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Molecular Identification and Clinicopathological Findings of Hepatozoon sp. Infection in a Cat: First Report from Turkey/Bir Kedide Hepatozoon sp. Enfeksiyonunun Molekuler Identifikasyonu ve Klinikopatolojisi: Turkiye'den Ilk Rapor.

INTRODUCTION

Hepatozoon species are parasites from the phylum of Apicomplexa with a definitive host (a blood-sucking invertebrate) and an intermediate host (a vertebrate) (1-3). There are more than 340 species of Hepatozoon and about 46 of them infect mammals (2, 3). Despite a few differences in the ecology and biology of these protozoa, the primary route of Hepatozoon species transmission to the vertebrate hosts is represented by the ingestion of the invertebrate host carrying mature oocysts (1, 4). Although the main vector of Hepatozoon spp. is the ixodid ticks, other blood-sucking invertebrates, fleas, and mosquitoes have the potential to transmit the infection. However, the vectors of feline hepatozoonosis are not exactly known (1).

Feline hepatozoonosis has been described from various countries, including Brazil, France, India, Israel, Nigeria, Portugal, Thailand, South Africa, Spain, USA, and Italy (3, 4). Three Hepatozoon species have been reported in felines: Hepatozoon felis, H. canis, and H. silvestris (2, 4, 5). A small number of epidemiological surveys have also been published (4, 6-8). Depending on the cat lifestyle, geographical area, diagnostic method used, and type of samples tested, the prevalence of infection was found to range from 0.6% to 36.8% using different methods across various regions (2-5).

Two forms of the parasite have been found in the cat: intracellular gamonts in neutrophils and monocytes and meronts in several tissues (3). Hepatozoonosis is usually diagnosed by detecting the gamonts in blood smears and by identifying meronts in biopsy specimens (3). However, the parasitemia degree is low in cats (less than 1%) (9). Hence, it is often overlooked in blood smears. In recent years, with the development of molecular techniques, PCR has been started to be used for the diagnosis of Hepatozoon spp. in cats. Molecular methods are more sensitive and specific than the blood smear method for the detection of Hepatozoon spp. (1, 3).

In Turkey, although hepatozoonosis is widespread in dogs (10-13), feline hepatozoonosis has not yet been documented. This case is the first report of molecular identification of Hepatozoon spp. infection in a domestic cat from Turkey.

CASE REPORT

A five-year-old female cat was presented to the Veterinary Teaching Hospital, Adnan Menderes University, with a 1-month history of inappetence and weight loss. The cat was living indoors with access to the outdoors. Vaccination and antiparasitic treatment program of the cat were irregular. The cat was fed with commercial food and did not have any serious health problems until 3 weeks ago. Three weeks before the presentation, the cat was treated for a hepatic/post hepatic icterus in a private clinic, but no improvement was observed. In a physical examination of the cat presented to our hospital, weakness, depression, anorexia, fever (40.5[degrees]C), lymphadenopathy, tachycardia, icteric mucous membranes, and abdominal distension were observed.

Three blood samples were collected from the cephalic vein for hematological, biochemical, and molecular examinations and for the detection of feline leukemia virus (FeLV) antigen and antibodies to feline immunodeficiency virus (FIV). The hematological abnormalities were moderate anemia and neutrophilic leukocytosis (Table 1). The serum biochemical abnormalities included elevated direct and total bilirubin concentrations (Table 1). The FIV antigen test by using rapid diagnostic commercial test kit (Fassisi[R] FeLFIV, Germany) was positive. To assess the cause of the abdominal distension, abdominal radiography and ultrasonography were performed. The radiography revealed increased soft tissue density in the proximal abdomen and numerous dilated bowel loops filled with gas and feces. The ultrasonography of the cranial abdomen showed marked hepatomegaly and a normal gallbladder structure. Caudal abdominal ultrasonography was unremarkable. Giemsa-stained blood smears were prepared and examined under the light microscope. The blood smears revealed Hepatozoon spp. gamonts within the neutrophils (Figure 1). The gamonts measured 9.72[+ or -]0.80 urn in length and 4.17[+ or -]0.09 um in width. The slides were also used to examine leukocyte differentiation, erythrocyte morphology, and other infectious agents.

Written informed consent was obtained from cat's owner who participant in this case. PCR was performed to confirm Hepatozoon sp. infection (Figure 2). The DNA was extracted from each of two ethylenediaminetetraacetic acid-containing blood samples using the Promega Wizard genomic DNA extraction kit (Madison, WI, USA) following the manufacturer's instructions. Two microliters of extracted DNA were used for PCR amplification. Primers HepF (5'-ATA-CAT-GAG-CAA-AAT-CTC-AAC-3') and HepR (5'-CTT-ATT-ATT-CCA-TGC-TGC-AG-3'; Thermo Electron GmbH, Germany), which amplify a 666 bp fragment of the 18S rRNA gene of Hepatozoon spp., were used according to Inokuma et al., (14). The PCR product was electrophoresed on a 1.5% agarose gel and visualized under ultraviolet light.

Following the diagnosis, intravenous fluid therapy, doxycycline (5 mg/kg/once a day), lactulose (5 mL/cat, PO, twice a day), ursodeoxycholic acid (10 mg/kg once a day), B complex vitamins (1.0 mL/cat, once a day), and vitamin C (200 mg/cat, once a day) were prescribed. Prescribed drugs were not administered by the cat owner because the cat had escaped and she died. A postmortem pathological examination could not be performed.

DISCUSSION

Tick-borne diseases are caused by a vast number of agents, including viruses, bacteria, and parasites. Although these diseases are more common in dogs, they are rarely reported in cats because cats are more resistant to tick-borne diseases. This situation is related to the specific behavior of cats and the mechanical removal of ticks from their body surface during grooming. Also, cats are likely to have innate resistance or adaptation to infection, restriction of the disease progression, or the transmission of the infectious agents (15, 16).

Hepatozoon infection in a domestic dog was first described by the Nevzat (17) in Turkey. The first clinical case of H. canis infection in a dog was reported by Voyvoda et al. (18). Several epidemiological surveys have revealed a high prevalence of H. canis in dogs in Turkey (10-13). The studies indicated that the prevalence of H. canis infection in dogs varies as 22.3-54.3%. Although canine hepatozoonosis caused by H. canis occurs commonly, Hepatozoon spp. in cats have not been reported in Turkey. We report the first case of Hepatozoon sp. infection in a cat in Turkey in this case.

Feline hepatozoonosis is usually associated with outdoor access where contact with ticks is more frequent compared to strictly indoor cats (2, 3, 5). In our case, we found no ticks on clinical examination, but since the cat lived in outdoor access, she might have had contact with ticks. The transmission of Hepatozoon in cats is not yet fully understood. However, Aktas (19) detected H. felis DNA in Rhipicephalus sanguineus tick in Turkey. Unlike other tick-borne protozoan and bacterial disease, Hepatozoon infection in a dog occurs by the ingestion of the infected ticks, and it is presumed that cats are infected by the same route (15).

In cats, the infection has a low virulence, mostly subclinical, and there is scarce clinical information on the disease (2, 5, 20). As in dogs with hepatozoonosis, the identification of a characteristic clinical syndrome in feline hepatozoonosis is difficult. The most common clinical findings of feline hepatozoonosis are nonspecific, including fever, anorexia, weakness, weight loss, lymphadenopathy, lethargy, and icterus (2, 14, 15). The clinical signs of the cat presented here were similar to those reported for cats with Hepatozoon sp. infection by other investigators (9, 20). The presence of co-infections with FeLV or FIV, other causes of immunosuppression, and other vectorborne pathogens have frequently been described in feline hepatozoonosis (5, 21). Similar with those reports, FIV and possible immunosuppression related to FIV could also play an important role in this case. A few studies have indicated that co-infections may enhance the virulence of Hepatozoon spp. (9, 22). Also, immunosuppression may increase the degree of parasitemia (2, 23).

In the present case, the results of laboratory tests revealed neutrophilic leukocytosis, moderate anemia, and bilirubinemia. Anemia and leukocytosis with neutrophilia in our case were similar with those previously reported (3, 9), and those alterations could be related to secondary necrosis and inflammation in the spleen, lymph nodes, liver, and lungs because of meronts observed in many tissues, such as the skeletal muscle, myocardium, lung, liver, pancreas, bone marrow, and lymph node (3, 9). Elevated serum total and direct bilirubin levels detected in our case may be related to the presence of developmental stages of the agent in the liver and hemolysis.

Nonspecific and broad clinical findings of the disease make it difficult to confirm the diagnosis based exclusively on clinical assessment. Hence, definitive diagnosis of feline hepatozoonosis is mainly performed by the determination of Hepatozoon spp. gametocytes within neutrophils and monocytes in stained blood smears. Because of low parasitemia, PCR should be considered the diagnostic test of choice for confirming Hepatozoon infection (3). In this case, the diagnosis was confirmed by the detection of gamonts in Giemsastained blood smears and by PCR.

CONCLUSION

This case is the first molecular identification of Hepatozoon sp. in a cat from Turkey. This infection should be considered in cats with inappetence, weight loss, anemia, icterus, and leukocytosis. Further studies are needed to understand hepatozoonosis in cats in Turkey.

Peer-review: Externally peer-reviewed.

Author Contributions: Concept--G.E.T., B.U.; Design--G.E.T.; Supervision--B.U.; Resources--G.E.T., G.B.; Materials--G.E.T., S.B.; Data Collection and/or Processing--S.B., C.D., G.B.; Analysis and/or Interpretation--G.E.T., S.B..; Literature Search--C.D., G.B.; Writing Manuscript--G.E.T., B.U.; Critical Review--G.E.T., S.B., B.U.

Conflict of Interest: Authors have no conflicts of interest to declare.

Financial Disclosure: The authors declared that this study has received no financial support.

Hakem Degerlendirmesi: Dis bagimsiz.

Yazar Katkilari: Fikir--G.E.T., B.U.; Tasarim--G.E.T.; Denetleme--B.U.; Kaynaklar--G.E.T., B.U.; Malzemeler--G.E.T., S.B.; Veri Toplanmasi ve/veya Islemesi--S.B., C.D., G.B.; Analiz ve/veya Yorum--G.E.T., S.B.; Literatur Taramasi--C.D., G.B.; Yaziyi Yazan--G.E.T., B.U.; Elestirel Inceleme--G.E.T., S.B., B.U.

Cikar Catismasi: Yazarlar cikar catismasi bildirmemislerdir.

Finansal Destek: Yazarlar bu calisma icin finansal destek almadiklarini beyan etmislerdir.

REFERENCES

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(21.) Maia C, Ferreira A, Nunes M, Vieira ML, Campino L, Cardoso L. Molecular detection of bacterial and parasitic pathogens in hard ticks from Portugal. Ticks Tick Borne Dis 2015; 5: 409-14. [CrossRef]

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Gulten Emek Tuna (1) [iD], Serkan Bakirci (2) [iD], Ceren Dinler (1) [iD], Gizem Battal (3) [iD], Bulent Ulutas (1) [iD]

(1) Department of Internal Diseases, Adnan Menderes University School of Veterinary Medicine, Aydin, Turkey

(2) Department of Parasitology, Adnan Menderes University School of Veterinary Medicine, Aydin, Turkey

(3) Therapy Pet Hospital, Izmir, Turkey

Cite this article as: Tuna GE, Bakirci S, Dinler C, Battal G, Ulutas B. Molecular Identification and Clinicopathological Findings of Hepatozoon sp. Infection in a Cat: First Report from Turkey. Turkiye Parazitol Derg 2018; 42(4): 286-9.

Received: 02.06.2018

Accepted: 08.08.2018

Gelis Tarihi: 02.06.2018

Kabul Tarihi: 08.08.2018

A part of the study was presented at the International Turkish Veterinary Internal Medicine Congress.

Bu calismanin bir kismi Uluslararasi Turk Veteriner Dahiliye Kongresi'nde sunulmustur.

Corresponding Author / Sorumlu Yazar: Gulten Emek Tuna E.mail: emektuna@adu.edu.tr

DOI: 10.5152/tpd.2018.5886
Table 1. Hemato-biochemical findings of the cat naturally infected with
Hepatozoon spp.

Parameters                                 Values

WBC (x[10.sup.3] cells/[micro]L)            59.10
Lymphocytes (x[10.sup.3] cells/[micro]L)     0.50
Monocytes (x[10.sup.3] cells/[micro]L)       2.83
Neutrophils (x[10.sup.3] cells/[micro]L)    54.38
Lymphocytes (%)                              0.8
Monocytes (%)                                4.4
Neutrophils (%)                             92.0
RBC (x[10.sup.6] cells/[micro]L)             3.84
Hemoglobin (g/dL)                            6.4
Hematocrit (%)                              18.19
MCV (fL)                                    47
MCH (p/g)                                   16.5
MCHC (g/dL)                                 34.9
Platelets (x[10.sup.3] cells/[micro]L)     771
Glucose (mg/dL)                             75
AST (U/L)                                   13
ALT (U/L)                                   21
ALP (U/L)                                  170
GGT (U/L)                                    9
Total protein (g/dL)                         6
Albumin                                      2.4
Direct Bilirubin (mg/dL)                     1.9
Total Bilirubin (mg/dL)                      4.76
Urea (mg/dL)                                42
Creatinine (mg/dL)                           0.75

WBC: white blood cells; RBC: red blood cell; MCV: mean corpuscular
volume; MCH: mean corpuscular hemoglobin; MCHC: mean corpuscular
hemoglobin concentration; AST: aspartate aminotransferase; ALT: alanine
aminotransferase; GGT: gamma-glutamyltransferase
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Title Annotation:Case Report
Author:Tuna, Gulten Emek; Bakirci, Serkan; Dinler, Ceren; Battal, Gizem; Ulutas, Bulent
Publication:Turkish Journal of Parasitology
Date:Dec 1, 2018
Words:2669
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