SNP-based real-time pyrosequencing as a sensitive and specific tool for identification and differentiation of <it>Rickettsia</it> species in <it>Ixodes ricinus</it> ticks

Abstract Background Rickettsioses are caused by pathogenic species of the genus Rickettsia and play an important role as emerging diseases. The bacteria are transmitted to mammal hosts including humans by arthropod vectors. Since detection, especially in tick vectors, is usually based on PCR with genus-specific primers to include different occurring Rickettsia species, subsequent species identification is mainly achieved by Sanger sequencing. In the present study a real-time pyrosequencing approach was established with the objective to differentiate between species occurring in German Ixodes ticks, which are R. helvetica, R. monacensis, R. massiliae, and R. felis. Tick material from a quantitative real-time PCR (qPCR) based study on Rickettsia-infections in I. ricinus allowed direct comparison of both sequencing techniques, Sanger and real-time pyrosequencing. Methods A sequence stretch of rickettsial citrate synthase (gltA) gene was identified to contain divergent single nucleotide polymorphism (SNP) sites suitable for Rickettsia species differentiation. Positive control plasmids inserting the respective target sequence of each Rickettsia species of interest were constructed for initial establishment of the real-time pyrosequencing approach using Qiagen’s PSQ 96MA Pyrosequencing System operating in a 96-well format. The approach included an initial amplification reaction followed by the actual pyrosequencing, which is traceable by pyrograms in real-time. Afterwards, real-time pyrosequencing was applied to 263 Ixodes tick samples already detected Rickettsia-positive in previous qPCR experiments. Results Establishment of real-time pyrosequencing using positive control plasmids resulted in accurate detection of all SNPs in all included Rickettsia species. The method was then applied to 263 Rickettsia-positive Ixodes ricinus samples, of which 153 (58.2%) could be identified for their species (151 R. helvetica and 2 R. monacensis) by previous custom Sanger sequencing. Real-time pyrosequencing identified all Sanger-determined ticks as well as 35 previously undifferentiated ticks resulting in a total number of 188 (71.5%) identified samples. Pyrosequencing sensitivity was found to be strongly dependent on gltA copy numbers in the reaction setup. Whereas less than 101 copies in the initial amplification reaction resulted in identification of 15.1% of the samples only, the percentage increased to 54.2% at 101-102 copies, to 95.6% at >102-103 copies and reached 100% samples identified for their Rickettsia species if more than 103 copies were present in the template. Conclusions The established real-time pyrosequencing approach represents a reliable method for detection and differentiation of Rickettsia spp. present in I. ricinus diagnostic material and prevalence studies. Furthermore, the method proved to be faster, more cost-effective as well as more sensitive than custom Sanger sequencing with simultaneous high specificity.</p

A 10-year surveillance of Rickettsiales (Rickettsia spp. and Anaplasma phagocytophilum) in the city of Hanover, Germany, reveals Rickettsia spp. as emerging pathogens in ticks

Abstract Background Rickettsiales (Rickettsia spp. and Anaplasma phagocytophilum) transmitted by ticks are considered (re-)emerging pathogens posing a risk to public health. Nevertheless, year-long monitoring studies on prevalences of these pathogens in questing ticks to contribute to public health risk assessment are rare. Methods The current study extends previous prevalence surveillances (2005 and 2010) by 2015 to a 10-year monitoring. Therefore, 2100 questing Ixodes ricinus were collected from April to October 2015 at ten different recreation sites in the city of Hanover, Germany, to determine potential changes in tick infection rates with Rickettsiales. Results Of the collected ticks, 288 were adult females, 285 adult males and 1527 nymphs. Overall, 3.8% (79/2100) of ticks were infected with A. phagocytophilum, 50.8% (1066/2100) with Rickettsia spp. and 2.2% (46/2100) with both pathogens. Statistical analyses revealed stagnating A. phagocytophilum infection rates over the 10-year monitoring period, whereas Rickettsia infections increased significantly (33.3% in 2005 and 26.2% in 2010 vs 50.8% in 2015). This increase was also characterized by prominent seasonality with higher prevalences from July to October. Conclusions As increased tick infection rates result in an increased risk for public health, the long-term data reported here provide significant implications for the understanding of progressing Rickettsiales distribution in ticks and essentially contribute to reliable public health risk assessments

Neurotoxocarosis: marked preference of Toxocara canis for the cerebrum and T. cati for the cerebellum in the paratenic model host mouse

BACKGROUND: Infective larvae of the worldwide occurring zoonotic roundworm T. canis exhibit a marked affinity to the nervous tissues of paratenic hosts. In humans, most cases of neurotoxocarosis are considered to be caused by larvae of T. canis as T. cati larvae have rarely been found in the CNS in previous studies. However, direct comparison of studies is difficult as larval migration depends on a variety of factors including mouse strains and inoculation doses. Therefore, the present study aims to provide a direct comparison of both roundworm species in mice as a model for paratenic hosts with specific focus on the CNS during the acute and chronic phase of disease to provide a basis for further studies dealing with neurotoxocarosis. METHODS: C57Bl/6J mice were infected with 2000 embryonated T. canis and T. cati eggs, respectively as well as Balb/c mice infected with T. cati eggs only. On 8 time points post infection, organs were removed and microscopically examined for respective larvae. Special focus was put on the CNS, including analysis of larval distribution in the cerebrum and cerebellum, right and left hemisphere as well as eyes and spinal cord. Additionally, brains of all infection groups as well as uninfected controls were examined histopathologically to characterize neurostructural damage. RESULTS: Significant differences in larval distribution were observed between and within the infection groups during the course of infection. As expected, significantly higher recovery rates of T. canis than T. cati larvae were determined in the brain. Surprisingly, significantly more T. canis larvae could be found in cerebra of infected mice whereas T. cati larvae were mainly located in the cerebellum. Structural damage in brain tissue could be observed in all infection groups, being more severe in brains of T. canis infected mice. CONCLUSIONS: The data obtained provides an extensive characterization of migrational routes of T. canis and T. cati in the paratenic host mouse in direct comparison. Even though to a lesser extent, structural damage in the brain was also caused by T. cati larvae and therefore, the potential as pathogenic agents should not be underestimated

Abnormal neurobehaviour and impaired memory function as a consequence of <i>Toxocara canis</i>- as well as <i>Toxocara cati</i>-induced neurotoxocarosis

<div><p>Background</p><p>Neuroinvasive larvae of the worldwide occurring zoonotic roundworms <i>Toxocara canis</i> and <i>T</i>. <i>cati</i> may induce neurotoxocarosis (NT) in humans, provoking a variety of symptoms including cognitive deficits as well as neurological dysfunctions. An association with neuropsychological disorders has been discussed. Similar symptoms have been described in <i>T</i>. <i>canis</i>-infected mice, whereas data on <i>T</i>. <i>cati</i>-induced NT are rare. Therefore, it was aimed to obtain insights into the impact on neurobehaviour as well as progression of neurological symptoms and behavioural alterations during the course of NT directly comparing <i>T</i>. <i>canis</i>- and <i>T</i>. <i>cati</i>-infected mice as models for human NT.</p><p>Methodology/Principal findings</p><p>C57BL/6 mice were orally infected with 2000 embryonated <i>T</i>. <i>canis</i> or <i>T</i>. <i>cati</i> eggs, respectively, the control group received tap water. Mice were screened weekly for neurobehavioural alterations and memory function starting one day prior infection until 97 days post infection (pi; <i>T</i>. <i>canis</i>-infection) and day 118 pi (<i>T</i>. <i>cati</i>-infection, uninfected control). Mostly motoric and neurological parameters were affected in <i>T</i>. <i>canis</i>-infected mice starting day 20 pi with severe progression accompanied by stereotypical circling. In contrast, <i>T</i>. <i>cati</i>-infected mice mostly showed reduced response to sudden sound stimulus (indicator for excitability) and flight behaviour starting day 6 pi. Interestingly, enhanced grooming behaviour was observed exclusively in <i>T</i>. <i>cati</i>-infected mice, indicating a possible role of neurotransmitter dysregulation. Reduced exploratory behaviour and memory impairment was observed in both infection groups with delayed onset and less severe progression in <i>T</i>. <i>cati</i>- compared to <i>T</i>. <i>canis</i>-infected mice.</p><p>Conclusions/Significance</p><p>Results highlight the need to consider <i>T</i>. <i>cati</i> beside <i>T</i>. <i>canis</i> as causative agent of human NT. Findings provide valuable hints towards differences in key regulatory mechanisms during <i>T</i>. <i>canis</i>- and <i>T</i>. <i>cati</i>-induced NT, contributing to a comprehensive picture and consequently a broader understanding of NT, which will aid in developing strategies towards prevention in addition to novel diagnostic and therapeutic approaches.</p></div

Shifts in Borrelia burgdorferi (s.l.) geno-species infections in Ixodes ricinus over a 10-year surveillance period in the city of Hanover (Germany) and Borrelia miyamotoi-specific Reverse Line Blot detection

Abstract Background Lyme borreliosis caused by spirochetes of the Borrelia burgdorferi (sensu lato) complex is still the most common tick-borne disease in Europe, posing a considerable threat to public health. The predominant vector in Europe is the widespread hard tick Ixodes ricinus, which also transmits the relapsing fever spirochete B. miyamotoi as well as pathogenic Rickettsiales (Anaplasma phagocytophilum, Rickettsia spp.). To assess the public health risk, a long-term monitoring of tick infection rates with the named pathogens is indispensable. Methods The present study is the first German 10-year follow-up monitoring of tick infections with Borrelia spp. and co-infections with Rickettsiales. Furthermore, a specific Reverse Line Blot (RLB) protocol for detection of B. miyamotoi and simultaneous differentiation of B. burgdorferi (s.l.) geno-species was established. Results Overall, 24.0% (505/2100) of ticks collected in the city of Hanover were infected with Borrelia. In detail, 35.4% (203/573) of adult ticks [38.5% females (111/288) and 32.3% males (92/285)] and 19.8% nymphs (302/1527) were infected, representing consistent infection rates over the 10-year monitoring period. Geno-species differentiation using RLB determined B. miyamotoi in 8.9% (45/505) of positive ticks. Furthermore, a significant decrease in B. afzelii and B. spielmanii infection rates from 2010 to 2015 was observed. Co-infections with Rickettsia spp. and A. phagocytophilum increased between 2010 and 2015 (7.3 vs 10.9% and 0.3 vs 1.1%, respectively). Conclusions Long-term monitoring is an essential part of public health risk assessment to capture data on pathogen occurrence over time. Such data will reveal shifts in pathogen geno-species distribution and help to answer the question whether or not climate change influences tick-borne pathogens

Alterations in activity patterns during the course of neurotoxocarosis.

<p>(A) uninfected control mice, (B) <i>T</i>. <i>canis</i>-infected mice, (C) <i>T</i>. <i>cati</i>-infected mice. Individual bars represent 100% of recorded activity with specific activities presented as respective fractions. Asterisks indicate significant differences in activities of the respective infection group compared to the uninfected controls. Note that intense reduction of “rearing on wall” and “rearing” in both infection groups did not allow presentation of asterisks in respective bars. Significantly reduced rearing on wall was additionally observed days 83–97 pi in <i>T</i>. <i>canis</i>-infected mice (B) and day 118 pi in <i>T</i>. <i>cati</i>-infected mice (C). Also, significantly reduced rearing was observed in <i>T</i>. <i>canis</i>-infected mice (B) days 6–20 pi and 34–97 pi days as well as in <i>T</i>. <i>cati</i>-infected mice (C) days 27–48 pi and 90–97 pi.</p

Assessment of sensorimotor function in <i>Toxocara canis-</i> and <i>T</i>. <i>cati</i>-infected mice.

<p>(A) Time-to-contact. Time required for mice to notice piece of tape on their front paws. (B) Time-to-remove. Time required for mice to remove piece of tape from their front paws. Ends of boxes define the 25th and 75th percentiles, with a line at the median and error bars defining the 10th and 90th percentiles. Asterisks indicate significant differences of the respective infection group compared to the uninfected control. *p≤0.05; **p≤0.001; ***p≤0.0001.</p

Altered health and behavioural parameters during the course of <i>Toxocara cati</i>-induced neurotoxocarosis.

<p>Arrows indicate if the parameter was increased or decreased in at least 25% of mice per group. Colours specify the severity of the presented alteration based on the group mean difference from the standard score (= 2), whereby colour categories represent the percentage deviation from the standard score. If less than 25% of mice per group showed behavioural alterations, no alteration was indicated.</p

Histological sections of <i>Toxocara</i>-infected mouse brains.

<p>(A) <i>T</i>. <i>canis</i>- and (B) <i>T</i>. <i>cati</i>-infected mouse brain section. Solid arrows indicate <i>Toxocara</i> larvae and dashed arrow indicates accumulation of gitter cells.</p