INTRODUCTION
Hunters, who spend time in forested and wild areas, may have contact with many causative agents of zoonoses through exposure to ticks, dead rodents, and birds, as well as excreta/secretions of wild animals or contaminated food, water, and soil1. Hence, this population is at sig- nificant risk of contracting infection with different patho- gens like Borrelia burgdorferi sensu lato (s.l.), Anaplasma phagocytophilum, Babesia spp., tick-borne encephalitis virus, Bartonella spp., Francisella tularensis, Echinococ- cus spp., or hantaviruses2, which are etiologic agents of diseases associated with severe health and socioeconomic consequences1.
Ticks are important vectors of zoonoses, among which Lyme borreliosis is the most widespread zoonotic dis- ease in the northern hemisphere. Spirochaetes from the
B. burgdorferi s.l. complex cause over 10,000 cases of Lyme borreliosis per year in Poland [12,759 cases in 2013, incidence per 100,000 (CI: 33.1); and 13,875 cases in 2014 (CI: 36)]3. The infection risk depends on the density of vectors, contribution of infected ticks in the local tick population, and the number of animals that are hosts for the vectors and reservoirs of the bacteria. The disease is a significant epidemiological problem due to its long-last- ing asymptomatic course, multisystem involvement, and serious consequences of untreated infection1.
Another important tick-borne disease is human granu- locytic anaplasmosis caused by the bacterium A.
phagocytophilum, which is an obligate parasite of neu- trophil granulocytes. The disease manifestations in hu- mans are not specific (influenza-like illness symptoms such as headache, fever, and muscle pains). The course of the disease is usually mild and self-limiting or even
Serological surveillance of vector-borne and zoonotic diseases among hunters in eastern Poland
Malgorzata Tokarska-Rodak
1, Dorota Plewik
2, Aleksander J. Michalski
3, Marcin Kolodziej
3, Alicja Melgies
3, Anna Panczuk
4, Henryk Konon
1& Marcin Niemcewicz
31Department of Public Health, Institute of Health Science, 2Innovation Research Centre, Pope John Paul II State School of Higher Education in Biala Podlaska; 3Military Institute of Hygiene and Epidemiology, Biological Threats Identification and Countermeasure Center, Pulawy;
4Department of Physiotherapy, Institute of Physical Education and Physiotherapy, Pope John Paul II State School of Higher Education in Biala Podlaska, Poland
ABSTRACT
Background & objectives: Many etiological agents of zoonoses are considered as significant biological hazard to people visiting forested areas frequently, for instance, hunters. They may be exposed to ticks, rodents, and birds as well as excreta/secretions of wild animals or contaminated water and soil. Hence, this population is at risk of contracting infection with pathogens such as Borrelia burgdorferi sensu lato (s.l.), Anaplasma phagocytophilum, Babesia spp., tick-borne encephalitis virus, Bartonella spp., Francisella tularensis, Echinococcus spp., or hantaviruses. The aim of the study was to assess the seroprevalence of zoonotic agents, viz. A. phagocytophilum, hantaviruses, and Echinococcus spp., with special regard to B. burgdorferi s.l., among hunters in Lubelskie Voivodeship (eastern Poland).
Methods: Serum samples collected from 134 hunters from Lubelskie Voivodeship were analyzed with the use of immunological techniques (enzyme-linked immunosorbent assay, line immunoblot test, and indirect fluorescence assay) for the presence of antibodies against the agents.
Results: Specific antibodies were detected in 66% of the tested individuals. Antibodies against B. burgdorferi s.l.
(39%), A. phagocytophilum (30%), hantaviruses (9%), and Echinococcus spp. (8%) were detected individually or as mixed results.
Interpretation & conclusion: The results confirm that there is a risk of exposure to different pathogens in the forested areas in eastern Poland and that hunters are highly vulnerable to infection with the examined zoonotic agents. A significant proportion of co-occurring antibodies against different pathogens was noticed. Thus, hunters have to take special care of their health status evaluation and mitigate the exposure risk by using adequate prophylaxis measures.
Key words Anaplasma phagocytophilum; Borrelia burgdorferi s.l.; eastern Poland; Echinococcus spp.; hantaviruses; hunters;
seroprevalence
asymptomatic, thus specific diagnosis is rare and the num- ber of reported cases is limited. In rare severe cases, liver malfunction, respiratory failure, and neurological com- plications may develop2.
Hantaviruses from the family Bunyaviridae are caus- ative agents of rodent-borne diseases that are newly re- ported in Poland3. European species can cause infections with a varied course: from completely asymptomatic, through mild to severe, including a life-threatening course.
They are carried by a specific asymptomatically infected rodent host and transmitted to humans through aerosolized excreta of infected rodents, direct contact, or bites4. The endemic focus of hantavirus diseases has recently been described in southeastern Poland5 and the occurrence of hantaviruses in local wild rodents has also been con- firmed6, but the viruses have been found in other regions of Poland as well7–8.
Another important disease associated with the forest environment is echinococcosis caused by parasites from the genus Echinococcus. In Europe, Echinococcus granulosus, E. ortleppi, E. canadensis, and E. interme- dius species cause hydatid echinococcosis and E.
multilocularis causes alveolar echinococcosis9-10. The disease is reported rather rarely (39 cases in 2013, CI:
0.10; 48 cases in 2014, CI: 0.12)3. These parasites circu- late among the intermediate hosts (sheep, cattle, moose, camels, pigs, etc) and definitive hosts (dogs, wolves, foxes, raccoon dogs, and other canidae). Infections of humans, who are rare intermediate hosts, are generally food-borne10. The symptoms usually occur after a few years affecting different organs—Liver, brain, lungs, or other organs. Moreover, in the case of alveolar echino- coccosis, the infection symptoms are similar to those re- lated to malignant liver tumor development11.
All these pathogens may develop apparent, clinically distinguishable symptoms, but cases with a mild course may be neglected and not reported. Hunters seem to be an underexamined population, which has not been stud- ied in Poland so far. This social group is heterogeneous in terms of social provenance, experience in hunting, and average time spent in the forest environment. The aim of the study was to analyze the occurrence of specific anti- bodies against selected pathogens among hunters, who are at risk to be exposed. The results may reveal the scale of contact with these pathogens and emphasize the ne- cessity of conducting regular individual check ups of their health status and applying appropriate prophylaxis and mitigation measures.
Laboratory diagnosis of Lyme borreliosis is a com- plex process, which basically comprises serological de-
tection of various anti-B. burgdorferi s.l. antibodies. An additional aim of the study was to assess the usefulness of different bacterial antigens in diagnostic tests.
MATERIAL & METHODS Study group
Blood samples were collected in Lubelskie Voivodeship, eastern Poland from October 2014 to April 2015. Subsequently, serum was obtained for further analy- sis. The study group consisted of 134 hunters aged be- tween 23 and 80 yr (average age 53 yr, SD 11.4), which included 124 men (93%) and 10 women (7%).
Serological analysis
All serological assays were carried out and results were interpreted according to the manufacturers’ instruc- tions. The ELISA test (anti-Borrelia ELISA IgM and anti- Borrelia plus VlsE ELISA IgG, Euroimmun, Germany) was applied for general screening of anti-B. burgdorferi s.l. IgM and IgG. The wells were coated with mixed anti- gens of B. burgdorferi s.s., B. afzelii, and B. garinii and recombinant protein VlsE. Results above 22 relative units/
ml (RU/ml) were considered as positive, below 16 RU/
ml as negative, and between 16 and 22 RU/ml as border- line.
Western blot (Wb) (anti-Borrelia Euroline-WB IgM/
IgG; Euroimmun, Germany) was used for confirmatory diagnosis of Lyme borreliosis for the positive and bor- derline results. The test stripes comprised immobilized antigens of B. afzelii (p83, p41, p39/BmpA, p31/OspA, p30, p25/OspC, p21, p19 and p17/DbpA) as well as a chip with recombinant antigen VlsE. EuroLinescan (Euroimmun, Germany) software was used for readout of the results. Additionally, antibodies against an extended pool of B. burgdorferi antigens (BBA36, BBO323, CRASP3 and pG) were analyzed by Borrelia Line IgG Line Immunoblot (Sekisui Virotech, USA).
The presence of anti-Echinococcus IgG was detected by ELISA (NovaLisa Echinococcus IgG, NovaTec Immunodiagnostica, Germany). Microtiter wells were coated with Echinococcus antigens. Samples were con- sidered as positive, if the absorbance value was higher than 10% over the cut-off or as negative if the absorbance value was lower than 10% over the cut-off. Samples with an absorbance value of 10% above or below the cut-off were interpreted as borderline.
The samples were tested for the presence of anti-A.
phagocytophilum IgG by immunofluorescence antibody (IFA) test (A. phagocytophilum IFA IgG antibody kit,
Fuller Laboratories, USA). Solid-phase antigens contain- ing fixed THP-1 cells were infected with A.
phagocytophilum. Patients’ sera were diluted to 1:80. A positive reaction was seen as sharply defined green fluo- rescent inclusions in the cytoplasm of infected cells. The fluorescence intensity of the positive control diluted to 1:640 was considered as the cut-off value.
The samples were also tested for the presence of anti- hantavirus IgG against Hantaan virus (HNTV), Puumala virus (PUUV), Dobrava-Belgrade virus (DOBV), Seoul virus (SEOV), and Saaremaa virus (SAAV) by IFA (IIFT Hantavirus Mosaic 2: Eurasia IgG, Euroimmun, Ger- many). Based on the intensity of fluorescence, the scores were considered as positive (++, +), borderline (±) or negative (–). Positive and borderline samples were sub- sequently tested by qualitative ELISA (anti-Hantavirus Pool 1 “Eurasia” ELISA IgG, Euroimmun, Germany) for HNTV, DOBV, and PUUV. Wells were coated with re- combinant viral antigens. Results >1.1 were considered as positive, <0.8 were negative, and results between 0.8 and 1.1 as borderline.
The Bioethical Committee of Medical University of Lublin authorized the project (permission No. KE-0254/
177/2014). Free and informed consent was obtained from the participants. The data obtained were analyzed statis- tically using Statistica v.10 software.
RESULTS
General screening: The screening revealed that there were 89 (66%) seropositive samples (84 men and 5 women, average age 54 yr, SD 12) out of the 134 exam- ined hunters while 45 (34%) patients from the study group (40 men and 5 women, average age 54 yr, SD 9.2) were negative.
The B. burgdorferi screening ELISA test revealed 51 (38%) negative samples for anti-B. burgdorferi s.l.
IgG, whereas 83 (62%) samples were positive or border- line. The results for IgM were as follows: 119 negative (89%) and 15 borderline or positive samples (11%). The Wb test, which was performed for the positive and bor- derline samples, showed positive or borderline results for 51 (38%) of the IgG samples and 3 (2%) of the IgM samples. The detailed results about the classes of the de- tected antibodies are presented in Figs. 1 and 2.
The anti-B. burgdorferi s.l. IgM antibodies detected by the Wb test were against p25/OspC, whereas the anti- B. burgdorferi s.l. IgG antibodies found in the examined samples were against various proteins. For four samples (8% of the positive or borderline samples), positive re- sults were determined on the basis of the presence of only
anti-VlsE IgG, whereas the other 32 (60%) positive se- rum samples contained IgG against two or more antigens.
The most common IgGs were as follows: anti-p17 (26 samples, 50% of the positive or borderline IgG samples), anti-VlsE (21 samples, 40%) anti-p19 (19 samples, 37%), anti-p83 (18 samples, 35%), and anti-p39 (14 samples, 27%). The presence of IgG for all the antigens from the extended pool (BBA36, BBO323, Crasp3 and pG) was confirmed for 16 samples (30%) by an additional Wb test;
however, all of these samples were previously determined as positive in the first test.
Antibodies (IgG), only against A. phagocytophilum were detected in 21 samples (16%) and while 19 (14%) samples were detected as mixed results. Anti-hantavirus antibodies (IgG) were detected in 44 (33%) samples by IFA (positive or borderline results), but only 9 (7%) and 3 (2%) of them were confirmed by ELISA as positive and as borderline results, respectively. There were eight single results and four mixed results. Anti-Echinococcus
Fig. 1:The analyses of antibodies anti-B. burgdorferi s.l. – ELISA and Wb results (Figures in parentheses indicate percentages).
Fig. 2:Classes of detected antibodies anti-B. burgdorferi s.l. (Figures in parentheses indicate percentages).
The presented results of the investigations conducted on the hunters’ population in Poland can be useful for assessment of the risk posed to them.
The risk of exposure to pathogens transmitted by ticks, e.g. B. burgdorferi s.l. or A. phagocytophilum, depends on the density of population of infected ticks and their hosts. The presence of anti-B. burgdorferi s.l. antibodies among forestry workers has frequently been studied. The antibodies were detected among 5–23% of forestry work- ers in Italy, 14–20% in France, 8–43% in Germany, 24%
in Slovenia, 9% in Romania, 11% in Turkey, and 37% in Hungary. In turn, this value was as high as 20–60% of examined forestry workers in Poland1. The results of the study (40% of seropositive individuals among the exam- ined hunters) seem to be coherent with results obtained from farmers, i.e. another exposed group in Lubelskie Voivodeship (38.5%); however, the results are substan- tially higher than those in the less exposed populations, e.g. blood donors (12.5%) from the region13. Thus, the study reconfirmed that occupational or environmental conditions have an impact on exposure to pathogens and human infections.
Recommendations for laboratory medical diagnosis of Lyme borreliosis issued by the National Chamber of Medical Diagnostic in Poland comprise two-stage tests:
quantitative ELISA (II or III generation) and subsequent qualitative Wb for both classes of antibodies (IgM–early phase or IgG–late phase). In the case of borderline re- sults, the tests have to be repeated in near future14. Posi- tive laboratory results are required for notification of a confirmed case of Lyme borreliosis in the late phase;
however, laboratory findings only are not sufficient to start borreliosis treatment—they have to be supplemented with the clinical picture of the disease14-15. In this study, 43 of the 98 ELISA positive or borderline samples (44%) were not confirmed by the Wb tests. Other researchers have also reported that results of ELISA assays were not confirmed by the Wb test in 68% of the clinically sus- pected cases16. The double stage approach seems to im- prove the disease diagnosis and limit false positive re- sults, which leads to application of unnecessary antibiotic therapy.
The European Union Concerted Action on Lyme Borreliosis (EUCALB) analysis has revealed the useful- ness of the following antigens in laboratory diagnosis of B. burgdorferi s.l.: OspC and p41 for IgM and p83/100, p58, p41, p39, OspC, p17/DbpA, and VlsE for IgG15. They are important markers of immunological response in a specific disease phase. For example, anti-OspC IgM (with lack of IgG) is significant in the diagnosis of the early stage of Lyme borreliosis with manifestation of promi-
Fig. 3:Results of antibody prevalence for investigated zoonotic infections, Lubelskie Voivodeship, 2014–15 (Figures in parentheses indicate percentages).
Fig. 4:Results of antibody co-prevalence for investigated zoonotic infections.
antibodies (IgG) were detected in 11 (8%) samples at a borderline level. There were four single results and six mixed results. The occurrence of antibodies against single pathogens in the analyzed serum samples, and mixed re- sults are presented in Fig. 3. Antibodies against B.
burgdorferi and A. phagocytophilum were detected in 14 samples (10%). Further, there were 7 (5%) and 3 (2%) samples with concomitant antibodies against two and three examined pathogens, respectively. The detailed mixed results are presented in Fig. 4.
DISCUSSION
The hunters’ population in Poland has not been a sub- ject of serosurveillance studies in terms of immunologi- cal response against zoonotic pathogens. In contrast, spe- cific serosurveillance studies among hunters have been performed in some countries, e.g. in Austria, antibodies against B. burgdorferi s.l., Ehrlichia spp., Brucella abor- tus, Leptospira interrogans, F. tularensis, Puumala vi- rus, E. multilocularis, E. granulosus, and Toxocara spp., encephalomyocarditis virus were detected12.
nent clinical symptoms. Only one such sample was found in this study. On the other hand, in the progress of immu- nological response, the presence of IgG against other an- tigens is more important, e.g. p83, VlsE, p17/DbpA or p39. Indeed, IgG antibodies against p17 (50%), VlsE (40%), p19 (37%), p83 (35%), and p39 (27%) were re- markably detected and thus their diagnostic usefulness was confirmed. Hofmann et al17 studied late Lyme borreliosis and reported higher prevalence of antibodies against BBO323 (90%), BBA36 (67%) and Crasp3 (38%).
A pool of in vivo synthesized antigens—BBA36, BBO323, Crasp3 and pG18 were also used in this study.
Positive results (anti-BBA36 in 23%, anti-Crasp3 in 13%, and anti-BBO323 in 10%) were reported only for samples that were positive for the basic panel of antigens. There- fore, the application of additional antigens did not pro- vide an additional value to the results of study.
The seroprevalence of antibodies against A.
phagocytophilum has been studied in Poland previously on various populations and regions. Surprisingly, the re- sults were quite divergent, i.e. 3.5–4.9% among healthy adults in the northeastern regions of Poland19, 9.4% among blood donors in Lubelskie Voivodeship13, and 3.9%
among foresters in Podlaskie20, 12% in Lubelskie Voivodeship13, and as high as 29.6% in eastern Poland21. In this study 30% of the hunters, confirmed the higher prevalence in the study area. Similarly, divergent results were reported in Greece, i.e. 7.3% prevalence of antibod- ies against A. phagocytophilum was reported among healthy blood donors in Greek Macedonia22, and 21.4%
among inhabitants of Crete23. Additionally, the higher prevalence in the hunter population compared to the blood donors13, 22 may indicate that environmental exposure associated with forest seems to be a crucial factor for in- fection with this pathogen.
Hantaviruses are newly emerging pathogens in Po- land related to a wild rodents’ reservoir5. Detailed research done in Subcarpathia between 2004 and 2011 among hu- man population and animal reservoir proved that this area was endemic for hantavirus diseases (HVD)5–6. The oc- currence of HVD has been reported for a few years by the National Institute of Public Health in Warsaw (13 cases in 2007, 7 in 2008, 5 in 2009, 6 in 2010, 8 in 2011, 3 in 2012, 8 in 2013, 54 in 2014, and 6 in 2015). Therefore, hantaviruses need to be monitored in the country3. How- ever, only selected populations have been studied so far in Poland. The seropositivity among forestry workers in Lubelskie Voivodeship was reported to range from 1.9 to 6.5%7. The overall seroprevalence of hantaviruses in an- other European country—Latvia rates 4.2%24. The results of this study (9%) are coherent with these data and sup-
port the thesis that hantaviruses are an important envi- ronmental threat to social groups associated with the for- est environment in Poland.
It was assumed that samples should be examined by more than one method due to possibility of false positive results25. Indeed, in this study, 44 positive samples were determined by IFA but only 12 of them were confirmed by ELISA. Moreover, a seroneutralization assay is re- quired for final confirmation of the etiological agent spe- cies, due to significant cross-reactivity among antibodies raised against various hantavirus species.
According to data from the European Food Safety Authority (EFSA), 811 echinococcosis cases in total were reported in the EU in 2013, including 39 cases in Poland.
Echinococcosis is a rare disease and the epidemiological situation in Poland is stable. However, the increasing number of cases of alveolar echinococcosis across Eu- rope is alarming due to the more severe course of the infection and worse prognosis26. The research conducted among Austrian hunters has shown 5 and 11% seroposi- tivity for E. multilocularis and E. granulosus, respec- tively12. Zukiewicz-Sobczak et al21 estimated seroposi- tivity for E. granulosus among Polish foresters at 3.2%.
The present study results revealed a comparable preva- lence of anti-Echinococcus antibodies, i.e. 11 samples (8%) were determined as borderline results. Kern et al27 reported that gun dogs actively participating in hunting are the most important risk factor for occurrence of multilocular echinococcosis. The animals roaming around the forest may be infected as a definite host and thus they might become a source of infections for intermediate hosts—hunters and their families27.
This study has confirmed the co-presence of antibod- ies against the studied pathogens. This phenomenon might be a result of infections that occurred in the past. The study reported 21 (16%) samples containing antibodies against two agents, mainly B. burgdorferi s.l. and A.
phagocytophilum, as well as three samples with antibod- ies against additional agents (hantaviruses or Echinococ- cus). This phenomenon has already been reported on other populations in Poland13, 28. The most prominent correla- tion was found between B. burgdorferi s.l. and A.
phagocytophilum in exposed populations (4.2% forest- ers and 2.6% farmers). An increased number of co-preva- lence was reported in 7% patients suspected of infection with B. burgdorferi s.l. from eastern Slovakia29. But re- sults of this study indicated greater co-prevalence (13%
of the analyzed samples) than that from the previous re- ports. This phenomenon may be important in the com- plex diagnosis of disease in the case of simultaneously ongoing infections. The clinical picture and course of
Lyme borreliosis in humans may be more difficult to in- terpret in the case of mixed infections with A.
phagocytophilum. The difficulty arises from the complex- ity of host immunological response in mixed infection, which can impede diagnostic process and treatment.
The results of the present study have proved that the studied population of hunters is a group with significant
“immunological history” of exposure to all the pathogens studied. These results are especially important given the fact that these zoonoses, i.e. Lyme borreliosis, HVD, ana- plasmosis, or echinococcosis may have an asymptomatic course throughout the disease or in certain phases only.
Thus, hunters have to take into account the exposure risk in the case of illnesses with non-specific symptoms or during periodical medical health status evaluations. More- over, they should respect safety rules and principles of behaviour. These include adequate prophylaxis measures (protective clothing, repellents), general hygiene precau- tions, especially when forest vegetation or wild meat are used for consumption, and appropriate veterinary care over gun dogs.
Research on the seroprevalence particularly on ex- posed populations is very useful to evaluate the risk re- lated to specific pathogens. These data may be used to localize regions at risk and identify emerging and re- emerging diseases. Jaenson et al30 suggest that increased number of cases of tick-borne diseases, including Lyme borreliosis, anaplasmosis, and others might be expected in palearctic countries due to climatic changes, i.e. milder winters, longer vegetation period, and increased number of hosts30. Climate changes also influence hantavirus in- fection—more food and shorter periods between mast years may increase the number of rodents and environ- mental exposure risk to humans6. Studies on emerging and re-emerging pathogens in Poland are indispensable for public health.
CONCLUSION
The study revealed that hunter population in Lubelskie Voivodeship is vulnerable to Lyme borreliosis, echino- coccosis, anaplasmosis, and HVD as evident by the sig- nificant co-occurrence of multiple antibodies against tick- borne diseases and other zoonoses on the studied population. The study provided arguments for improve- ment of the evaluation of hunters’ health status and pro- phylaxis and mitigation measures.
Diagnosis of Lyme borreliosis must be based on two- steps diagnostic schemes: The results of the ELISA, and Western blot assays. Application of antigen proteins like- BBA36, BBO323, Crasp3, and pG in the serological as-
say does not provide any additional value in the general serosurveillance studies.
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Correspondence to: Ms Dorota Plewik,Pope John Paul II State School of Higher Education in Biala Podlaska, Innovation Research Centre, Sidorska 105, 21-500 Biala Podlaska, Poland.
E-mail: [email protected]
Received: 27 April 2016 Accepted in revised form: 29 September 2016
