The first case of feline cytauxzoonosis in Germany: clinical description and molecular confirmation

Cytauxzoonosis is gaining clinical importance in recent years, being described as an emerging tick-borne disease of domestic and wild felids caused by protozoans of the genus Cytauxzoon . While in the Americas the condition is described as a fatal disease, in Europe, reports on the clinical expression of the infection are scarce. This study describes the first case of Cytauxzoon sp. infection in a domestic cat in Germany. Clinical and laboratory findings, and molecular and DNA sequencing results were collected from a domestic cat suspected for cytauxzoonosis after the microscopic examination of a blood smear. the


Conclusion
This study provides the first case of molecularly confirmed Cytauxzoon sp. infection in a domestic cat in Germany and describes the clinical picture of the disease in association with FIV infection. These findings suggest that cytauxzoonosis should be considered as a differential diagnosis in cases of anaemia in domestic outdoor cats, particularly in areas where wild felid populations are present.

Background
A tick-borne and Babesia-like pathogen in felids, Cytauxzoon felis, is gaining clinical importance in recent years, although its occurrence outside the Americas is not reported.
Domestic cats infected with C. felis generally develop a peracute and highly fatal disease [1].
The European isolates of Cytauxzoon seem to be less virulent than C. felis and the clinical manifestations appear to be associated with immune-mediated diseases or secondary infections [5,11]. Due to non-clinical carrier state as well as non-specific clinical signs like lethargy, anorexia, anaemia, fever, weight loss, tachycardia, tachypnoea, diarrhoea, 4 and vomiting, clinical reports for Cytauxzoon sp. infections in Europe are scarce [7][8][9][10].
The present paper reports the first molecular confirmation of Cytauxzoon sp. infection in Germany and provides a detailed clinical picture of the disease in association with feline immunodeficiency virus (FIV) infection.

Patient
In March 2017, a 6-year-old male domestic cat was referred to a veterinary facility in Saarlouis (49.31°N, 6.75°E), a small city located in the south-west part of Germany, with unspecific clinical signs (detailed case description is provided in the results section). The cat had an outdoor lifestyle and had never travelled abroad.

Clinical blood analyses
In March 2017, a complete blood count and serum clinical biochemical analysis (including the assessment of liver, kidney, and pancreas function) were performed at IDEXX reference laboratory in Ludwigsburg, Germany (Table 1). Symmetric dimethylarginine (SDMA) was measured using a commercially available high-throughput immunoassay (IDEXX SDMA Test, IDEXX Laboratories Inc.) [22]. No urine examination was performed.

Microscopic examination of blood smears
May Grünwald-Giemsa-stained blood smears were prepared from peripheral blood and were evaluated for the presence of intraerythrocytic piroplasms.  [24]. Furthermore, a nested and a conventional PCR protocol were used for the amplification of the partial 18S rRNA gene of the phylum Apicomplexa, with the primer pairs BTH-1F/BTH-1R, GF2/GR2 and 7549/7548, respectively, following previously published reaction procedures and protocols [3,25].
PCR products were visualised by electrophoresis in a 2% agarose gel. After purification, the amplicons were submitted for sequencing on both strands (at Macrogen, Netherlands).
Consensus sequences were compared to the sequences available in GenBank™ dataset by

Haematology and blood biochemistry
In March 2017, the haematological analyses revealed azotaemia with markedly increased SDMA, hypercreatinemia, as well as hyperphosphatemia and hypoalbuminemia.
Furthermore, a mild non-regenerative anaemia was present. Abnormal laboratory findings are shown in Table 1. The results indicated decreased glomerular filtration rate, likely due to kidney disease. Urinalysis was not performed to assess specific gravity, protein concentration or sediment.
In January 2016, the hematological and clinical chemistry variables had been within normal limits. FeLV and FCoV tests were negative, but a positive FIV antibody test was obtained.

Blood smear microscopy
Blood smear examination in March 2017 revealed morphologically unremarkable leukocytes, while erythrocytes displayed no significant anisocytosis or polychromasia.
Blood smear evaluation revealed round to oval signet-ring shaped organisms inside the erythrocytes, with a lightly basophilic cytoplasm and a basophilic nucleus usually located in an eccentric position (Additional file 1: Fig. 1).
One to four merozoites were observed within individual red blood cells. The mean length of the well-defined forms was 1.2 ± 0.2 µm and the measured width was 1.0 ± 0.2 µm. The intensity of parasitaemia was estimated at 32.95%. The intracellular parasites were morphologically suggestive for a piroplasm. Moreover, the typical signet ring-shaped organisms could be clearly differentiated from feline haemotropic mycoplasma due to their evident large nuclear area (see also [1]).
Microscopic examination of the blood smear in January 2016 revealed no blood cell 8 morphologic abnormalities and no microorganisms.

Discussion
The study reported herein describes the clinical picture, laboratory findings and diagnostic The cat patient in the current report was also diagnosed with a FIV infection, being presented in a critical condition a few days following clinical examination due to suspected kidney disease. Since in dogs, clinical cases of babesiosis and theilerioses are known to cause acute or chronic nephropathy or glomerulonephritis [27,28], the renal disturbances observed in this patient could have been attributed to the piroplasmid infection, to FIV or both. Previous studies have confirmed that FIV infection can induce the accumulation of immune complexes in renal tissue, therefore, a causative relationship between FIV infection and glomerulonephritis has been posited [29][30][31][32]. In the light of this information and the laboratory results (hypoalbuminemia pointing toward a renal loss of protein, increased creatinine and phosphate values as well as a very high SDMA value) one could postulate that the patient had glomerular disease due to immune-complex deposition in terms of a membranoproliferative glomerulonephritis. Other potential causes of hypalbuminaemia in this patient would include decreased production, other causes of loss (i.e. blood loss, protein-losing enteropathy) or haemodilution. In cases of renal injury, SDMA serum concentration increases earlier than creatinine, remaining elevated also in cases of chronic kidney disease (on average with 40% reduction of glomerular filtration rate, compared with up to 75% reduction needed to increase the creatinine value) [33].
Regarding the pathogenesis of FIV infection, cats remain asymptomatic for several years until functional immunodeficiency (by increasing the susceptibility to secondary infections and neoplasia) and/or immune-mediated diseases will eventually translate into clinical manifestation around 4 to 6 years of age or older [31]. In the present case, the cat did not display any noticeable symptoms at 5 years of age, approximately 1 year before merozoites resembling Cytauxzoon sp. were detected in high numbers in the blood smear.
Therefore, it can be presumed that, as a result of immunosuppression caused by FIV, the piroplasm contributed as an opportunistic factor triggering the clinical picture.
Furthermore, several reports have identified a positive relationship between FIV infection or other immune-mediated conditions and cytauxzoonosis in Europe [5,11]. The clinical role of Cytauxzoon sp. in domestic cats without immunosuppression and coinfections remains debatable, as the information about pathogenesis and clinical involvement is limited. Although some cases of clinical illness and fatal outcome have been described [7,10,11], the majority of Cytauxzoon sp. infected cats were apparently healthy, with only few animals showing mild anaemia syndrome [5,8,9,11].
As the molecular confirmation of Cytauxzoon sp. infection was established post-mortem, no specific anti-piroplasm treatment was applied. Although, in C. felis infection, the recommended therapeutic protocol consists of atovaquone and azithromycin [34], the optimal therapy for Cytauxzoon sp. infection remains unknown due to the lack of controlled clinical studies. In a recently published case report, three 2 month old kittens infected with Cytauxzoon sp. were treated with a combination of atovaquone and azithromycin, with a putative success [11]. Also, previously published Cytauxzoon sp.
infections in domestic cats have been medicated with various antiprotozoal drugs, including imidocarb dipropionate and doxycycline [7][8][9][10]. Clinical studies in Europe reported that a single imidocarb dipropionate administration was not successful in treating cytauxzoonosis, although the addition of doxycycline cleared parasitaemia in one cat [7,9].
Based on the sequencing results performed on the partial 18S rRNA gene amplification product, Cytauxzoon sp. detected in the present case revealed a high homology (99-100%) with Cytauxzoon sp. reported in European domestic felids [7,11] and with Cytauxzoon manul (99.80%) described from Pallas' cats from Mongolia [3]. In addition, the sequence displayed a similarity of 95% to C. felis 18S rRNA partial sequences deposited in GenBank®.
Microscopic examination of the blood smear revealed a degree of parasitaemia of approximately 33%. Other clinical studies showed lower levels of parasitaemia [, 7-9, 15], while similar percentages of piroplasm-infected erythrocytes were observed in the blood smear of three cats with cytauxzoonosis from Switzerland [11].

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A significant association between the detection of Cytauxzoon sp. DNA in European domestic cats and outdoor lifestyle has been found, particularly in rural areas [5,8] [20], Bosnia and Herzegovina [21], and Spain [12,14].
Moreover, Germany has one of the biggest European populations of Felis silvestris, that is concentrated in the low altitude mountain areas [38]. Studies on wild cat distribution suggest that Saarlouis city is surrounded by an abundant population [39]. Consequently, considering that they might live in close proximity to urban and rural areas and crossbreed with domestic cats, it is possible that wild cats play a central role in the transmission of Cytauxzoon sp. in west Germany, as in probably all of Europe. Ixodes ricinus is the most commonly found tick in Germany [40], and it has already been hypothesized to be a possible vector for Cytauxzoon sp. [19].
This study describes, to the best of our knowledge, the first case of molecularly confirmed Cytauxzoon sp. infection in Germany.

Ethics approval and consent to participate
All clinical procedures were done as part of the diagnostic workup and treatment of the cat.

Consent for publication
Not applicable.

Availability of data and material
The datasets supporting the conclusions of this article are included within the article.

Competing interests
The authors declare that they have no competing interests.

Funding
Part of the work was supported by the UEFISCDI Grant Agency Romania, grant number PCCDI 57/2018 and by the University of Agricultural Sciences and Veterinary Medicine of Cluj-Napoca, Romania.

Authors' contributions
LCP wrote the manuscript and performed the microscopical analyses, molecular assays and sequence analyses of the sample. GS, MG, JG and NP were responsible for the serum/blood analyses, microscopical examination of the blood smear and the initial molecular diagnosis. BG performed anamnesis, clinical examination and treatment. ADM and NP supervised and critically revised the manuscript for important intellectual content.
NP drafted the original manuscript. All authors read and approved the final manuscript.