Opisthorchis felineus first autochthonous case in Austria

doi:10.21203/rs.3.rs-5084320/v1

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Opisthorchis felineus first autochthonous case in Austria

https://doi.org/10.21203/rs.3.rs-5084320/v1

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Opisthorchis felineus is a feline pathogen with zoonotic potential that can cause opisthorchiasis and cholangiocarcinoma in humans. In Europe, endemic areas particularly include Eastern Europe and parts of Asia, such as Russia, Ukraine, and Kazakhstan. However, the parasite has also been sporadically detected in Germany and northern Poland. A seven-year-old female neutered European shorthair cat without any travel history presented in May 2023 with weight loss, anorexia and vomiting. The cat presented increased liver enzyme activity, hyperbilirubinemia and hyperammonemia, which was consistent with the suspected diagnosis of cholangitis with subsequent hepatoencephalopathy. Opisthorchis felineus was detected via routine cytological examination of bile smears, and PCR confirmed O. felineus. This is the first report of autochthonous O. felineus infection in Austria.

liver fluke infestation

opisthorchiosis

Opisthorchis felineus

zoonosis

feline liver fluke

Opisthorchis felineus, belonging to the family Opistorchiidae, are large liver flukes amenable to human opisthorchiasis. Human liver flukes are endemic in Eurasia, and the WHO estimated that a subtotal of 1.51 million people are infected with O. felineus there (1). In Southeast Asia, other Opisthorchiidae can be found in up to 50% of free-roaming cats. Reports of clinical disease in cats are rare, but this may be underreported. In Europe, O. felineus has been reported in several countries, but to our knowledge, no reports of cats infected with O. felineus in Austria exist (2–5). In Siberia, this liver fluke is an important zoonotic pathogen with a reported prevalence exceeding 80% in humans in some regions. Reports of this specific liver fluke are rare in cats, and the lifecycle is likely maintained by wild carnivores, as it has been found in various species, including the abundant red fox (Vulpes vulpes). Mammals serve as definitive hosts for this parasite, where the fluke migrates to the liver and eggs are shed into feces (6). The fluke requires two intermediate hosts: snails, which constitute the first intermediate host, and cyprinid fishes, which constitute the second host. The consumption of raw fish is the main route of infection for new definitive hosts. Most cats are asymptomatic, and clinical symptoms, if present, are highly variable and nonspecific. Jaundice, dehydration, hepatomegaly, ascites, inappetence, vomiting, diarrhea and weight loss are among them. The clinical symptoms resemble those of lymphocytic cholangitis. Parasite burden, the time of infection and the animal’s response to parasite infection interact in complex ways, influencing the overall health and clinical presentation of the infected cat (7). Even death can occur with chronic infections or a high parasite burden, causing hepatic failure and extrahepatic biliary duct obstruction (8).. Laboratory changes may include mild nonregenerative anaemia, eosinophilia, lymphocytosis, hyperbilirubinemia, and increased ALT and ALP activities (4). Abdominal radiographs and ultrasounds are often unremarkable and/or nonspecific. Findings may include distended bile ducts, hyperechoic and thickened gallbladder walls, gallbladder “sludge”, hepatomegaly or diffuse cystic liver changes (9). The diagnosis is made via fecal flotation or sedimentation analysis, which is compromised by intermittent egg shedding, low parasite burdens and variable fecal analysis methods (10). Diagnostic sensitivity can be increased by analysis of bile obtained by percutaneous US-guided cholecystocentesis (9). Ten to 20 µL of bile in a direct smear preparation is considered sufficient to identify eggs (11). Praziquantel is the anthelmintic of choice for treating O. felineus. Dosages range from 20–40 mg/kg/SQ, IM or PO as a single dose or every 24 hrs for 3–5 days (8, 12). In cats infected with feline immunodeficiency virus, concurrent bacterial cholangitis with Escherichia coli has been reported (9). Liver fluke infestations are rarely associated with biliary cystadenoma or cholangiocarcinoma in cats (13, 14).

Cytology

Percutaneous ultrasound-guided cholecystocentesis and fine needle aspiration of the liver were performed. Routine hematoxylin and eosin (H&E) staining and bacteriological culture of the liver and bile were performed.

Molecular analysis

Bile fluid was examined under a light microscope, and DNA was extracted via a commercial DNA extraction kit (QIAamp PowerFecal Pro DNA Kit; QIAGEN, Hilden, Germany) according to the manufacturer’s instructions with adaptation. A Tissue Lyser II (QIAGEN, Hilden, Germany) was used for mechanical homogenization. To obtain a fragment of the mitochondrial cytochrome c oxidase subunit I (COI) gene with 638 nucleotide positions, PCR was performed via the primers COINeodFw (5’-TTT ACT TTG GAT CAT AAG CG-3’) and COINeodRv (5’-CCA AAA AAC CAA AAC ATA TGT TGA A-3’) (15) with the following amplification temperature profile: initial denaturation at 95°C for 2 min; 35 cycles of 95°C, 48°C, and 72°C for 1 min; and a final extension at 72°C for 5 min. The PCR product was run on 2% agarose gels stained with Midori Green. The PCR product was sent to Microsynth Austria GmbH (Vienna, Austria) for purification and sequencing in both directions. The electropherograms were inspected by eye via BioEdit (16). The sequences were compared to available sequences via the GenBank nucleotide basic local alignment search tool. For phylogenetic analysis, nucleotide sequences of Opisthorchiidae (taxid: 6196) available in the NCBI GenBank database were searched with the blastn function via the sequence obtained in this study. The sequences were aligned and cut to the same length via BioEdit (16). Maximum likelihood (ML) and Bayesian inference (BI) trees were calculated on the basis of the alignment, including 310 sequences (507 nucleotide positions). The sequences were collapsed to haplotypes via DAMBE v.7.0.5.1 (Xia and Xie 2001), resulting in 83 haplotypes. A sequence of Haplorchis taichui (GenBank accession number: KF214770) was used as an outgroup. An ML bootstrap consensus tree (1000 replicates) was calculated via the W-IQTREE web server (http://iqtree.cibiv.univie.ac.at/; (17), applying the model K3Pu + F + I + G4, which was suggested as the best fit for the data set in the model test according to the corrected Akaike information criterion. The BI trees were calculated via MrBayes v.3.2.7 (18), applying the next complex model GTR + I + G, because the same model was not available in this program. The analysis was run for 10⁶ generations (number of chains: 4), with sampling every 1000th tree. The first 25% of the trees were discarded as burn-in, and a 50% majority rule consensus tree was calculated on the basis of the remaining 7500 trees.

Results

Figure 1, 2: Seven-year-old female neutered European shorthair cat presented with severe weight loss and inappetence.

A seven-year-old female neutered European shorthair cat was presented to the emergency service for small animals at Vetmeduni Vienna in May 2023 because of severe weight loss, vomiting and ongoing inappetence (Figs. 1, 2). The cat has been owned since it was a kitten and comes from a farm in Lower Austria. The cat was neither regularly vaccinated nor dewormed, had no history of periods abroad and was held as an indoor-only cat. The cat has been fed conventional cat food and has never had access to raw fish or fish scraps. The other two cats in the household appeared clinically normal. Preliminary treatment by the referring veterinarian included cefovecin, meloxicam, and mirtazapine.

In the clinical examination, the cat showed mildly icteric mucous membranes and moderately reduced skin elasticity. A blood test was subsequently performed, revealing an unremarkable complete blood count. The activities of liver enzymes, such as ALT and ALP, as well as the bilirubin concentration were increased. To test liver function, the whole blood ammonia concentration was measured and found to be significantly increased (Table 1). Serum amyloid A was within the reference range at 2 mg/L (reference range < 5.0 mg/L). Prior to further diagnostic assessment, a coagulation status assessment was performed, where the prothrombin time was significantly prolonged to 14.5 seconds (reference range 8.0–10.0 sec). The partial thromboplastin time (PTT) and thrombin time (TZ) were within the reference range. The Combo + Snaptest® (Idexx Laboratories, Westbrook, ME, USA) for infection with feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) had negative results.

Table 1

Blood chemistry – May and June 2023
Parameter	Value		From	To
	May 2023	June 2023
ALP (U/L)	464	48		< 30.00
ALT (U/L)	293	56		< 100.00
Bilirubin (mg/dL)	7.41	0.14		< 0.2
Total Protein (g/dL)	7.21	7.69	6.00	7.50
Albumin g/dL	3.28	3.61	2.60	4.60
Ammonia (µmol/L)	129	28		< 60.00

For further diagnostic work-up, an abdominal ultrasound was performed by a European board-certified radiologist. The liver was significantly enlarged with homogenous parenchyma. The gallbladder wall had an irregular lining, and the common bile duct was 2.2 mm wide (< 4.0 mm) without any signs of obstruction (Fig. 3). On the basis of these findings, consent from the owner was sought to perform US-guided fine needle aspiration of the liver and cholecystocentesis for cytologic and bacteriologic examination. Liver cytology revealed numerous clusters of swollen hepatocytes with signs of fatty degeneration. In some areas, neutrophilic infiltrates were observed. Upon inspection of bile fluid smears, operculated, colourless parasitic objects measuring 26.8–31 were identified. The size of the O. felineus egg was 13.4–14.6 µm, which is consistent with the egg size of O. felineus (Fig. 4).

For further confirmation that O. felineus was involved, PCR was performed. The DNA sequence obtained was 100% identical to an O. felineus sequence documented in a cat from Russia (GenBank accession number: NC011127) and has been uploaded to BoldSystems® (Process ID: PAVEA251-23) and GenBank (accession number: OR486049). In the phylogenetic tree, the sequence was within a clade with other O. felineus found in fish (Fig. 5).

The cat was treated at a constant rate infusion with praziquantel (5 mg/kg) every 24 hrs over three days. For symptomatic therapy, 1 mg/kg maropitant q24 hrs, ondansetron 0.2 mg/kg q8 hrs, S-adenine-methionine 20 mg/kg q24 hrs, ursodeoxycholic acid 10 mg/kg q24 hrs, marbofloxacin 2 mg/kg q24 hrs, and lactulose 150 mg/cat q8 hrs were given. After the first dose of praziquantel, the cat developed anisocoria with an absent menace response and pupillary reflex, as well as generalized tremor and a head tilt consistent with hepatoencephalic syndrome. To prevent the progression of hepatic lipidosis, the cat was fed via a nasogastric tube. The neurological symptoms were self-limiting, and a significant improvement was observed after three days. The cat started eating on its own and was discharged home after a total of eight days. At the one-month follow-up examination, the cat had already gained one kilogram of body weight, and the clinical examination was unremarkable. The laboratory test results improved, and the ammonia concentration was within the reference limit. The pooled stool sample collected over three days was negative for O. felineus.

To our knowledge, this is the first case report of an autochthonous infection with O. felineus in a cat in Austria. However, the source of infection of our patient remains unclear because the cat has been adopted as a kit from a farm in lower Austria without access to raw freshwater fish and was kept indoors only from adoption onwards without any travel history to endemic areas. The predominant endemic area for O. felineus is Russia, with approximately 1.22 million people infected in the 1990s (1). A large outbreak of Opistorchiosis has been reported after the ingestion of raw fish from lakes Bolsena and Bracciano in Italy (19). In humans, infections with O. felineus are occasionally reported in other parts of the world because of immigration from endemic countries, consumption of raw or undercooked freshwater fish or import of these from endemic areas (1). Cats are considered important reservoir hosts for human opisthorchiasis, particularly in areas with a high prevalence of infection(5). In dogs, travel history or import from endemic regions may be important risk factors; however, in cats, these factors might play a minor role. Additionally, our patient has no history of periods abroad. The clinical symptoms of cats usually correlate with worm burden. If a cat has a high parasite load, it will exhibit symptoms such as lethargy, diarrhea, and ocular and nasal discharges. Age, sex and breed are not related to infection (2). Cats in endemic areas are usually raised without annual health checks or deworming. The prevalence of Opisthorchiidae is 30–50% in free-roaming cats, whereby cats may act as reservoir hosts (4, 2). One possible source of infection for cats is the consumption of self-caught fish from natural water reservoirs or fish scraps from village households (12). In our case, the cat was adopted from a farm in Lower Austria and reportedly has never eaten raw fish or fish scraps. Additionally, there was no large water source in the surrounding area of the farm. Therefore, the source of liver fluke infestation remains unclear. However, it is possible that the cat became infected as a kitten, as it was kept indoors thereafter. There are published data showing that metacercariae can remain in fish muscle and subcutaneous tissue for up to 7 years or more (21). However, whether adult O. felineus can survive this long period in the mammalian host is unknown. Even though the cat owners stated that they had not fed raw or frozen fish and had neither engaged in fishing as a profession nor as a hobby, it cannot be completely ruled out that the cat became infected at a later timepoint. Another rather unlikely possibility could be an unnoticed inadequate processing of commercial feed. These are speculations; the exact route of infection in this cat remains unclear. Further studies on the prevalence of O. felineus in Austria and the longevity of adult trematodes are necessary to estimate the probability of the source of infection.

Nonetheless, our cat exhibited an acute course, which could have been triggered by concurrent hepatic encephalopathy (lethargy, inappetence) and superimposed hepatic lipidosis. Ultrasound revealed a mildly distended common bile duct (CBD) without signs of obstruction. Thickening of the gallbladder wall and distension of the common bile duct without obstruction are ultrasonographic findings associated with cholecystitis and cholangitis, regardless of etiology. The known ultrasonographic findings in the literature indicate that the upper limit for the CBD diameter in normal and icteric cats is up to 4 mm. A CBD diameter greater than 5.0 mm is suggestive of extrahepatic bile duct obstruction (21, 22). However, gallbladder wall thickening and distension of the CBD are pathognomonic for parasitic infection. A case series published by Haney et al. in 2006 revealed that all cats with liver fluke infections had a significantly enlarged CBD both on ultrasound and in pathohistology, which exhibited consequent inflammation (23). The diagnosis of a feline liver fluke infestation is typically made through examination of a pooled stool sample. In our case, the parasite eggs were found in the bile. Köster et al. evaluated percutaneous US-guided cholecystocentesis and bile analysis for the detection of Platynosomum spp. and reported that bile egg counts were more sensitive in diagnosing platynosomosis than were fecal egg counts (9). Since the diagnosis was made on the second day of the cat's hospital stay, there was also not enough stool available at that time for a significant stool examination. Therefore, diagnosing through the bile was significantly faster, although it was associated with a greater risk of bleeding and leakage of bile (24). The anthelmintic drug of choice for treating O. felineus is praziquantel at a dosage range of 20–40 mg/kg as a single dose or every 24 hrs for 3–5 days. Praziquantel is considered a safe drug, and in a study by Sereerak et al., no side effects were observed in healthy cats given a single dose of 40 mg/kg (8, 12). Our patient developed significant neurological symptoms, including blindness and generalized tremors, after the first dose of 5 mg/kg q24 hrs for three days. Since praziquantel is metabolized primarily by the liver, its administration could temporarily exacerbate hepatic encephalopathy, leading to pronounced neurological symptoms in cats. Although studies have shown that a single dose of praziquantel at 30 mg/kg in dogs with fasciolosis only achieved a cure rate of 20%, our patient's pooled stool sample was negative after one month (25). Since the cat was clinically normal and had already gained one kilogram within the month, repeat percutaneous US-guided cholecystocentesis was deemed unnecessary. Since O. felineus is shed only intermittently in feces, there is a possibility of a false-negative stool sample. At the time of writing the case report, the cat continues to do well and appears clinically normal.

This case report presents the first autochthonous infection of a cat with O. felineus in Austria. Veterinarians should consider infection with O. felineus in cats with a suspected diagnosis of cholangitis, despite Austria not being considered an endemic area for O. felineus. This case report highlights that fine-needle aspiration of the gallbladder is essential as an advanced diagnostic procedure in cats with suspected cholangitis, as the diagnosis could not have been correctly made otherwise, and infection with O. felineus would have been missed. In cats diagnosed with O. felineus and associated hepatoencephalic syndrome, caution should be taken with the dosage of praziquantel, as in our patient, its administration led to pronounced neurological symptoms. To determine the prevalence of O. felineus in Austria, follow-up studies involving cats, humans, and fish are necessary to increase awareness of this zoonotic parasite.

O. felineus

Opisthorchis felineus

ALT

alanine transaminase

ALP

alkaline phosphatase

perorally

FIV

feline immunodeficiency virus

FeLV

feline leukemia virus

CBD

common bile duct

Ethics approval and consent to participate

were not applicable.

Consent for publication

The owner of the cat was informed and gave her consent for publication of the obtained data.

Competing interests

The authors declare that they have no competing interests.

Author details

(1) University Hospital for Small Animals, Department for Companion Animals and Horses, University of Veterinary Medicine Vienna, Vienna, Austria

(2) Institute of Parasitology, Department of Biological Sciences and Pathobiology, University of Veterinary Medicine Vienna, Vienna, Austria

(3) Diagnostic Imaging, Department for Companion Animals and Horses, University of Veterinary Medicine Vienna, Vienna, Austria

(4) Clinical Pathology Platform, University of Veterinary Medicine, 1210 Vienna, Austria

Funding

Open access funding for this article was provided by the University of Veterinary Medicine Vienna (Vetmeduni Vienna).

Author Contribution

LMK: Data acquisition (medical care), drafting of the manuscript. MSU: Data acquisition (molecular analysis), editing of the manuscript; YV: Data acquisition (abdominal ultrasound), editing of the manuscript; IS: Data acquisition (cytology), editing the manuscript; NLZ: Data acquisition (medical care), editing the manuscript. All the authors read and approved the final manuscript.

Acknowledgement

The authors thank Josef Harl for his supervision in generating the phylogenetic tree and Bita Shahi Barogh for the technical support in the molecular analysis.

Availability of data and materials

Additional data can be provided on request.

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Opisthorchis felineus first autochthonous case in Austria

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Version 1

Abstract

Figures

Background

Methods

Cytology

Molecular analysis

Discussion

Conclusion

Abbreviations

Declarations

Author details

Funding

Author Contribution

Acknowledgement

Availability of data and materials

References

Additional Declarations

Supplementary Files

Status:

Version 1