vet

1. Efficacy of Atovaquone and Azithromycin or Imidocarb Dipropionate
in Cats with Acute Cytauxzoonosis
L.A. Cohn, A.J. Birkenheuer, J.D. Brunker, E.R. Ratcliff, and A.W. Craig
Background: Imidocarb or a combination of atovaquone and azithromycin (A&A) has been suggested for treatment of cats
with cytauxzoonosis, but neither has been prospectively evaluated for efficacy.
Hypothesis/Objectives: That survival to hospital discharge is improved by treatment with A&A as compared with imido-
carb.
Animals: Eighty acutely ill cats with Cytauxzoon felis infection treated at one of 18 veterinary clinics in 5 states.
Methods: An open-label, randomized prospective study compared survival in cats treated with atovaquone (15 mg/kg PO
q8h) and azithromycin (10 mg/kg PO q24h) or imidocarb (3.5 mg/kg IM). All received heparin, fluids, and supportive care.
Clinical and clinicopathologic data from initial presentation were collated. Parasitemia was quantified (n 5 79) and pathogens
genotyped (n 5 60). Logistic regression was used to determine the impact of treatment group on the primary endpoint, survival
to hospital discharge or death. Covariants were analyzed by rank-sum testing.
Results: Of 53 cats treated with A&A, 32 (60%) survived to discharge while only 7 of 27 cats (26%) treated with imidocarb
survived (P 5 .0036; odds ratio 7.2, 95% CI 2.2, 24). Cats with a lower parasitemia were more likely to survive, as were cats with
higher white blood cell counts and lower total bilirubin. Unique pathogen genotypes were identified from 15 cats, while geno-
type isolated from 21 cats had been described previously. There were multiple pathogen genotypes identified in 24 cats.
Conclusions and Clinical Importance: Survival to discharge was more likely in cats treated with A&A as compared with
imidocarb, although case fatality rate remained high.
Key words: Antimicrobials; Clinical Trials; Microbiology; Molecular biology; Parasitology; PCR assays; Protozoa.
Cytauxzoonosis is an acute-onset disease of cats
caused by the apicomplexan parasite Cytauxzoon
felis, which is transmitted by the bite of a tick vector.
Historically considered a fatal infection in domestic cats,
in recent years a number of cats that survived the disease
have been described.1–7
Cats that survive the disease can
overcome a severe illness with or without specific antimi-
crobial drug therapy, or persistent infection can be
discovered in healthy cats with no known preceding ill-
ness. The severe acute illness is believed to be caused by
the schizogenous phase of parasite development during
which parasite-distended mononuclear cells cause occlu-
sion of blood vessels. After several days, merozoites are
released from ruptured cells and taken up by red blood
cells (RBCs), producing the classic signet ring shaped
merozoites frequently used to diagnose the infection on
blood smear. While the schizogenous phase of infection
only appears to last for days to weeks, merozoite infected
RBCs can be detected for long periods in cats that sur-
vive acute infection.
To date, no antiprotozoal therapy has been demon-
strated to alter the disease course in acutely infected cats.
Survival was not enhanced in experimentally infected cats
treated with parvaquone or buparvaquone.6
Case reports
exist of survival in naturally infected cats treated with dimi-
nazene aceturate, an antiprotozoal agent used for the
treatment of babesiosis and trypanosomiasis, but the drug
is not approved for use in the United States.2
Despite a lack
of proven efficacy, the standard therapy for cytauxzoonosis
has become imidocarb dipropionate.1,8
A combination of
an antimalarial drug atovaquone with the antibacterial
drug azithromycin has been successful in the management
of other haemoprotozoan infections that were notoriously
difficult to treat.9,10
This led the authors to investigate the
utility of this drug combination for the treatment of
cytauxzoonosis. Some efficacy was demonstrated in a pre-
liminary, uncontrolled trial of naturally infected cats.a
Further, the combination was more effective than imido-
carb dipropionate alone in reducing the parasitemia of
healthy but persistently infected cats.b
We hypothesized
that treatment of naturally infected, ill cats with atovaqu-
one and azithromycin (A&A) would result in improved
survival as compared with treatment of an equivalent
group of cats with imidocarb dipropionate. An open label,
From the Department of Veterinary Medicine and Surgery, Uni-
versity of Missouri, Columbia, MO (Cohn); Department of Clinical
Sciences, North Carolina State University, Raleigh, NC (Birken-
heuer); Oklahoma State University, Stillwater, OK (Brunker); Fair
Grove Veterinary Services, Fair Grove, MO (Ratcliff); and the
Lebanon Animal Hospital, Lebanon, TN (Craig). This study was de-
signed jointly at the University of Missouri (UMC) and North
Carolina State University (NCSU). An abstract of this work was
presented at the ACVIM Forum in Anaheim, CA, June 2010. It was
managed and data collated at UMC. Genetic testing and PCR testing
were conducted at NCSU.
Corresponding author: L. A. Cohn, University of Missouri, 900 E.
Campus Drive, Columbia, MO 65211; e-mail: cohnl@missouri.edu.
Submitted July 8, 2010; Revised October 5, 2010; Accepted
October 7, 2010.
Copyright r 2010 by the American College of Veterinary Internal
Medicine
10.1111/j.1939-1676.2010.0646.x
Abbreviations:
A&A atovaquone and azithromycin
CT cycle threshold
IQR interquartile range
ITS internal transcribed spacer
NCSU North Carolina State University
PCR polymerase chain reaction
RBC red blood cells
UMC University of Missouri
VMTH Veterinary Medical Teaching Hospital
J Vet Intern Med 2011;25:55–60

2. imbalanced randomized block designed prospective clinical
trial was conducted to assess the validity of this hypothesis.
Materials and Methods
Study Design
The goal of this multicenter, open-label parallel-group study with
imbalanced block randomization (2:1) was to compare efficacy of
treatment with A&A or imidocarb for acute cytauxzoonosis in natu-
rally infected cats. Cats were treated in a variety of clinical settings
ranging from small, single veterinarian rural clinics lacking 24-hour
care to university-associated veterinary medical teaching hospitals
(VMTH). The vast majority of case recruitment occurred during the
spring and summer of 2009, although 5 cases were enrolled before 2009.
Enrollment Criteria
Cats included in the study were presented for evaluation of an
acute illness with signs suggestive of cytauxzoonosis, including fe-
ver, lethargy, anorexia, or icterus and cytologic demonstration of
suspected piroplasms within RBC or schizonts in either blood
mononuclear cells or from aspirates of the lymph nodes, liver, or
spleen. Cytologic evaluation was conducted by the veterinarian
responsible for enrolling the cat.
Participating veterinary clinics were solicited from areas consid-
ered endemic for C. felis based on expressed interest in study
participation. All participating veterinarians agreed to abide by
provided instructions for evaluation and treatment, including com-
pliance with a predetermined order of randomization. Once a
veterinarian or veterinary clinic agreed to participate, they were
supplied with study drugs adequate to treat 3 cats, detailed instruc-
tions, order of randomization, and owner consent forms.
At presentation, the treating veterinarian was responsible for ex-
plaining the study to the cat’s owner, including potential benefits
and harms and the option to withdraw from the study. Study drug
was supplied free of charge and additional monetary compensation
was provided to offset some of the costs of veterinary care. Owners
were invited to contact the lead investigator with questions and were
asked to sign an informed consent document before inclusion of
their cat in the study.
Exclusion criteria were applied both at the time of enrollment
and retrospectively. Cats presented with body temperature o37.81C
(
o100.01F) or in a moribund condition were excluded immediately.
Cats were excluded retrospectively if C. felis DNA could not be de-
tected by polymerase chain reaction (PCR), if medical records were
not made available to the investigators, or if there was a failure to
adhere to randomization order or treatment instructions.
All procedures and consent forms were reviewed and approved
by the animal care and use committees of the University of Missouri
(UMC), North Carolina State University (NCSU), and Oklahoma
State University.
Randomization and Allocation
An imbalanced blocked randomization of 2 : 1 (A&A and imido-
carb, respectively) was used. Three pieces of paper, 2 marked with
‘‘A&A’’ and one with ‘‘imidocarb,’’ were folded and placed in a bowl.
For most cats, a technician at UMC withdrew the papers one at a
time to determine the order of treatment for the 1st 3 cats enrolled at
each participating veterinary clinic; the clinic was supplied with this
order at the same time they were supplied with study drugs. When the
clinic began treating the 3rd cat enrolled, they contacted the techni-
cian who repeated the procedure and supplied additional drug and a
new randomization order for the next 3 cats enrolled, repeating the
procedure as necessary. For 15 cats treated at 1 VMTH (NCSU),
randomization was determined in identical fashion onsite.
Test Treatments
Immediately after enrollment treatment was begun with either a
combination of A&A or with imidocarb. Atovaquonec
was admin-
istered PO or by means of enteral feeding tube at a dose of 15 mg/kg
q8h for 10 days. Administration along with a fatty meal was sug-
gested but not required. Azithromycind
was administered PO or by
means of enteral feeding tube at a dose of 10 mg/kg q24h for 10
days. Cats that received imidocarb were pretreated with atropine
(0.05 mg/kg) SC 15 minutes before injection of 3.5 mg/kg imidocarb
dipropionatee
IM. Injection was repeated in the same fashion 7 days
later.
Concomitant Treatments
Besides treatment with study drug, therapeutic management was
largely left to the discretion of the treating veterinarian, with 2
exceptions. All cats received unfractionated heparin (200 U/kg) SC
q8h for the duration of hospitalization, but the duration of
hospitalization (and therefore heparin use) was decided by the vet-
erinarian treating the cat based on their clinical judgment. Provided
guidelines suggested that hospital discharge was appropriate when
the cat was afebrile and eating voluntarily. Similarly, all cats
received intravenous crystalloid fluids initially, but fluid type, vol-
ume, and duration were left to the treating veterinarian. Guidelines
suggested the use of any of several types of balanced saline solutions
at a rate of 50–100 mL/kg/d. As with heparin administration, dis-
continuation of fluid therapy was left to the discretion of the
treating veterinarian. Adjunctive and supportive care including
blood transfusion, the use of analgesics, antibiotics, antiemetics,
and appetite stimulants was allowed and all such treatments were
documented. Placement of esophagostomy tubes was specifically
approved to ease administration of medication as well as to provide
nutritional support.
Clinical Evaluation
Available clinical information varied. In all cases, historical in-
formation was requested in regards to signalment, housing (indoor/
outdoor), duration of illness, ectoparasite application, health of
other cats within the household, and clinical signs prompting the
veterinary visit. Information requested from the initial physical ex-
amination included the cat’s temperature, pulse and respiratory
rate, body condition score, and any other identified abnormality.
Anticoagulated whole blood was collected for attempted PCR de-
tection of C. felis. Although assessment of CBC, serum biochemical
profile, coagulation status, and retroviral testing were suggested,
they were not required for participation. When tests were repeated,
only results from the initial assessment were recorded. Additional
recorded information included the primary endpoint (survival to
discharge or death), the duration of hospitalization, and all forms of
therapy administered. Copies of all medical records were sent to one
of the investigators (L.A.C.), who collated and verified the data by
dual entry.
Molecular and Genetic Testing
The persons conducting all molecular and genetic testing were
unaware of assigned treatment. Anticoagulated blood was sent
directly to NCSU for PCR detection of C. felis using previously
published methods with minor modifications.11
Briefly, each reac-
tion consisted of 12.5 mL 2 PCR master mix,f
7 mL water, 50 pmoL
of each oligonucleotide primer, and 5 mL sample. Thermal cycling
conditions were 981C for 30 seconds followed by 45 cycles 951C for
5 seconds and 601C for 5 seconds. Melting curve analysis was initi-
ated at 751C and data were captured at increasing increments of
0.51C for 30 time points. Internal transcribed spacer (ITS) region
56 Cohn et al

3. genotyping was performed as described previously with minor mod-
ifications.3,7,12
Each 50 mL reaction contained a 1 concentration of
PCR buffer II, 1.25 U of taq polymerase,g
50 pmoL of each primer,
1.5 mM MgCl, 200 mmoL of each dNTP, 33.5 mL of water, and 5 mL
of each sample. Thermal cycling conditions were: 951C for 5 min-
utes followed by 45 cycles 951C for 45 seconds, 541C for 45 seconds,
and 721C for 45 seconds. ITS amplicons were visualized by ethidium
bromide staining and ultraviolet light transillumination after elec-
trophoresis in a 1.5% agarose gel. ITS amplicons were purified and
sequenced bidirectionally. Each chromatogram was inspected and
contigs were assembled by commercially available software pack-
age.h
A threshold for calling secondary peaks within each
chromatogram was set at 50%. For each PCR assay, positive (pre-
viously characterized C. felis samples) and negative (no template)
controls were used. Standard precautions were used to prevent
amplicon carryover.
Statistical Analysis
The primary endpoint with respect to treatment efficacy was
survival to hospital discharge, or death (including euthanasia). Lo-
gistic regression was applied to the dichotomous outcome by the
LOGISTIC procedure for primary analysis. The Hosmer-Leme-
show statistic was used to assess the model fit. Descriptive statistics
were also determined. Two categorical variables were tested for in-
dependence by Chi-square tests while categorical groups were
compared with each other on numeric variables (temperature, total
bilirubin, PCV, white blood cell [WBC] count, cycle threshold [CT])
by Wilcoxon’s rank-sum tests. Calculations were performed by
standard statistical software,i
and significance was set at P o .025,
with marginal significance at P o .05 but P 4 .025.
Results
Demographics
Eighteen veterinary clinics, including 2 university-
associated VMTH and 16 private practice clinics,
participated in recruitment of 97 cats with suspected
cytauxzoonosis from 5 states. A median of 3 cats per
clinic were enrolled (range, 1–20). Of the 97 cats, 17 were
later excluded due to either a negative PCR result for C.
felis (n 5 11) or failure to adhere to study protocol (6). Of
the 80 cats included in statistical analysis of outcome, 53
received AA and 27 received imidocarb. Twenty of
these cats were treated at a VMTH and 60 received treat-
ment at a private practice. Cats included in the analysis
were recruited from Missouri (n 5 28), Tennessee (21),
North Carolina (15), Arkansas (10), and Oklahoma (6).
Breed, age, and sex were documented. Most cats were
domestic short hair or mixed breeds (n 5 71), while oth-
ers were domestic long haired (6), Siamese (2), or Bengal
(1). Cats ranged in age from 6 months to 12 years, with a
mean of 3.6 2.9 years; for 12 cats, age was recorded
simply as ‘‘adult.’’ Forty-three cats were male (10 intact,
33 neutered) and 35 were female (2 intact, 30 neutered, 3
unknown), and the sex of 2 cats was not recorded.
Baseline Clinical Evaluation
Findings on history and physical examination were
nonspecific. All infected cats had exposure to the out-
doors. The most common historical complaints were
lethargy (n 5 78), and anorexia (60). Vomiting (usually
once or twice) was reported in 6 cats. Other complaints
included unsteady gait (3), abnormal behavior (1), abor-
tion (1), and hematuria (1). The most common
abnormalities on physical examination included hyper-
thermia (temperature 439.21C [102.51F]; n 5 72),
icterus (31), elevated nictatans (31), dehydration (22),
the presence of ticks (22), tachypnea (respiratory rate
440 breaths per minute; 20), tachycardia (heart rate
4200 beats per minute; 13), pallor (9), murmur (8),
vocalization (5), discomfort on abdominal palpation (5),
lymphadenomegaly (5), and splenomegaly (5). Abortion,
stupor, gallop rhythm, muscle wasting, ear mites, ab-
scess, and disorientation were each found in a single cat.
Temperature range in the 78 cats with legible recorded
temperature was 38.3–41.71C (101.0–107.01F). The mean
temperature of cats randomized to receive AA (104.5
1.21F; 40.31C) was identical to that of cats randomized to
receive imidocarb (104.5 1.11F; 40.31C).
Initial laboratory findings, other than piroplasms or
schizonts, were also nonspecific and did not differ be-
tween treatment groups. Abnormalities on CBC (Table
1) included leukopenia (WBC o 5 103
/mL; n 5 43 of
73) and anemia (PCV o 26%; 40 of 74). While
thrombocytopenia was noted on 59 samples, adequate
information about platelet clumping was not available to
assess the validity of this finding. Common biochemical
abnormalities included hyperbilirubinemia (total biliru-
bin 4 0.5 mg/dL; 37 of 50), hyperglycemia (glucose
4150 mg/dL; 35 of 55), and hypocalcemia (Ca o 9.0
mg/dL; 32 of 47) (Table 1). Total solids or total protein
below the lower limits of the reference range (
o6.0 g/dL)
Table 1. Initial laboratory findings in cats with cytauxzoonosis treated with either AA or imidocarb dipropionate.
All Cats AA Imidocarb
PCV (%) 25.5 5.9 (74) 25.95 6.3 (49) 24.3 4.5 (25)
WBC 103
/mL 5.1 2.8 (73) 5.26 2.6 (48) 4.53 3.0 (25)
Total solids or protein (g/dL) 6.6 1.0 (51) 6.7 1.0 (35) 6.3 0.7 (16)
Total bilirubin (mg/dL) 2.3 2.0 (50) 2.15 1.8 (36) 2.74 2.4 (14)
Glucose (mg/dL) 169 43 (55) 169 42 (38) 169 45 (17)
Calcium (mg/dL) 8.4 0.9 (47) 8.4 0.9 (32) 8.1 0.8 (15)
BUN (mg/dL) 29.0 21.3 (56) 27.5 18.4 (38) 32.2 26.5 (18)
ALT (U/L) 68.3 99.4 (55) 58.9 65.2 (37) 102 143 (18)
WBC, white blood cell; AA, atovaquone and azithromycin; ALT, alanine transaminase; BUN, blood urea nitrogen.
Mean SD (n).
57
Cytauxzoonosis Treatment of Cats

4. were detected in 12 of 51 cats. Although frequently
assayed, blood urea nitrogen and ALT were rarely out-
side of reference ranges. Thirty-eight cats underwent
retroviral testing; only a single cat was positive for feline
leukemia virus.
Because of limitations of sample size and number of
cats tested for specific variables, only temperature, PCV,
WBC count, and total bilirubin were statistically evalu-
ated as potential covariants influencing outcome. Neither
temperature nor PCV had a significant relationship to
survival or death in hospital. Rank-sum tests comparing
treatment relative to WBC (P 5 .0014) or total bilirubin
(P 5 .031) did indicate significance or marginal signifi-
cance. Cats that survived were likely to have a higher
WBC count (Fig 1) and lower total bilirubin (Fig 2) than
cats that died.
Concomitant and Adjunctive Therapies
A variety of supportive and adjunctive therapies were
administered. Thirty cats received only the drugs required
for study participation. Blood transfusions were adminis-
tered to 9 cats. Antimicrobials administered in addition to
the study drugs included cefazolin (n 5 11), enrofloxacin
(9), ampicillin-sulbactam (7), doxycycline (5), amoxicillin-
clavulanate (3), amoxicillin (2), ampicillin (2), and penicillin
(2), and 1 cat each received marbofloxacin, clindamycin,
and gentamicin. Several cats received analgesic, antiinflam-
matory, or antipyretic drugs. These included buprenor-
phine (14), meloxicam (10), ketoprofen (5), fentanyl (1),
dexamethasone (3), prednisone/prednisolone (3), and meta-
mizole (1). Some cats additionally received antiemetic
medications, gastric protectants, or appetite stimulants.
These included odansetron (5), dolasetron (3), maropitant
(3), metoclopramide (3), famotidine (3), ranitidine (1),
sucralfate (2), mirtazpine (2), cyproheptadine (2), or intra-
venous diazepam (1). Other concomitant drug therapies
included B vitamins (3), and lactulose (3), and to 1 cat each
glucosamine, SAMe, drontal, fenbendazole, nitenpyram,
flumazenil, furosemide, glucosamine, enalapril, and Delta
albeplex.j
It was rare for cats treated at private practices to
receive more than a single adjunctive therapy (usually an
analgesic or antibiotic), while all cats treated at VMTH
received from 2 to 14 additional therapies during hospital-
ization. Esophagostomy feeding tubes were placed in 8 cats
treated at a VMTH.
Effect of Therapy on Outcome
Of 80 cats included in data analysis, 39 survived and 41
died. Of the 41 that died, 5 were euthanized because of
severe clinical deterioration and moribund condition.
Twenty-four of 41 cats that died did so the day of or the
day after presentation for care; only 3 cats died or were
euthanized more than 3 days after presentation. Of the 53
cats treated with AA, 32 (60%) survived and 21 (40%)
died before hospital discharge. Of the 27 cats treated with
imidocarb, 7 survived (26%) and 20 died (74%). Survival
was greater in cats treated with AA than in cats treated
with imidocarb (P 5 .0036; odds ratio 7.2; 95% CI 2.2,
24.0).
Parasitemia
CT determined from real-time PCR was used as an
estimate of parasitemia in 79 cats. There was no differ-
ence between CT from cats randomized to treatment
with AA as compared with treatment with imidocarb
(Table 2; P 5 .79). Parasitemia was lower in survivors as
compared with nonsurvivors for cats receiving either
treatment (Table 2; P 5 .0012). The odds ratios for
this continuous variable suggest that for each unit of in-
crease in CT, odds for survival were 1.3 times higher
(95% CI 1.1, 1.5). Sample size limitations prevented sta-
tistical analysis of treatment and CT interaction, but
mean CT was lower (therefore parasitemia higher) in
Fig 1. Box-plot of WBC counts in cats that survived to hospital
discharge or died in hospital. Each box represents the interquartile
range (IQR) from the 25th to 75th percentile. The line inside the box
represents the median, ‘‘1’’ represents the mean, and ‘‘’’ represent
statistical outliers (values more than 1.5 IQR beyond the 1st and 3rd
quartiles). Whiskers extend to the maximum and minimum values
within 1.5 IQR above or below the 75th and 25th percentile. WBC
count was significantly higher in cats, which survived to discharge
(P 5 .0014).
Fig 2. Box-plot of total bilirubin in cats that survived to hospital
discharge or died in hospital. Each box represents 25th–75th per-
centile of values, while the line represents the median and the ‘‘1’’
represents the mean. Range is indicated by the whiskers. Total
bilirubin was marginally lower in cats that survived to discharge
(P 5 .031).
58 Cohn et al

5. survivors treated with AA than in survivors treated
with imidocarb.
ITS Genotype of Pathogen
ITS genotyping of the pathogen was attempted in 64 of
80 cats sampled. A complete genotype (ie both ITS1 and
ITS2) could be unambiguously resolved for 36 of 64 cats.
For 15 of 36, unique ITS genotypes not previously de-
scribed were detected while 21 of 36 corresponded with
previously described ITS genotypes.3,7,12
Of these, 14
corresponded with ITSA/ITSc, (Genbank accession
EU450802/EU450804), 5 corresponded to ITSj, (Gene-
bank accession FJ536426/FJ536418) and 2 corresponded
with ITSC (Genbank accession EU450803/EU450804).
Of the cats infected with pathogen of the ITSA/ITSc ge-
notype, 8 received AA (7 survived) and 6 received
imidocarb (2 survived). Of the cats infected with the ITSj
genotype 3 received AA (all survived) and 2 received
imidocarb (1 survived). Both cats infected with ITSC
genotype received AA; 1 survived and 1 died.
For 24 of 67 cases, the sequence chromatograms were
consistent with the presence of 2 or more genotypes with
insertions or deletions in 1 or both ITS regions. For 4 of
67 cases resolution of the ITS regions was not possible
because of poor sequence quality. Attempts to further
classify the ITS genotypes (ie, cloning and sequencing of
multiple clones) from these samples were not pursued.
Discussion
This study demonstrates that cats with acute cytauxzo-
onosis treated with AA had improved survival to hos-
pital discharge (60%) as compared with a similar
population treated with imidocarb dipropionate (26%).
The relatively small study size precluded statistical
evaluation of all potential clinical or laboratory predic-
tors of treatment outcome. Subjective evaluation of
demographic data, historical, and physical findings did
not suggest important or obvious differences between
cats that survived infection and those that died. Most
cats, regardless of outcome, were young adults with ex-
posure to the outdoors and vague clinical signs of
lethargy and anorexia. Similarly, the mean initial
temperature of survivors was identical to that of non-
survivors, and cats with either outcome demonstrated
dehydration or icterus. While initial PCV was unrelated
to outcome, cats with higher initial WBC count
and lower total bilirubin were statistically more likely to
survive. Unfortunately, important overlap and modest
biological differences in values between survivors and
nonsurvivors invalidate these parameters as outcome
predictors.
Parasitemia, as determined by real-time PCR, did not
differ in cats treated with AA or imidocarb. However,
cats administered either treatment protocol were more
likely to survive in the face of a lower parasitemia. The
effect of parasitemia was partially mitigated by treatment
with AA, suggesting that cats with a higher parasitemia
may be more likely to survive if treated with AA. The
contributions of the life stages (merozoites or schizonts)
to the overall parasitemia cannot be determined by a
PCR assay detecting genomic DNA. Each infected RBC
typically contains a single merozoite with a single ge-
nome copy, while each infected macrophage contains a
multinucleated schizont that may contain thousands of
genome copies. Therefore, clinicians should not attempt
to determine a prognosis based on the number of me-
rozoite infected RBCs.
Reports have suggested differences in survival based on
geographic region, prompting speculation that genetic
differences in pathogen virulence occur in geographic
proximity.1,3,12
While our study was not designed to iden-
tify geographic associations with outcome, similar
proportions of cats lived or died in each state (data not
shown). Genotypic variation in pathogens was investi-
gated by analysis of noncoding regions of the rRNA
operons known as ITS regions 1 and 2. Prior investigation
of pathogens in domestic cats and bobcats has identified
distinct pathogen genotypes. In our study, 24 pathogens
could not be resolved to a single genotype because of the
presence of insertions or deletions in 1 or both ITS re-
gions. These results might reflect polymorphisms in
multiple copies of the rRNA operons or coinfection with
multiple genotypes.7
Although initial studies seemed to
suggest that certain genotypes (ITSa/ITSc) were more
likely to be associated with a less pathogenic variant of C.
felis than other genotypes (ITSC), it now seems unlikely
that ITS genotype can predict pathogenicity.3,7,12
Cats in this study received treatment in any of several
veterinary clinics using a number of different adjunctive
therapies. As predicted before the study, adjunctive thera-
pies tended to be similar between all cats treated at a given
clinic. For instance, clinics tended to give all cats or no cats
adjunctive antibiotics, and when antibiotics were used the
same type of antibiotic was usually used in all cats from a
single clinic. Similarly, clinics tended to use the same type
of analgesic (or no analgesic) in treated cats. Randomiza-
tion of cats at each clinic to a given treatment protocol by
block was designed to minimize impact of treatment vari-
ation by ensuring that no single clinic would treat large
numbers of cats with only 1 of the 2 study protocols. The
variety of treatments applied in a variety of clinic types re-
sulted in a study that more closely mimicked a realistic
application of treatment than would a study conducted en-
tirely within a single practice. However, nonuniformity of
both data collection and treatments can also be viewed as a
Table 2. Cycle threshold from real-time PCR in 79 cats
with cytauxzoonosis treated with either AA or imido-
carb dipropionate.
AA Imidocarb All Treatments
Survivors 26.5 4.3 31.3 2.7 27.4 4.4
(17–37.5) (28.1–36.7) (17–37.5)
Nonsurvivors 24.6 4.1 24.4 2.7 24.5 3.4
(19.8–36.0) (20.9–31.5) (19.8–36.0)
All outcomes 25.8 4.24 26.2 4.0 25.9 4.2
(17–37.5) (20.9–36.7) (17–37.5)
AA, atovaquone and azithromycin.
Mean SD (range).
P 5 .0012.
59
Cytauxzoonosis Treatment of Cats

6. limitation of the study in that effects of concomitant treat-
ments may have altered outcome.
The open label nature of the study allowed the intro-
duction of bias. Masking was considered impractical
because 1 protocol involved daily oral medication whereas
the other involved injections. Additionally, pretreatment
of cats with atropine would be easily distinguished from
pretreatment with placebo based on observable reactions
to the medication. Outcome bias was minimized by using
the simple end point of survival or death during hospital-
ization. Investigators who conducted genetic analysis of
pathogens from sampled cats were unaware of treatment
arm, thereby minimizing bias regarding genotypic results
or pathogen burden in any given cat.
Because no placebo treatment was included in the trial,
it is impossible to speculate whether treatment with
imidocarb dipropionate offered any advantage over sup-
portive care alone. All of the veterinarians participating
in our trial had routinely treated infected cats with imido-
carb in years past. Most of these veterinarians espoused a
belief that very few cats treated with imidocarb survived
the acute illness, a belief that prompted their participa-
tion in the trial. While both our study and multiple case
reports document survival of cats treated with imido-
carb, anecdotal mention is made in a retrospective report
of 18 surviving infected cats (only 1 of which was treated
with imidocarb) that imidocarb lacked efficacy in the
treatment of experimentally induced cytauxzoonosis.1,2,5
This randomized prospective treatment trial demon-
strated improved survival in cats with acute cytaux-
zoonosis treated with AA as compared with treatment
with imidocarb dipropionate. Nonetheless, mortality re-
mained high with 40% of AA treated cats dying in
hospital. The course of illness with cytauxzoonosis is swift,
and most cats that succumbed did so shortly after presen-
tation for care. It may well be that even effective anti-
protozoal therapy cannot reverse the course of clinical
illness. Efforts must be focused on prevention of disease,
either through minimization of exposure to tick vectors or
through development of chemoprophylaxis or vaccination.
Footnotes
a
Birkenheuer AJ, Cohn LA, Levy MG, et al. Atovaquone and
azithromycin for the treatment of Cytauxzoon felis. J Vet Intern
Med 2008;22:703 (abstract)
b
Cohn LA, Birkenheuer AJ, Ratcliff E. Comparison of two drug
protocols for clearance of Cytauxzoon felis infections. J Vet Intern
Med 2008;22:704 (abstract)
c
Mepron, GlaxoSmithKline, Research Triangle Park, NC
d
Azithromycin suspension, Greenstone LLC, Peapack, NJ
e
Imizol, Intervet Schering-Plough Animal Health, Summit, NJ
f
SsoFast EvaGreen Supermix Bio-Rad, Hercules, CA
g
Amplitaq gold, Applied Biosystems by Life Technologies, Carls-
bad, CA
h
ContigExpress, Vector NTI Advance 11.0, Invitrogen by Life
Technologies, Carlsbad, CA
i
SAS v9; SAS Institute Inc, Cary, NC
j
Delta albeplex, Pfizer Animal Health, New York, NY
Acknowledgments
Cats were enrolled and received treatment at 18 animal
clinics in Missouri (Fair Grove Veterinary Services; All
Creatures Veterinary Hospital; Animal Clinic of West
Plains; Best Friends Animal Hospital; Cross Creek Ani-
mal Hospital; Emergency Veterinary Clinic of Southwest
Missouri; Gainesville Veterinary Clinic; Mid Ozark
Animal Health Center), Arkansas (Russellville Animal
Clinic; Eureka Springs Animal Hospital; Southwind An-
imal Hospital; Sugar Creek Animal Hospital),
Oklahoma (Animal Care Central; Oklahoma State Uni-
versity-VMTH), Tennessee (Lebanon Animal Hospital;
DeKalb Animal Clinic; VCA Animal Care Center), and
North Carolina (NCSU).
The work was supported by a charitable entity, which
prefers to remain anonymous.
The authors acknowledge all of the veterinarians par-
ticipating in the care of these cats, as well as the expert
technical assistance of Ms Victoria Catto and Mrs Ginny
Dodam.
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