Heidi Hemmilä

Experimental Infection of Voles with Francisella tularensis Indicates Their Amplification Role in Tularemia Outbreaks

Tularemia outbreaks in humans have been linked to fluctuations in rodent population density, but the mode of bacterial maintenance in nature is unclear. Here we report on an experiment to investigate the pathogenesis of Francisella tularensis infection in wild rodents, and thereby assess their potential to spread the bacterium. We infected 20 field voles (Microtus agrestis) and 12 bank voles (Myodes glareolus) with a strain of F. tularensis ssp. holarctica isolated from a human patient. Upon euthanasia or death, voles were necropsied and specimens collected for histological assessment and identification of bacteria by immunohistology and PCR. Bacterial excretion and a rapid lethal clinical course with pathological changes consistent with bacteremia and tissue necrosis were observed in infected animals. The results support a role for voles as an amplification host of F. tularensis, as excreta and, in particular, carcasses with high bacterial burden could serve as a source for environmental contamination.

Detection of Francisella tularensis in voles in Finland.

Francisella tularensis is a highly virulent intracellular bacterium causing the zoonotic disease tularemia. It recurrently causes human and animal outbreaks in northern Europe, including Finland. Although F. tularensis infects several mammal species, only rodents and lagomorphs seem to have importance in its ecology. Peak densities of rodent populations may trigger tularemia outbreaks in humans; however, it is still unclear to which extent rodents or other small mammals maintain F. tularensis in nature. The main objective of this study was to obtain information about the occurrence of F. tularensis in small mammals in Finland. We snap-trapped 547 wild small mammals representing 11 species at 14 locations around Finland during 6 years and screened them for the presence of F. tularensis DNA using PCR analysis. High copy number of F. tularensis-specific DNA was detected in tissue samples of five field voles (Microtus agrestis) originating from one location and 2 years. According to DNA sequences of the bacterial 23S ribosomal RNA gene amplified from F. tularensis-infected voles, the infecting agent belongs to the subspecies holarctica. To find out the optimal tissue for tularemia screening in voles, we compared the amounts of F. tularensis DNA in lungs, liver, spleen, and kidney of the infected animals. F. tularensis DNA was detectable in high levels in all four organs except for one animal, whose kidney was F. tularensis DNA-negative. Thus, at least liver, lung, and spleen seem suitable for F. tularensis screening in voles. Thus, liver, lung, and spleen all seem suitable for F. tularensis screening in voles. In conclusion, field voles can be heavily infected with F. tularensis subsp. holarctica and thus potentially serve as the source of infection in humans and other mammals.

Phylogeography of Francisella tularensis subspecies holarctica in Finland, 1993-2011.

Abstract Background: Finland repeatedly reports some of the highest incidences of tularaemia worldwide. To determine genetic diversity of the aetiologic agent of tularaemia, Francisella tularensis, a total of 76 samples from humans (n = 15) and animals (n = 61) were analysed. Methods: We used CanSNPs and canINDEL hydrolysis or TaqMan MGB probes for the analyses, either directly from the clinical tissue samples (n = 21) or from bacterial isolates (n = 55). Results: The genotypes of the strains were assigned to three previously described basal subspecies holarctica clades. The majority of strains (n = 67) were assigned to B.12, a clade reported to dominate in Scandinavia and Eastern Europe. A single strain was assigned to clade B.4, previously reported from North America, Europe and China. The remaining strains (n = 8) were members of clade B.6. Importantly, new diversity was discovered in clade B.6. We describe two newly designed TaqMan MGB probe assays for this new B.6 subclade B.70, and its previously identified sister clade B.11, a clade dominantly found in Western Europe. Conclusions: The high genetic diversity of F. tularensis subspecies holarctica present in Finland is consistent with previous findings in Sweden. The results suggest a northern and southern division of the B.6 subclade B.10, where B.11 predominates in Western and Central Europe and B.70 is found in Fennoscandia. Further research is required to define whether the vast diversity of genotypes found is related to different habitats or reservoir species, their different postglacial immigration routes to Fennoscandia, or dynamics of the reservoir species.

Phylogeography of Francisella tularensis subspecies holarctica in Finland, 1993-2011

Abstract Background: Finland repeatedly reports some of the highest incidences of tularaemia worldwide. To determine genetic diversity of the aetiologic agent of tularaemia, Francisella tularensis, a total of 76 samples from humans (n = 15) and animals (n = 61) were analysed. Methods: We used CanSNPs and canINDEL hydrolysis or TaqMan MGB probes for the analyses, either directly from the clinical tissue samples (n = 21) or from bacterial isolates (n = 55). Results: The genotypes of the strains were assigned to three previously described basal subspecies holarctica clades. The majority of strains (n = 67) were assigned to B.12, a clade reported to dominate in Scandinavia and Eastern Europe. A single strain was assigned to clade B.4, previously reported from North America, Europe and China. The remaining strains (n = 8) were members of clade B.6. Importantly, new diversity was discovered in clade B.6. We describe two newly designed TaqMan MGB probe assays for this new B.6 subclade B.70, and its previously identified sister clade B.11, a clade dominantly found in Western Europe. Conclusions: The high genetic diversity of F. tularensis subspecies holarctica present in Finland is consistent with previous findings in Sweden. The results suggest a northern and southern division of the B.6 subclade B.10, where B.11 predominates in Western and Central Europe and B.70 is found in Fennoscandia. Further research is required to define whether the vast diversity of genotypes found is related to different habitats or reservoir species, their different postglacial immigration routes to Fennoscandia, or dynamics of the reservoir species.

Severe Ocular Cowpox in a Human, Finland

To the Editor: We describe cowpox with corneal involvement in a 31-year-old atopic woman who lived in southern Finland and was unvaccinated for smallpox. In August 2009, she noticed irritation and edema in her right eye and sought care from a local physician; she started topical antimicrobial drug therapy and oral cephalexin 2 days later. Over the following week, fever developed (37.6°C –39.0°C), edema developed on half her face, the eye became increasingly painful, and visual acuity decreased. The conjunctiva was severely chemotic and hyperemic, but the cornea was clear and the other eye unaffected. Microbiologic samples taken from the eye 11 days after onset showed neither bacteria nor respiratory viruses. Orbital tomography results were normal. The patient was hospitalized, and broad-spectrum intravenous antimicrobial treatment (meropenem, vancomycin, valacyclovir, and fluconazole) was started, combined with topical corticosteroids and antimicrobial drugs. Within 2 weeks, the conjunctiva showed necrosis, and epithelial erosions appeared in the lower cornea, but visual acuity normalized (Technical Appendix Figure, panels A, B). A strong cytopathic effect was observed in Vero cells infected with conjunctival swab (Technical Appendix Table 1), but the virus was unidentifiable by routine methods. In electron microscopy, cell culture and tear fluid samples contained particles with typical orthopoxvirus (OPV) morphology. PCRs for hemagglutinin (1) and 14-kDa genes (2) verified OPV infection. Additional PCRs and sequencing confirmed zoonotic cowpox virus (CPXV) with strain designation FIN/K2009. Nucleotide sequences of the hemagglutinin, thymidine kinase, and A-type inclusion body protein genes were identical to those of CPXV strains T2000 and E1989 previously identified in Finland (3). In phylogenetic analysis (Figure), CPXV/FIN/K2009 clustered with strains from Austria and shared ancestry with vaccinia virus. OPV IgG and IgM were detected by immunofluorescence assay (3) in serum samples up to 5 months after symptom onset (Technical Appendix Table 1). Figure Phylogenetic tree of orthopoxviruses constructed on the basis of the hemagglutinin gene; boldface indicates the CPXV strain infecting the patient described in this article. The phylogeny shows that the sequence derived from this patient represents a locally ... The patient was started on intravenous polyclonal gammaglobulin and topical trifluorothymidine with in vitro anti-OPV effects; nevertheless, corneal erosions enlarged, corneal stromal edema ensued, and intraocular pressure increased (online Technical Appendix Figure, panel C), suggesting trabeculitis. Topical autologous serum drops had no effect. Periorbital edema slowly resolved, but corneal erosions persisted. Amniotic membrane transplantation (AMT) (4) was performed 2.5 months after onset. The inferior cornea melted, and the cornea lost transparency (online Technical Appendix Figure, panel D). AMT was repeated twice at 1-month intervals because of corneal thinning. At 3.5 months after symptom onset, tecovirimat (400 mg 2×/d) was given orally for 14 days. Despite treatment, ocular OPV PCR test results remained positive until 9 months after onset (Technical Appendix Table 1), and corneal melting progressed (Technical Appendix Figure, panel E). Corneal collagen cross-linking and a fourth AMT were performed at 5 months after onset with partial success (Technical Appendix Figure, panel F). At 1 year after symptom onset, corneal limbal stem cell deficiency with deep corneal neovascularization was evident. Autologous limbal stem cell transplantation from the patient’s other eye and another AMT were performed, resulting in stable corneal surface 2 months later (Technical Appendix Figure, panel G). Neovascularization regressed, the cornea cleared, and vision improved (Technical Appendix Figure, panels H, I). Cowpox is transmitted to humans sporadically from rodents or cats (5). We snap-trapped 23 wild rodents from the yard of the patient’s home and from an adjacent meadow and trapped 136 rodents from 3 other regions 30–100 km from the patient’s home (Technical Appendix Table 2). We also collected 8 environmental samples from the patient’s storehouse. In accordance with the Finnish Act on Use of Animals for Experimental Purposes (62/2006) and the Finnish Animal Experiment Board’s later decision (May 16, 2007), the animal capture technique used is not an animal experiment and requires no ethics license. Diluted blood for IFA was collected from all rodents (6), and DNA was extracted from rodent liver and lungs and from environmental samples. One vole and 1 mouse from the meadow were seropositive for OPV; however, no CPXV DNA was amplifiable in the samples from the liver, lungs, or environment (online Technical Appendix Table 2). CPXV infection may manifest in severe ocular forms along with self-limiting cutaneous pocks (5). Our patient had keratitis with no other identifiable cause but CPXV. Culture and PCR from early conjunctival samples and serology confirmed the etiologic diagnosis. Our case and that of another report (7) highlight the challenges of treating cowpox keratitis. Topical and systemic antiviral drugs and AMT appear ineffective during the acute phase. Corneal melting and scarring continued as long as CPXV was observed and until combined limbal stem cell and AMT treatment had favorable outcomes. Anamnesis of therapy-resistant keratitis should include information on rodent contacts. We dated the infection to mid-August (incubation 7–21 days). Catching OPV-IgG–positive rodents close to the patient’s home 2 months after onset showed that OPVs were circulating in the local rodent population and indicated the putative role of CPXV-infected voles as the source of infection. The latest cowpox outbreak in Central Europe involved several humans and pets (8). This patient was born in 1977, after Finland ceased smallpox vaccinations. Declining cross-reactive smallpox-vaccination immunity enables emergence of unusual cowpox infections in humans (9). Technical Appendix.Diagnostic findings of patient, laboratory findings of sampling from rodents and environment, and images of progressive disease in eye of patient with ocular cowpox, Finland. Click here to view.(150K, pdf)

Monitoring biothreat agents (Francisella tularensis, Bacillus anthracis and Yersinia pestis) with a portable real-time PCR instrument

In the event of suspected releases or natural outbreaks of contagious pathogens, rapid identification of the infectious agent is essential for appropriate medical intervention and disease containment. The purpose of this study was to compare the performance of a novel portable real-time PCR thermocycler, PikoReal™, to the standard real-time PCR thermocycler, Applied Biosystems® 7300 (ABI 7300), for the detection of three high-risk biothreat bacterial pathogens: Francisella tularensis, Bacillus anthracis and Yersinia pestis. In addition, a novel confirmatory real-time PCR assay for the detection of F. tularensis is presented and validated. The results show that sensitivity of the assays, based on a dilution series, for the three infectious agents ranged from 1 to 100 fg of target DNA with both instruments. No cross-reactivity was revealed in specificity testing. Duration of the assays with the PikoReal and ABI 7300 systems were 50 and 100 min, respectively. In field testing for F. tularensis, results were obtained with the PikoReal system in 95 min, as the pre-PCR preparation, including DNA extraction, required an additional 45 min. We conclude that the PikoReal system enables highly sensitive and rapid on-site detection of biothreat agents under field conditions, and may be a more efficient alternative to conventional diagnostic methods.

Molecular characterization of adenoviruses among finnish military conscripts

Although adenoviruses were identified as important respiratory pathogens many years ago, little information is available concerning the prevalence of different adenovirus serotypes, which are circulating and causing epidemics in Finnish military training centers. Over a period of five years from 2008 to 2012, 3577 respiratory specimens were collected from military conscripts presenting with symptoms compatible with acute respiratory tract infection. Upon initial testing for certain respiratory viruses by real‐time PCR, 837 of these specimens were identified as adenovirus‐positive. For 672 of these specimens, the serotype of the adenovirus responsible was successfully determined by DNA sequencing. Serotypes 1, 2, 3, and 4 were detected in 1, 3, 181, and 487 samples, respectively. Adenovirus epidemics were observed during each year of this study. Based on these findings, adenovirus vaccination should be considered for military conscripts in the Finnish Defence Forces. J. Med. Virol. 88:571–577, 2016.

Molecular characterization of adenoviruses among finnish military conscripts.

Although adenoviruses were identified as important respiratory pathogens many years ago, little information is available concerning the prevalence of different adenovirus serotypes, which are circulating and causing epidemics in Finnish military training centers. Over a period of five years from 2008 to 2012, 3577 respiratory specimens were collected from military conscripts presenting with symptoms compatible with acute respiratory tract infection. Upon initial testing for certain respiratory viruses by real‐time PCR, 837 of these specimens were identified as adenovirus‐positive. For 672 of these specimens, the serotype of the adenovirus responsible was successfully determined by DNA sequencing. Serotypes 1, 2, 3, and 4 were detected in 1, 3, 181, and 487 samples, respectively. Adenovirus epidemics were observed during each year of this study. Based on these findings, adenovirus vaccination should be considered for military conscripts in the Finnish Defence Forces. J. Med. Virol. 88:571–577, 2016.

Comparison of four commercial DNA extraction kits for the recovery of Bacillus spp. spore DNA from spiked powder samples

Bacillus spp. include human pathogens such as Bacillus anthracis, the causative agent of anthrax and a biothreat agent. Bacillus spp. form spores that are physically highly resistant and may remain active over sample handling. We tested four commercial DNA extraction kits (QIAamp DNA Mini Kit, RTP Pathogen Kit, ZR Fungal/Bacterial DNA MiniPrep, and genesig Easy DNA/RNA Extraction kit) for sample inactivation and DNA recovery from two powders (icing sugar and potato flour) spiked with Bacillus thuringiensis spores. The DNA was analysed using a B. thuringiensis-specific real-time PCR assay. The detection limit was 3×10(1)CFU of spiked B. thuringiensis spores with the QIAamp DNA Mini, RTP Pathogen, and genesig Easy DNA/RNA Extraction kits, and 3×10(3)CFU with the ZR Fungal/Bacterial DNA MiniPrep kit. The results showed that manual extraction kits are effective and safe for fast and easy DNA extraction from powder samples even in field conditions. Adding a DNA filtration step to the extraction protocol ensures the removal of Bacillus spp. spores from DNA samples without affecting sensitivity.