Introduction

Parasitic nematodes of the order Ascaridida are considered important for veterinary and human medicine worldwide, not only because of the long-term viability of their highly resistant eggs but also because of the somatic migration of their larvae in a wide host range. Ascaris, Baylisascaris and Toxocara species are considered the most common cause of clinical larva migrans in animals and humans, which can be associated with severe or fatal disease. Baylisascaris transfuga (Rudolphi, 1819) represents the most common ascarid species in Carpathian brown bears (Ursus arctos arctos, Linné, 1758) in Slovakia (Štrkolcová et al. 2018). Recently, the brown bear population in Central Europe has been recovering thanks to species conservation, and thus, B. transfuga can spread along with thriving of its host. B. transfuga appears to have primarily a direct life cycle, but can lead to larva migrans syndrome in accidental hosts. The risk of clinical disease from B. transfuga larva migrans in aberrant hosts is considered low (Sapp et al. 2017). To date, there are two recent reports from natural conditions in wild rodents (Bugmyrin and Spiridonov 2019) and moose (Hoberg et al. 2018), with further sporadic information from experiments in laboratory animals. B. transfuga larvae have been shown to be capable of migrating in mice, guinea pigs, Mongolian gerbils and chickens (Matoff and Komandarev 1965; Papini et al. 1993, 1996; Sato et al. 2004). In these studies, high doses of B. transfuga embryonated eggs (1000–5000 or up to 30,000) were used for experimental inoculations with different observations of the clinical condition and behaviour of the larvae in different hosts. Compared to related Baylisascaris species, especially the raccoon roundworm B. procyonis (Stefanski and Zarnowski, 1951), larva migrans of B. transfuga is generally regarded less pathogenic (Papini and Casarosa 1994; Sato et al. 2004), and no human case of larva migrans caused by this species has been reported so far (Bauer 2013).

The aim of the present study is to compare the outcomes of two infectious doses of B. transfuga eggs in laboratory mice and the development of larva migrans including clinical manifestation, and to evaluate the pathogenicity of the lower infectious dose for mice aberrant hosts.

Material and methods

Experimental inoculation

Adult B. transfuga nematodes were obtained from the small intestine of an adult male roadkill brown bear from Malá Fatra National Park (Lipovec locality), Slovakia and provided by the State Nature Conservancy of the Slovak Republic. Eggs were obtained from adult females by dissection of their uteri and incubated at laboratory temperature (21 °C) for 30 days to obtain embryonated eggs, which were microscopically examined to assess their viability and counted.

Thirty-five subadult laboratory mice (females, CD1 strain) were housed in animal facility at the Department of Pathological Morphology and Parasitology, University of Veterinary Sciences Brno in accordance with Czech legislation (Act No 246/1992 Coll., on the Protection of Animals from Cruelty). The mice were kept on commercial pelleted food and water ad libitum. All experimental procedures were performed in accordance with Protocol 10–2015 approved by UVS and Central Commission for Animal Welfare, Czech Republic. Mice were divided into 3 experimental groups. The first group included 15 mice; each mouse was inoculated with a dose of 200 embryonated eggs (ID 200). The second group consisted of 15 mice, each inoculated with 2 000 embryonated eggs (ID 2000). All mice were inoculated orally by gavage with purified and counted viable eggs of B. transfuga suspended in drinking water (0.2 ml/mouse). The third group of 5 mice served as uninfected negative control. All mice were checked daily for 8 weeks after inoculation by observation in their home cage (activity, observable abnormalities of general health and well-being) for the presence of clinical sings of the disease. Dead or euthanized mice were dissected and their organs [eyes, brain, heart, lungs, liver, jejunum, kidneys, spleen and skeletal muscles (triceps, quadriceps)] were processed for histopathological examination: fixed in 10% buffered formalin, embedded in paraffin, sectioned and stained with haematoxylin and eosin.

Molecular analyses

Total DNA was isolated from adult B. transfuga females using a NucleoSpin Tissue kit (Macherey Nagel, Düren, Germany) following the manufacturer’s instructions. The DNA markers used for identification were as follows: internal transcribed spacers 1, 5.8S and internal transcribed spacer 2 (ITSs); Cytochrome c oxidase subunit I (COI) and 28S ribosomal RNA gene (28S). The primers used are shown in Table 1. All polymerase chain reactions (PCR) were prepared in a final volume of 25 µl containing 12.5 µl of PCRBIO Taq Mix Red (PCR Biosystems), 20 pmol of forward and reverse primer, 8.5 µl of PCR water and 2 µl of the DNA (10–20 ng). Cycling conditions are described in Table 2. PCR products were visualized on a 2% agarose gel stained with Midori Green Advance (Elisabeth Pharmacon, Czech Republic). PCR amplicons were purified with the Gel/PCR DNA Fragments Extraction Kit (Geneaid Biotech, Taiwan) and sequenced commercially by Macrogen (Amsterdam, The Netherlands) using the indicated PCR primers.

Table 1 PCR primers used in this study
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Table 2 PCR cycling conditions used for different markers of DNA Baylisascaris transfuga
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ITSs that yielded sequences with mixed chromatograms were subsequently cloned using pGEM®-T Easy Vector Systems (Promega Corporation, USA). The cloned plasmid DNA was purified from bacterial culture using the GenElute™ Plasmid Miniprep Kit (Sigma-Aldrich, USA) and sequenced using universal T7/SP6 primers. Sequences were edited and assembled using the Geneious Prime software. Contigs were compared with those in the GenBank database by BLASTn analysis (http://blast.ncbi.nlm.nih.gov/Blast.cgi).

Differences in parasite invasion in particular tissues were analysed using the chi-square test.

Results

In the group of mice infected with ID 200, three individuals died between DPI (days post inoculation) 2–10, and twelve mice were euthanized between DPI 7–56. The second group of mice infected with ID 2000 included seven mice that were found dead between DPI 6 and 7; the remaining eight mice were euthanized between DPI 7–9 due to severe clinical symptoms of infection (Table 3). Overall, 10 out of 30 (33,3%) of the infected mice died after experimental infection. All five mice in the negative control group showed no clinical symptoms and were euthanized sequentially: the first mouse at DPI 7, the second mouse at DPI 14 and remaining three mice at DPI 56.

Table 3 Mice experimentally inoculated with Baylisascaris transfuga eggs. D found dead, E euthanized, DPI days post infection, ID infectious dose (number of embryonated eggs used for experimental inoculation), S symptoms (x = found dead with no symptoms previously observed, + mild symptoms, +  + moderate symptoms, +  +  + severe symptoms, no no symptoms observed at all), PF pathological findings (A autolysis, N no findings during necropsy, Y macroscopic pathological findings at necropsy), L larva found, dark grey colour inflammatory changes. Mice 19, 28, 33–35 served as negative controls with no symptoms/pathological findings
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Six mice (ID 200) were asymptomatic, four showed mild clinical symptoms such as roughened fur and moderate symptoms such as laboured breathing and five mice developed severe symptoms such as apathy, torticollis, immobility and ascites along with laboured breathing and roughened fur. Three mice also showed slowing of movements on the left side only. In the group of ID 2000 mice, seven mice died peracutely without clinical symptoms being observed and eight mice had to be euthanized due to apathy, atypical posture and respiratory problems (accelerated laboured abdominal breathing). The last euthanized mouse with ID 2000 developed severe clinical symptoms such as apathy, inability to move, ascites, also with laboured breathing and roughened fur.

Histological examination of ID 200 mice revealed larval stages in only some of the organs examined (liver, brain, skeletal muscles, lung, jejunum and spleen; Table 3). However, in the group of ID 2000 mice, larvae were found larvae in all organs examined except the spleen (Table 3). Overall, the most invaded organ was the liver, where the presence of larvae was statistically significant compared to other organs examined (p < 0.05). Moderately infected organs were lung, brain, skeletal muscles and jejunum and least frequently larvae were detected in the eyes, heart, kidneys and spleen (p < 0.05).

In general, acute, subacute and subchronic histopathological changes were observed in mice from DPI 7. Between DPI 10 and 56 subchronic and chronic changes were detected, 56 DPI only chronic inflammatory changes and fibrosis were noted.

Pathological changes in the liver of mice examined up to DPI 9 included acute focal changes with inflammatory infiltrates (neutrophils), multifocal chronic active inflammation with abscesses and focal necrosis with a mixed inflammatory infiltrate of macrophages, lymphocytes, plasmatic cells and neutrophils. Larvae were detected in the liver parenchyma and vessels. Mice examined between DPI 10 and 56 showed predominantly chronic active inflammation with abscesses and necrosis, moreover focal or diffuse vacuolar and hydrophic dystrophy of hepatocytes with larvae in the liver parenchyma. Histopathological changes in the liver were observed in 10 of 15 ID 200 mice and in 13 of ID 2000 mice.

Histopathological changes were found in the lungs of 11 ID 200 mice and 13 ID 2000 mice. Findings included haemorrhage, multifocal chronic purulent pneumonia, fibroplasia, inflammatory infiltrates with macrophages and eosinophils. Histopathological changes of various intensity were detected from DPI 7; subchronic and chronic inflammatory changes were observed between 10 and 56 DPI.

Forelimb and hindlimb skeletal muscle exhibited focal chronic active inflammation and fibroplasia with inflammatory infiltrates containing neutrophils, lymphocytes, macrophages and central larvae in mice examined by DPI 9. In mice examined at the 10th and 14th DPI, histopathology showed abscesses in skeletal muscles and chronic inflammation with fibroplasia and larvae in fibrotic tissue at the 56th DPI. Inflammatory changes were detected in 4 (triceps) and 7 (quadriceps) ID 200 mice and in 2 (triceps) and 3 (quadriceps) ID 2000 mice.

Chronic inflammatory infiltrates were present in the kidneys in DPI 7–10 in 2 ID 200 mice and 4 ID 2000 mice.

Despite the presence of larvae, brain tissue was without pathological changes in most mice; abscesses were detected only in two symptomatic mice (euthanized 10 DPI and 56 DPI), and in two asymptomatic mice (56 DPI), a focal-mixed inflammatory infiltrate with neutrophils, eosinophils, lymphocytes and plasmatic cells was observed.

The heart was without pathological changes in most mice; only two ID 200 mice 10 DPI and 56 DPI showed a focal chronic active infiltrate in the myocardium.

In the spleen, reactive hyperplasia of lymphoid follicles was observed in mice between DPI 10 and 56 in 11 ID 200 mice and two ID 2000 mice. The jejunum and eyes were without pathological changes in all examined mice. Table 3 shows that inflammatory changes occurred in the lung, kidney, liver, brain, skeletal and cardiac muscle tissues were present in some mice without findings of larvae, indicating a tissue reaction to the previous presence or migration of larvae. Histological sections of B. transfuga larvae in the mouse brain (Fig. 1a), heart (Fig. 1b), liver (Fig. 1c) and lung (Fig. 1d) are depicted.

Fig. 1
figure 1

Histological sections of Baylisascaris transfuga larvae in tissues from experimentally infected mice, HE stain, scale = 50 µm. a Brain tissue with larval stages without local pathological changes (mouse no 3, ID 2000, 6 DPI), b myocardium with larva located between cardiomyocytes, without pathological changes of the host tissue (mouse no 3, ID 2000, 6 DPI), c liver parenchyma with larva in dilated hepatic capillary lumen (mouse no 7, ID 2000, 7 DPI), d lung with larvae in peribronchial connective tissue with mixed inflammatory infiltrate (mouse No. BT5, ID 2000, 6 DPI)

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The final sequences of 28S, COI and ITSs were obtained and deposited in the GenBank database under accession numbers MW026407–MW026411. All sequences obtained showed identity with the sequences of B. transfuga available in the GenBank database (Table 4).

Table 4 Comparison of the Baylisascaris transfuga sequences from the GenBank database showing identity with the sequences of the isolate used in this study, deposited in the GenBank database under accession numbers MW026407–MW026411
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Discussion

The present results indicate a high pathogenicity of B. transfuga from the European brown bear for experimentally infected mice. To simulate possible natural conditions under which Baylisascaris eggs may be spread in the environment, a lower dose (200 embryonated eggs) was used to be compared with the higher dose (2000 eggs) similar to those used in previous experimental studies (Matoff and Komandarev 1965; Papini et al. 1996; Sato et al. 2004).

In mice, Matoff and Komandarev (1965) reported that larvae of B. transfuga migrate by three routes: (1) in the intestinal wall, where they encyst; (2) through the intestinal wall into the peritoneal cavity and into the mesenteries, abdominal and thoracic organs; and (3) from the intestinal wall into the heart, lungs and skeletal muscles via the bloodstream or lymphatic circulation. In experimentally inoculated mice, larvae were found in the central nervous system (Sprent 1955; Matoff and Komandarev 1965; Sato et al. 2004) rarely if ever resulting in clinical symptoms/neurological disorders. To induce neurological manifestations by B. transfuga larva migrans, Papini and Casarosa (1994) inoculated mice with high doses of infectious eggs and observed neurological symptoms in 2.5% of mice inoculated with 3000 eggs, 6.7% at a dose of 4000 eggs and in 20% of mice inoculated with 50,000 eggs. Ocular larva migrans in mice after administration of 3500 infectious B. transfuga eggs was reported by Papini et al. (1996).

In contrast to previous experiments in mice, in which only isolated clinical symptoms or neurological disorders occured (Sprent 1955; Matoff and Komandarev 1965; Sato et al. 2004), the mice in our study showed clinical symptoms in most cases, regardless of the lower or higher infectious dose. However, the mice that received a higher dose of 2000 eggs were more clinically affected. The symptoms of dyspnoea and ascites corresponded to the histological findings in the lungs and liver. Some mice showed symptoms such as slowed movements on the left side and torticollis, suggestive of neurological disorders. Nevertheless, in most of them, with the exception of two cases, the larvae found in their brains did not trigger any tissue reaction in the brain. These results are in contradiction with previous studies in which higher infectious doses of 3000 to 50,000 eggs were required to induce neurological manifestation in mice (Papini and Casarosa 1994) and in which the larvae in mice brains were surrounded or embedded by granulomatous reactions in the brains of mice (Sato et al. 2004). The infective dose of 2000 eggs used by Sato et al. (2004) resulted in one case of constant circling and three cases of motor incoordination in the infected mice; only one case was fatal, all other mice recovered completely within a week. The host response to the migrating Baylisascaris larvae is thought to be critical for the development of clinical symptoms. In the case of encapsulation of migrating larvae in the brain, infected rodents did not show neurological symptoms, but non-immobilized larvae in the brain caused neurological manifestations (Sheppard and Kazacos 1997; Sato et al. 2004). We observed different outcomes in the clinical manifestation of infection in mice compared to previous studies (Papini and Casarosa 1994; Sato et al. 2004), which may be due to differences in the immune response and the ability to encapsulate migrating larvae between hosts. Symptomatic infection in aberrant hosts caused by larva migrans would lead to an increase in Baylisascaris transmission in the wild as infected host become weakened or die and are more easily preyed upon or captured (Page 2013).The target regions of 28S, ITS1 and 2 (ITSs) and cox1 and 2, which were tested by Testini et al. 2011, showed the highest interspecific difference with B. procyonis in the case of ITS1; 28S rDNA and cox1 sequences showed high intraspecific similarity and high interspecific variations compared to other Baylisascaris species available in GenBank; the cox2 sequence showed low similarity with B. transfuga sequences available in GenBank. Molecular analysis of five markers selected in our study confirmed morphological identification of the species as B. transfuga, but only on the basis of 28S, it was not possible to distinguish B trasfuga from B. schroederi and B. ailuri. Thus, among the markers tested, COI and ITSs seem to be suitable for the identification of B. transfuga.

B. transfuga appears to be a very thriving parasite species in areas inhabited by bears. Nevertheless, the transmission pattern and the role of the aberrant host under natural conditions are still unclear. While B. transfuga was considered to be of low pathogenicity to aberrant hosts (Sprent 1955; Matoff and Komandarev 1965; Sato et al. 2004), the results of our study revealing high pathogenicity to mice are contributing to the knowledge of the relationships between B. transfuga and its aberrant hosts.