This manuscript represents a portion of a thesis submitted by Dr Lewis to the University of Missouri as partial fulfillment of the requirements for a Master of Science degree. This work was presented as a research abstract at the 2012 ACVIM Forum in New Orleans, LA.
The treatment trial was performed at the University of Missouri, with PCR samples processed at North Carolina State University.
Corresponding author: L.A. Cohn, Department of Veterinary Medicine and Surgery, College of Veterinary Medicine, University of Missouri, 900 E. Campus Dr, Columbia, MO 65211; e-mail: firstname.lastname@example.org
Cytauxzoon felis is a hemoprotozoal parasite that causes substantial morbidity and mortality during the acute phase of infection in cats. However, cats that survive the acute illness remain persistently infected and may serve as a reservoir for the tick-transmitted pathogen.
We investigated the ability of the antiprotozoal compound diminazene diaceturate to eliminate the pathogen from naturally infected C. felis carriers.
Seven healthy, chronically infected domestic cats housed in a research setting.
Prospective clinical trial. Cats were treated in a masked fashion with diminazene diaceturate (3 mg/kg) or placebo IM in a series of 2 injections 7 days apart. Clearance of the organism was assessed by light microscopy and real-time polymerase chain reaction (PCR) at 0, 3, 6, and 10 weeks. In addition, cats were monitored for behavioral changes or for changes on physical examination, CBC, plasma biochemical profile, and urinalysis periodically. Cats that remained parasitemic at the end of 10 weeks were switched to the alternative treatment and similarly monitored for an additional 10 weeks.
Adverse events associated with treatment were limited to self-resolving hypersalivation and injection site soreness; the former was ameliorated by premedication with atropine. Parasite burden, as assayed by both light microscopy and real-time PCR, was similar between diminazene- and placebo-treated cats.
Conclusions and Clinical Relevance
Diminazene diaceturate was unable to eliminate the pathogen or decrease parasite burden in healthy, chronically infected cats.
Cytauxzoon felis is a hemoprotozoan parasite that causes substantial morbidity and mortality in cats. The life cycle of the parasite relies on an appropriate tick vector feeding on an infected host and later feeding on a second, susceptible felid to transmit the pathogen. Cats that survive initial infection remain persistently parasitemic without illness.[2-4] Although once considered uniformly fatal, it has become apparent that some cats survive illness whereas others survive infection with no known illness.[2-5] Chronic carriers are capable of transmitting infection through the appropriate Amblyomma americanum tick vector.
The drug diminazene is used as either of 2 salts (aceturate or diaceturate) to treat a variety of protozoal diseases in domestic livestock throughout the world. Five of 6 cats survived acute cytauxzoonosis after treatment with diminazene aceturate at 2 mg/kg IM. Although the drug is not Food and Drug Administration approved for use in any species in the United States, it is possible to obtain the drug for a specific animal patient under Compassionate Use Protocols. Although such importation is impractical for cats suffering acute cytauxzoonosis, it should be possible for the treatment of chronic carriers were it to be demonstrated to be efficacious in eliminating the pathogen.
Despite improved treatments, feline cytauxzoonosis remains a disease with high morbidity and mortality. We hypothesized that diminazene, administered in a 2-dose protocol equivalent to that used for the treatment of acute cytauxzoonosis, could safely eliminate C. felis infection from naturally infected carrier cats as assessed by light microscopy and PCR. Were this treatment to prove effective, it could not only be used to decrease the chance that recovered cats could serve as an indirect source of infection for others, but would provide impetus for further study of the drug for the treatment of acute cytauxzoonosis.
Seven adult domestic cats (2 female intact, 2 female spayed, 3 male castrated) were donated for the study of cytauxzoonosis by their owners. All cats were from C. felis endemic areas of northern or central Arkansas, had extensive outdoor exposure, and all were suspected or confirmed to harbor the pathogen before donation. One cat had been owned for only 2 months, but the others had been owned for several (up to 5) years. None of the cats had a known history of illness and none had received antiprotozoal medications. All cats appeared to be young to middle-aged adults, and weighed from 3.2 to 5.8 kg (mean, 4.8 ± 0.95 kg).
At the time of donation, each cat underwent a physical examination along with feline leukemia (FeLV) and feline immunodeficiency virus (FIV) testing,1 CBC, biochemical profile, urinalysis, and species-specific PCR testing for C. felis. No important abnormalities were identified on physical examination, biochemical profile, or urinalysis, and all cats were negative for retroviral infection. All cats were positive for C. felis by both PCR and microscopic review of stained blood smears. Analysis of a nearly full-length (excluding unreadable sequence at the ends of the amplicons) 18S rRNA gene from the parasites, as amplified by a PCR assay with the same primers but different cycling conditions, confirmed their identity as North American C. felis. Specifically, when compared with published full-length C. felis 18S sequences in Genbank, nucleotide variability was limited to secondary nucleotide peaks (corresponding to position 256 on Genbank accession AY679105.1) with both cytosine and thymine detected, and to single nucleotide alterations compared with Genbank accessions AY531524 and L19080.1, but identical to accessions AY679105.1 and AF399930. All cats were vaccinated with FVRCP and rabies vaccines, and treated with an antihelminthic (fenbendazole, 50 mg/kg PO or emodepside/praziquantel2 topically 15 mg/60 mg per cat) and topical antiparasitic (fipronil3). Animals were cared for according to the principles outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and were acclimated to housing in an accredited facility for a minimum of 30 days before initiation of the study. All animal use was approved by the University of Missouri Animal Use and Care Committee (protocol number 6665).
A commercially prepared product of diminazene diaceturate4 (70 mg/mL) and phenazone (375 mg/mL) in sterile water was stored as per manufacturer directions. The cats were arbitrarily assigned to receive either drug (n = 3) or placebo (n = 4) for the 1st phase of treatment. Any cat that remained positive on light microscopy or PCR at the end of the first 10 weeks was then switched to the alternative treatment.
Cats were given either 3 mg/kg of diminazene diaceturate or an equivalent volume of sterile saline in the epaxial muscles or the biceps femoris on days 1 and 8 of each treatment phase. Owing to their fractious nature, 2 cats required sedation with ketamine (10–15 mg/kg) administered IM before treatment or blood sample collection. Because excessive ptyalism was noted in several cats immediately after the 1st treatment, all animals subsequently were pretreated with atropine (0.03 mg/kg) SC 15 minutes before drug or placebo administration.
Cats were monitored for 10 weeks after each phase of the study. Physical examination, CBC with careful blood smear review, biochemistry profile, urinalysis, and C. felis PCR were performed before the first treatment, then again at weeks 3 and 10. At week 6, the same tests were performed with the omission of the biochemistry profile and urinalysis. For the first 2 hours after each injection, cats were monitored for changes in heart or respiratory rates, and the injection site was palpated. The attitude and appetite of all cats was observed twice daily for 1 week after each injection, and once daily for the study duration. At study completion, the 2 fractious intact female cats were euthanized and necropsies were performed.
Quantitative CBC, plasma biochemistry profile, and urinalysis were performed in routine fashion by the University of Missouri Veterinary Medical Diagnostic Laboratory. In addition, a trained technician thoroughly reviewed Wright-Giemsa stained blood smears for piroplasms and counted the number of piroplasms per 1,500 erythrocytes.
Real-time PCR was performed in triplicate on DNA extracted from anticoagulated blood using a previously published technique; positive and negative controls were run with each assay. Briefly, each reaction consisted of 12.5 μL 2× PCR master mix,5 7 μL water, 50 pmol of each oligonucleotide primer, and 5 μL sample. Thermal cycling conditions were as follows: 98°C for 30 seconds followed by 45 cycles, 95°C for 5 seconds, and 60°C for 5 seconds. Melting curve analysis was initiated at 75°C and data were captured at increasing increments of 0.5°C for 30 time points. Average cycle threshold (Ct) was used as an estimate of parasitemia. Typical precautions (eg, disposable gloves, clean to dirty workflow, physical separation of pre- and post-PCR samples) were used to prevent amplicon carryover.
Adverse events were minor and self-limiting. Ptyalism was minimal or absent after atropine pretreatment. Transient tachypnea without respiratory distress developed in 1 cat after both drug and placebo injection, and in 2 different cats after drug or placebo injection. Injection site soreness was noted in a single cat after diminazene administration, but resolved by the next day. Vomiting was not seen within hours of injection, but during the entire 10 weeks cats treated with both diminazene (n = 3) and placebo (n = 3) vomited on occasion. All cats retained normal appetite and attitude through the entire study course.
Hematologic abnormalities were limited to the presence of low numbers of piroplasms and neutropenia. A single sample from week 10 was not available for CBC as a result of technical issues. None of the cats became anemic. Moderate neutropenia was noted in 1 cat before study entry, and this cat continued to have neutrophil counts just above or below the lower end of the reference range for the duration of the study (nadir, 1.55; day of study entry, 1.73; reference range, 2.5–12.5 × 103/μL). A bone marrow aspirate obtained before entry confirmed marked myeloid hypoplasia, and although occasional RBC containing piroplasms were identified in the marrow, schizonts were never seen. Bone marrow from this cat was negative by IFA for FeLV. Although the cat was negative by PCR for Ehrlichia and Anaplasma, a trial course of doxycycline (10 mg/kg/day) was administered for 3 weeks without improvement in neutrophil count before diminazene treatment. Mild neutropenia (2.07–2.48 × 103/μL) was detected in 2 other cats at 3 separate time points, 3 weeks after diminazene treatment (n = 2) or 3 weeks after placebo (n = 1). Neutropenia has not been identified previously as a consequence of chronic C. felis infection, and was not present in all of the cats. Although it is possible that chronic C. felis infection may cause neutropenia in some cats, it is also possible that another comorbid condition went undetected, or that for the most affected cat, the low neutrophil count was normal. Despite demonstrated moderate neutropenia, this cat has remained well for 18 months.
Biochemical profiles and urinalyses were unremarkable from most cats at most time points. Mild hyperglycemia was detected from both semiferal cats after both treatment and placebo treatments, likely as a stress response unrelated to drug therapy. In a single cat, mild hypokalemia (2.8 mEq/L; reference range, 3.0–4.7 mEq/L) was noted at several time points after both diminazene and placebo. Plasma alanine transaminase (ALT) activity was increased (110 U/L; reference range, 18–77 U/L) in 1 cat 3 weeks after diminazene injection. Hepatotoxicity of diminazene has been reported in dogs and camelids, but has not been previously reported in cats.[9-11] Histologic evaluation of 2 cats necropsied 10 weeks after diminazene administration, including the cat that had increased ALT activity, did not disclose any pathologic changes in the liver. Proteinuria (2+ on dipstick testing, 100 mg/dL on sulfosalicylic acid testing) detected in a single concentrated urine sample (specific gravity, 1.069) may have been of lower urinary origin as that sample also contained Capillaria eggs, RBC, epithelial cells, and occasional white blood cells.
Piroplasms were observed in low numbers on every blood smear from every cat at every time point, with a maximum of 3 piroplasms per 1,500 erythrocytes. Because feline erythrocytes can remain in the circulation for approximately 70 days, the study was continued for 10 weeks (enough time that any RBC containing parasites that might have been killed by treatment should have been cleared from circulation).
PCR is a more sensitive means of detecting the pathogen than blood smear review, and the Ct values from real-time PCR offer a semiquantitative measure of the degree of parasitemia. Because of tube mislabeling, samples for real-time PCR were unavailable for 2 cats at week 10. Otherwise, all cats remained C. felis positive by PCR at all time points, and Ct values were similar in the treatment and placebo groups (Fig 1).
Gross lesions were not identified on necropsy of the 2 euthanized cats. Histologic evidence of mild focal lymphocytic pancreatitis and hypertrophic cardiomyopathy was observed in 1 cat. Although it is possible that either diminazene diaceturate may have induced cardiomyopathy or that the finding was somehow related to C. felis infection, it is more likely an unrelated finding because hypertrophic cardiomyopathy has been reported in 15% of apparently normal cats. In the other cat, mild pancreatic amyloidosis was noted but was unlikely to be related to either infection or treatment. No C. felis schizonts were noted in the tissues from either cat.
We demonstrated that 3 mg/kg diminazene diaceturate given IM in 2 doses 1 week apart was unable to eliminate parasitemia in chronic C. felis carrier cats. The dosage chosen was similar to the dosage of diminazene used in a series of 6 cats, 5 of which survived acute cytauxzoonosis. In that study, 2 doses of 2 mg/kg diminazene aceturate were administered 7 days apart. Correcting for difference in molecular weight between the 2 diminazene salts, our dosage of diminazene diaceturate was equivalent to 2.6 mg/kg diminazene aceturate.
Although cytauxzoonosis was once believed to be nearly uniformly fatal in domestic cats, it is now obvious that some portion of domestic cats survive acute infection to become chronic carriers. As with the cats donated for this study, there may be no known antecedent illness suggesting that these cats, like the bobcat (the natural reservoir host), experienced a limited schizogenous phase of infection with only mild illness. Much less is known about the chronic stage of infection than the acute stage. Animals with chronic infection appear well, but it is possible that there may be important sequela to such infections. Although anemia never occurred in the cats studied here, it is possible that parasitized RBC might predispose to hemolytic anemia or that chronic infection and antigenemia could lead to type III hypersensitivity reactions.
Even if chronic C. felis infection does not lead to disease sequela for the individual cat, it might be of importance to the spread of disease. Once they are infected, domestic cats may remain persistently parasitemic.[2, 3] These parasitemic cats might pose an indirect threat to other cats in the area. We now know that domestic cats are capable of transmitting the pathogen to a vector tick that can in turn transmit infection to another susceptible cat. Cytauxzoonosis is an emerging disease with an expanding geographic range. Although the bobcat (Lynx rufus) is the natural reservoir host, it seems plausible that chronically parasitemic domestic cats might play a role in this geographic expansion. In fact, the cats used in this study were donated by owners who did not want chronic carriers on their property for the fear that they increased the risk of infection for other cats in the area. Effective treatment would potentially enable these cats to have remained with their owners.
Although diminazene diaceturate was ineffective at eliminating C. felis from chronic carriers, it may still be efficacious in the treatment of acute cytauxzoonosis. At a dosage of 3 mg/kg administered IM 2 times 7 days apart, adverse effects were transient and mild. Future studies using diminazene to treat cats with acute cytauxzoonosis infections are warranted.
The authors thank Matt Haight, RVT for assistance with sample collection.
Funding: Funding was provided by a nonprofit organization that wishes to remain anonymous and a grant from the University of Missouri – College of Veterinary Medicine.
Conflict of Interest Declaration: Authors disclose no conflict of interest.
SNAP FeLV/FIV Combo test, IDEXX Laboratories, Westbrook, ME