Tularemia From Veterinary Occupational Exposure.

Tularemia is a disease caused by Francisella tularensis, a highly infectious bacteria that can be transmitted to humans by direct contact with infected animals. Because of the potential for zoonotic transmission of F. tularensis, veterinary occupational risk is a concern. Here, we report on a human case of tularemia in a veterinarian after an accidental needlestick injury during abscess drainage in a sick dog. The veterinarian developed ulceroglandular tularemia requiring hospitalization but fully recovered after abscess drainage and a course of effective antibiotics. To systematically assess veterinary occupational transmission risk of F. tularensis, we conducted a survey of veterinary clinical staff after occupational exposure to animals with confirmed tularemia. We defined a high-risk exposure as direct contact to the infected animal's body fluids or potential aerosol inhalation without use of standard personal protective equipment (PPE). Survey data included information on 20 veterinary occupational exposures to animals with F. tularensis in 4 states. Veterinarians were the clinical staff most often exposed (40%), followed by veterinarian technicians and assistants (30% and 20%, respectively). Exposures to infected cats were most common (80%). Standard PPE was not used during 80% of exposures; a total of 7 exposures were categorized as high risk. Transmission of F. tularensis in the veterinary clinical setting is possible but overall risk is likely low. Veterinary clinical staff should use standard PPE and employ environmental precautions when handling sick animals to minimize risk of tularemia and other zoonotic infections; postexposure prophylaxis should be considered after high-risk exposures to animals with suspected or confirmed F. tularensis infection to prevent tularemia.

First European report of Francisella tularensis subsp. holarctica isolation from a domestic cat.

Francisella tularensis subsp. holarctica is a select agent causing life-threatening tularemia. It has been isolated from humans and animals, mainly lagomorphs and rodents, rarely other wild carnivore species. Increasing numbers of human tularemia cases have been reported during the last 5 years in Switzerland. Here we report the first isolation of Francisella tularensis subsp. holarctica from a domestic cat in Europe and compare its genome sequence with other Swiss isolates. The cat isolate shows a close phylogenetic relationship with a contemporary hare isolate from close geographic proximity, indicating a possible epidemiological link.

Francisella tularensis in Swedish predators and scavengers.

Tularaemia is a zoonotic disease, in Europe caused by Francisella tularensis subsp. holarctica. Many lagomorphs and a variety of small rodents are wildlife species prone to develop clinical disease, while predators and scavengers are relatively resistant and may serve as sentinels. Blood samples from 656 Swedish wild predators and scavengers were serologically investigated using slide agglutination and microagglutination. In the slide agglutination test, 34 seropositive animals were detected, and they were found among all species investigated: brown bear (Ursus arctos), Eurasian lynx (Lynx lynx), raccoon dog (Nyctereutes procyonoides), red fox (Vulpes vulpes), wild boar (Sus scrofa), wolf (Canis lupus) and wolverine (Gulo gulo). Due to haemolysis the microagglutination test was more difficult to read at low titres, and only 12 animals were classified as seropositive. F. tularensis subsp. holarctica was detected by a polymerase chain reaction in lymphatic tissues of the head in one brown bear, one red fox and one wolf. The significance of this finding regarding possible latency of infection is not clear. In conclusion, the results of this study indicate that all predator and scavenger species included in this study may serve as sentinels for tularaemia in Sweden. Their role as reservoirs is unclear.

Detection of Coxiella burnetii and Francisella tularensis in Tissues of Wild-living Animals and in Ticks of North-west Poland.

This work presents results of the research on the occurrence of Coxiella burnetii and Francisella tularensis in the tissues of wild-living animals and ticks collected from Drawsko County, West Pomeranian Voivodeship. The real-time PCR testing for the pathogens comprised 928 samples of animal internal organs and 1551 ticks. The presence of C. burnetii was detected in 3% of wild-living animals and in 0.45-3.45% (dependent on collection areas) of ticks. The genetic sequences of F. tularensis were present in 0.49 % of ticks (only in one location - Drawa) and were not detected in animal tissues. The results indicate respectively low proportion of animals and ticks infected with C. burnetii and F. tularensis .This work presents results of the research on the occurrence of Coxiella burnetii and Francisella tularensis in the tissues of wild-living animals and ticks collected from Drawsko County, West Pomeranian Voivodeship. The real-time PCR testing for the pathogens comprised 928 samples of animal internal organs and 1551 ticks. The presence of C. burnetii was detected in 3% of wild-living animals and in 0.45­3.45% (dependent on collection areas) of ticks. The genetic sequences of F. tularensis were present in 0.49 % of ticks (only in one location ­ Drawa) and were not detected in animal tissues. The results indicate respectively low proportion of animals and ticks infected with C. burnetii and F. tularensis.

Francisella tularensis ssp. holarctica in Ringtail Possums, Australia.

The occurrence of Francisella tularensis outside of endemic areas, such as North America and Eurasia, has been enigmatic. We report the metagenomic discovery and isolation of F. tularensis ssp. holarctica biovar japonica from diseased ringtail possums in Sydney, Australia. This finding confirms the presence of F. tularensis in the Southern Hemisphere.

Enhanced solid-phase recombinase polymerase amplification and electrochemical detection.

Recombinase polymerase amplification (RPA) is an elegant method for the rapid, isothermal amplification of nucleic acids. Here, we elucidate the optimal surface chemistry for rapid and efficient solid-phase RPA, which was fine-tuned in order to obtain a maximum signal-to-noise ratio, defining the optimal DNA probe density, probe-to-lateral spacer ratio (1:0, 1:1, 1:10 and 1:100) and length of a vertical spacer of the probe as well as investigating the effect of different types of lateral spacers. The use of different labelling strategies was also examined in order to reduce the number of steps required for the analysis, using biotin or horseradish peroxidase-labelled reverse primers. Optimisation of the amplification temperature used and the use of surface blocking agents were also pursued. The combination of these changes facilitated a significantly more rapid amplification and detection protocol, with a lowered limit of detection (LOD) of 1 · 10-15 M. The optimised protocol was applied to the detection of Francisella tularensis in real samples from hares and a clear correlation with PCR and qPCR results observed and the solid-phase RPA demonstrated to be capable of detecting 500 fM target DNA in real samples. Graphical abstract Relative size of thiolated lateral spacers tested versus the primer and the uvsx recombinase protein.

Harmonizing methods for wildlife abundance estimation and pathogen detection in Europe-a questionnaire survey on three selected host-pathogen combinations.

BACKGROUND: The need for wildlife health surveillance as part of disease control in wildlife, domestic animals and humans on the global level is widely recognized. However, the objectives, methods and intensity of existing wildlife health surveillance programs vary greatly among European countries, resulting in a patchwork of data that are difficult to merge and compare. This survey aimed at evaluating the need and potential for data harmonization in wildlife health in Europe. The specific objective was to collect information on methods currently used to estimate host abundance and pathogen prevalence. Questionnaires were designed to gather detailed information for three host-pathogen combinations: (1) wild boar and Aujeszky's disease virus, (2) red fox and Echinococcus multilocularis, and (3) common vole and Francisella tularensis. RESULTS: We received a total of 70 responses from 19 European countries. Regarding host abundance, hunting bags are currently the most widely accessible data source for widely distributed mid-sized and larger mammals such as red fox and wild boar, but we observed large differences in hunting strategies among countries as well as among different regions within countries. For small rodents, trapping is the method of choice, but practical applications vary among study sites. Laboratory procedures are already largely harmonized but information on the sampled animals is not systematically collected. CONCLUSIONS: The answers revealed that a large amount of information is available for the selected host-pathogen pairs and that in theory methods are already largely harmonized. However, the comparability of the data remains strongly compromised by local differences in the way, the methods are applied in practice. While these issues may easily be overcome for prevalence estimation, there is an urgent need to develop tools for the routine collection of host abundance data in a harmonized way. Wildlife health experts are encouraged to apply the harmonized APHAEA protocols in epidemiological studies in wildlife and to increase cooperation.

Environmental surveillance during an outbreak of tularaemia in hares, the Netherlands, 2015.

Tularaemia, a disease caused by the bacterium Francisella tularensis, is a re-emerging zoonosis in the Netherlands. After sporadic human and hare cases occurred in the period 2011 to 2014, a cluster of F. tularensis-infected hares was recognised in a region in the north of the Netherlands from February to May 2015. No human cases were identified, including after active case finding. Presence of F. tularensis was investigated in potential reservoirs and transmission routes, including common voles, arthropod vectors and surface waters. F. tularensis was not detected in common voles, mosquito larvae or adults, tabanids or ticks. However, the bacterium was detected in water and sediment samples collected in a limited geographical area where infected hares had also been found. These results demonstrate that water monitoring could provide valuable information regarding F. tularensis spread and persistence, and should be used in addition to disease surveillance in wildlife.

[EPIZOOTOLOGICAL AND EPIDEMIOLOGICAL CHARACTERISTICS OF TULAREMIA IN GEORGIA].

The appropriate data as well as archival materials about the spread of Tularemia disease have been studied over of period from 1991-2014 in Georgia in order to evaluate the current situation in Kartli lowland foci as well as the main reasons of disease agent circulation and maintenance in the study area. 54 (24.6%) out of 220 suspicious cases were laboratory confirmed during the study period. Most of confirmed cases - 32 (59.26%) have been detected in Shida Kartli focus among the old population during the outbreaks in the winter period. Tularemia causative agent has been isolated from the environmental object on 87 occasions, 50 (57.5%) of them have been found in Shida Kartli and 8 (9.2%) - in Kvemo Kartli lowland. Existence of rodents and ixodes ticks in Kartli lowland greatly supports keeping up Tularemia foci in this area. The common vectors and kind of storage of disease are considered to be different species of ixodes ticks especially D. marginatus, Haem. Punctata, Haem. Sulcata. It should be noted that reduction of intensity of Tularemia agent partially should be linked to the reduction frequency of planned and intentional researches during the last period of time.

High and novel genetic diversity of Francisella tularensis in Germany and indication of environmental persistence.

In Germany tularemia is a re-emerging zoonotic disease. Therefore, we investigated wild animals and environmental water samples for the presence and phylogenetic diversity of Francisella tularensis in the poorly studied Berlin/Brandenburg region. The phylogenomic analysis of three isolates from wild animals revealed three new subclades within the phylogenetic tree of F. tularensis [B.71 from a raccoon dog (Nyctereutes procyonoides); B.74 from a red fox (Vulpes vulpes), and B.75 from a Eurasian beaver (Castor fiber albicus)]. The results from histological, PCR, and genomic investigations on the dead beaver showed that the animal suffered from a systemic infection. Indications were found that the bacteria were released from the beaver carcass into the surrounding environment. We demonstrated unexpectedly high and novel phylogenetic diversity of F. tularensis in Germany and the fact that the bacteria persist in the environment for at least one climatic season. These findings support a broader host species diversity than previously known regarding Germany. Our data further support the assumption derived from previous serological studies of an underestimated frequency of occurrence of the pathogen in the environment and in wild animals. F. tularensis was isolated from animal species not previously reported as natural hosts in Germany.

Francisella Tularensis Clades B.FTN002-00 and B.13 Are Associated With Distinct Pathology in the European Brown Hare (Lepus europaeus).

Tularemia is a severe disease caused by Francisella tularensis This bacterium has a major pathogenic potential in countless animal species as well as in humans. Despite the relatively significant body of literature available on this microorganism, many questions are still open concerning its biological cycle in the environment, the pathology and pathogenesis of the disease, the possible routes of infection in animals, and the pathologic and ecological relevance of the distinct phylogenetic clusters of F. tularensis In order to address these questions, we have thoroughly characterized the pathology and microbiology of terminally ill European brown hares (Lepus europaeus) infected with F. tularensis subsp. holarctica, collected in Switzerland from 2012 to 2014. F tularensis isolates were typed by defining their phylogenetic clusters. We showed that the pathology associated with F. tularensis subsp. holarctica belonging to the clade B.FTNF002-00 is different from that previously reported to be associated with the clade B.13. In particular, strains of the clade B.FTNF002-00 were almost invariably associated with splenitis and hepatitis and not with the polyserositis affecting pleura, pericardium, and kidney reported in the literature for infections caused by the clade B.13. We describe findings suggesting that the ports of entry for the bacteria might be the respiratory and digestive routes.

Tularemia among free-ranging mice without infection of exposed humans, Switzerland, 2012.

The animals primarily infected by Francisella tularensis are rapidly consumed by scavengers, hindering ecologic investigation of the bacterium. We describe a 2012 natural tularemia epizootic among house mice in Switzerland and the assessment of infection of exposed humans. The humans were not infected, but the epizootic coincided with increased reports of human cases in the area.

The status of tularemia in Europe in a one-health context: a review.

The bacterium Francisella tularensis causes the vector-borne zoonotic disease tularemia, and may infect a wide range of hosts including invertebrates, mammals and birds. Transmission to humans occurs through contact with infected animals or contaminated environments, or through arthropod vectors. Tularemia has a broad geographical distribution, and there is evidence which suggests local emergence or re-emergence of this disease in Europe. This review was developed to provide an update on the geographical distribution of F. tularensis in humans, wildlife, domestic animals and vector species, to identify potential public health hazards, and to characterize the epidemiology of tularemia in Europe. Information was collated on cases in humans, domestic animals and wildlife, and on reports of detection of the bacterium in arthropod vectors, from 38 European countries for the period 1992-2012. Multiple international databases on human and animal health were consulted, as well as published reports in the literature. Tularemia is a disease of complex epidemiology that is challenging to understand and therefore to control. Many aspects of this disease remain poorly understood. Better understanding is needed of the epidemiological role of animal hosts, potential vectors, mechanisms of maintenance in the different ecosystems, and routes of transmission of the disease.

Live attenuated mutants of Francisella tularensis protect rabbits against aerosol challenge with a virulent type A strain.

Francisella tularensis, a Gram-negative bacterium, is the causative agent of tularemia. No licensed vaccine is currently available for protection against tularemia, although an attenuated strain, dubbed the live vaccine strain (LVS), is given to at-risk laboratory personnel as an investigational new drug (IND). In an effort to develop a vaccine that offers better protection, recombinant attenuated derivatives of a virulent type A strain, SCHU S4, were evaluated in New Zealand White (NZW) rabbits. Rabbits vaccinated via scarification with the three attenuated derivatives (SCHU S4 ΔguaBA, ΔaroD, and ΔfipB strains) or with LVS developed a mild fever, but no weight loss was detected. Twenty-one days after vaccination, all vaccinated rabbits were seropositive for IgG to F. tularensis lipopolysaccharide (LPS). Thirty days after vaccination, all rabbits were challenged with aerosolized SCHU S4 at doses ranging from 50 to 500 50% lethal doses (LD50). All rabbits developed fevers and weight loss after challenge, but the severity was greater for mock-vaccinated rabbits. The ΔguaBA and ΔaroD SCHU S4 derivatives provided partial protection against death (27 to 36%) and a prolonged time to death compared to results for the mock-vaccinated group. In contrast, LVS and the ΔfipB strain both prolonged the time to death, but there were no survivors from the challenge. This is the first demonstration of vaccine efficacy against aerosol challenge with virulent type A F. tularensis in a species other than a rodent since the original work with LVS in the 1960s. The ΔguaBA and ΔaroD SCHU S4 derivatives warrant further evaluation and consideration as potential vaccines for tularemia and for identification of immunological correlates of protection.

Seroprevalence, Blood Chemistry, and Patterns of Canine Parvovirus, Distemper Virus, Plague, and Tularemia in Free-Ranging Coyotes (Canis latrans) in Northern New Mexico, USA.

Wildlife diseases have implications for ecology, conservation, human health, and health of domestic animals. They may impact wildlife health and population dynamics. Exposure rates of coyotes (Canis latrans) to pathogens such as Yersinia pestis, the cause of plague, may reflect prevalence rates in both rodent prey and human populations. We captured coyotes in north-central New Mexico during 2005-2008 and collected blood samples for serologic surveys. We tested for antibodies against canine distemper virus (CDV, Canine morbillivirus), canine parvovirus (CPV, Carnivore protoparvovirus), plague, tularemia (Francisella tularensis), and for canine heartworm (Dirofilaria immitis) antigen. Serum biochemistry variables that fell outside reference ranges were probably related to capture stress. We detected antibodies to parvovirus in 32/32 samples (100%), and to Y. pestis in 26/31 (84%). More than half 19/32 (59%) had antibodies against CDV, and 5/31 (39%) had antibodies against F. tularensis. We did not detect any heartworm antigens (n = 9). Pathogen prevalence was similar between sexes and among the three coyote packs in the study area. Parvovirus exposure appeared to happen early in life, and prevalence of antibodies against CDV increased with increasing age class. Exposure to Y. pestis and F. tularensis occurred across all age classes. The high coyote seroprevalence rates observed for CPV, Y. pestis, and CDV may indicate high prevalence in sympatric vertebrate populations, with implications for regional wildlife conservation as well as risk to humans via zoonotic transmission.

German Francisella tularensis isolates from European brown hares (Lepus europaeus) reveal genetic and phenotypic diversity.

BACKGROUND: Tularemia is a zoonotic disease caused by Francisella tularensis that has been found in many different vertebrates. In Germany most human infections are caused by contact with infected European brown hares (Lepus europaeus). The aim of this study was to elucidate the epidemiology of tularemia in hares using phenotypic and genotypic characteristics of F. tularensis. RESULTS: Cultivation of F. tularensis subsp. holarctica bacteria from organ material was successful in 31 of 52 hares that had a positive PCR result targeting the Ft-M19 locus. 17 isolates were sensitive to erythromycin and 14 were resistant. Analysis of VNTR loci (Ft-M3, Ft-M6 and Ft-M24), INDELs (Ftind33, Ftind38, Ftind49, RD23) and SNPs (B.17, B.18, B.19, and B.20) was shown to be useful to investigate the genetic relatedness of Francisella strains in this set of strains. The 14 erythromycin resistant isolates were assigned to clade B.I, and 16 erythromycin sensitive isolates to clade B.IV and one isolate was found to belong to clade B.II. MALDI-TOF mass spectrometry (MS) was useful to discriminate strains to the subspecies level. CONCLUSIONS: F. tularensis seems to be a re-emerging pathogen in Germany. The pathogen can easily be identified using PCR assays. Isolates can also be identified within one hour using MALDI-TOF MS in laboratories where specific PCR assays are not established. Further analysis of strains requires genotyping tools. The results from this study indicate a geographical segregation of the phylogenetic clade B.I and B.IV, where B.I strains localize primarily within eastern Germany and B.IV strains within western Germany. This phylogeographical pattern coincides with the distribution of biovar I (erythromycin sensitive) and biovar II (erythromycin resistance) strains. When time and costs are limiting parameters small numbers of isolates can be analysed using PCR assays combined with DNA sequencing with a focus on genetic loci that are most likely discriminatory among strains found in a specific area. In perspective, whole genome data will have to be investigated especially when terrorist attack strains need to be tracked to their genetic and geographical sources.

Tularaemia seroprevalence of captured and wild animals in Germany: the fox (Vulpes vulpes) as a biological indicator.

A total of 2475 animals from Germany, both captive and wild, were tested for antibodies against Francisella tularensis to obtain more knowledge about the presence of this pathogen in Germany. An indirect and a competitive ELISA served as screening methods, positive and inconclusive samples were confirmed by Western blot. Of the zoo animals sampled between 1992 and 2007 (n = 1122), three (0·3%) were seropositive. The seroconversion of a hippopotamus in Berlin Zoo was documented. From 1353 serum samples of wild foxes (Vulpes vulpes), raccoon dogs (Nyctereutes procyonoides) and wild boars (Sus scrofa), collected between 2005 and 2009 in the federal state of Brandenburg (surrounding Berlin), a total of 101 (7·5%) tested positive for antibodies to F. tularensis lipopolysaccharide. Our results indicate a higher seroprevalence of F. tularensis in wildlife in eastern Germany than commonly assumed. Furthermore, we found foxes and raccoon dogs to be biological indicators for tularaemia.

Tularaemia in a brown hare (Lepus europaeus) in 2013: first case in the Netherlands in 60 years.

Tularaemia has not been reported in Dutch wildlife since 1953. To enhance detection, as of July 2011, brown hares (Lepus europaeus) submitted for postmortem examination in the context of non-targeted wildlife disease surveillance, were routinely tested for tularaemia by polymerase chain reaction (PCR). Francisella tularensis subspecies holarctica infection was confirmed in a hare submitted in May 2013. The case occurred in Limburg, near the site of the 1953 case. Further surveillance should clarify the significance of this finding.

[Monitoring the natural foci of tularemia on Wrangel Island].

Long-term annual monitoring of the natural foci of tularemia was first made on Wrangel Island. The objects of the investigation were pellets of birds-myophages, blood samples from rodents, and excrements from carnivorous mammals. A total of 2626 biological samples were examined in the period 2002 to 2011. A serological test was ascertained to be the most effective method for the detection of tularemia epizooties; polymerase chain reaction should be used as an additional technique to examine blood samples, as well as rodent tubular bone debris taken from the pellets. Tularemia epizooties were registered in the populations of two species of lemmings every year, except in 2003. An intensive diffuse tularemia epizooty was first detected in this area, which emerged in 2019, peaked by spring 2011, and covered most of the island. The antigen of tularemia pathogen was identified in 43.46% of the samples under examination,which is a high quantitative indicator of the intensity of an epizootic process. The fact that positive samples are annually found in the same areas of the island suggests that the causative agent is steadily and long preserved in the parasitic system. The availability of stable and active natural tularemia foci on Wrangel Island calls for preventive measures, particularly vaccination of risk groups coming to the island to conduct researches.