1. Introduction
Cytauxzoonosis is an often-fatal disease of domestic cats caused by
Cytauxzoon felis, a tick-borne hemoprotozoal pathogen of felids. In the United States (U.S.), cytauxzoonosis cases most frequently occur in southeastern and south-central regions. This protozoal organism has a complex lifecycle that includes asexual reproduction in felid hosts and both asexual and sexual reproduction in competent ixodid tick vectors [
1] (
Figure 1). Briefly,
C. felis sporozoites are transferred via tick saliva into a felid host during a blood meal. Once within the felid host, sporozoites enter monocytes and begin replicating asexually (schizogony) forming many 1–2 μm diameter signet ring merozoites. When the monocyte ruptures, these merozoites are released into the blood, where they enter host erythrocytes and begin either replicating asexually (merogony) or develop into gametocytes. Ticks become infected when they feed on an infected felid host and ingest gametocytes. The ingested gametocytes undergo sexual reproduction to form zygotes in the lumen of the tick gut. Zygotes invade the tick gut epithelium, transform into kinetes, and migrate to the salivary glands where they transform into sporozoites. Transstadial maintenance of the parasite through the larvae-to-nymph or nymph-to-adult ecdysis is required for the transmission of the infectious sporozoites to a new felid host during the subsequent tick bloodmeal. The competent biological transmission vectors of
C. felis in the U.S. include
Dermacentor variabilis (American dog tick) and
Amblyomma americanum (Lone star tick) [
2,
3,
4,
5]. The range of
D. variabilis encompasses the eastern U.S. as well as focal regions in the west [
6,
7]. The continuously expanding range of
A. americanum covers the southeastern and mid-central U.S., largely overlapping with the range of
D. variabilis [
8,
9]. Larval and nymphal
D. variabilis prefer to feed on small to medium-sized mammals, whereas adults feed on larger mammals, including cats. In contrast,
A. americanum are less discriminating, such that all life stages will seek out and feed on many mammals, including cats. Because of intensifying populations and its aggressive nature and willingness to feed on cats at all life stages, it is likely that
A. americanum is a more significant vector of
C. felis compared to
D. variabilis in areas where both tick species reside.
Clinical cytauxzoonosis cases are most commonly observed in domestic cats (
Felis catus), which serve as intermediate host carriers for
C. felis along with other members of the Felidae family. Domestic cat cytauxzoonosis cases typically peak in late spring and early fall when the
A. americanum adult and nymph life stages are most active [
11]. For many decades, domestic cats were considered a dead-end host, as those with observed clinical disease commonly died [
12,
13,
14]. Clinical disease signs for acute cytauxzoonosis appear during the schizogenous phase of
C. felis asexual replication and can cause severe illness and death [
2,
12]. Cats commonly present with anorexia, depression, lethargy, pyrexia, dehydration, pale mucous membranes, and splenomegaly 11–13 days post infection (dpi) [
2,
15,
16]. As the disease progresses, the pyrexia resolves and drops to subnormal, with icterus and dyspnea developing shortly thereafter. Complete blood count and serum biochemistry changes are seen around 13 dpi and can include variable cytopenias, hypoalbuminemia, hyperglycemia, and increased ALT (alanine aminotransferase) and total bilirubin [
2,
14,
16]. Intra-erythrocytic signet rings (also known as piroplasms) and/or schizont-laden monocytes are often observed at the feathered edge of thin blood smear preparations [
2]. The organs most affected include the lung, liver, spleen, and lymph nodes [
13,
14,
16,
17,
18]. Death occurs around 21 dpi due to inflammatory cytokine-mediated injury of multiple organs, interstitial pneumonia, hypoxic injury, and disseminated intravascular coagulation [
2,
11,
12,
17,
18,
19,
20]. If initiated early in the disease process, treatment for ten days with a combination of atovaquone (15 mg/kg) and azithromycin (10 mg/kg) may result in the recovery of up to 60% of patients [
21]. For survivors, clinical signs begin to resolve around 23–24 dpi, after which they become subclinical infected carriers that act as reservoirs for further
C. felis transmission via competent tick vectors [
2,
22]. Domestic cat carriers of
C. felis may be more common than previously thought, as we recently identified
C. felis carrier infection in 25% of cats in eastern Kansas [
10]. Similar clinical signs and disease progression have been reported with
Cytauxzoon spp. in South America, Europe, and Asia [
23,
24,
25,
26].
Only a few studies have examined the incidence and risk factors associated with acute cytauxzoonosis cases in U.S. domestic cats; however, the conclusions of these studies vary. For example, investigation into cytauxzoonosis incidence trends varied from stable [
11] to increasing [
27], while evaluation of specific risk factors varied from identifying a predisposition of disease in young male cats [
16] to tick climate and habitat conditions being more predictive of the disease rather than cat age and gender [
28]. Anecdotal reports of increasing acute cytauxzoonosis cases, as well as heightened public awareness of this disease, makes understanding cytauxzoonosis and associated patient risk factors important for mitigating disease transmission among high-risk cat populations. To address these concerns in eastern Kansas, the objectives of this study were to: (i) determine if acute cytauxzoonosis cases in eastern Kansas increased between 2006–2019; and (ii) examine if specific feline risk factors are correlated with acute disease or disease outcomes, by performing a retrospective records search and review of acute cytauxzoonosis cases submitted to the Kansas State Veterinary Diagnostic Laboratory (KSVDL). Based on the high density of
A. americanum ticks in eastern Kansas and feline risk factors reported as important by cytauxzoonosis studies in other states, we hypothesized that cases of acute cytauxzoonosis would: (i) show an increasing trend over time; (ii) peak in seasons of peak
A. americanum activity; and (iii) be reported more commonly in young, intact, male cats. Updated information on acute cytauxzoonosis incidence and risk factors will help identify and focus
C. felis education and intervention efforts for high-risk domestic cat populations.
4. Discussion
In
C. felis endemic regions, acute cytauxzoonosis is one of the most fatal feline diseases. Several retrospective studies have demonstrated a bimodal pattern of seasonal cytauxzoonosis cases that correlates with
A. americanum activity; young male cats being over-represented [
8,
11,
16,
27,
28,
33]. As a disease with a high fatality rate, increased public concern, and practitioner belief of increased incidence, understanding factors contributing to acute cytauxzoonosis can help focus efforts on preventing
C. felis transmission among high-risk cat populations. In the present retrospective study, we demonstrate: (1) acute cytauxzoonosis case incidence in domestic cats from eastern Kansas has an overall stable trend; (2) acute cytauxzoonosis cases are more common in the spring and summer seasons; (3) young male cats are more likely to be diagnosed with acute cytauxzoonosis; and (4) schizont and/or intra-erythrocytic signet ring identification on histology or blood smear samples are reliable identifiers of acute cytauxzoonosis when associated with typical cytauxzoonosis clinical signs.
This study did not support anecdotal reports of an increasing acute cytauxzoonosis case trend in eastern Kansas cats based on KSVDL case sample submissions over the study period (
Figure 2). Rather, the anecdotal reports of increasing acute cytauxzoonosis cases may be more attributed to individual year-to-year case fluctuations instead of a general increasing trend over the evaluated 14-year period (2006–2019). This finding agrees with a study that determined there was no significant year-to-year difference in 232 acute cytauxzoonosis cases identified over a 12-year period (1995–2006) in Oklahoma [
11]. In contrast, another study determined there was an overall increase in acute cytauxzoonosis cases over a 10-year period (2001–2011) in western Kentucky [
27]. However, the latter study consisted of only 56 cases and no statistical analysis method was reported. It is important to note that all three studies, including ours, were based on retrospectively evaluated data with limited patient details and histories. The discrepancy between anecdotal reports of increasing case numbers and data presented here may be due to a variety of reasons. Firstly, more clinicians may be diagnosing cytauxzoonosis with in-hospital microscopic blood smear evaluations versus sending out samples to a diagnostic laboratory, like KSVDL. Alternatively, an increased awareness of the disease may make the number of cases appear more prominent in the eyes of the clinicians, diagnosticians, and/or owners, while not actually being increased. Thirdly, vector tick populations—especially
A. americanum—may be increasing or expanding their ranges with changing environmental conditions, increasing clinician index of suspicion and resulting in more suspected
C. felis infection diagnoses without definitive diagnosis. Lastly, there may be an increasing incidence of acutely infected cats surviving infection without veterinary care, while the incidence of those presented to veterinary hospitals may be unchanged. Interestingly, with regard to this last point, we recently conducted a study investigating the prevalence of
C. felis carrier (reservoir) cats in eastern Kansas and found that 25.8% of sampled domestic cats were actively infected with
C. felis [
10]. As such, more cats may survive acute cytauxzoonosis than previously expected, thus contributing to an expanding domestic cat reservoir population. The extent of
C. felis genetic diversity and how these differences may affect virulence, transmissibility, and potential for treatment success is largely unexplored, with investigations hampered by a lack of methods to preserve or manipulate
C. felis isolates outside of living cat or tick hosts. Attempts at identifying genetic markers of virulence have yet to find definitive virulence determinants [
21,
23,
34,
35,
36].
A seasonal pattern of acute cytauxzoonosis incidence in domestic cats from eastern Kansas demonstrated a bimodal distribution, similar to those reported in other studies, peaking in early summer (June) with a smaller peak in early fall (September) [
11,
16,
27,
28]. This pattern corresponds with
A. americanum peak activity in this area, which is predominantly dependent on environmental conditions like diurnal temperature range, precipitation, and humidity [
8,
28]. Adult ticks are most active in early spring to mid-summer, nymphs in late spring to mid-summer as well as late summer to early fall, and larvae in late summer to early fall [
8]. This would suggest that
C. felis may have been transmitted to the cats in this study predominantly by adults/nymphs in early summer with fewer transmissions occurring in early fall by nymphs. Previous tick transmission studies have demonstrated that both adult and nymphal
A. americanum can successfully transmit
C. felis [
2,
11]. However, since tick findings or tick preventative use practices were not identified in case records, further evaluation regarding the tick vector was not possible in this study. In addition, late spring through early fall may be times when cats spend more time outdoors actively roaming, resulting in a greater chance of encountering tick vectors. The combined seasonal cat and tick activity could explain why cats in our study area with evidence of fever, malaise, icterus, and anorexia were more likely to be diagnosed with acute cytauxzoonosis in spring/summer than other diseases with a similar presentation. A larger, long-range, prospective study evaluating both predisposing environmental conditions and habitat niches would provide insight into the timing of increased tick activity, which could be used to anticipate or forecast times of increased risk for
C. felis transmission, as well as other tick-borne diseases.
As expected, young cats in their first and second year of life were more likely to have samples submitted and diagnosed with acute cytauxzoonosis. Our finding that young cats had a predisposition for acute cytauxzoonosis diagnosis supports previous data regarding age distribution for cats diagnosed with acute cytauxzoonosis [
16] and tick infestation [
33]. The first study, conducted in the mid-Atlantic states, found cats diagnosed with acute cytauxzoonosis (
n = 34) had a mean age of 4 years (range 2 months to 14 years) [
16]. Interestingly, another study investigating tick infestation of cats found cats with tick infestations were a mean age of 4.4 years (range 18 days to 18 years) (
n = 336), with ticks recovered more frequently on young cats [
33]. Combined, both studies support our study results of most commonly observing acute cytauxzoonosis cases in younger cats. In our study, a greater proportion of sample submissions came from young cats, for which there are several possible explanations. Firstly, young cats may explore their environment more aggressively and/or more frequently than older cats, resulting in potentially greater exposure to
C. felis infected ticks. Secondly, owners may be more likely to present samples from younger cats with illness or an unexpected death, thinking an older cat died of old age. Thirdly, older cats may lack the energy or drive to return home when feeling ill and expire where their body is less likely to be retrieved. Lastly, older cats maybe be more likely to have encountered and survived a previous
C. felis challenge, resulting in an asymptomatic reservoir stage. It is reasonable to assume that most cats that contract cytauxzoonosis do so through their outdoor activities because that is where the tick vector is most commonly located.
Among cats diagnosed with acute cytauxzoonosis in our study, male cats were over-represented (
Table 4), supporting a previous study that also found male cats to be over-represented (20/31; 64.5% male cats in other study) [
16]. Interestingly, male cats were also previously found to be more likely to have tick infestations than female cats (59% and 41%, respectively) [
33]. At one time it was believed that male cats had territories up to ten times larger than female cats, which could explain these findings [
37]. However, more recent studies determined there is no significant difference in home range sizes between male and female cats [
38,
39,
40]. As such, it is unclear why male cats had a higher incidence of disease compared to female cats in our study and others. A larger sample size as well as more complete patient background information would have been helpful in more thoroughly evaluating acute cytauxzoonosis risk factors; however, we were limited by the information provided in the case records. Overall, our findings regarding acute cytauxzoonosis seasonality and feline age/sex predilections in Kansas provide additional support to age and sex being important risk factors.
Not surprisingly, most of the sample submissions in this study came from owned cats (96.5%), with rare rescue/rescinded or feral cat samples. Since owned cats generally benefit from a stronger human–animal bond, more diagnostics are performed either to understand the cause of a pet’s demise or to ascertain if a zoonotic or infectious potential exists for the owner and/or other household pets. Additionally, 26.5% (45/170) of the patient records evaluated included mention of multiple cats in the household dying of acute cytauxzoonosis or a cat that died in a similar but undiagnosed manner. These pet owners may be more sensitive to the potential for this disease in their pets, resulting in increased diagnostic efforts. Feral cats, barn cats, and/or outdoor “pets” which are fed but lack a strong human–animal bond with the owners are less likely to be submitted for diagnoses due to cost and/or difficulty in obtaining samples. This population of “wild” cats may also stray further afield, dying far from the owned property, or the property owners may be less attentive to these cats’ cause of death or disappearance. Rescue and humane society organizations have limited medical care budgets and are more likely to perform on-site diagnostics for treatment decisions, and less likely to submit tissue samples to an outside diagnostic laboratory after death.
Rapid, patient-side diagnostics are not currently available for cytauxzoonosis diagnosis. Instead, the diagnosis of acute cytauxzoonosis is most commonly accomplished by a review of blood smears by a clinical pathologist or a review of tissue histology after necropsy. A diagnosis of acute cytauxzoonosis via identification of
C. felis schizonts or intra-erythrocytic signet rings in blood smears, or schizonts in histology tissue samples, was equally diagnostic (
Figure 6). Additionally, the density of intra-erythrocytic signet ring forms seen in blood smears did not determine the severity of this disease as nearly all cats in this cohort died or were euthanized (99.3%) due to their illness. Different methods and criteria have been used in studies to diagnose acute cytauxzoonosis, including the identification of: (i) schizonts only [
28]; (ii) schizonts or intra-erythrocytic signet rings [
27]; or (iii) either schizonts or intra-erythrocytic signet rings with expected clinical signs [
11]. Schizonts, when identified, are specific for acute cytauxzoonosis because this is the first life stage that develops in the feline host after the tick inoculates the sporozoites (
Figure 1). In this study, observation of schizonts was noted in all case records where histologic tissue samples were evaluated (
Figure 6C). That said, tissue sampling for histologic evaluation is far more costly and invasive than obtaining a blood sample, and requires a sedated, anesthetized, or deceased patient. Additionally, it does not demonstrate/confer a diagnostic advantage over a blood smear sample. Indeed, schizonts are not always identified on blood smear evaluation (
Figure 6B). Approximately half of our blood smear samples lacked schizont identification. Piroplasms, 1–2 μm diameter intra-erythrocyte signet rings, are found in both the schizogenous and erythrocytic phase of cytauxzoonosis and are too small to be reliably seen on histology (
Figure 6A). Blood smear cases lacking schizonts in this study had either multiple intra-erythrocytic signet rings per erythrocyte (active merogony), a high density of intra-erythrocytic signet rings, or occasional intra-erythrocytic signet rings in patients exhibiting clinical signs consistent with acute cytauxzoonosis. These presenting cats were unlikely to be merely a
C. felis reservoir, which can also have low parasite loads (generally 0.045–1.27% infected erythrocytes), because they also exhibited clinical signs of cytauxzoonosis [
22]. In subacute and early acute infections, intra-erythrocytic signet rings and schizonts may not be immediately apparent on blood smears. Blood or tissue sample submissions to a diagnostic laboratory for pathologist evaluation takes time that these patients may not have. Ideally, a rapid chairside test for in-house veterinary use would be developed to identify these cases very early in the disease process, allowing for early treatment initiation that may prove lifesaving for many of these cats.
The differential diagnosis for the nonspecific clinical signs seen in cytauxzoonosis (lethargy, fever, icterus, dyspnea, +/− anemia) could include cholangiohepatitis, triaditis, pancreatitis, hepatic lipidosis, sepsis, immune-mediated hemolytic anemia, toxins causing oxidative damage (e.g.,
Allium spp,, acetaminophen), neoplasia, and infectious agents (tularemia, feline infectious peritonitis, hemotropic mycoplasma), to name a few [
41,
42]. When intra-erythrocytic hemoparasites with a signet ring or piroplasm appearance are seen in cats, there are three main differential diagnoses: (i)
Cytauxzoon felis, (ii)
Mycoplasma hemofelis, and (iii)
Babesia felis.
C. felis, discussed herein, typically presents with a variable non-regenerative anemia, intra-erythrocytic signet rings, rare to occasional signet rings in the background, and/or schizonts at the feathered edge of blood smears or on histology of tissue samples.
M. hemofelis commonly presents with a strong regenerative anemia, and signet ring and/or coccoid forms of epierythrocytic organism located on the red cells and/or in the background [
41].
B. felis presents with a regenerative anemia and intra-erythrocytic signet rings often arranged in tetrads [
42]. Considering our study area is within the U.S.,
B. felis is unlikely to be the cause of infection in these cats as
B. felis has not been reported in the U.S. However, it should be considered for any feline patient with a travel history to Africa, particularly the southern coastal regions [
42]. While
M. hemofelis and
Babesia spp. PCR is readily available for differentiation,
C. felis PCR is less available. That said, identification of either schizonts and/or intra-erythrocytic signet rings via histology or blood smear samples are equally diagnostic for acute cytauxzoonosis when associated with typical acute cytauxzoonosis clinical signs for cats living in or near endemic areas. In locations where these feline piroplasm diseases overlap, DNA sequencing may be necessary to resolve piroplasm species identity.