High infection rate of zoonotic Eucoleus aerophilus infection in foxes from Serbia

The respiratory capillariid nematode Eucoleus aerophilus (Creplin, 1839) infects wild and domestic carnivores and, occasionally, humans. Thus far, a dozen of human infections have been published in the literature but it cannot be ruled out that lung capillariosis is underdiagnosed in human medicine. Also, the apparent spreading of E. aerophilus in different geographic areas spurs new studies on the epidemiology of this nematode. After the recognition of the first human case of E. aerophilus infection in Serbia, there is a significant merit in enhancing knowledge on the distribution of the nematode. In the present work the infection rate of pulmonary capillariosis was investigated in 70 red foxes (Vulpes vulpes) from the northern part of Serbia by autopsy. The estimated infection rate with Eucoleus aerophilus was 84%. In contrast, by copromicroscopic examination only 38% of foxes were positive. In addition, 10 foxes were investigated for the closely related species in nasal cavity, Eucoleus boehmi, and nine were positive. Our study demonstrates one of the highest infection rates of pulmonary capillariosis in foxes over the world.


Introduction
Nematodes of the family Capillariidae Neveu-Lemaire, 1936 are a very large group with not yet completely accepted taxonomy [16,32]. Among species ranked in this family, Eucoleus aerophilus (Creplin, 1839) Dujardin, 1845 (syn. Capillaria aerophila or Thominx aerophilus) affects trachea and main bronchi of canids, felids and some omnivorous animals [19]. Although several morphological descriptions have been provided for different biological stages of E. aerophilus [3,5,18,23,32], several other aspects of this nematode are still poorly known. Indeed, this is the case of geographic distribution, clinical significance and actual zoonotic potential [31]. Also, knowledge on the biological cycle of this nematode is scanty and, for instance, the route of transmission to vertebrate hosts remains to be understood. In general it is thought that animals become infected by ingesting environmental larvated eggs [31] but it has been also hypothesized, but never demonstrated, that larval E. aerophilus might require the passage through earthworms to become infective for cats and foxes (Borovkova, 1947 cited in [18] In the past few years some evidences have suggested that E. aerophilus is spreading in several geographic areas and that there is an increase of cross-infections between wild and domestic animals [7-9, 30, 31]. Indeed, it has been preliminarily shown that some E. aerophilus populations are shared between domestic pets and wildlife, e.g. red fox (Vulpes vulpes) and beech-marten (Martes foina (Erxleben) [9]).
Such changes are a likely effect of the increase of fox populations in peri-urban and urban areas and of movements of wild and companion animals around regions. In fact, an increase of the red fox population has been documented in Europe after the success of the oral vaccination against rabies [4,33]. The role of the red fox as reservoir for parasites may be now of relevance especially in suburban areas, e.g. for multilocular echinococcosis and other parasitic diseases of veterinary (e.g. angiostrongylosis) and human (e.g. trichinellosis) concern [11,15,21,22,31].
With regard to Serbia, E. aerophilus is circulating in cats and foxes [12,13] and, interestingly, the last case of human capillariosis documented in the literature is from the same country. More specifically, an infected woman showed respiratory symptoms miming bronchial carcinoma and blood eosinophilia [12]. Considering all the aforementioned background, especially the potential risk for human health which causes new interest in capillariid nematodes, the aim of this work was to investigate the infection rate of E. aerophilus in red foxes of Serbia.

Study area
In winter season from 2008 to the end of 2011, foxes (n = 118) hunted in the framework of a rabies control campaign were collected from the whole territory (21,506 square km) of the Vojvodina province of Serbia (45°15 N, 19°50 0 E). This province is crossed by the Danube river, two tributaries (Tisza and Sava) and many canals of the Danube-Tisza-Danube system (Pannonian Basin) on the left side and the Fruska Gora mountain on the right. The climate is continental.

Sampling
All foxes were adults with a body weight ranging from 4.0 to 8.1 kg. Carcasses were opened and the tracheas of 70 foxes, from the larynx to the main bronchi bifurcation, were collected and preserved in 30% ethanol. Tracheas were then opened on the anterior side by scissors and parasites retrieved were collected under a stereomicroscope and wet-mounted in glycerine-ethanol or lactophenol. The nasal cavity of 10 foxes was opened through the palate, and the nasal mucosa was scrapped and conserved in 30% ethanol. Faecal samples were collected during the necropsy of the 118 foxes and tested by the glycerine flotation method, respectively. Parasites collected were counted, photographed by a Leica microscope and measured by the ''ImageJ'' free program (http://imagej.nih.gov/ij/). Adult stages of nematodes recovered from trachea and bronchi of the foxes were identified at the species level by morphological keys [17,23]. Five adult female and three male E. aerophilus voucher specimens were deposited in the Zoological collection of the Department of Biology and Ecology, University of Novi Sad, Serbia (Registration No. NEM-0037).

Results
Adults of E. aerophilus (i.e. total of 817) were detected in 59 foxes (84%), with an average worm burden of 14 (range 1-71) (Figures 1 and 2). Out of 70 examined foxes, 32% harboured more than 14 adult worms of E. aerophilus with no relevant difference in parasitic burden between foxes collected from different mountainous regions or the Pannonian basin. The sex was assessed for 390 out of 817 examined worms because some E. aerophilus specimens were damaged during the collection. The sex ratio between male and female worms was 1:2.56.
The results of the copromicroscopic examinations on faecal samples collected at the necropsy are reported in Table 1.

Discussion
The infection rate of E. aerophilus infection (84%) herein detected in foxes from the Pannonian and the Fruska Gora Mountain regions (Vojvodina province) is indeed high. Interestingly, along with the results (88%) recently obtained from foxes of Norway [6], this study demonstrated the highest infection rate of pulmonary capillariosis in wild canids in Europe.
In the Zagreb region (Croatia) which shows a similar landscape to the central part of Serbia, about 300 km on the West, the prevalence of E. aerophilus in foxes was only 4.7% with a highest worm burden of five worms [20]. In Italy, the infection rate of E. aerophilus detected in the trachea of foxes was 7.0% in the Tuscany region [14]. In the Netherlands and Hungary E. aerophilus was found in 46.8% and 66% of dissected foxes [1,27]. An infection rate by ''Capillaria spp.'' of 22.4% was found in foxes from Slovakia by copromicroscopic investigation [15].
Albeit the high prevalence of E. aerophilus in foxes from Serbia could be due to the high sensitivity of the diagnostic methods used, most of the aforementioned studies have relied on trachea dissection as well. A great discrepancy between copromicroscopic and necroscopical findings of capillariids in the present study demonstrates that copromicroscopy may fail in terms of diagnostic sensitivity for this infection in foxes. Therefore, factors other than the diagnostic approach should be implicated in influencing such a high infection rate by lung capillariosis in foxes from Serbia.
The high humidity of the habitat of Vojvodina, the South part of Pannonian Basin, with many canals between the Danube and Tisza rivers, and many small tributaries of the Sava river in the Fruska Gora Mountain could favour parasite's dissemination. In this habitat, the fox density is of over 0.2 per square km and the high humidity can favour earthworms' dispersion. Also, competitive pressure and other diseases (e.g. sarcoptic mange) can enhance the susceptibility of foxes to nematodes [24] and the increase of reports of capillariosis in companion animals suggests that E. aerophilus is spreading in several areas. Epidemiological data (e.g. range of hosts and geographic distribution) of E. aerophilus in Europe are poorly known, thus at the moment it is difficult to assess to what degree this parasite may be spreading or what influence global warming or other factors may have on the current distribution [31]. On the other hand, it is known that wild foxes act as reservoirs and amplifiers of several canid nematodes, thus they can re-enforce    23 20 environmental contamination and risk of infection for domestic dogs and humans as well [29]. There is still not an agreement of the taxonomy of capillariid nematodes. Key morphological characters (i.e. eggs, oesophageal structure and genital organs of adult stages) allow the identification of E. aerophilus but, sometimes its identification at the species level and the differentiation from E. boehmi may be difficult.
While Christenson [5] did not detect spicule and spicule sheath in male E. aerophilus, more recent line drawings of this very characteristic organ were published by Butterworth and Beverley-Burton [3], Moravec [17] and Romashov [23]. The present work confirms the presence of these morphological features, which is of key diagnostic relevance for the identification at the species level of male E. aerophilus.
The stichocyte number, higher in E. aerophilus than in E. boehmi, can help to distinguish these two species between them, even though in some cases, the number of stichocytes may overlap. In fact in the present study, one E. boehmi female showed 35 stichocytes as an E. aerophilus female. According to Moravec [17], E. boehmi and E. aerophilus females have 30-32 and 42-46 stichocytes, respectively, whereas, according to Romashov [23], the number of stichocytes in the female is 32-36 for E. boehmi and 35-49 for E. aerophilus. Greater variations of the stichocyte number were reported in E. aerophilus males, i.e. 42-55 [23], or 43-50 [17]. The present study revealed that the number of stichocytes may be useful in the identification of Eucoleus spp. but it is not absolute criteria. The localization of adult stages could also greatly help in distinguishing between E. aerophilus (in trachea and bronchi) and E. boehmi (in nasal cavities).
Another issue warranting further investigations is the size of the eggs. While some textbooks report a length of up to 83 lm for E. aerophilus eggs, e.g. Soulsby [26], Bowman et al. [2], Taylor et al. [28], recent studies have demonstrated that the length of E. aerophilus eggs from dogs and cats is unwaveringly less than 70 lm [7,8,32]. Interestingly, some ova collected from the uterus of adult E. aerophilus in the present study were found to be longer and this could be from different reasons. It could be that eggs undergo further development and modifications when they are passed with the faeces, or the existence of different morphotypes within the same species E. aerophilus and with different host affiliations could be hypothesized. Recent molecular studies have shown that different haplotypes of E. aerophilus may be either shared between domestic and wild carnivores or are affiliated to specific hosts [9]. Further studies are necessary for evaluating the distribution of E. aerophilus in wildlife and pets cohabiting the same geographic areas in order to elucidate the phylogeography of different parasite populations and for verifying the possible existence of different morphotypes [8,9].
Under a clinical standpoint, the pathogenic role of E. aerophilus in wildlife is not well recognized because it is ranging from low to high and E. aerophilus has been also considered as an agent of massive mortality in farmed silver foxes [25]. In the present work, a very low inflammatory response was found around the worms (data not shown) which can be the result of a low pathogenicity or an immunosuppression in the mucosa caused by the parasite in these animals. A past work carried out in six experimentally infected foxes showed that animals may develop bronchopneumonia with cough and other signs of heavy respiratory infection, till death in some cases (Borovkova, cited in [25]). In pets, this parasite may cause bronchovesicular sounds, respiratory inflammation, sneezing, wheezing, chronic moist or dry cough, (broncho)-pneumonia respiratory failure and heavy parasite burdens may lead to mortality [10,31]. More studies are also necessary to evaluate the actual role of E. aerophilus in causing undiagnosed respiratory distresses in companion dogs and cats.
Another topic which needs to be better elucidated is the actual role of E. aerophilus in causing lung diseases in humans. Indeed, only 12 cases of infection have been published in the literature [13], but it is likely that the infection is underdiagnosed because the clinical signs may overlap a plethora of respiratory diseases which may be self-limiting or may resolve after non-specific treatments.