlichia phagocytophila genogroup (by nested PCR targeted the 16S rRNA gene) revealed the presence of Ehrlichia sp. in I. ricinus from the central part of Latvia. Obtained data on TBD vectors and the epidemiological situation during the last decade indicates the complex impact of different factors influencing TBE and LB morbidity in Latvia.

Prevalence of tick-borne pathogens in ticks collected from migratory birds in Latvia

Migratory birds act as hosts and long-distance vectors for several tick-borne infectious agents. Here, feeding Ixodes ticks were collected from migratory birds during the autumn migration period in Latvia and screened for the presence of epidemiologically important non-viral pathogens. A total of 93 DNA samples of ticks (37 larvae and 56 nymphs) removed from 41 birds (order Passeriformes, 9 species) was tested for Lyme borreliosis spirochaetes, Anaplasma phagocytophilum, Rickettsia spp., and Babesia spp. Borrelia burgdorferi DNA was detected in 18% of the tick samples, and a majority of infected ticks were from thrush (Turdus spp.) birds. Among the infected ticks, Borrelia valaisiana was detected in 41% of cases, Borrelia garinii in 35%, and mixed Bo. valaisiana and Bo. garinii infection in 24%. Anaplasma phagocytophilum DNA was detected in 2% of ticks, R. helvetica in 12%, and Babesia spp. pathogens in 4% of ticks. Among these samples, 3 Babesia species were identified: Ba. divergens, Ba. microti, and Ba. venatorum. Coinfection with different pathogens that included mixed infections with different Borrelia genospecies was found in 20% of nymphal and 3% of larval Ixodes ticks. These results suggest that migratory birds may support the circulation and spread of medically significant zoonoses in Europe.

Identification of Three Clinically Relevant Borrelia burgdorferi Sensu Lato Genospecies by PCR-Restriction Fragment Length Polymorphism Analysis of 16S-23S Ribosomal DNA Spacer Amplicons

ABSTRACT We report the results of a study of the prevalences of three clinically relevant Borrelia burgdorferi sensu lato genospecies (Borrelia burgdorferi sensu stricto, Borrelia afzelii, and Borrelia garinii) in 1,040 questing Ixodes ticks from all regions of Latvia, where Lyme borreliosis is endemic. The prevalences of Borrelia in Ixodes ricinus and Ixodes persulcatus were 22.6 and 27.9%, respectively. Molecular typing of B. burgdorferi from infected ticks was performed by restriction fragment length polymorphism (RFLP) analysis of PCR-amplified fragments of the 16S-23S (rrs-rrlA) rRNA intergenic spacer by using species-specific primers and subsequent sequencing. The dominant Borrelia species in both Ixodes species was B. afzelii. In addition, different restriction patterns of B. garinii and B. afzelii were also identified. This study demonstrates that the 16S-23S rRNA PCR-RFLP typing method is simple, sensitive, and fast and that it allows one to differentiate among B. burgdorferi species and subspecies with various degrees of pathogenic potential directly in ticks. These features are important in monitoring Lyme disease.

Latin-American-Mediterranean lineage of Mycobacterium tuberculosis: Human traces across pathogen's phylogeography.

Currently, Mycobacterium tuberculosis isolates of Latin-American Mediterranean (LAM) family may be detected far beyond the geographic areas that coined its name 15years ago. Here, we established the framework phylogeny of this geographically intriguing and pathobiologically important mycobacterial lineage and hypothesized how human demographics and migration influenced its phylogeography. Phylogenetic analysis of LAM isolates from all continents based on 24 variable number of tandem repeats (VNTR) loci and other markers identified three global sublineages with certain geographic affinities and defined by large deletions RD115, RD174, and by spoligotype SIT33. One minor sublineage (spoligotype SIT388) appears endemic in Japan. One-locus VNTR signatures were established for sublineages and served for their search in published literature and geographic mapping. We suggest that the LAM family originated in the Western Mediterranean region. The most widespread RD115 sublineage seems the most ancient and encompasses genetically and geographically distant branches, including extremely drug resistant KZN in South Africa and LAM-RUS recently widespread across Northern Eurasia. The RD174 sublineage likely started its active spread in Brazil; its earlier branch is relatively dominated by isolates from South America and the derived one is dominated by Portuguese and South/Southeastern African isolates. The relatively most recent SIT33-sublineage is marked with enigmatic gaps and peaks across the Americas and includes South African clade F11/RD761, which likely emerged within the SIT33 subpopulation after its arrival to Africa. In addition to SIT388-sublineage, other deeply rooted, endemic LAM sublineages may exist that remain to be discovered. As a general conclusion, human mass migration appears to be the major factor that shaped the M. tuberculosis phylogeography over large time-spans.

Autochthonous canine babesiosis caused by Babesia canis canis in Latvia

This is the first report of confirmed canine babesiosis in Latvia supporting the observed geographical expansion of this disease. Between 2009 and 2011 three dogs which have not traveled outside of Latvia were diagnosed with babesiosis. Hematological analysis and serological tests for granulocytic anaplasmosis, ehrlichiosis and borreliosis were negative (Idexx SNAP 4Dx test). Peripheral blood erythrocytes of the three dogs contained large Babesia that were identified as Babesia canis canis by PCR. Sequences of partial 18S rRNA gene were 98-100% similar to the sequences of B. canis canis isolated from dogs in other European countries. We conclude that these are the first autochthonous canine babesiosis cases reported from Latvia.

Genotypic and phenotypic characteristics of aminoglycoside-resistant Mycobacterium tuberculosis isolates in Latvia.

Mutations causing resistance to aminoglycosides, such as kanamycin (KAN), amikacin (AMK), and streptomycin, are not completely understood. In this study, polymorphisms of aminoglycoside resistance influencing genes such as rrs, eis, rpsL, and gidB in 41 drug-resistant and 17 pan-sensitive Mycobacterium tuberculosis clinical isolates in Latvia were analyzed. Mutation A1400G in rrs gene was detected in 92% isolates with high resistance level to KAN and diverse MIC level to AMK. Mutations in promoter region of eis were detected in 80% isolates with low-level MIC of KAN. The association of K43R mutation in rpsL gene, a mutation in the rrs gene at position 513, and various polymorphisms in gidB gene with distinct genetic lineages of M. tuberculosis was observed. The results of this study suggest that association of different controversial mutations of M. tuberculosis genes to the drug resistance phenotype should be done in respect to genetic lineages.

Structure of an outer surface lipoprotein BBA64 from the Lyme disease agent Borrelia burgdorferi which is critical to ensure infection after a tick bite.

Lyme disease is a tick-borne infection caused by the transmission of Borrelia burgdorferi from infected Ixodes ticks to a mammalian host during the blood meal. Previous studies have shown that the expression of B. burgdorferi surface-localized lipoproteins, which include BBA64, is up-regulated during the process of tick feeding. Although the exact function of BBA64 is not known, this lipoprotein is critical for the transmission of the spirochete from the tick salivary glands to the mammalian organism after a tick bite. Since the mechanism of development of the disease and the functions of the surface lipoproteins associated with borreliosis are still poorly understood, the crystal structure of the B. burgdorferi outer surface lipoprotein BBA64 was solved at 2.4 Å resolution in order to obtain a better insight into the pathogenesis of B. burgdorferi and to promote the discovery of novel potential preventive drugs against Lyme disease. In this study, the crystal structure of BBA64 was also compared with that of the paralogous protein CspA (also referred to as BbCRASP-1, CRASP-1 or BBA68). CspA is the complement regulator-acquiring surface protein-1 of B. burgdorferi; its structure is known, but its function apparently differs from that of BBA64. It is demonstrated that unlike the homologous CspA, BBA64 does not form a homodimer. Their differences in function could be explained by divergence in their amino-acid sequences, electrostatic surface potentials and overall tertiary structures. The C-terminal part of BBA64 has a different conformation to that of CspA; the conformation of this region is essential for the proper function of CspA.

Association between Anaplasma phagocytophilum seroprevalence in dogs and distribution of Ixodes ricinus and Ixodes persulcatus ticks in Latvia

Anaplasma phagocytophilum has been detected in ticks in Latvia; however, this is the first study to investigate this pathogen in dogs in Latvia. The aims of this study were: (i) to determine A. phagocytophilum seroprevalence in dogs, (ii) to correlate A. phagocytophilum seroprevalence in dogs with the geographic distribution of the tick species Ixodes ricinus and Ixodes persulcatus, and (iii) to determine if seroprevalence for A. phagocytophilum is higher in dogs with clinical signs suggestive of canine granulocytic anaplasmosis (CGA). Peripheral venous blood samples were collected from 3 dog groups: (i) clinically healthy dogs (HD, n=400), (ii) clinically healthy hunting dogs (HHD, n=41), and (iii) dogs with a clinical suspicion of anaplasmosis (SD, n=29). Sampling was carried out in regions inhabited by I. ricinus (IR), I. persulcatus (IP), and in regions where both tick species were present (M). SNAP 4Dx test (IDEXX) was used to detect antibodies against A. phagocytophilum in the blood of all dogs; nested PCR was performed in selected dogs of the SD group. Seroprevalence for A. phagocytophilum was calculated and correlated with the prevalent tick species in the region. A. phagocytophilum seroprevalence was 11.0% in HD, 12% in HHD, and 17% in SD with no significant differences among groups. In the IR region, seroprevalence was 12.5% (34/272) while seroprevalence in the M region was 17% (13/76), and both were significantly higher than the seroprevalence of 2% in the IP region (2/93; p<0.0005). One CGA case was diagnosed. We conclude that A. phagocytophilum seroprevalence in Latvia is within the range reported from other European countries. CGA should be included in the differential list in Latvian dogs with appropriate clinical signs and laboratory abnormalities, especially in I. ricinus habitat areas.

Dynamics of Telomere Length in Different Age Groups in a Latvian Population

The shortening of telomeres with ageing is a well-documented observation; however, the reported number of nucleotides in telomeres varies between different laboratories and studies. Such variability is likely caused by ethnic differences between the populations studied. Until now, there were no studies that investigated the variability of telomere length in a senescent Latvian population of the most common mitochondrial haplogroups, defined as H (45%), U (25%), Y chromosomal N1c (40%) and R1a1 (40%). Telomere length was determined in 121 individuals in different age groups, including a control group containing individuals of 20-40 years old and groups of individuals between 60-70 years old, 71-80 years old, 81-90 years old, and above 90 years old. Telomere length was determined using the Southern blot telomeric restriction fragment assay (TRF). Decreased telomere length with ageing was confirmed, but a comparison of centenarians and individuals between 60-90 years of age did not demonstrate a significant difference in telomere length. However, significant variability in telomere length was observed in the control group, indicating probable rapid telomere shortening in some individuals that could lead up to development of health status decline appearing with ageing. Telomere length measured in mononuclear blood cells (MNC) was compared with the telomere length measured in whole peripheral white blood cells (WBC) using TRF. Telomere length in MNC was longer than in WBC for the control group with individuals 20 to 40 years old; in contrast, for the group of individuals aged 65 to 85 years old, measured telomere length was shorter in MNC when compared to WBC.

Structural characterization of CspZ, a complement regulator factor H and FHL-1 binding protein from Borrelia burgdorferi

Borrelia burgdorferi is the causative agent of Lyme disease and is found in two different types of hosts in nature – Ixodes ticks and various mammalian organisms. To initiate disease and survive in mammalian host organisms, B. burgdorferi must be able to transfer to a new host, proliferate, attach to different tissue and resist the immune response. To resist the hosts immune response, B. burgdorferi produces at least five different outer surface proteins that can bind complement regulator factor H (CFH) and/or factor H‐like protein 1 (CFHL‐1). The crystal structures of two uniquely folded complement binding proteins, which belong to two distinct gene families and are not found in other bacteria, have been previously described. The crystal structure of the CFH and CFHL‐1 binding protein CspZ (also known as BbCRASP‐2 or BBH06) from B. burgdorferi, which belongs to a third gene family, is reported in this study. The structure reveals that the overall fold is different from the known structures of the other complement binding proteins in B. burgdorferi or other bacteria; this structure does not resemble the fold of any known protein deposited in the Protein Data Bank. The N‐terminal part of the CspZ protein forms a four‐helix bundle and has features similar to the FAT domain (focal adhesion targeting domain) and a related domain found in the vinculin/α‐catenin family. By combining our findings from the crystal structure of CspZ with previous mutagenesis studies, we have identified a likely binding surface on CspZ for CFH and CFHL‐1.