Cytauxzoonosis is an emerging, tick-borne hemoparasitic disease of felidae family. It is caused by Cytauxzoon spp. which belong to the phylum Apicomplexa, class Sporozoasida, order Piroplasmida, family Theileriidae and genus Cytauxzoon. Feline cytauxzoonosis was first reported in Missouri, USA by Wagner in the year 1976. There are very few published reports of Cytauxzoonosis in domestic cats from India. Management of Cytauxzoonosis is generally difficult due to complex pathogenicity as well as refractory behaviour of the pathogen to chemotherapeutic agents.  Prompt and comprehensive treatment is required for cytauxzoonosis because it is a rapidly progressive disease (Sherrill and Cohn, 2015). Several drugs with antiprotozoal property have been employed for therapy of clinically ill cats. It included buparvaquone, diamenazene aceturate, doxycycline, atovaquone, azithromycin, imidocarb etc. Paravaquone and buparavaquone, antiprotozoal drugs used successfully to treat bovine theileriosis, completely failed against experimentally induced cytauxzoonosis (Motzel and Wagner, 1990). Greene et al. (1999) successfully treated five of six naturally infected domestic cats in a trial using diminazene aceturate. Cohn et al. (2011) in their study found 60% survival in infected cats that were given the combination of atovaquone and azithromycin while it was mere 26% with imidocarb dipropionate. Presently, combination of atovaquone and azithromycin is considered standard for cytauxzoonosis treatment in cats.
       
Feline cytauxzoonosis on whole is less explored in India, especially when it comes to treatment aspect. The standard combination of atovaquone and azithromycin is still not 100% successful in achieving survival of ill cats. Further, atovaquone is an expensive drug and less used in veterinary sector in India. Thus, there is a need of alternative chemotherapy for treating cytauxzoonosis in domestic cats. Imidocarb is easily available drug and was previously used to successfully treat a single case of cytauxzoonosis affected cat in India (Halder and Gupta, 2021). Antimicrobials having antiprotozoal actions, such as clindamycin, doxycycline and metronidazole, were shown efficient against Babesia spp. in humans and dogs (Suzuki et al., 2007). Almendros et al. (2020) reported the effective action of combination therapy using doxycycline, clindamycin and metronidazole, against Babesia gibsoni infection in dogs where atovaquone and azithromycin failed to eliminate the infection on a polymerase chain reaction (PCR) test. But there is no literature that documents the use of these drugs in feline babesiosis (Ayoob et al., 2010). Hence a controlled study was designed to study the efficacy of triple drug combination and imidocarb dipropionate against feline piroplasmid Cytauxzoon organism.
               
Imidocarb dipropionate is a carbanilide derivative, has antiprotozoal activity and is available only for veterinary use. Proposed mechanisms of action for imidocarb dipropionate are interference in utilisation or production of polyamines by parasites as found for Trypanosoma brucei, starvation of parasite resulting from blockage of inositol entry into parasite containing erythrocytes as found for Babesia organism and binding with nucleic acids of DNA of susceptible organisms causing damage to DNA to inhibit cellular repair and replication (Plumb, 2015). Doxycycline and clindamycin are thought to kill eukaryotic parasites by targeting ribosomes in the apicoplast (plastid) organelle where it inhibits prokaryotic-like translation (Okada et al., 2020). Metronidazole on entering the target cell is reduced to unstable intermediates that interacts with bacterial or protozoal DNA, causing loss of helical DNA structure and breakage of strand. This inhibits nucleic acid synthesis leading to death of the cell (Weir and Le, 2023). Oral administration of metronidazole for longer than 7 days consecutively or total daily dosage greater than 50 mg/kg has the risk of causing neurotoxicity and disruption of DNA within peripheral mononuclear cells in cats. But these toxicities are reversible on discontinuation of the drug (Sekis et al., 2009). The present study was undertaken to evaluate efficacy as well as any untoward effects (if any) in clinical cases of cytauxzoonosis in domestic cats.

Study design and setting
 
The cat cases presented at the Veterinary Clinical Complex, Bihar Veterinary College, Patna were screened for cytauxzoonosis based on previously defined symptoms namely anorexia, vomition, fever, icterus, dyspnea, tachycardia, lethargy, dehydration, vocalization and diarrhoea. Further, the disease was confirmed by microscopic blood smear and PCR test. The study design was discussed with the owner of the animal and included in this study after obtaining their consent. Twelve affected cats were selected for clinical trial and were divided into two treatment groups T1 and T2 having 6 cats each. A control group T3 was made with six clinically healthy cats which gave negative microscopic and PCR test for cytauxzoonosis. Cats in T1 group received imidocarb dipropionate @ 3.5 mg/kg deep intramuscular two doses 14 days apart. Pheniramine maleate @1-2 mg/cat was administered intramuscular before imidocarb injection to tackle any adverse reactions of it. In group T2 triple drug combination of doxycycline @10 mg/kg PO sid for 14 days, clindamycin @ 12.5mg/kg PO sid for 14 days and metronidazole benzoate@ 25 mg/kg PO sid for 7 days. Supportive therapy with fluid, liver support and hematinic were common to all and given as per the condition of the case. The data on physiological parameters and hemato-biochemical findings were recorded on 0, 7, 14 and 21 days of treatment. Also, blood smear and PCR test was performed on all these days. Blood samples were collected from medial saphenous vein or cephalic vein in EDTA vial (1 ml) and plain vial (2 ml). Comparative efficacy of the two protocols in treatment of cytauxzoonosis in cats was based on clinical recovery, absence of piroplasm stage in blood smear examination and improvement in hemato-biochemical parameters.
 
Molecular analysis
 
Genomic DNA from clinically ill cats was isolated using 300 µL whole blood sample and HiPurA® SPP blood DNA isolation kit (HIMEDIA) following the manufacturer’s instructions. The isolated DNA sample was stored at -20^oC for further use. Molecular detection of piroplasms was carried out using a conventional PCR assay using the sense primer 5ʹCCAGCAGCCGCGGTAATT3ʹ and the antisense primer 5ʹCTTTCGCAGTAGTTYGTCTTAACAAATCT3ʹ that amplify a fragment of ~373 bp of the 18S rRNA gene (Diaz-Reganon et al., 2017). PCR reactions were carried out in a final volume of 25 μL containing 12.5 μl of Taq PCR Master Mix (2 × Premix) containing Taq DNA Polymerase, optimized PCR buffer and dNTPs, 1.0 μl of each primer (Forward and Reverse), 2 μl of the DNA template and 8.5 μl nuclease free water.
       
The thermal cycling conditions consisted of an initial denaturation: 94^oC for 3 min 35 cycles of 94^oC for 30 s 64^oC for 45 s 72^oC for 30 s Final extension: 72^oC for 7 min. All reactions were performed using an automated thermal cycler (BIO-RAD, T100). The amplicons were visualized under UV illumination in gel documentation system after electrophoresis of 10 µL of the reaction solution in a 2% agarose Gel.
 
Statistical analysis
 
The data obtained were statistically analysed by the IBM SPSS 29.0 standard version for Windows. Two-way ANOVA was used for comparing the mean values of the three treatment groups with the control group and within the groups on different days of assessment. Variables with P≥0.05 were considered as statistically “non-significant.” variables with P<0.05 were considered as statistically “significant”.

On microscopic blood smear examination both bipolar safety pin-like piroplasms (Fig 1) and round signet-ring shaped (Fig 2) were found in this study. Concentration of parasitaemia was graded mild in all the cases. The cats in the imidocarb group (T1) and triple drugs (T2) group became microscopically negative on day 21(Table 1). However, with both of these protocols PCR gave positive result (band of 373 bp, Fig 3) on all the days of assessment.









The body temperature, respiratory rate and heart rate (Table 2) were increased significantly (P<0.05) in both the groups on the day of presentation. Significant improvement was observed from 0 to 7^th day reaching the normal level in both the treatment groups. The mean hemoglobin, haematocrit, TEC, platelet counts (Table 3) were significantly (P<0.05) lower from the control group on 0 day. The values increased significantly (P<0.05) from 0 to 14^th day in imidocarb group while in triple drug group the increase was non-significant (P≥0.05). The mean total leukocyte count (TLC) (Table 3) did not show any significant changes (P≥0.05) in any of the treatment groups as compared to healthy control. However, mild leucocytosis and leukopenia were observed on referring to individual cases. Treatment brought non-significant changes in both the groups. The ALT activity (Table 4) were found to be significantly higher (P<0.05) in both the treatment groups from the healthy control on day 0. Significant decrease (P<0.05) in ALT was noted in imidocarb treated group from 0 to 14^th day level. Triple drug combination also showed decrease but non-significantly. Mean total protein concentration (Table 4) was non-significantly (P≥0.05) lower in both the treatment groups as compared to healthy control on 0 day. The mean albumin concentration (Table 4) was significantly decreased (P<0.05) in both the treatment groups as compared to healthy control. Imidocarb significantly increased (P<0.05) albumin concentration on 14^th day of treatment whereas triple drug did not show any significant (P≥0.05) improvement. There were no significant changes (P≥0.05) in mean total bilirubin values (Table 4) between and within groups. But there was considerable rise in two cats, one from each group that improved well with the treatment. One cat from group T1 had total bilirubin value 4.6 mg/dL on 0 day which lowered to 2.8, 1.5 and 0.9 mg/dL on 7^th, 14^th and 21^st day of the treatment respectively (Fig 4 and 5). Similarly, total bilirubin level in a cat in group T2 improved from 4 mg/dL to 3, 1.4 and 1 mg/dL on 7^th, 14^th and 21^st day of treatment respectively (Fig 6 and 7). However, effect of treatment could not be comprehended statistically on the mean bilirubin values. Blood Urea Nitrogen (BUN) and creatinine did not show significant alterations in affected cats of any of the group (Table 5).
















       
Both the treatment modalities appear safe for renal and hepatic function as there was no evidence of derailment in levels of renal and hepatic enzymes after treatment (Table 4 and 5). The safety of drugs in terms of pre and post administration effects on the physiological and behavioural factors of cats is presented in Table 6.


       
Recurrence of illness after a clinical recovery was reported with triple drug combination (after 45 days) in three of cats and in one of the imidocarb treated cat (after 60 days).
       
Imidocarb dipropionate and the combination of doxycycline, clindamycin and metronidazole gave negative microscopic blood smear test in all cats on day 21. Thus, both were equally effective in terms of clearing erythro-parasitemia. However, both the drugs were not able to sterilise the infection of cytauxzoonosis giving positive PCR results. Thus, the cats once infected may recover from the clinical illness but remain to act as carrier. Harvey et al., (2007) found that almost all cougars, bobcats and domestic cats recovering from acute C. felis infection continued to be parasitaemic for life. However, Carli et al., (2014) thoroughly studied cytauxzoonosis infection in a free-ranging cat with multiple evaluations post-treatment with doxycycline and imidocarb dipropionate and it gave negative PCR result on all these days.
       
Elevated body temperature, respiratory rate and heart rate was consistent with the findings of Cohn et al., (2011). The remission of these vital parameters to normal on 7th day was parallel in both groups. It could be attributed to antipyretic treatment and lowered infection.
       
Mild to moderate anemic state of infected cats was due to immune mediated erythro-phagocytosis and was also reported in previous studies (Carli et al., 2022).
       
Thrombocytopenia as evidenced in present and other studies (Alho et al., 2016) is believed to be due to DIC and inflammation led coagulation (Conner et al., 2015).
       
There is no uniform pattern in shift of leukocyte count in present work similar to previous studies. Moghaddam et al., (2020) found leukocytosis, indicative of acute infection. Naidenko et al., (2022) reported leukopenia indicating chronic infection. While Carli et al. (2022) did not find any significant changes in total leucocyte, neutrophil and lymphocyte counts in their case-control study.
       
Higher liver enzyme activity in infected cats was alike reports of Alho et al., (2016) and Nentwig et al., (2018). This signifies hepatic damage that could be both hypoxic (due to anemia) and infarctive (due to occlusion of blood vessels by parasite distended mononuclear cells).
       
Hyperbilirubinemia was detected but not an obvious finding in the present study. Such variation can also be concluded from the findings of Carli et al., (2022) who found the total bilirubin level within the normal range in the infected cats while Cohn et al., (2020) reported hyperbilirubinemia.
       
Low mean albumin concentration was also observed by Moghaddam et al., (2020) similar to present study. Vasculitis, liver dysfunction and protein losing nephropathy and enteropathy are the possible causes for hypoproteinemia and hypoalbuminemia (Frontera-Acevedo et al., 2013).
       
Imidocarb dipropionate gave significant result in improving the hemato-biochemical parameters within 21 days of treatment. However triple drug combination showed non-significant improvement during this period. Thus, triple drug had slower action than imidocarb in normalising the hemato-biochemical alterations. Due to the lengthy protocol of triple drug combination, there could be possibility of discontinuation of the treatment by the owners once the symptoms resolved. Besides, complaint of mild diarrhoea was given in many cases under this treatment protocol. But overall no serious side effects or mortality could be recorded in cats that received any of the treatment protocols.
       
Kier and Greene (1998) claimed a 50% survival rate using imidocarb in cats suffering from naturally occurring cytauxzoonosis. Cohn et al., (2011) in their study found mere 26% survival in cats that were treated with imidocarb dipropionate. In a case report of cytauxzoonosis in a kitten from India treatment was attempted using doxycycline but the kitten collapsed ultimately (Varshney et al., 2009). The severely affected cats that died during the treatment duration was excluded from the present study. Thus, we could not conclude the efficacy in terms of survival percentage. Recurrence of illness in few cats from both the groups could be either new infection or recrudescence of previously lowered infection with the treatment.

Imidocarb dipropionate as well as combination of doxycycline, clindamycin and metronidazole combination brought clinical remission. However, improvement in morbidity and hemato-biochemical parameters were more rapid in imidocarb treated cats. No serious side effects or mortality could be recorded in cats that received the treatment protocols used in this study. But none of these treatment protocols could sterilize the infection and rendered the cats as carrier for life.

The authors are highly thankful to the Dean, Bihar Veterinary College, Dean Post graduate studies, Bihar Animal Science University Patna for providing necessary facility for research work. The financial assistance provided to the first author in the form of University fellowship, for her post graduate degree programme is thankfully acknowledged.
 
Ethics approval
 
The experimental protocol was approved by institutional animal ethical committee, Bihar Animal Science University, India (vide letter no- F.26-1/2022-23/DR).

The authors have no relevant financial or non-financial interests to disclose.