Novel Protozoans in Austria Revealed through the Use of Dogs as Sentinels for Ticks and Tick-Borne Pathogens

We previously isolated and cultivated the novel Rickettsia raoultii strain Jongejan. This prompted us to ask whether this strain is unique or more widely present in Austria. To assess this issue, we retrospectively screened ticks collected from dogs in 2008. Of these collected ticks, we randomly selected 75 (47 females and 28 males) Dermacentor reticulatus, 44 (21 females, 7 males, and 16 nymphs) Haemaphysalis concinna, and 55 (52 females and 3 males) ticks of the Ixodes ricinus complex. Subsequently, these ticks were individually screened for the presence of tick-borne pathogens using the reverse line blot hybridization assay. In our current study, we detected DNA from the following microbes in D. reticulatus: Anaplasma phagocytophilum, Borrelia lusitaniae, Borrelia spielmanii, Borrelia valaisiana, and R. raoultii, all of which were R. raoultii strain Jongejan. In H. concinna, we found DNA of a Babesia sp., Rickettsia helvetica, and an organism closely related to Theileria capreoli. Lastly, I. ricinus was positive for Anaplasma phagocytophilum, Borrelia afzelii, Borrelia burgdorferi sensu stricto, Borrelia garinii/Borrelia bavariensis, B. lusitaniae, B. spielmanii, B. valaisiana, Candidatus Neoehrlichia mikurensis, Rickettsia helvetica, Rickettsia monacensis, and Theileria (Babesia) microti DNA. The detection of DNA of the Babesia sp. and an organism closely related to Theileria capreoli, both found in H. concinna ticks, is novel for Austria.

The Population Structure of Borrelia lusitaniae Is Reflected by a Population Division of Its Ixodes Vector

Populations of vector-borne pathogens are shaped by the distribution and movement of vector and reservoir hosts. To study what impact host and vector association have on tick-borne pathogens, we investigated the population structure of Borrelia lusitaniae using multilocus sequence typing (MLST). Novel sequences were acquired from questing ticks collected in multiple North African and European locations and were supplemented by publicly available sequences at the Borrelia Pubmlst database (accessed on 11 February 2020). Population structure of B. lusitaniae was inferred using clustering and network analyses. Maximum likelihood phylogenies for two molecular tick markers (the mitochondrial 16S rRNA locus and a nuclear locus, Tick-receptor of outer surface protein A, trospA) were used to confirm the morphological species identification of collected ticks. Our results confirmed that B. lusitaniae does indeed form two distinguishable populations: one containing mostly European samples and the other mostly Portuguese and North African samples. Of interest, Portuguese samples clustered largely based on being from north (European) or south (North African) of the river Targus. As two different Ixodes species (i.e., I. ricinus and I. inopinatus) may vector Borrelia in these regions, reference samples were included for I. inopinatus but did not form monophyletic clades in either tree, suggesting some misidentification. Even so, the trospA phylogeny showed a monophyletic clade containing tick samples from Northern Africa and Portugal south of the river Tagus suggesting a population division in Ixodes on this locus. The pattern mirrored the clustering of B. lusitaniae samples, suggesting a potential co-evolution between tick and Borrelia populations that deserve further investigation.

Wild canids as hosts for ticks and tick-borne zoonotic pathogens in Serbia

Wild canids are recognized as important reservoir hosts for several vector-borne pathogens and are a good sentinel species for epidemiological surveillance. Identifying the reservoir hosts for particular zoonotic pathogens in a specific area enables the implementation of adequate measures in order to prevent the occurrence of zoonotic diseases in humans and domestic animals. The present review aims to summarize the role of wild canids in the epidemiology of tick-borne zoonoses in Serbia based on previously published papers and to discuss the available data from an epidemiological point of view. The results of previously published studies indicate that wild canids are potential reservoir hosts for several zoonotic tick-borne pathogens in Serbia, and hosts for several tick species which are proven competent vectors. Based on molecular studies conducted so far, the presence of Anaplasma phagocytophilum, was confirmed in jackals while the presence of Candidatus Neoehrlichia micurensis (FU98), Borrelia burgdorferi sensu stricto, Borrelia garinii, and Borrelia lusitaniae was proven by PCR in analyzed red foxes. There is a need for continuous epidemiological monitoring of diseases caused by pathogens previously confirmed in wild canids. Also, further research is needed to reveal the role of other wild and domestic animals in the epidemiology of tick-borne zoonoses in Serbia.

Candidatus Neoehrlichia sp. (FU98) and Borrelia burgdorferi Sensu Lato in Red Foxes (Vulpes vulpes) from Serbia

Human activities such as deforestation, urbanization, and environmental pollution lead to a reduction in the spatial boundary between wild animals, domestic animals and humans. These activities increase the risk for the emergence of pathogens from the sylvatic cycle in the population of domestic animals and humans. Foxes are recognized as potential reservoirs for a number of bacterial pathogens of medical and public health concern. The aim of the present study was to investigate the prevalence and spatial distribution of bacterial tick-borne pathogens from the Anaplasmataceae family, Borrelia burgdorferi sensu lato (s.l.), Rickettsia spp., Coxiella burnetii, Francisella tularensis, Bartonella spp., in the red fox population from Serbia and to discuss the obtained results from the epidemiological point of view. Legally hunted red foxes (Vulpes vulpes) from 14 localities in Serbia were included in the study and spleen samples from 129 animals were tested with conventional PCR assays for the presence of bacterial tick-borne pathogens. DNA of Candidatus Neoehrlichia sp. (FU98), Borrelia burgdorferi sensu stricto, Borrelia lusitaniae, and Borrelia garinii was detected in 6 (4.7%), 1 (0.8%), 2 (1.6%) and 1 (0.8%) animals, respectively. Co-infection by Candidatus Neoehrlichia sp. (FU98) and B. garinii was detected in one animal. All samples were negative for other tested bacterial tick-borne pathogens. The results of the present study indicate the potential role of foxes in natural cycles of Candidatus Neoehrlichia sp. (FU98) and causative agents of Lyme borreliosis in the investigated areas. Further research is required to elucidate the role of foxes in the epidemiology of these and other tick-borne zoonotic pathogens in the Republic of Serbia.

Borrelia lusitaniae Infection Mimicking Headache, Neurologic Deficits, and Cerebrospinal Fluid Lymphocytosis

Headache with neurologic deficits and cerebrospinal fluid lymphocytosis (HaNDL) is a rare headache syndrome included in the Classification of Headache of the International Headache Society as a “headache attributed to non-infectious inflammatory intracranial disease.” We report one 15-year-old patient with clinical history and cerebrospinal fluid findings compatible with the diagnosis of HaNDL in whom Borrelia lusitaniae was identified in cerebrospinal fluid by polymerase chain reaction.

Tick-borne pathogen detection in midgut and salivary glands of adult Ixodes ricinus

Background: Tick midgut and salivary glands represent the primary organs for pathogen acquisition and transmission, respectively. Specifically, the midgut is the first organ to have contact with pathogens during the blood meal uptake, while salivary glands along with their secretions play a crucial role in pathogen transmission to the host. Currently there is little data about pathogen composition and prevalences in I. ricinus midgut and salivary glands. The present study investigated the presence of 32 pathogen species in the midgut and salivary glands of unfed I. ricinus males and females using high-throughput microfluidic real-time PCR. Such an approach is important for enriching the knowledge about pathogen distribution in distinct tick organs which should lead to a better understanding I. ricinus-borne disease epidemiology. Results: Borrelia lusitaniae, Borrelia spielmanii, and Borrelia garinii, were detected in both midgut and salivary glands suggesting that the migration of these pathogens between these two organs might not be triggered by the blood meal. In contrast, Borrelia afzelii was detected only in the tick midgut. Anaplasma phagocytophilum and Rickettsia helvetica were the most frequently detected in ticks and were found in both males and females in the midgut and salivary glands. In contrast, Rickettsia felis was only detected in salivary glands. Finally Borrelia miyamotoi and Babesia venatorum were detected only in males in both midgut and salivary glands. Among all collected ticks, between 10 and 21% of organs were co-infected. The most common bacterial co-infections in male and female midgut and salivary glands was R. helvetica/A. phagocytophilum and R. helvetica/B. lusitaniae respectively. Conclusions: Analysing tick-borne pathogen (TBP) presence in specific tick organs enabled us to (i) highlight contrasting results with well-established transmission mechanism postulates, (ii) venture new hypotheses concerning pathogen location and migration from midgut to salivary glands, and (iii) suggest other potential associations between pathogens not previously detected at the scale of the whole tick. This work highlights the importance of considering all tick scales (i.e. whole ticks vs organs) to study TBP ecology and represents another step towards improved understanding of TBP transmission.

Evidence for Borrelia bavariensis Infections of Ixodes uriae within Seabird Colonies of the North Atlantic Ocean

ABSTRACT The first report of members of the spirochete genus Borrelia in the seabird tick, Ixodes uriae, and seabird colonies occurred during the early 1990s. Since then, Borrelia spp. have been detected in these ticks and seabird colonies around the world. To date, the primary species detected has been Borrelia garinii, with rare occurrences of Borrelia burgdorferi sensu stricto and Borrelia lusitaniae. During our research on Borrelia and I. uriae in seabird colonies of Newfoundland and Labrador, Canada, we have identified Borrelia bavariensis in I. uriae. To our knowledge, B. bavariensis has previously been found only in the Eurasian tick species Ixodes persulcatus and Ixodes ricinus, and it was believed to be a rodent-specific Borrelia ecotype. We found B. bavariensis within I. uriae from three seabird colonies over three calendar years. We also reanalyzed B. garinii sequences collected from I. uriae from Eurasian seabird colonies and determined that sequences from two Russian seabird colonies likely also represent B. bavariensis. The Canadian B. bavariensis sequences from I. uriae analyzed in this study cluster with previously described sequences from Asia. Overall, this is an important discovery that illustrates and expands the range of hosts and vectors for B. bavariensis, and it raises questions regarding the possible mechanisms of pathogen dispersal from Asia to North America. IMPORTANCE To our knowledge, this is the first documentation of B. bavariensis outside Eurasia. Additionally, the bacterium was found in a marine ecosystem involving the seabird tick I. uriae and its associated seabird hosts. This indicates that the epizootiology of B. bavariensis transmission is much different from what had been described, with this species previously believed to be a rodent-specific ecotype, and it indicates that this pathogen is established, or establishing, much more widely.

Approaches for Reverse Line Blot-Based Detection of Microbial Pathogens in Ixodes ricinus Ticks Collected in Austria and Impact of the Chosen Method

ABSTRACT Ticks transmit a large number of pathogens capable of causing human disease. In this study, the PCR-reverse line blot (RLB) method was used to screen for pathogens in a total of 554 Ixodes ricinus ticks collected from all provinces of Austria. These pathogens belong to the genera Borrelia, Rickettsiae, Anaplasma/Ehrlichia (including “Candidatus Neoehrlichia”), Babesia, and Coxiella. The pathogens with the highest detected prevalence were spirochetes of the Borrelia burgdorferi sensu lato complex, in 142 ticks (25.6%). Borrelia afzelii (80/142) was the most frequently detected species, followed by Borrelia burgdorferi sensu stricto (38/142) and Borrelia valaisiana (36/142). Borrelia garinii/Borrelia bavariensis, Borrelia lusitaniae, and Borrelia spielmanii were found in 28 ticks, 5 ticks, and 1 tick, respectively. Rickettsia spp. were detected in 93 ticks (16.8%): R. helvetica (39/93), R. raoultii (38/93), R. monacensis (2/93), and R. slovaca (1/93). Thirteen Rickettsia samples remain uncharacterized. “Candidatus Neoehrlichia mikurensis,” Babesia spp. (B. venatorum, B. divergens, B. microti), and Anaplasma phagocytophilum were found in 4.5%, 2.7%, and 0.7%, respectively. Coxiella burnetii was not detected. Multiple microorganisms were detected in 40 ticks (7.2%), and the cooccurrence of Babesia spp. and “Candidatus Neoehrlichia mikurensis” showed a significant positive correlation. We also compared different PCR-RLBs for detection of Borrelia burgdorferi sensu lato and Rickettsia spp. and showed that different detection approaches provide highly diverse results, indicating that analysis of environmental samples remains challenging. IMPORTANCE This study determined the wide spectrum of tick-borne bacterial and protozoal pathogens that can be encountered in Austria. Surveillance of (putative) pathogenic microorganisms occurring in the environment is of medical importance, especially when those agents can be transmitted by ticks and cause disease. The observation of significant coinfections of certain microorganisms in field-collected ticks is an initial step to an improved understanding of microbial interactions in ticks. In addition, we show that variations in molecular detection methods, such as in primer pairs and target genes, can considerably influence the final results. For instance, detection of certain genospecies of borreliae may be better or worse by one method or the other, a fact of great importance for future screening studies.