two tales of cytauxzoon felis infections in domestic cats · cytauxzoon species harbored by the...

Two Tales of Cytauxzoon felis Infectionsin Domestic Cats

Jin-Lei Wanga Ting-Ting Lia Guo-Hua Liuac Xing-Quan Zhuac Chaoqun Yaob

State Key Laboratory of Veterinary Etiological Biology Key Laboratory of Veterinary Parasitology of GansuProvince Lanzhou Veterinary Research Institute Chinese Academy of Agricultural Sciences Lanzhou GansuProvince Peoples Republic of Chinaa Department of Biomedical Sciences and One Health Center forZoonoses and Tropical Veterinary Medicine Ross University School of Veterinary Medicine Basseterre St KittsWest Indiesb College of Veterinary Medicine Hunan Agricultural University Changsha Hunan ProvincePeoples Republic of Chinac

SUMMARY 861INTRODUCTION 862THE PARASITES AND THEIR LIFE CYCLE 862

Cytauxzoon Species 862Parasite Life Cycle 863Transmission 865Host Specificity 867Tick Vectors 867

PHYLOGENETIC CLASSIFICATION 869EPIDEMIOLOGY WORLDWIDE DISTRIBUTION SEASONALITY AND RISK

FACTORS 870LETHAL INFECTIONS IN BOBCATS AND OTHER WILD FELIDS 872NONFATAL AND CLINICALLY NORMAL INFECTIONS IN THE DOMESTIC CAT 872PATHOGENESIS AND CLINICAL FEATURES 874IMMUNOLOGY 875DIAGNOSIS 876

Microscopic Diagnosis 876Clinical Diagnosis 876Immunologic Diagnosis 877Molecular Diagnosis 878

DISEASE TREATMENT AND PREVENTION 878MOLECULAR DIVERSITY OF THE PATHOGEN CORRELATION BETWEEN PARASITES

AND CLINICAL OUTCOMES 879CONCLUDING REMARKS AND FUTURE PERSPECTIVES 880ACKNOWLEDGMENTS 881REFERENCES 881AUTHOR BIOS 884

SUMMARY Cytauxzoonosis is an emerging infectious disease that affects wild felids aswell as the domestic cat it is caused by the apicomplexan protozoan parasites belong-ing to the genus Cytauxzoon Cytauxzoon felis is the species of major concern whosetransmission occurs via the bite of an infected tick Cytauxzoonosis of the domestic cathas historically been considered uniformly fatal with a short course of illness and mostdomestic cats die within 9 to 15 days postinfection However increasing evidence of do-mestic cats surviving C felis infection suggests the existence of different strains with var-ious levels of pathogenicity Although wild felids are considered natural reservoirs forthis parasite a number of studies suggest that domestic cats that have survived nonle-thal infections may serve as an additional reservoir The current article comprehensivelyreviews the parasite and its life cycle geographic distribution genetic variability andpathogenesis as well as host immunology and the diagnosis treatment and preventionof infection in the domestic cat This information should provide a basis for better un-derstanding the parasite as well as the pathogenesis of the disease

KEYWORDS Cytauxzoon bobcat cytauxzoonosis domestic cat wild felid

Published 21 June 2017

Citation Wang J-L Li T-T Liu G-H Zhu X-Q YaoC 2017 Two tales of Cytauxzoon felis infections indomestic cats Clin Microbiol Rev 30861ndash885httpsdoiorg101128CMR00010-17

Copyright copy 2017 American Society forMicrobiology All Rights Reserved

Address correspondence to Xing-Quan Zhuxingquanzhu1hotmailcom or Chaoqun Yaochyaorossvetedukn

REVIEW

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October 2017 Volume 30 Issue 4 cmrasmorg 861Clinical Microbiology Reviews

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INTRODUCTION

Cytauxzoonosis an emerging tick-borne disease of domestic cats and wild felids iscaused by the protozoa belonging to the genus Cytauxzoon Cytauxzoon felis the

agent of cytauxzoonosis of major concern belongs to the phylum Apicomplexa classSporozoasida order Piroplasmorida and family Theileriidae (1ndash8) For many years itwas endemic exclusively to North America ie to the southern southeastern andmid-Atlantic states of the United States (9ndash16) Only recently has it been reported inSouth America ie in Brazil and a few other identified species have been reported inEurope and other geographic regions (17ndash28) The most common natural host for Cfelis is the bobcat (Lynx rufus) Upon infection bobcats generally experience a shortcourse of non-life-threatening illness followed by a full recovery They serve as a naturalreservoir for the pathogen (10 29 30) In contrast infected domestic cats (Felis catus)usually succumb to infections within 9 to 15 days postinfection (pi) Infected domesticcats show clinical signs such as pyrexia anorexia dehydration depression icterus andhepatosplenomegaly They usually die within 24 to 48 h upon presentation to veteri-narians in the presence or absence of supportive therapy and experimental therapeuticregimens against this protozoan (2ndash6 11ndash13 15 31 32) However recent research hasindicated that this scenario can also present the other way around That is somebobcats suffer from severe acute cytauxzoonosis sometimes even leading to deathand in contrast the domestic cat can be clinically normal or carry the parasite for a longperiod upon recovery from acute cytauxzoonosis (14 17 33ndash37) It appears that themore we learn about the parasite and the disease the more questions are being raisedand need to be answered

Due to the severity of feline cytauxzoonosis and its geographic expansion it iscrucially important to raise awareness of the disease among veterinarians pet ownersand the general public Thus the current article reviews the parasite and its life cyclegeographic distribution genetic variability and pathogenesis as well as host immu-nology and the diagnosis treatment and prevention of infection in the domestic cat

THE PARASITES AND THEIR LIFE CYCLECytauxzoon Species

Feline cytauxzoonosis was first reported in 1976 in Missouri with four fatal cases (1)The disease has been endemic mainly in the domestic cat in the southern andsoutheastern United States which is considered ground zero of the disease (2ndash6) Thedisease has also been reported from other geographic regions such as South AmericaEurope Africa and Asia (17ndash28) The species and nomenclature of the causativeprotozoa have been a topic of debate for over a decade due to the indistinguishablemorphological features of the intraerythrocytic merozoites commonly called piro-plasms In general intraerythrocytic piroplasms have a diameter of 1 to 2 m and mayappear in several different forms (2ndash6 38) The most common form is a round ldquosignetringrdquo form (Fig 1A) (16) Bipolar oval ldquosafety pinrdquo and round anaplasmoid bodies are lesscommon whereas tetrads are occasionally discovered in Giemsa-stained blood smears(2ndash6 38) However genomic data (httppiroplasmadborgpiro) and DNA sequenceshave revealed differences that are sufficient enough to differentiate the causativeprotozoan agents at the species level and finally to allow for species classificationwithin the genus (7 8 39ndash43)

The genus Cytauxzoon is closely related to Theileria (43) The piroplasm stages in theerythrocytes of mammalian hosts are morphologically similar making it very difficult ifnot impossible to distinguish them The two were originally thought to differ from oneanother by the location of schizogony within the host cells Schizogony of Cytauxzoonspp occurs in macrophages whereas that of Theileria spp takes place in lymphocytes(2ndash6 38 44) However Theileria spp were later found to also infect macrophages (4546) Macrophages containing mature Cytauxzoon schizonts are greatly increased in sizeto up to 250 m in diameter and are often associated with endothelial cells ofcapillaries of many internal organs (Fig 1B) (16) which contributes to capillary occlu-sion resulting in tissue damage and organ failure (2ndash6 15 32 44)

Wang et al Clinical Microbiology Reviews

October 2017 Volume 30 Issue 4 cmrasmorg 862


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Cytauxzoon species harbored by the domestic cat and wild felids include C felis Cmanul and a Cytauxzoon species of undetermined status (Fig 2) In the present reviewthe latter is referred to as the Cytauxzoon sp European strain C manul naturally occursin free-ranging Pallasrsquos cats (Otocolobus manul) in Mongolia (19 20) The Cytauxzoon spEuropean strain was first found in a domestic cat with clinical signs in Spain (8) It wasalso discovered in France and Italy again in the domestic cat (22 47 48) It was furtherdetected in Iberian lynxes (Lynx pardinus) in Spain (23 49 50) More recently theprotozoan was found in Romania in four Eurasian lynxes (Lynx lynx) and 12 wild cats(Felis silvestris) with a collective prevalence of 625 (51)

The 18S rRNA genes of these protozoa have been sequenced The difference betweenC felis and C manul is 149 The difference between C felis and the Cytauxzoon spEuropean strain is 173 The difference between C manul and the Cytauxzoon spEuropean strain is 039 (19 20) Therefore it is plausible that the latter two in the OldWorld may represent the same species which would carry the name C manul if this issupported by more evidence (Fig 2) (51) Nevertheless the current review focuses on C felisin the New World

Parasite Life Cycle

The life cycle of C felis requires a tick vector and a felid host in which the sexual andasexual reproduction processes respectively occur These processes are not yet fullyunderstood especially the sexual reproduction within the tick vector Bobcats (Lynxrufus) serve as a natural reservoir host in the southern and southeastern United StatesThey generally experience non-life-threatening illness and recover fully from the infec-tion (30) They often carry intraerythrocytic merozoites commonly known as piro-plasms after recovering from infection Upon ingestion by a suitable vector ieAmblyomma americanum the lone star tick or Dermacentor variabilis the American dogtick gametocytes are released into the tickrsquos gut after digestion of erythrocytes anddevelop into ray bodies also known as gametes (52) A male gamete fertilizes a female

FIG 1 (A) Intraerythrocytic piroplasms (dark arrows) in a blood smear from an infected domestic cat (B)Numerous schizont-laden macrophages (white arrows) in a splenic blood vessel Both original micro-graphs were taken with a 100 oil immersion lens (Reprinted from reference 16 with permission ofElsevier)

Feline Cytauxzoonosis Clinical Microbiology Reviews



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one resulting in the formation of a diploid zygote Zygotes mature into motile haploidkinetes that invade the salivary gland in which sporozoites form as a result ofsporogony (52) These sporozoites are infective to a feline host such as a bobcat or adomestic cat Therefore these hard tick vectors serve as the definitive (final) hosts (Fig3) (52) Only nymphs and adults are infective to felids since there is no evidence oftransovarial transmission Upon inoculation into a domestic cat or a wild felid during ablood meal by a tick vector sporozoites directly enter host macrophages by a mech-anism that has yet to be elucidated They undergo asexual reproduction by schizogonyresulting in formation of mature schizonts The latter release numerous merozoitesupon rupture of the infected macrophages (52) Subsequently merozoites infect eryth-

FIG 2 Phylogenetic analysis based on the 18S rRNA gene sequences of C felis C manul and the Cytauxzoon spEuropean strain from domestic and wild animals The sequences of the 18S rRNA genes were aligned using MAFFT7122 and the phylogenetic tree topology was obtained from a maximum likelihood analysis by use of PhyML 31with the GTR G substitution model selected by jModelTest 202 The strain name GenBank accession numberhost and area of origin are listed Numbers at the nodes indicate bootstrap support obtained by repeating theanalysis 100 times Theileria parva was used as an outgroup




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rocytes and undergo asexual reproduction by binary fission yielding merozoites in thered blood cells (RBCs) Merozoites egress from the erythrocytes and invade new onesfollowed by asexual reproduction This cycle continues repeatedly in the feline hostTherefore the domestic cat and wild felids serve as intermediate hosts for Cytauxzoonspp (Fig 3) (52)

Transmission

Most studies on transmission were originally performed by inoculation of animalswith peripheral blood andor tissue homogenates of the lymph nodes liver spleen orlungs of C felis-infected animals In an experiment aimed to study interspecies trans-mission of cytauxzoonosis both domestic and wild animals were used These included9 species of laboratory animals (mouse Mus musculus [ICR] nude mouse M musculus[BALBc-nu] rat Rattus norvegicus [Sprague-Dawley] gerbil Meriones unguiculatus[Mongolian] hamster Mesocricetus auratus [golden] guinea pig Cavia porcehus [Hart-ley] chinchilla Chinchilla laniger [mixed colors] rabbit Oryctolagus cuniculus [NZW]and squirrel monkey Saimiri sciureus [Bolivian]) 4 species of domestic farm animals(cattle Bos taurus [Angus] sheep Ouis aries [mixed breed] goat Capra hircus [mixed

FIG 3 Schematic life cycle of Cytauxzoon felis The life cycle of C felis requires a hard tick vector as a definitive host and a felid host as anintermediate host Both Dermacentor variabilis and Amblyomma americanum are competent vectors Since there is no transovarial transmissiononly nymphs and adults are infective to felids All felids are capable hosts but no other mammals are Bobcats and domestic cats are the mostcommon hosts After inoculation by a tick vector during a blood meal (1) sporozoites directly enter macrophages of a felid host in which theyundergo schizogony (2) resulting in formation of mature schizonts that rupture the infected macrophages and release copious merozoites (3)Numerous enlarged schizont-laden macrophages occlude small blood vessels of various organs which is responsible for acute cytauxzoonosisMerozoites enter erythrocytes (4) undergo binary fission (5) and ultimately kill their host cells resulting in release of merozoites (6) Merozoitesenter other erythrocytes (7) and the asexual cycle continues Some intraerythrocytic merozoites differentiate into gametocytes which areinfectious for a tick vector (8) In the tick host the gametocytes develop into ray bodies (9) that fuse to form a diploid zygote (10) in the gutZygotes mature into motile haploid kinetes (11) that enter the tickrsquos salivary glands where they undergo multiple fission to produce numeroussporozoites that are infectious for a felid host (12)




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breed] and swine Sus scrofa [mixed breed]) 17 wildlife species (coyote Canis latrans[southern Missouri] red fox Vulpes fulua [northern Missouri] striped skunk Mephitismephitis [northwestern Missouri] raccoon Procyon lotor [central Missouri] woodchuckMarmota monax [central Missouri] yellow-bellied marmot Marmota flaviventris [centralMissouri] opossum Didelphis marsupialis [central Missouri] ground squirrel Citellustridecemlineatus [eastern Kansas] gray squirrel Sciurus carolinensis [central Missouri]prairie meadow vole Microtus ochrogaster [central Missouri] white-footed mousePeromyscus maniculatus [central Missouri] little brown bat Myotis lucifugus [centralMissouri] eastern cottontail rabbit Sylvilagus floridanus [central Missouri] white-taileddeer Odocoileus virginianus [central Missouri] ocelot Felis pardalis mountain lion Felisconcolor and bobcat Lynx rufus rufus [eastern bobcat]) cat (Felis domesticus [mixedbreed]) and dog (Canis familiaris [mixed breed]) (53) These animals were individuallyinoculated with blood andor tissue homogenates from domestic cats which wereexperimentally infected with C felis and were euthanatized due to severe cytauxzoo-nosis A Florida bobcat died 2 weeks after inoculation with typical clinical signs ofcytauxzoonosis prior to its death An eastern bobcat developed no overt clinical signsof cytauxzoonosis although it carried a persistent parasitemia No animals of the otherspecies except a sheep with persistent low parasitemia developed cytauxzoonosisclinically or subclinically as confirmed by blood smear or histopathologic or necropsyexaminations Nevertheless domestic cats inoculated with the blood from this sheepdid not develop any clinical cytauxzoonosis or parasitemia (53) A subsequent studyshowed that splenectomized sheep inoculated with blood and tissue homogenatesprepared from a fatally C felis-infected cat did not develop any clinical signs of diseaseor parasitemia (54) These studies collectively demonstrated that sheep are not sus-ceptible to C felis infection One plausible explanation for the sheep with persistentparasitemia is a concomitant infection with another species of piroplasm that ismorphologically indistinguishable from C felis

It is worthwhile to include a brief discussion of the schizogonous phase in bobcatsthe natural reservoir of C felis In one experiment two C felis-free bobcats were eachfed 400 adult D variabilis ticks These ticks were infected with C felis in the nymphalstage by taking a blood meal on a naturally infected splenectomized bobcat Numerousschizont-filled macrophages were observed in the prescapular lymph node of onebobcat 11 days after tick attachment This bobcat died 8 days later with typical clinicalsigns and microscopic evidence of cytauxzoonosis However no schizonts were foundin the same lymph node of the second bobcat on the 30th day after tick attachmentFurthermore 10 free-ranging bobcats with natural infection by C felis were confined for1 to 12 months before euthanasia for microscopic examination of schizonts in the liverspleen lungs and lymph nodes No schizonts were found in any examined tissues ofany bobcats Therefore only a limited schizogony appears to occur in bobcats (30)Even this brief schizogony presented in the blood mononuclear cells may lead to deathof the domestic cat after inoculation into the peripheral blood from clinically normalinfected bobcats (Lynx rufus) or other wild felines For example among four domesticcats that received a parenteral inoculation of blood from C felis-infected but clinicallynormal bobcats one domestic cat died of a cytauxzoonosis 18 days after inoculationwith schizont-laden macrophages that was mainly located in lymph nodes and lungsThe other three developed parasitemia 5 to 14 days after inoculation as shown bymicroscopy of blood smears but they remained clinically normal No schizonts werefound in any tissues of two of the three clinically normal cats during necropsy 2 monthsafter inoculation (10) The third cat was subsequently challenged parenterally on day 40postinoculation with the blood of a domestic cat dying of experimentally inducedcytauxzoonosis This domestic cat showed manifestation of cytauxzoonosis and waseuthanized at 9 days postchallenge (10) Another similar study also showed that onedomestic cat died 12 days after inoculation with the blood of a Florida panther (Felisconcolor coryi) infected with C felis (55)

Several studies have shown that inoculation of peripheral blood from a parasitemiccat into an uninfected domestic cat can result only in a detectable erythroparasitemia




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in the recipient with no clinical signs For example after inoculation of an uninfecteddomestic cat with the whole blood of an infected domestic cat the recipient showeda detectable parasitemia in erythrocytes starting at 8 days postinoculation that waspersistent for at least 18 months Furthermore PCR and sequencing confirmed theidentity of the parasites as C felis Nevertheless the domestic cat was clinically normalthroughout this entire course (14) In another study six domestic cats were randomlydivided into two groups that were subcutaneously injected with either dexamethasoneor sterile water Afterwards they were inoculated intravenously (iv) with C manul inthe whole blood of a Pallasrsquos cat (Otocolobus manul) (56) Although they were clinicallyhealthy these domestic cats harbored low levels of parasites in the blood by 9 dayspostinoculation They were further challenged with C felis schizonts to study whetherthey were protected by the prior exposure to C manul A seventh healthy domestic catserving as a control receiving no inoculation of C manul was injected only with sterilewater prior to the challenge with C felis schizonts All seven domestic cats developedcytauxzoonosis with typical clinical signs at 5 days postchallenge and the diagnosiswas further confirmed by histopathology at necropsy (56)

Taken together these transmission studies indicate that inoculation of uninfecteddomestic cats with C felis schizonts causes cytauxzoonosis and death of recipient catsIn contrast inoculation of peripheral blood containing only piroplasms does not resultin schizogony or illness Additionally domestic cats receiving intraerythrocytic piro-plasms did not have adequate protection against a subsequent challenge with schi-zonts from an infected felid or sporozoites from a tick vector This has practical valuefor vaccine development ie determining which parasitic life cycle stage should betargeted for vaccination

Can the parasite be transmitted congenitally from queens to kittens Two clinicallynormal queens who had lived mostly outdoors in rural Arkansas were found to bepositive for the parasite by microscopic review of blood smears and PCR One queendelivered one live kitten and the other queen gave birth to two litters with seven andsix live-born kittens Both microscopy and PCR were performed to detect C felisparasites in these 14 apparently healthy kittens before 12 weeks of age All kittenstested negative for the parasites This experiment indicates that C felis failed to betransmitted vertically from mother to offspring (57)

Host Specificity

In addition to infections in domestic cats and bobcats C felis infection has also beenreported for other wild felines A survey in Florida showed that the prevalences of Cfelis in Texas cougars (Puma concolor stanleyana) and Florida panthers (Puma concolorcoryi) were 39 and 36 respectively The cougars and panthers were asymptomaticbut carried intraerythrocytic piroplasms and infection with C felis did not appear tohave a negative impact on their hematologic parameters (58) Furthermore C felisinfection has also been diagnosed for ocelots (Leopardus paradise) jaguars (Pantheraonca) lions (Panthera leo) and tigers (Panthera tigris) (34 35 58ndash63)

Among other Cytauxzoon species C manul has been detected in Pallasrsquos cats(Otocolobus manul) and lions (Panthera leo) (19 20 27) The Cytauxzoon sp Europeanstrain has been found in domestic cats meerkats (Suricata suricatta) wild cats (Felissilvestris) Eurasian lynxes (Lynx lynx) and Iberian lynxes (Lynx pardinus) (21 23 28 6465) Collectively only the domestic cat and wild felids are suitable hosts of Cytauxzoonspp (2ndash6)

Tick Vectors

Members of the two other genera in the order Piroplasmorida ie Theileria andBabesia use a tick vector in their life cycle Therefore it is reasonable to assume this tobe true for C felis as well Laboratory-raised D variabilis nymphs were allowed to feedon a bobcat (Lynx rufus) naturally infected with C felis under controlled laboratoryconditions Three and 8 weeks after molting the adult ticks fed on splenectomizeddomestic cats The cats died at 13 to 17 days pi with confirmed cytauxzoonosis (29)




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As mentioned in the life cycle section two C felis-negative bobcats were fed on by Cfelis-infected adult D variabilis ticks The data from the experiment indicated that Dvariabilis ticks are capable of transmitting C felis to bobcats (30) A similar experimentwas carried out with domestic cats In this case the D variabilis adults arose fromnymphs that had fed on domestic cats with experimental infection by C felis They thenwere allowed to feed on C felis-negative domestic cats The latter developed cytaux-zoonosis (44) Collectively these experiments unequivocally showed that D variabilis isa vector of C felis that is capable of transmitting the protozoan from bobcats todomestic cats between bobcats and between domestic cats at least under experi-mental conditions Currently little is known about the tick vector of C manul and theCytauxzoon sp European strain It is almost certain that those parasites do not use Dvariabilis as a vector because its geographic distribution is limited to North America(66)

The effectiveness of D variabilis as a vector in the areas of endemicity under naturalconditions cannot be determined from such experiments The natural vector capacityof arthropod vectors is influenced by many factors such as vector species vectorpopulation vector susceptibility to a pathogen the pathogenrsquos effect on the vectorvector life span and vector blood preference to name a few A confirmed vector underlaboratory conditions does not always hold up as a natural vector

An epidemiological study of ticks was conducted by means of PCR and sequencingIn total 1631 ticks were collected in 2007 to 2009 from hosts such as humans domesticanimals and wildlife in areas where cytauxzoonosis is endemic including KentuckyGeorgia Tennessee and Texas Nevertheless bobcats (Lynx rufus) and domestic catswere not included except for one feral domestic cat with only one D variabilis tick Anoverall prevalence of 13 was found for C felis in D variabilis ticks (9702 ticks) Incontrast no C felis-positive ticks were found among 743 A americanum 99 Ambly-omma cajennense 16 Amblyomma maculatum 4 Ixodes scapularis and 1 Ixodes wooditick and 64 unidentified Amblyomma sp nymphs (67)

Similarly a total of 1362 ticks were collected either individually or in pools fromdogs and domestic cats in Missouri where feline cytauxzoonosis is endemic The tickswere analyzed by PCR for the 18S rRNA gene and internal transcribed spacer 1 (ITS-1)sequence of C felis Three A americanum nymphs were positive which yielded aprevalence of 09 and indicated that A americanum could be a potential vector for theprotozoan while no other tick species including D variabilis were found to be positivefor C felis (68) Further A americanum nymphs were allowed to feed on a domestic catwith a clinically normal infection by C felis that was confirmed by a blood smearshowing intraerythrocytic piroplasms PCR amplification of the 18S rRNA gene andsequencing The resulting adult ticks after molting were allowed to feed on a Cfelis-negative domestic cat The cat developed typical clinical signs of cytauxzoonosis at11 days pi However it resolved the clinical signs by 23 days pi and remained clinicallynormal until day 74 pi when the experiment ended Interestingly the domestic catsthat were fed on by three other tick species namely D variabilis Ixodes scapularis andRhipicephalus sanguineus never became clinically ill Necropsy of the domestic cat thatwas fed on by D variabilis revealed no evidence of C felis infection (69) Later the sameauthors performed another similar experiment with an expanded number of domesticcats Four domestic cats each were used in the A americanum and D variabilis groupsthis time Again all four cats fed on by A americanum ticks became sick with clinicalsigns of cytauxzoonosis and survived the infection In contrast none of the four catsfed on by D variabilis became ill or PCR positive (70) Further a cross-sectional studywas carried out to determine the minimum infection rate of C felis in ticks by collectingunengorged A americanum and D variabilis ticks from wild habitats in an area ofendemicity A americanum ticks were positive for C felis in both the adult and nymphalstages with 06 of males (1178 ticks) 15 of females (3197 ticks) and 08 ofnymphs (3393 nymphs) being positive No D variabilis ticks were found to be positivefor C felis (70) These data confirm that A americanum is a primary vector of C felisalthough it does not rule out D variabilis as a vector




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In a survey of bobcat infections in association with tick density in 13 US statesShock et al found that high densities of A americanum were associated with higherprevalences of C felis In contrast no such association existed between the density ofD variabilis and C felis prevalence (71) Altogether the data presented above confirmthat both D variabilis and A americanum are suitable vectors for C felis in NorthAmerica It appears that the latter has a higher vector capacity in nature Neverthelesstheir exact roles in maintaining and transmitting C felis infection in areas where it isenzootic remain to be explored

How long does it take for a tick vector to transmit C felis to a domestic cat upon itsattachment Can C felis be transmitted by the host ingesting a C felis-positive tick Arecent study aimed to answer these questions In the study 49 domestic cats wererandomly divided into seven groups with seven cats per group Five groups ofdomestic cats were each infested with 25 male-female pairs of C felis-positive Aamericanum adults per domestic cat for 12 18 24 36 or 48 h In the sixth group eachdomestic cat was fed the same number of C felis-infected adult ticks embedded incanned cat food In the seventh group 50 C felis-infected ticks were allowed to feed oneach cat until repletion Six of seven domestic cats in group 7 were determined to bepositive for C felis between 8 and 12 days after infestation One of seven cats in the48-h group ie group 5 was determined to be positive 12 days after infestation Noneof the domestic cats in the other groups were found to be positive for C felis Thesedata indicate that transmission of C felis by an A americanum tick requires a minimumof 36 h of attachment and that transmission cannot be achieved by the hostrsquos ingestionof C felis-infected ticks (72) Further studies are needed for other Cytauxzoon speciesand other ticks

Can tick attachment and transmission of C felis to a domestic cat be prevented bya collar and how effective is it In an experiment 10 domestic cats were each fittedwith a 10 imidacloprid and 45 flumethrin collar (Seresto Bayer Animal Health) andanother 10 domestic cats were used as untreated controls All cats were intact femalesof 6 months of age and were randomly assigned to either the control or treatmentgroup They were each exposed to 25 A americanum adult ticks that were experimen-tally infected by C felis No ticks were found on any of the treated cats In contrast theyattached to every domestic cat in the nontreated control group with geometric meansof 153 and 142 ticks at 24 and 48 h respectively Zero (0) and 9 of 10 (90) domesticcats in the collar-treated and nontreated control groups respectively were C felispositive It was concluded that the 10 imidacloprid and 45 flumethrin collareffectively prevented ticks from attaching feeding and transmitting C felis to domesticcats (73)

PHYLOGENETIC CLASSIFICATION

Parasites in the order Piroplasmorida are blood parasites of vertebrates and someof them cause serious diseases in domestic animals (2ndash6 74ndash78) The taxonomy of theorder Piroplasmorida is debatable with considerable confusion about the relationshipsamong the piroplasmid species and the classification method (7 8 39ndash42 77) Threegenera ie Babesia Theileria and Cytauxzoon are established based on morphologicalultrastructural and parasite life cycle characteristics such as host preference and thecell type(s) infected (4ndash8 39ndash42 77) All species of Babesia Theileria and Cytauxzoon aretransmitted by a tick vector However the mechanisms of transmission are different(2ndash6 52 74ndash78) During a blood meal by a suitable tick vector Babesia sp sporozoitesdirectly enter the host erythrocytes and develop into merozoites Merozoites undergobinary fission in the erythrocytes resulting in two or sometimes four daughter cells andthe erythrocytes break apart Each daughter enters another red blood cell and thiscycle repeats (74ndash77) In contrast Cytauxzoon and Theileria protozoa first infect mac-rophages andor lymphocytes in which they undergo schizogony resulting in ruptureof the infected host cells and release of numerous merozoites (2ndash6 52 78) Themerozoites enter erythrocytes and undergo asexual binary fission yielding merozoites




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Upon rupture of infected erythrocytes these merozoites infect other red blood cellsand the intraerythrocytic cycle continues (2ndash6 52 78)

There are differences in the development of Babesia Cytauxzoon and Theileria in atick vector The zygotes of Babesia species undergo multiplication and invade multipleorgans of the tick including the salivary glands and ovaries Thus parasites are passedfrom mother ticks to their offspring by transovarial transmission The sporozoites in thelarvae of these offspring ticks are infectious for mammalian hosts (74ndash77) HoweverCytauxzoon and Theileria species are limited to transstadial transmission Their zygotesinvade only the salivary glands to develop into infectious sporozoites Consequentlythe larvae are not infective to mammalian hosts due to a lack of sporozoites (2ndash6 5278) These life cycle characteristics make it possible to distinguish Babesia from Cytaux-zoon and Theileria species Although a previous study indicated that Theileria speciesinvade lymphocytes whereas Cytauxzoon species enter macrophages more studiesshow that Theileria can also infect macrophages (45 46) Therefore this classificationscheme for Cytauxzoon and Theileria species has limited value

In an attempt to resolve the controversy several studies have used molecularmethodology such as 18S rRNA gene sequencing to classify the order Piroplasmorida(39ndash42 77) Unfortunately they have made it no clearer if not more confusing becauseCytauxzoon species have been put into different phylogenetic positions within theorder Piroplasmorida (39ndash42 77) Recently the taxonomy of the order Piroplasmoridawas revisited based on the mitochondrial genome sequences and structures (43) Fivedistinct lineages within the order Piroplasmorida were identified based on phylogeneticanalysis of the nucleotide sequences of the concatenated mitochondrial genome andthe 18S rRNA gene The five lineages were further confirmed by analysis of cox1-encoded amino acid sequences and the mitochondrial genome structure as well as thebiology of Piroplasmorida organisms The Cytauxzoon (Cytauxzoon felis) and Theileria(Theileria annulata Theileria parva and Theileria orientalis) species were grouped into asingle lineage and the four previously identified lineages were the Babesia sensu strictolineage (Babesia sp Coco Babesia bigemina Babesia bovis Babesia caballi Babesia canisBabesia vogeli Babesia rossi and Babesia gibsoni) the western Babesia lineage (Babesiaconradae) the Babesia microti lineage (Babesia microti-like species Babesia microti andBabesia rodhaini) and the Theileria equi lineage (Theileria equi) (43) Although thecurrently assigned nomenclature provides important new evidence for elucidation ofthe evolutionary relationships of the order Piroplasmorida it has room for reconsider-ation and improvement Some clades may even have their nomenclature changed dueto the limited number of samples and species used in the recent phylogenetic analysis(43) So far only the C felis mitochondrial genome sequence and structure have beencharacterized whereas those of the other Cytauxzoon species such as the Cytauxzoonsp European strain remain unknown Thus further studies using larger numbers ofsamples and representative species within the order Piroplasmorida are warranted torefine their clade taxonomy

EPIDEMIOLOGY WORLDWIDE DISTRIBUTION SEASONALITY AND RISKFACTORS

The first fatal case of feline cytauxzoonosis in India was reported in 2009 Thediagnosis was based solely on clinical signs and microscopic demonstration of intra-erythrocytic piroplasms The domestic cat was a 4-month-old male kitten with anorexiaprostration anemia and pyrexia It collapsed on the 3rd day of reference even withapplication of a broad spectrum of antibiotics and supportive therapy (79) Accordingto the authors of the study this was also the first case of feline cytauxzoonosis causedby C felis in Asia which has yet to be confirmed since no molecular data werepresented The next reports in Asia were from Iran where a stray cat and a wild cat (Felissilvestris) were infected with C felis as confirmed by blood smear and molecularanalyses respectively (25 26) The stray cat exhibited clinical signs of cytauxzoonosiswith moderate anemia and the wild cat died after 4 days of treatment with piperacillin-tazobactam (Tazocin) and clindamycin (11 mgkg of body weight per os [po] every 12




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h [q12h]) (25 26) As mentioned later in this review C felis has been found in Brazil indomestic cats (18 80) and zoo lions (Panthera leo) (17) In addition it was also foundin a zoo tiger (Panthera tigris) in Germany (35) Therefore so far cytauxzoonosis causedby C felis has been found in North America (United States) South America (Brazil)Europe (Germany) and Asia (India and Iran) (Fig 4)

A retrospective study of feline cytauxzoonosis in domestic cats between 1998 and2006 in North Carolina (28 cases) South Carolina (3 cases) and Virginia (3 cases)determined that 32 of 34 cases were diagnosed between April and September (12) Asimilar study of 56 cases of feline cytauxzoonosis in western Kentucky between 2001and 2011 revealed a single peak in May with diagnoses made between March andOctober (16) A total of 232 cases from two veterinary hospitals in Stillwater OK from1995 to 2006 were collectively analyzed First there was no significant fluctuation inannual numbers of diagnosed cytauxzoonosis cases over the entire period Second thenumber of cases peaked in April May and June with a minor peak in August andSeptember (81) Clearly the peak time of diagnosis in the US regions of endemicity isMay and June

In an effort to determine different environmental conditions as risk factors for felinecytauxzoonosis Raghavan and colleagues used geographic information systems (GIS)(82) They used a cohort of 69 confirmed cases of cytauxzoonosis and 123 control caseswith negative microscopic findings for piroplasms yet similar clinical signs Significantrisk factors for cytauxzoonosis in the study region included total edge contrast indexgrassland coverage humidity during the 9th week prior to case arrival and an inter-action variable ie diurnal temperature range percent mixed-forest area Raghavanet al concluded that land-cover areas and climatic conditions favorable for tick pop-ulations were strong risk factors for feline cytauxzoonosis (82) Further analysis of thepotential ecological distribution of C felis in Missouri Oklahoma and Arkansas showedonce again that environmental niches suitable for tick breeding were more likely

FIG 4 Geographic distribution of Cytauxzoon infections in domestic cats and wild felids The countries where cases of cytauxzoonosis have beenreported are highlighted in yellow




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related to transmission of the parasite in the domestic cat (83) Environmental analysisshowed that cytauxzoonosis cases were more likely to occur in low-density residentialareas and were significantly associated with more wooded cover and closer proximityto natural or unmanaged areas (81) Considering that C felis requires a tick vector in itslife cycle these results confirm what would have been expected

LETHAL INFECTIONS IN BOBCATS AND OTHER WILD FELIDS

As mentioned earlier bobcats unlike the domestic cat often experience non-life-threatening illness followed by a recovery from C felis infections thereby serving as anatural reservoir (29 30) That being said severe and fatal cases of cytauxzoonosis inbobcats have been recorded in the literature A free-ranging bobcat cub (Lynx rufus)presenting to a veterinary clinic in Kansas was euthanized due to a moribund stateDuring necropsy multifocal atelectasis splenomegaly and pericardial effusion wereevident Microscopy of hematoxylin and eosin (HampE)-stained tissue sections revealedsubacute pulmonary thrombosis and greatly enlarged macrophages filled with schi-zonts within blood vessels in many tissues Pathological changes at both the gross andmicroscopic levels confirmed that cytauxzoonosis was the cause of the bobcat cubdeath (36) In addition a 7-year-old female white tiger (Panthera tigris) died of cytaux-zoonosis in Florida The tiger presented with a history of a 2-day duration of anorexialethargy mild dehydration fever and anemia She progressed rapidly the followingday to recumbency coma and death after developing icterus Detailed microscopyanalysis detected merozoites in RBCs and schizonts of C felis in macrophages of varioustissues Again clinical signs and intraerythrocytic piroplasms confirmed cytauxzoonosisas the cause of death (34)

Cases of fatal cytauxzoonosis in wild felids occur not only in the United States butalso in other geographic regions Fatal cases of cytauxzoonosis occurred in a lioness(Panthera leo) raised in captivity and her 6-month-old cub in a Brazilian zoo The lionessshowed weight loss depression anemia alopecia dark discolored urine tachypneanystagmus deafness and a staggering gait Necropsy revealed marked pulmonaryedema red and semiliquid intestinal content and slight gelatinous translucent edemain the mediastinum The cub presented with endocardial and pulmonary edemahemothorax and hepatic and splenic congestion and died soon afterwards Histo-pathological examination revealed schizont-filled macrophages in numerous tissuesand organs These were the first confirmed cases of fatal cytauxzoonosis in SouthAmerica (17) In 1984 a young Bengal tiger (Panthera tigris) in a German zoo died of anunknown protozoal infection The tiger was born and raised in the zoo The infectionwas retrospectively confirmed as fatal cytauxzoonosis by histologic and microscopicexamination (35)

Although the last two case reports are interesting caution should be taken ininterpretation of their significance Both cases were from wild felids in zoos Zoos oftenobtain exotic animals from other geographic regions including from North Americawhere C felis is endemic Although in both cases the wild felids were born and raisedin the countries where the cases were reported it seems premature to conclude thatcytauxzoonosis is endemic in Brazil and Germany There is a possibility that these zoosmight have imported C felis-infected animals from areas of endemicity and that thesecases were merely a result of tick transmissions that occurred on site Indeed theGerman zoo directly imported three young bobcats from the United States before theBengal tiger infection (4) However this might not be the case for Brazil as describedbelow

NONFATAL AND CLINICALLY NORMAL INFECTIONS IN THE DOMESTIC CAT

Although it is considered uniformly fatal (2ndash6) feline cytauxzoonosis in the domesticcat turns out to have a wide spectrum of clinical signs ranging from clinically normalthrough nonfatal to fatal manifestations In this section only clinically normal andnonfatal infections are reviewed A 1-year-old spayed female domestic cat with ahistory of 2 days of fever lethargy and anorexia was diagnosed with C felis infection




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by demonstration of intraerythrocytic piroplasms consistent with C felis at a level of05 parasitemia The cat received supportive therapy yet developed severe icterus andhad dark brown urine within 24 h Nevertheless the cat recovered fully was confinedindoors afterwards and was clinically normal for 25 years post-initial diagnosis Thiswas apparently the first documented case of a domestic cat recovering from a naturalinfection of C felis in Oklahoma (84) Since then more recovery cases of felinecytauxzoonosis have been reported in the literature These include 3 cases in Arkansasand Georgia and 18 cases in northwestern Arkansas and northeastern Oklahoma Allthese cases were confirmed by PCR and DNA sequencing (14 85)

Furthermore cross-sectional surveys of the domestic cat in the southern andsoutheastern United States have revealed many clinically normal infections in thedomestic cat In a survey of domestic cats in Florida (494) North Carolina (392) andTennessee (75) using PCR and DNA sequencing 04 00 and 13 of cats respec-tively were positive for C felis with an overall prevalence of 03 (85) In addition areal-time PCR analysis of blood samples collected from clinically healthy domestic catswith a high risk of C felis infections in Arkansas and Georgia showed that theprevalence rate of the parasite was 303 (2789 cats) (33) More recently a study of902 domestic cats in Arkansas Missouri and Oklahoma by PCR yielded a 62 preva-lence (37) A summary of data on nonfatal and clinically normal infections of thedomestic cat and other felids is shown in Table 1 Collectively these data unequivocallydemonstrate that these infections are anything but rare events in the regions ofendemicity of the southern and southeastern United States

TABLE 1 Cross-sectional studies of Cytauxzoon infections in asymptomatic domestic cats and other felids

Continentand country

Studyperiod (yr)a Animal species

Testmethod

Cytauxzoonspeciesb

No of positive animalsno of animals tested () Reference

North AmericaUSA 1999ndash2000 Domestic cat (Felis catus) PCR C felis 3961 (031) 85USA 2008ndash2010 Domestic cat (Felis catus) PCR C felis 2789 (303) 33USA 2008ndash2012 Domestic cat (Felis catus) PCR C felis 56902 (62) 37USA 2002 Bobcat (Lynx rufus) PCR C felis 1030 (333) 107USA 2004ndash2006 Bobcat (Lynx rufus) PCR C felis 569 (72) 107USA 1999ndash2010 Bobcat (Lynx rufus) PCR C felis 138696 (198) 71USA 2008ndash2010 Bobcat (Lynx rufus) PCR C felis 34133 (256) 33USA 2003ndash2015 Bobcat (Lynx rufus) PCR C felis 88125 (704) 121USA 1983ndash1997 Cougar (Puma concolor) Blood smear C felis 3391 (363) 58USA 1989ndash2005 Cougar (Puma concolor) PCR C felis 541 (122) 60USA 1999ndash2010 Cougar (Puma concolor) PCR C felis 17 (143) 71

South AmericaBrazil 2013 Domestic cat (Felis catus) PCR C felis 1151 (066) 80Brazil 2001ndash2004 Ocelot (Leopardus pardalis) PCR C felis 116 (63) 61Brazil 2006 Jaguar (Panthera onca) PCR C felis 19 (111) 62Brazil 2006 Ocelot (Leopardus pardalis) PCR C felis 629 (207) 62Brazil 2006 Puma (Puma concolor) PCR C felis 29 (222) 62Brazil 2000ndash2009 Jaguar (Panthera onca) PCR C felis 2930 (967) 63

AfricaSouth Africa 2011 Meerkat (Suricata suricatta) PCR Cytauxzoon sp 2646 (565) 28Zimbabwe NA Lion (Panthera leo) PCR C manul 286 (23) 27

EuropeSpain 2005ndash2008 Domestic cat (Felis catus) PCR Cytauxzoon sp 8644 (124) 122Spain 2003ndash2007 Iberian lynx (Lynx pardinus) PCR Cytauxzoon sp 2477 (312) 50Spain 2004ndash2006 Iberian lynx (Lynx pardinus) PCR Cytauxzoon sp 320 (15) 23Spain NA Wild cat (Felis silvestris) PCR Cytauxzoon sp 46 (667) 64France 2006ndash2007 Domestic cat (Felis catus) PCR Cytauxzoon sp 1116 (08) 47Italy 2007ndash2008 Domestic cat (Felis catus) PCR Cytauxzoon sp 27118 (229) 22Italy 2011ndash2014 Wild cat (Felis silvestris) PCR Cytauxzoon sp 321 (143) 65Romania 2014 Wild cat (Felis silvestris) PCR Cytauxzoon sp 612 (50) 51Romania 2014 Eurasian lynx (Felis silvestris) PCR Cytauxzoon sp 44 (100) 51

aNA not availablebThe Cytauxzoon species found in Europe is defined as the Cytauxzoon sp European strain in the text and in Fig 4




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Unexpectedly nonfatal and clinically normal infections of the domestic cat by C felisoccur in geographic regions other than the United States as well In Brazil 07 of 151cats (86 owned and 65 stray) were positive by PCR and DNA sequencing This Brazilianstrain clustered in the same clade as that for other C felis strains by phylogeneticanalysis (80) Another survey showed that 967 (2930 animals) of wild jaguars(Panthera onca) in Brazil were positive for C felis by PCR and DNA sequencing (63)These data along with the cases presented earlier for wild zoo felids (62) confirm thatC felis and cytauxzoonosis occur in Brazil (17) Consequently both North and SouthAmerica are regions of endemicity for C felis

Similarly the Cytauxzoon sp European strain was reported in clinically normaldomestic cats with prevalences ranging from 0 to 229 For example a study innortheastern Italy showed that the prevalence of Cytauxzoon sp infection was 229(27118 animals) for detection using PCR amplification of the 18S rRNA gene and 153(18118 animals) by microscopic examination of blood smear (22) In France 08 of116 domestic cats were positive for Cytauxzoon sp by PCR and DNA sequencing (47)

A recent study showed that subclinically infected domestic cats could transmitclinical cytauxzoonosis to a domestic cat with A americanum as a vector (69 70) Thisis very important because naturally infected domestic cats serving as an additionalreservoir would greatly increase the risk of exposure for domestic cats in general

PATHOGENESIS AND CLINICAL FEATURES

The pathogenesis of C felis infection is associated with the various life cycle stagesof the parasite and the host species (2ndash6) A host acquires an infection by the bite of aninfected tick During a blood meal an infected tick inoculates the infectious sporozoitesinto the dermis of a host where they invade endothelium-associated mononuclear cellsand progress to form schizonts (Fig 1B) (2ndash6 16) These large schizont-filled macro-phages occlude small vessels in various organs especially in the lungs spleen kidneysand liver (2ndash6 11ndash13 15 32 44 86ndash91) Vascular obstruction is one of the majorpathophysiologic mechanisms that lead to considerable pathological changes such ascirculatory impairment hemolytic anemia and multiorgan dysfunction (2ndash6 11ndash13 1532 44 86ndash91)

An infected domestic cat first shows clinical signs at approximately 5 to 14 days piwith these signs including fever inappetence depression lethargy dyspnea vomitingicterus tachycardia generalized pain and vocalization The sick cat experiences anonregenerative anemia thrombocytopenia and lymphopenia (2ndash6 92ndash95) Moribundcats commonly manifest recumbency hypothermia and vocalization in the terminalstages of disease Most of them appear to die from a shock-like state (2ndash6)

A 1-year-old neutered male domestic shorthair cat was euthanized due to rapidlyworsening clinical signs the day after being evaluated for a sudden onset of inappe-tence and lethargy Necropsy findings included noncollapsing lungs with multiple lociof hemorrhage a brownish yellow liver and an enlarged spleen with a meaty consis-tency (95) In the brain schizont-laden macrophages of increased size were foundoccluding leptomeningeal and parenchymal arterioles and venules as well as in thesmall capillaries throughout the gray and white matter and the choroid plexus Necro-sis including necrotic neurons was found in the gray or white matter with markedvacuolation (95) In a retrospective study of 148 cases archived between January 1995and June 2005 in the Oklahoma Animal Disease Diagnostic Laboratory Snider andcolleagues found overall moderate interstitial pneumonia mild alveolar macrophagenumbers mild intra-alveolar hemorrhage moderate neutrophil infiltration and mod-erate to severe vascular occlusion (89)

Although schizogony of C felis appears to be short most domestic cats die in lessthan 1 week from the onset of signs In contrast most bobcats survive through thisstage These findings suggest that schizogony of this parasite is responsible for theclinical manifestations of cytauxzoonosis in the domestic cat (2ndash6 29 30 86ndash95)

Mature schizonts rupture the enlarged macrophages and release large numbers ofmerozoites which infect erythrocytes resulting in the formation of the ring-shaped




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piroplasms (Fig 1A) (16) In general clinical signs occur prior to the appearance ofblood piroplasms At the erythrocytic stage the piroplasm undergoes multiplicationand dividing forms may be observed Although it is low and difficult to detect in earlyinfection parasitemia typically increases as the disease progresses with values rangingfrom 1 to as high as 4 (2ndash6) It may reach as high as 50 in some unusual cases(2ndash6) In the acute phase parasitemia revealed by blood smears is commonly detectedin the late stage of cytauxzoonosis usually 1 to 3 days before death (2ndash6 15 86ndash95)

Domestic cats that have recovered from the acute infection frequently maintainintraerythrocytic piroplasms for years In this chronic stage piroplasms themselvesappear to be relatively harmless and do not cause clinical signs Nevertheless thesedomestic cats may serve as additional reservoir hosts Cats with subclinical cytauxzoo-nosis usually have lower parasitemia than that of cats with acute infections with valuesranging from 0045 to 127 (2ndash6 96) Host immune status may affect parasitemiasince immunosuppression of a host increased its parasitemia (97)

IMMUNOLOGY

Despite decades of research very limited information has been gained regardingthe feline immune response to C felis infection A previous study showed that domesticcats inoculated intraperitoneally with bobcat peripheral blood containing the non-pathogenic piroplasms generated anti-Cytauxzoon antibodies Antibody levels tendedto be related to the degree of parasitemia High antibody titers were observed at ornear the peak of parasitemia (97) Another study demonstrated that domestic catsinoculated with Pallasrsquos cat blood containing C manul piroplasms did not develop fatalcytauxzoonosis These cats were not completely protected from a challenge with C felisschizonts (56) On the other hand domestic cats that experienced and survived acuteinfections were protected against challenge with virulent C felis schizonts No domesticcats have recrudesced with clinical cytauxzoonosis after recovery from the acuteinfection (98) Collectively these data suggest that the immune response to initialinfection by C felis schizonts is important for the domestic cat to develop sufficientimmunity to protect against C felis schizonts

In order to further characterize immune responses in the domestic cat Frontera-Acevedo et al (99) studied cats that died of cytauxzoonosis acutely ill C felis-infectedcats healthy survivors of C felis infection and healthy uninfected cats They showedthat the concentrations of tumor necrosis factor alpha (TNF-) and interleukin-1

(IL-1) were higher and the serum albumin concentrations significantly lower in catsthat died of cytauxzoonosis than in the cats of the other three groups The concen-tration of serum albumin a negative acute-phase protein decreased during the acuteinflammatory response CD18 an important leukocyte adhesion molecule was upregu-lated during inflammation and stimulated the release of some proinflammatory cyto-kines and chemokines such as TNF- IL-1 and IL-6 Compared to the healthyuninfected cats cats that succumbed to infection had the highest CD18 level with an116-fold increase acutely ill cats had a moderate CD18 level with a 90-fold increaseand healthy survivors had a 13-fold increase These data collectively indicate that Cfelis infection causes a robust systemic inflammatory response and that the immuneresponse plays an important role in the pathogenesis of this disease (99)

In addition to a systemic inflammatory response C felis infections also cause localinflammatory responses in the affected organs such as the spleen and the lungsInterstitial pneumonia was common in the domestic cats with fatal cytauxzoonosissuggesting that inflammation was likely a consequence of reaction to the proinflam-matory mediators that were released by the schizont-laden macrophages Immunohis-tochemistry analysis of C felis-infected cat lungs confirmed that the expression ofproinflammatory mediators such as TNF- IL-1 IL-6 inducible nitric oxide synthaseand major histocompatibility complex class II was significantly increased compared tothat in uninfected lungs Furthermore these proinflammatory mediators were distrib-uted in almost all the infected lung tissues particularly in the cytoplasm of schizont-laden macrophages (100) Taken together the data show that C felis infections activate




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the M1 macrophage response in infected macrophages with secretion and expressionof various proinflammatory cytokines and other molecules which plays a pivotal role inthe morbidity and mortality of cytauxzoonosis

DIAGNOSISMicroscopic Diagnosis

Diagnosis of C felis infection is made by various methods (2ndash6) Microscopicdetection of parasites in thin blood smears andor fine-needle aspirates with Giemsa orHampE staining is the most common method (2ndash6) In the acute disease phase indomestic cats in an area of endemicity diagnosis is based on a combination of clinicalsigns and the finding of intraerythrocytic piroplasms in a blood smear or the enlargedschizont-filled macrophages in fine-needle tissue aspirates The latter may be takenfrom the spleen liver lymph nodes or lungs It is worth keeping in mind that thesensitivity of blood smears is lower than that of tissue aspirates and that intraerythro-cytic piroplasms may appear days after the onset of clinical signs (2ndash6)

As just mentioned intraerythrocytic piroplasms are widely used for diagnosis ofcytauxzoonosis (2ndash6 101 102) C felis piroplasms may present in several differentshapes with the characteristic one being the ldquosignet ringrdquo shape This ring of 1 to 2 min diameter is characterized by a thick round nuclear chromatin at one point of the ringOther forms include a bipolar oval ldquosafety pinrdquo form that has two nuclear areas onopposite sides and the anaplasmoid round ldquodotsrdquo form which is less than 05 m indiameter Additional forms such as small and linear comma-shaped and tetrad formsmay be seen (2ndash6) However there are several biological and technical limitations tothis method First levels of circulating piroplasms are usually low and piroplasms maybe absent in early infection because clinical signs are caused by schizonts and oftenprecede intraerythrocytic piroplasms detectable by microscopy In general the level ofintraerythrocytic piroplasms continues to increase over the course of the diseaseHowever piroplasms are present in only about half of acute cases of feline cytauxzoo-nosis from either natural or experimental infection (2ndash6) Therefore a lack of piro-plasms in a blood smear does not necessarily rule out the disease In a suspected casethe preparation of daily blood smears is recommended Second these piroplasms aremorphologically indistinguishable from those of Babesia spp or Theileria spp HoweverBabesia spp such as Babesia felis and Babesia catis are not endemic in the UnitedStates (2ndash6 103) In addition fever and icterus are uncommon in cats infected withBabesia spp or Theileria spp Third the small and pleomorphic piroplasms may bemistaken as Howell-Jolly bodies stain precipitate water artifacts or even Mycoplasmahaemofelis (syn Haemobartonella felis) Howell-Jolly bodies appear as dark round dotsof various sizes They do not have the typical characteristic ring forms of C felispiroplasms nor do they possess cytoplasm Stain precipitate is usually observedbetween blood cells as well as overlying blood cells M haemofelis a bacterium isusually observed around the periphery of an infected erythrocyte and usually formdots chains and small rings but it does not contain a nuclear area and it is rarelylocated inside an erythrocyte in a ring form (104) (Fig 5) A well-made thin blood smearshould have blood cells spread out sufficiently to avoid overlying blood cells Addi-tionally slides should be clean and thoroughly air-dried before starting the stainingprocess to avoid the development of stain precipitate or water artifacts Last but notleast intraerythrocytic piroplasms may occasionally be found in clinically healthydomestic cats some of which are survivors of C felis infection The latter usuallymaintain low levels of parasitemia for years Observation of intraerythrocytic piroplasmsamong these survivors does not necessarily confirm that clinical signs are due tocytauxzoonosis because they may be infected with other disease-causing pathogensthat may mistakenly be identified as fatal cytauxzoonosis in the presence of piroplasms(2ndash6)

Clinical Diagnosis

Although there are no clinical signs specific for feline cytauxzoonosis icterus




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anorexia and lethargy are the most common manifestations In most cases domesticcats died within less than 1 week of clinical sign onset and these cats were usually ingood nutritional condition (2ndash6) Domestic cats with outdoor exposure in areas ofendemicity where tick infestation is common are more likely to be infected with C felisIn addition most cytauxzoonosis cases are reported in spring summer or early autumnwhile the number of fatal cytauxzoonosis cases is significantly decreased in hot and dryperiods of summer Thus an acute onset of icterus anorexia and lethargy in domesticcats in the epidemic areas should alert a veterinarian to possible cytauxzoonosis (2ndash6)

Complete blood counts and serum biochemical analyses though nonspecific arealso useful for diagnosis of acute cytauxzoonosis Abnormal hematologic parametersthat are supportive of a diagnosis of cytauxzoonosis include the presence of dissemi-nated intravascular coagulation (DIC) and pancytopenia such as low white blood cellcounts thrombocytopenia and lymphopenia (2ndash6 92ndash94) Biochemical abnormalitiesin some fatal cytauxzoonosis cases include prolonged prothrombin time low protein Cactivity hyperglycemia hyperbilirubinemia hypoalbuminemia hypocalcemia andmoderately increased alanine transaminase and aspartate aminotransferase levels (2ndash692ndash94) In addition hemolytic anemia is also a frequent finding in the acute phaseHowever these abnormal hematologic parameters do not occur in all cases (2ndash692ndash94)

Immunologic Diagnosis

Antibodies to Cytauxzoon spp do exist in the sera from experimentally infecteddomestic cats Due to the technical difficulty of generating purified antigens nodiagnostic kits for detection of antibodies to C felis are commercially available (97 105)On the other hand an indirect fluorescent-antibody (IFA) test using serum isolated froma recovered domestic cat detects C felis antigens in tissues of experimentally infecteddomestic cats although its specificity has yet to be determined However this IFA testis not very useful for diagnosis of infected but clinically normal cats and the naturalreservoir bobcats in which the titer of parasitic antigens is usually very low (105)Uilenberg et al (54) used piroplasm antigens of C felis to detect antibodies in infecteddomestic cats or bobcats This method has limited usage in acute cases because

FIG 5 Microscopic differentiation of Cytauxzoon felis from other common intraerythrocytic pathogens or particlesin a feline blood smear




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domestic cats have not yet developed antibodies or have only low titers of antibodybefore death

Molecular Diagnosis

An in situ hybridization (ISH) assay was developed to examine various tissues ofseven archived cases of feline cytauxzoonosis The digoxigenin-labeled probe targetedthe Babesia microti 16S rRNA-like gene which is 91 identical to that of C felis ISH is2 to 10 times more sensitive than HampE staining and has the advantage of applicationto archived tissue However ISH specificity is low and there is variation among differenttissuescells (106) The low specificity in this case may be due to use of a nonspecificprobe

Another molecular technique is PCR which has a high sensitivity and specificity (3368 102 107ndash111) So far PCR target genes include the C felis 18S rRNA gene (102) ITS-1and ITS-2 (33 68 101 108ndash110) and a multicopy mitochondrial gene (cox3) (111) PCRtargeting the cox3 gene was more sensitive than that amplifying the 18S rRNA genedue to a higher copy number (111) PCR assays are generally 1000-fold more sensitivethan microscopic examination of a blood smear PCR is especially applicable to confirmclinical suspicion of C felis infection in the absence of microscopic piroplasms and todetect infections in domestic cat survivors of acute cytauxzoonosis Further PCR assayespecially along with DNA sequencing can differentiate Cytauxzoon spp from Theileriaspp and Babesia spp even though they are morphologically indistinguishable (33 4368 102 108ndash111) Drawbacks of PCR include being incapable of differentiating acutedisease from chronic C felis infection and requiring a time frame of several hours foronsite performance or days for samples remotely mailed out to a diagnostic laboratoryThis delay of diagnosis is crucial since veterinarians cannot afford to lose these crucialhours in the acute phase of the disease

DISEASE TREATMENT AND PREVENTION

Historically cytauxzoonosis has been considered a uniformly fatal infection in thedomestic cat with almost 100 mortality (2ndash6) However recent studies have revealeddomestic cat survivors of C felis infection although they are infrequent Furthermorethe incidence of survival cases is increasing with recent advances in treatment andorthe possible existence of a less virulent strain of C felis as discussed below There iscurrently no routine veterinary care or therapy for feline cytauxzoonosis A variety ofantibiotics including clindamycin enrofloxacin and antiprotozoal agents have beentested for treatment of domestic cats experimentally or naturally infected with C feliswith various efficacies (2ndash6) The antiprotozoal agents parvaquone and buparvaquoneknown to be effective in therapy of bovine theileriosis are ineffective against experi-mental feline cytauxzoonosis (98) Diminazene aceturate has been used to treat babe-siosis and other protozoal diseases in the United States without approval of the USFood and Drug Administration Intramuscular (im) injection of diminazene aceturate ata dose of 2 mgkg successfully treated five of six naturally infected domestic cats in onetrial (96) However it failed to reduce parasite burdens in clinically healthy chronicallyor naturally infected domestic cats in another trial even at a higher dose of 4 mgkgim (112 113) Moreover these treated domestic cats experienced multiple adverseside effects so this treatment is not recommended for chronic carrier domestic cats(112 113)

Several cytauxzoonosis cases were successfully treated with imidocarb an antipro-tozoal drug used in the United States for treating canine babesiosis (14 48 114 115)An open-label randomized prospective study was undertaken to evaluate the efficacyof treatment of feline cytauxzoonosis with imidocarb (35 mgkg im) or a combinationof the antibiotics azithromycin (10 mgkg po q24h) and atovaquone (an antimalarialagent) (15 mgkg po q8h) (AampA) Thirty-two of 53 domestic cats (604) treated withAampA survived In contrast only 259 of domestic cats (727 cats) receiving imidocarbsurvived To date AampA is considered the most effective treatment for acute infection(114) However atovaquone is expensive and is difficult to access Atovaquone targets




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C felis cytochrome b and its efficacy is highly variable (116 117) AampA also fails to clearchronic parasitemia (114) Therefore more studies are needed to verify the efficacy ofAampA and to develop more effective and cheaper drugs

In addition to antiprotozoal regimens intensive supportive therapy is beneficialalthough there is no obvious evidence to support such a claim Because death appearssoon after the onset of illness immediate implementation of intensive supportivetherapy is highly recommended Infected domestic cats usually seem to be in pain soan analgesic therapy such as buprenorphine (001 mgkg given intravenously [iv] orpo q8h) is recommended (2 3) Antithrombotic therapies such as heparin (200 Ukggiven subcutaneously [sc] q8h) are administered to prevent DIC (2 3) Other support-ive therapies including intravenous crystalloid fluid therapy oxygen supplementationplasma supplementation blood cell transfusion and antiemetic therapy (maropitantcitrate 1 mgkg sc or po q24h) may be needed based on the manifestations(dehydration dyspnea anemia and vomiting) of the infected domestic cat (2ndash6)

Transmission of C felis to domestic cats is usually by a tick vector and cannot occurfrom one domestic cat to another by direct contact even when domestic cats are keptin a small cattery throughout the course of the disease or via the placenta (57)Currently no vaccines are commercially available although a vaccine candidate cf76was recently identified by comparative apicomplexan genomics (52) Minimizing ex-posure to the tick vector is the only effective way to prevent transmission of C felis tothe domestic cat Keeping cats indoors or implementing ectoparasite control onoutdoor cats should be advocated in the regions of endemicity As mentioned earliera collar containing 100 imidacloprid and 45 flumethrin (Seresto Bayer AnimalHealth) was effective at preventing transmission of C felis by preventing ticks fromattaching to cats (73) Transmission of C felis from a tick vector to a domestic catrequires more than 36 h (72) Therefore it should be beneficial to develop an anti-arachnid drug that kills ticks within a few hours of attachment

In transfusion medicine attention should be paid to detecting the presence ofCytauxzoon piroplasms in feline blood donors in the area of endemicity Inoculation ofpiroplasms by blood transfusion may lead to recipient cats becoming chronic carriersand serving as a reservoir (118 119)

MOLECULAR DIVERSITY OF THE PATHOGEN CORRELATION BETWEENPARASITES AND CLINICAL OUTCOMES

The increasing number of reports of domestic cat survival of C felis infectionssuggests the existence of distinct genotypes of C felis with different virulence poten-tials for domestic cats To test this hypothesis several genetic markers have been usedto study the genetic variability among C felis protozoan populations The first markeris the 18S rRNA gene However C felis 18S rRNA gene sequences from domestic catsthat survived infections were almost identical to those from domestic cats that died ofC felis infections (14) The second one is ITS-1 and ITS-2 These genes evolve morequickly than the 18S rRNA gene and are more likely to reveal C felis genotypicvariability (120) Genetic analysis of the C felis ITS regions showed that the ITS sequencevariability is high with many polymorphisms and suggested that there is a diversepopulation of C felis strains (33 108ndash110) A combination of ITS-1 and ITS-2 was usedto determine the genotypes So far more than 22 genotypes named ITSa to ITSv havebeen detected in C felis strains isolated from various domestic cats and wild felids(Table 2) First a study of the genetic variability of C felis isolates from 88 infecteddomestic cats in Arkansas and Georgia showed that a strong association existedbetween the C felis genotype and disease severity Specifically domestic cats infectedwith ITSa-type C felis had a high survival rate In contrast genotype ITSb was associatedwith a high fatality rate Moreover 47 of 48 ITSa isolates were from Arkansas and onlyone was from Georgia whereas all ITSb isolates were from Georgia These datademonstrated that ITS genotypes vary geographically (108) Another study of geneticvariability of archived C felis isolates from domestic cats in Georgia from 1995 to 2007found 11 different genotypes among 48 C felis samples with the most common




httpcmrasm

orgD

ownloaded from

genotype ITSo detected in only one fatality sample (109) Collectively these twostudies support the existence of diverse C felis populations and the coexistence ofvarious genotypes in one area Although ITSb was strongly associated with fatality asmentioned earlier an additional study including C felis isolates from 25 infected butclinically normal domestic cats revealed three genotypes ie ITSa ITSb and ITSc (33)Studies of the genetic variability of C felis strains isolated from bobcats also yieldeddiverse genotypes with some genotypes that were found only in bobcats (110) (Table2) Taken together the unique ITS phenotypes varied geographically and were suitablefor studying genetic diversity among C felis populations An association might existbetween ITS phenotypes and virulence potential eg ITSb is associated with highmortality However extreme caution should be taken in interpretation of such data atthis time since this phenotype exists in clinically healthy infected domestic cats as well(Table 2) (33 108ndash110 114) Further studies with a bigger sample size are warranted Asfar as markers other than ITS are concerned very little is known Whole-genomesequencing or identification of genetic variability in genes that encode key proteinsplaying roles in the pathogenicity of C felis may be the best way to understand geneticvariability and to investigate the pathogenicity and virulence of C felis

CONCLUDING REMARKS AND FUTURE PERSPECTIVES

Cytauxzoonosis is an emerging hemoprotozoal disease of the domestic cat and wildfelids and is caused by tick-borne Cytauxzoon spp Over the past 40 years knowledgeabout the disease and C felis has grown considerably The parasite is mainly endemicto North America and South America Although infections by C felis have historicallybeen considered uniformly fatal in the domestic cat and clinically self-limited in wild

TABLE 2 Information on Cytauxzoon felis genotypes and outcomes of infection

Genotypei

GenBank accessionno

No of animals

Study 1a Study 2b Study 3c Study 4d Study 5e

ITS-1 ITS-2Domesticcats Survivors

Archiveddomestic cats

Asymptomaticdomestic cats Bobcats Bobcats Pumas

Domesticcats

Survivors thatreceived drug

ITSa EU450802 EU450804 48 38 3 16 5 11 1 14 9f

ITSb EU450802 EU450805 21 4 8 8 0 1 0ITSc EU450803 EU450804 5 0 1 0 0 0 2 1g

ITSd GU581166 EU450805 0 1 0 0ITSe GU581167 GU581170 0 1 8 0ITSf EU450802 FJ536421 2 0 1 0 1 0 0ITSg GU581167 GU581171 0 8 2 0ITSh EU450802 GU581172 0 1 0 0ITSi EU450802 FJ536419 4 2 3 0 3 2 0ITSj FJ536425 EU450804 0 2 0 0ITSk FJ536425 GU581171 0 1 0 0ITSl GU581168 GU581171 0 1 0 0ITSm GU581169 GU581171 0 1 0 0ITSn EU450802 EU450806 1 0 0 0ITSo EU450802 FJ536418 1 0 27 0 0ITSp EU450802 FJ536420 1 0 0ITSq FJ536423 FJ536418 1 0 0ITSr FJ536424 FJ536418 1 0 0ITSs FJ536425 FJ536418 1 0 0ITSt FJ536426 FJ536418 1 0 0 5 4h

ITSu EU450802 FJ536422 1 0 0ISTv FJ536425 FJ536422 2 0 0 0aReference 108bReference 109cReference 33dReference 110eReference 114fSeven of eight domestic cats that received atovaquone and azithromycin and two of six domestic cats that received imidocarb survivedgOne of two domestic cats that received atovaquone and azithromycin survivedhAll three domestic cats that received atovaquone and azithromycin and one of two domestic cats that received imidocarb survivediGenotypes are designated based on the scheme from reference 110




httpcmrasm

orgD

ownloaded from

felids increasing evidence suggests that nonfatal infections occur in the domestic catand that fatal cases occur in wild felids It is plausible that the genetic differencesamong distinct C felis populations may cause various pathogenicities However thesewarrant further investigations Moreover the mechanisms of disease pathogenesisalternate tick vectors other than D variabilis and A americanum and host immuneresponses are unclear Currently there are no commercially available diagnostic kits forC felis infections no vaccines against C felis exist and prevention is limited to tickcontrol In addition C felis has yet to be cultured continuously in vitro which greatlyhinders the study of the parasitersquos biology and vaccine development Comprehensiveand systematic research is urgently needed to illuminate the ecology epidemiologybiology and possible control of this devastating disease

ACKNOWLEDGMENTSThis work was partially supported by the Fundamental Research Funds of the

Chinese Academy of Agricultural Sciences (grant Y2016JC05) and the AgriculturalScience and Technology Innovation Program (ASTIP) (grant CAAS-ASTIP-2014-LVRI-03)

We are very thankful to Mark Forman of the Ross University School of VeterinaryMedicine for proofreading the manuscript

CY and X-QZ conceived the review CY and J-LW wrote the first draft T-TL andG-HL helped in the preparation of the manuscript G-HL performed phylogenetictree analysis All authors read and approved the final version

REFERENCES1 Wagner JE 1976 A fatal cytauxzoonosis-like disease in cats J Am Vet

Med Assoc 168585ndash5882 Sherrill MK Cohn LA 2015 Cytauxzoonosis diagnosis and treatment of

an emerging disease J Feline Med Surg 17940 ndash948 httpsdoiorg1011771098612X15610681

3 Lloret A Addie DD Boucraut-Baralon C Egberink H Frymus TGruffydd-Jones T Hartmann K Horzinek MC Hosie MJ Lutz H MarsilioF Pennisi MG Radford AD Thiry E Truyen U Moumlstl K EuropeanAdvisory Board on Cat Diseases 2015 Cytauxzoonosis in cats ABCDguidelines on prevention and management J Feline Med Surg 17637ndash 641 httpsdoiorg1011771098612X15589878

4 Greene CE Meinkoth J Kocan AA 2006 Cytauxzoonosis p 722ndash733 InGreene CE (ed) Infectious diseases of the dog and cat 3rd ed SaundersElsevier St Louis MO

5 Meinkoth JH Kocan AA 2005 Feline cytauxzoonosis Vet Clin North AmSmall Anim Pract 3589ndash101 httpsdoiorg101016jcvsm200408003

6 Holman PJ Snowden KF 2009 Canine hepatozoonosis and babesiosisand feline cytauxzoonosis Vet Clin North Am Small Anim Pract 391035ndash1053 httpsdoiorg101016jcvsm200908002

7 Allsopp MT Cavalier-Smith T De Waal DT Allsopp BA 1994 Phylogenyand evolution of the piroplasms Parasitology 108147ndash152 httpsdoiorg101017S0031182000068232

8 Criado-Fornelio A Goacutenzalez-del-Riacuteo MA Buling-Sarantildea A Barba-Carretero JC 2004 The ldquoexpanding universerdquo of piroplasms Vet Para-sitol 119337ndash345 httpsdoiorg101016jvetpar200311015

9 Glenn BL Rolley RE Kocan AA 1982 Cytauxzoon-like piroplasms inerythrocytes of wild-trapped bobcats in Oklahoma J Am Vet Med Assoc1811251ndash1253

10 Glenn BL Kocan AA Blouin EF 1983 Cytauxzoonosis in bobcats J AmVet Med Assoc 1831155ndash1158

11 Hoover JP Walker DB Hedges JD 1994 Cytauxzoonosis in cats eightcases (1985ndash1992) J Am Vet Med Assoc 203455ndash 460

12 Birkenheuer AJ Le JA Valenzisi AM Tucker MD Levy MG BreitschwerdtEB 2006 Cytauxzoon felis infection in cats in the mid-Atlantic states 34cases (1998 ndash2004) J Am Vet Med Assoc 228568 ndash571 httpsdoiorg102460javma2284568

13 Hauck WN Snider TG III Lawrence JE 1982 Cytauxzoonosis in a nativeLouisiana cat J Am Vet Med Assoc 1801472ndash1474

14 Meinkoth J Kocan AA Whitworth L Murphy G Fox JC Woods JP 2000Cats surviving natural infection with Cytauxzoon felis 18 cases(1997ndash1998) J Vet Intern Med 14521ndash525

15 Meier HT Moore LE 2000 Feline cytauxzoonosis a case report and

literature review J Am Anim Hosp Assoc 36493ndash 496 httpsdoiorg10532615473317-36-6-493

16 Miller J Davis CD 2013 Increasing frequency of feline cytauxzoonosiscases diagnosed in western Kentucky from 2001 to 2011 Vet Parasitol198205ndash208 httpsdoiorg101016jvetpar201308012

17 Peixoto PV Soares CO Scofield A Santiago CD Franccedila TN Barros SS2007 Fatal cytauxzoonosis in captive-reared lions in Brazil Vet Parasitol145383ndash387 httpsdoiorg101016jvetpar200612023

18 Maia LM Cerqueira Ade M de Barros Macieira D de Souza AM MoreiraNS da Silva AV Messick JB Ferreira RF Almosny NR 2013 Cytauxzoonfelis and lsquoCandidatus Mycoplasma haemominutumrsquo coinfection in aBrazilian domestic cat (Felis catus) Rev Bras Parasitol Vet 22289 ndash291httpsdoiorg101590S1984-29612013000200049

19 Ketz-Riley CJ Reichard MV Van den Bussche RA Hoover JP MeinkothJ Kocan AA 2003 An intraerythrocytic small piroplasm in wild-caughtPallasrsquos cats (Otocolobus manul) from Mongolia J Wildl Dis 39424 ndash 430httpsdoiorg1075890090-3558-392424

20 Reichard MV Van Den Bussche RA Meinkoth JH Hoover JP Kocan AA2005 A new species of Cytauxzoon from Pallasrsquo cats caught in Mongoliaand comments on the systematics and taxonomy of piroplasmids JParasitol 9420 ndash 426 httpsdoiorg101645GE-384R

21 Luaces I Aguirre E Garciacutea-Montijano M Velarde J Tesouro MA SaacutenchezC Galka M Fernaacutendez P Sainz A 2005 First report of an intraerythro-cytic small piroplasm in wild Iberian lynx (Lynx pardinus) J Wildl Dis41810 ndash 815 httpsdoiorg1075890090-3558-414810

22 Carli E Trotta M Chinelli R Drigo M Sinigoi L Tosolini P Furlanello TMillotti A Caldin M Solano-Gallego L 2012 Cytauxzoon sp infection inthe first endemic focus described in domestic cats in Europe VetParasitol 183343ndash352 httpsdoiorg101016jvetpar201107025

23 Millaacuten J Naranjo V Rodriacuteguez A de la Lastra JM Mangold AJ de laFuente J 2007 Prevalence of infection and 18S rRNA gene sequencesof Cytauxzoon species in Iberian lynx (Lynx pardinus) in Spain Parasi-tology 134995ndash1001 httpsdoiorg101017S003118200700248X

24 Alho AM Silva J Fonseca MJ Santos F Nunes C de Carvalho LMRodrigues M Cardoso L 2016 First report of Cytauxzoon sp infectionin a domestic cat from Portugal Parasit Vectors 9220 httpsdoiorg101186s13071-016-1506-5

25 Rassouli M Sabouri S Goudarzi A Parsa M 2015 Cytauxzoon felis in astray cat in Iran Comp Clin Pathol 2475ndash77 httpsdoiorg101007s00580-013-1858-6

26 Zaeemi M Razmi GR Khoshnegah J 2015 The first detection of Cytaux-




httpcmrasm

orgD

ownloaded from

zoon felis in a wild cat (Felis silvestris) in Iran Comp Clin Pathol 24181ndash184 httpsdoiorg101007s00580-014-1898-6

27 Kelly P Marabini L Dutlow K Zhang J Loftis A Wang C 2014 Moleculardetection of tick-borne pathogens in captive wild felids ZimbabweParasit Vectors 7514 httpsdoiorg101186s13071-014-0514-6

28 Leclaire S Menard S Berry A 2015 Molecular characterization ofBabesia and Cytauxzoon species in wild South-African meerkats Para-sitology 142543ndash548 httpsdoiorg101017S0031182014001504

29 Blouin EF Kocan AA Glenn BL Kocan KM Hair JA 1984 Transmissionof Cytauxzoon felis Kier 1979 from bobcats Felis rufus (Schreber) todomestic cats by Dermacentor variabilis (Say) J Wildl Dis 20241ndash242httpsdoiorg1075890090-3558-203241

30 Blouin EF Kocan AA Kocan KM Hair J 1987 Evidence of a limitedschizogonous cycle for Cytauxzoon felis in bobcats following exposureto infected ticks J Wildl Dis 23499 ndash501 httpsdoiorg1075890090-3558-233499

31 Glenn BL Stair EL 1984 Cytauxzoonosis in domestic cats report of twocases in Oklahoma with a review and discussion of the disease J AmVet Med Assoc 184822ndash 825

32 Jackson CB Fisher T 2006 Fatal cytauxzoonosis in a Kentucky cat (Felisdomesticus) Vet Parasitol 139192ndash195 httpsdoiorg101016jvetpar200602039

33 Brown HM Lockhart JM Latimer KS Peterson DS 2010 Identificationand genetic characterization of Cytauxzoon felis in asymptomatic do-mestic cats and bobcats Vet Parasitol 172311ndash316 httpsdoiorg101016jvetpar201004041

34 Garner MM Lung NP Citino S Greiner EC Harvey JW Homer BL 1996Fatal cytauxzoonosis in a captive-reared white tiger (Panthera tigris)Vet Pathol 3382ndash 86 httpsdoiorg101177030098589603300111

35 Jakob W Wesemeier HH 1996 A fatal infection in a Bengal tigerresembling cytauxzoonosis in domestic cats J Comp Pathol 114439 ndash 444 httpsdoiorg101016S0021-9975(96)80018-1

36 Nietfeld JC Pollock C 2002 Fatal cytauxzoonosis in a free-rangingbobcat (Lynx rufus) J Wildl Dis 38607ndash 610 httpsdoiorg1075890090-3558-383607

37 Rizzi TE Reichard MV Cohn LA Birkenheuer AJ Taylor JD Meinkoth JH2015 Prevalence of Cytauxzoon felis infection in healthy cats fromenzootic areas in Arkansas Missouri and Oklahoma Parasit Vectors813 httpsdoiorg101186s13071-014-0618-z

38 Simpson CF Harvey JW Carlisle JW 1985 Ultrastructure of the intra-erythrocytic stage of Cytauxzoon felis Am J Vet Res 461178 ndash1180

39 Criado-Fornelio A Martinez-Marcos A Buling-Sarantildea A Barba-CarreteroJC 2003 Molecular studies on Babesia Theileria and Hepatozoon insouthern Europe Part II Phylogenetic analysis and evolutionary historyVet Parasitol 114173ndash194

40 Lack JB Reichard MV Van Den Bussche RA 2012 Phylogeny andevolution of the Piroplasmida as inferred from 18S rRNA sequences IntJ Parasitol 42353ndash363 httpsdoiorg101016jijpara201202005

41 Sivakumar T Hayashida K Sugimoto C Yokoyama N 2014 Evolutionand genetic diversity of Theileria Infect Genet Evol 27250 ndash263 httpsdoiorg101016jmeegid201407013

42 Allsopp MT Allsopp BA 2006 Molecular sequence evidence for thereclassification of some Babesia species Ann N Y Acad Sci 1081509 ndash517 httpsdoiorg101196annals1373076

43 Schreeg ME Marr HS Tarigo JL Cohn LA Bird DM Scholl EH LevyMG Wiegmann BM Birkenheuer AJ 2016 Mitochondrial genomesequences and structures aid in the resolution of Piroplasmidaphylogeny PLoS One 11e0165702 httpsdoiorg101371journalpone0165702

44 Kocan AA Kocan KM Blouin EF Mukolwe SW 1992 A redescription ofschizogony of Cytauxzoon felis in the domestic cat Ann N Y Acad Sci653161ndash167 httpsdoiorg101111j1749-66321992tb19639x

45 Spooner RL Innes EA Glass EJ Millar P Brown CG 1988 Bovinemononuclear cell lines transformed by Theileria parva or Theileria an-nulata express different subpopulation markers Parasite Immunol 10619 ndash 629 httpsdoiorg101111j1365-30241988tb00249x

46 Spooner RL Innes EA Glass EJ Brown CG 1989 Theileria annulata andT parva infect and transform different bovine mononuclear cells Im-munology 66284 ndash288

47 Criado-Fornelio A Buling A Pingret JL Etievant M Boucraut-Baralon CAlongi A Agnone A Torina A 2009 Hemoprotozoa of domestic ani-mals in France prevalence and molecular characterization Vet Parasitol15973ndash76 httpsdoiorg101016jvetpar200810012

48 Carli E Trotta M Bianchi E Furlanello T Caldin M Pietrobelli M

Solano-Gallego L 2014 Cytauxzoon sp infection in two free rangingyoung cats clinicopathological findings therapy and follow up TurkiyeParazitol Derg 38185ndash189 httpsdoiorg105152tpd20143540

49 Millaacuten J Candela MG Palomares F Cubero MJ Rodriacuteguez A Barral Mde la Fuente J Almeriacutea S Leoacuten-Vizcaiacuteno L 2009 Disease threats to theendangered Iberian lynx (Lynx pardinus) Vet J 182114 ndash124 httpsdoiorg101016jtvjl200804005

50 Meli ML Cattori V Martiacutenez F Loacutepez G Vargas A Simoacuten MA Zorrilla IMuntildeoz A Palomares F Loacutepez-Bao JV Pastor J Tandon R Willi BHofmann-Lehmann R Lutz H 2009 Feline leukemia virus and otherpathogens as important threats to the survival of the critically endan-gered Iberian lynx (Lynx pardinus) PLoS One 4e4744 httpsdoiorg101371journalpone0004744

51 Gallusovaacute M Jirsovaacute D Mihalca AD Gherman CM DrsquoAmico G QablanMA Modryacute D 2016 Cytauxzoon infections in wild felids fromCarpathian-Danubian-Pontic space further evidence for a differentCytauxzoon species in European felids J Parasitol 102377ndash380 httpsdoiorg10164515-881

52 Tarigo JL Scholl EH McK Bird D Brown CC Cohn LA Dean GA LevyMG Doolan DL Trieu A Nordone SK Felgner PL Vigil A BirkenheuerAJ 2013 A novel candidate vaccine for cytauxzoonosis inferred fromcomparative apicomplexan genomics PLoS One 8e71233 httpsdoiorg101371journalpone0071233

53 Kier AB Wightman SR Wagner JE 1982 Interspecies transmission ofCytauxzoon felis Am J Vet Res 43102ndash105

54 Uilenberg G Franssen FF Perieacute NM 1987 Relationships betweenCytauxzoon felis and African piroplasmids Vet Parasitol 2621ndash28httpsdoiorg1010160304-4017(87)90073-2

55 Butt MT Bowman D Barr MC Roelke ME 1991 Iatrogenic transmissionof Cytauxzoon felis from a Florida panther (Felix concolor coryi) to adomestic cat J Wildl Dis 27342ndash347 httpsdoiorg1075890090-3558-272342

56 Joyner PH Reichard MV Meinkoth JH Milne VE Confer AW Kocan AAHoover JP 2007 Experimental infection of domestic cats (Felis domes-ticus) with Cytauxzoon manul from Pallasrsquo cats (Otocolobus manul) VetParasitol 146302ndash306 httpsdoiorg101016jvetpar200703001

57 Lewis KM Cohn LA Birkenheuer AJ 2012 Lack of evidence for perinataltransmission of Cytauxzoon felis in domestic cats Vet Parasitol 188172ndash174 httpsdoiorg101016jvetpar201202019

58 Rotstein DS Taylor SK Harvey JW Bean J 1999 Hematologic effects ofcytauxzoonosis in Florida panthers and Texas cougars in Florida J WildlDis 35613ndash 617 httpsdoiorg1075890090-3558-353613

59 Lewis KM Cohn LA Downey ME Whitney MS Birkenheuer AJ 2012Evaluation of Cytauxzoon felis infection status in captive-born wildfelids housed in an area endemic for the pathogen J Am Vet MedAssoc 2411088 ndash1092 httpsdoiorg102460javma24181088

60 Yabsley MJ Murphy SM Cunningham MW 2006 Molecular detectionand characterization of Cytauxzoon felis and a Babesia species in cou-gars from Florida J Wildl Dis 42366 ndash374 httpsdoiorg1075890090-3558-422366

61 Filoni C Catatildeo-Dias JL Cattori V Willi B Meli ML Correcirca SH MarquesMC Adania CH Silva JC Marvulo MF Ferreira Neto JS Durigon EL deCarvalho VM Coutinho SD Lutz H Hofmann-Lehmann R 2012 Surveil-lance using serological and molecular methods for the detection ofinfectious agents in captive Brazilian neotropic and exotic felids J VetDiagn Invest 24166 ndash173 httpsdoiorg1011771040638711407684

62 Andreacute MR Adania CH Machado RZ Allegretti SM Felippe PA Silva KFNakaghi AC Dagnone AS 2009 Molecular detection of Cytauxzoon sppin asymptomatic Brazilian wild captive felids J Wildl Dis 45234 ndash237httpsdoiorg1075890090-3558-451234

63 Furtado MM Taniwaki SA Metzger B Dos Santos Paduan K OrsquoDwyerHL de Almeida Jaacutecomo AT Porfiacuterio GE Silveira L Sollmann R TocircrresNM Ferreira Neto JS 2017 Is the free-ranging jaguar (Panthera onca) areservoir for Cytauxzoon felis in Brazil Ticks Tick Borne Dis 8470 ndash 476httpsdoiorg101016jttbdis201702005

64 Barandika JF Espiacute A Oporto B del Cerro A Barral M Povedano IGarciacutea-Peacutereza L Hurtado A 2016 Occurrence and genetic diversity ofpiroplasms and other apicomplexa in wild carnivores Parasitol Open2e6 httpsdoiorg101017pao20164

65 Veronesi F Ravagnan S Cerquetella M Carli E Olivieri E Santoro APesaro S Berardi S Rossi G Ragni B Beraldo P Capelli G 2016 Firstdetection of Cytauxzoon spp infection in European wildcats (Felissilvestris silvestris) of Italy Ticks Tick Borne Dis 7853ndash 858 httpsdoiorg101016jttbdis201604003




httpcmrasm

orgD

ownloaded from

66 Araya-Anchetta A Busch JD Scoles GA Wagner DM 2015 Thirty yearsof tick population genetics a comprehensive review Infect Genet Evol29164 ndash179 httpsdoiorg101016jmeegid201411008

67 Shock BC Moncayo A Cohen S Mitchell EA Williamson PC Lopez GGarrison LE Yabsley MJ 2014 Diversity of piroplasms detected inblood-fed and questing ticks from several states in the United StatesTicks Tick Borne Dis 25373ndash380 httpsdoiorg101016jttbdis201401003

68 Bondy PJ Jr Cohn LA Tyler JW Marsh AE 2005 Polymerase chainreaction detection of Cytauxzoon felis from field-collected ticks andsequence analysis of the small subunit and internal transcribed spacer1 region of the ribosomal RNA gene J Parasitol 91458 ndash 461 httpsdoiorg101645GE-374R

69 Reichard MV Meinkoth JH Edwards AC Snider TA Kocan KM BlouinEF Little SE 2009 Transmission of Cytauxzoon felis to a domestic cat byAmblyomma americanum Vet Parasitol 161110 ndash115 httpsdoiorg101016jvetpar200812016

70 Reichard MV Edwards AC Meinkoth JH Snider TA Meinkoth KR HeinzRE Little SE 2010 Confirmation of Amblyomma americanum (AcariIxodidae) as a vector for Cytauxzoon felis (Piroplasmorida Theileriidae)to domestic cats J Med Entomol 47890 ndash 896

71 Shock BC Murphy SM Patton LL Shock PM Olfenbuttel C Beringer JPrange S Grove DM Peek M Butfiloski JW Hughes DW Lockhart JMBevins SN VandeWoude S Crooks KR Nettles VF Brown HM PetersonDS Yabsley MJ 2011 Distribution and prevalence of Cytauxzoon felis inbobcats (Lynx rufus) the natural reservoir and other wild felids inthirteen states Vet Parasitol 175325ndash330 httpsdoiorg101016jvetpar201010009

72 Thomas JE Ohmes CM Payton ME Hostetler JA Reichard MV 2017Minimum transmission time of Cytauxzoon felis by Amblyomma ameri-canum to domestic cats in relation to duration of infestation andinvestigation of ingestion of infected ticks as a potential route oftransmission J Feline Med Surg 11098612X17691172 httpsdoiorg1011771098612X17691172

73 Reichard MV Thomas JE Arther RG Hostetler JA Raetzel KL MeinkothJH Little SE 2013 Efficacy of an imidacloprid 10flumethrin 45collar (Serestoreg Bayer) for preventing the transmission of Cytauxzoonfelis to domestic cats by Amblyomma americanum Parasitol Res112(Suppl 1)S11ndashS20 httpsdoiorg101007s00436-013-3277-7

74 Schuster FL 2002 Cultivation of Babesia and Babesia-like bloodparasites agents of an emerging zoonotic disease Clin Microbiol Rev15365ndash373 httpsdoiorg101128CMR153365-3732002

75 Homer MJ Aguilar-Delfin I Telford SR III Krause PJ Persing DH 2000Babesiosis Clin Microbiol Rev 13451ndash 469 httpsdoiorg101128CMR133451-4692000

76 Uilenberg G 2006 Babesiamdasha historical overview Vet Parasitol 1383ndash10 httpsdoiorg101016jvetpar200601035

77 Schnittger L Rodriguez AE Florin-Christensen M Morrison DA 2012Babesia a world emerging Infect Genet Evol 121788 ndash1809 httpsdoiorg101016jmeegid201207004

78 Bishop R Musoke A Morzaria S Gardner M Nene V 2004 Theileriaintracellular protozoan parasites of wild and domestic ruminants trans-mitted by ixodid ticks Parasitology 129(Suppl)S271ndashS283 httpsdoiorg101017S0031182003004748

79 Varshney JP Deshmukh VV Chaudhary PS 2009 Fatal cytauxzoonosisin a kitten Intas Polivet 10392ndash393

80 Andreacute MR Herrera HM Fernandes SJ de Sousa KC Gonccedilalves LRDomingos IH de Macedo GC Machado RZ 2015 Tick-borne agents indomesticated and stray cats from the city of Campo Grande state ofMato Grosso do Sul midwestern Brazil Ticks Tick Borne Dis 6779 ndash786httpsdoiorg101016jttbdis201507004

81 Reichard MV Baum KA Cadenhead SC Snider TA 2008 Temporaloccurrence and environmental risk factors associated with cytauxzoo-nosis in domestic cats Vet Parasitol 152314 ndash320 httpsdoiorg101016jvetpar200712031

82 Raghavan RK Almes K Goodin DG Harrington JA Jr Stackhouse PW Jr2014 Spatially heterogeneous land coverland use and climatic riskfactors of tick-borne feline cytauxzoonosis Vector Borne Zoonotic Dis14486 ndash 495 httpsdoiorg101089vbz20131496

83 Mueller EK Baum KA Papes M Cohn LA Cowell AK Reichard MV 2013Potential ecological distribution of Cytauxzoon felis in domestic cats inOklahoma Missouri and Arkansas Vet Parasitol 192104 ndash110 httpsdoiorg101016jvetpar201210008

84 Walker DB Cowell RL 1995 Survival of a domestic cat with naturallyacquired cytauxzoonosis J Am Vet Med Assoc 2061363ndash1365

85 Haber MD Tucker MD Marr HS Levy JK Burgess J Lappin MR Birken-heuer AJ 2007 The detection of Cytauxzoon felis in apparently healthyfree-roaming cats in the USA Vet Parasitol 146316 ndash320 httpsdoiorg101016jvetpar200702029

86 Wightman SR Kier AB Wagner JE 1977 Feline cytauxzoonosis clinicalfeatures of a newly described blood parasite disease Feline Pract723ndash26

87 Kier AB Wagner JE Kinden DA 1987 The pathology of experimentalcytauxzoonosis J Comp Pathol 97415ndash 432 httpsdoiorg1010160021-9975(87)90020-X

88 Harvey JW Dunbar MR Norton TM Yabsley MJ 2007 Laboratoryfindings in acute Cytauxzoon felis infection in cougars (Puma concolorcougar) in Florida J Zoo Wildl Med 38285ndash291 httpsdoiorg1016381042-7260(2007)038[0285LFIACF]20CO2

89 Snider TA Confer AW Payton ME 2010 Pulmonary histopathology ofCytauxzoon felis infections in the cat Vet Pathol 47698 ndash702 httpsdoiorg1011770300985810364527

90 Clarke LL Rissi DR 2015 Neuropathology of natural Cytauxzoon felisinfection in domestic cats Vet Pathol 521167ndash1171 httpsdoiorg1011770300985814564986

91 Ferris DH 1979 A progress report on the status of a new disease ofAmerican cats cytauxzoonosis Comp Immunol Microbiol Infect Dis1269 ndash276 httpsdoiorg1010160147-9571(79)90028-6

92 Kocan AA Blouin EF Glenn BL 1985 Hematologic and serum chemicalvalues for free-ranging bobcats Felis rufus (Schreber) with reference toanimals with natural infections of Cytauxzoon felis Kier 1979 J WildlDis 21190 ndash192 httpsdoiorg1075890090-3558-212190

93 Franks PT Harvey JW Shields RP Lawman MJP 1988 Hematologicfindings in experimental feline cytauxzoonosis J Am Anim Hosp Assoc24395ndash 401

94 Conner BJ Hanel RM Brooks MB Cohn LA Birkenheuer AJ 2015Coagulation abnormalities in 5 cats with naturally occurring cytaux-zoonosis J Vet Emerg Crit Care (San Antonio) 25538 ndash545 httpsdoiorg101111vec12326

95 Aschenbroich SA Rech RR Sousa RS Carmichael KP Sakamoto K 2012Pathology in practice Cytauxzoon felis infection J Am Vet Med Assoc240159 ndash161 httpsdoiorg102460javma2402159

96 Greene CE Latimer K Hopper E Shoeffler G Lower K Cullens F 1999Administration of diminazene aceturate or imidocarb dipropionate fortreatment of cytauxzoonosis in cats J Am Vet Med Assoc 215497ndash500

97 Cowell RL Fox JC Panciera RJ Tyler RD 1988 Detection of anticytaux-zoon antibodies in cats infected with a Cytauxzoon organism frombobcats Vet Parasitol 2843ndash52 httpsdoiorg1010160304-4017(88)90017-9

98 Motzel SL Wagner JE 1990 Treatment of experimentally inducedcytauxzoonosis in cats with parvaquone and buparvaquone Vet Para-sitol 35131ndash138 httpsdoiorg1010160304-4017(90)90122-R

99 Frontera-Acevedo K Balsone NM Dugan MA Makemson CR Sellers LBBrown HM Peterson DS Creevy KE Garner BC Sakamoto K 2013Systemic immune responses in Cytauxzoon felis-infected domestic catsAm J Vet Res 74901ndash909 httpsdoiorg102460ajvr746901

100 Frontera-Acevedo K Sakamoto K 2015 Local pulmonary immune re-sponses in domestic cats naturally infected with Cytauxzoon felis VetImmunol Immunopathol 1631ndash7 httpsdoiorg101016jvetimm201410012

101 Weisman JL Woldemeskel M Smith KD Merrill A Miller D 2007 Bloodsmear from a pregnant cat that died shortly after partial abortion VetClin Pathol 36209 ndash211 httpsdoiorg101111j1939-165X2007tb00212x

102 Birkenheuer AJ Marr H Alleman AR Levy MG Breitschwerdt EB 2006Development and evaluation of a PCR assay for the detection ofCytauxzoon felis DNA in feline blood samples Vet Parasitol 137144 ndash149 httpsdoiorg101016jvetpar200512007

103 Ayoob AL Prittie J Hackner SG 2010 Feline babesiosis J Vet EmergCrit Care (San Antonio) 2090 ndash97 httpsdoiorg101111j1476-4431200900493x

104 Tasker S Lappin MR 2002 Haemobartonella felis recent developmentsin diagnosis and treatment J Feline Med Surg 43ndash11 httpsdoiorg101053jfms20010155

105 Shindel N Dardiri AH Ferris DH 1978 An indirect fluorescent antibodytest for the detection of Cytauxzoon-like organisms in experimentallyinfected cats Can J Comp Med 42460 ndash 465




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106 Susta L Torres-Velez F Zhang J Brown C 2009 An in situ hybridizationand immunohistochemical study of cytauxzoonosis in domestic catsVet Pathol 461197ndash1204 httpsdoiorg101354vp08-VP-0132-B-FL

107 Birkenheuer AJ Marr HS Warren C Acton AE Mucker EM HumphreysJG Tucker MD 2008 Cytauxzoon felis infections are present in bobcats(Lynx rufus) in a region where cytauxzoonosis is not recognized indomestic cats Vet Parasitol 153126 ndash130 httpsdoiorg101016jvetpar200801020

108 Brown HM Berghaus RD Latimer KS Britt JO Rakich PM Peterson DS2009 Genetic variability of Cytauxzoon felis from 88 infected domesticcats in Arkansas and Georgia J Vet Diagn Invest 2159 ndash 63 httpsdoiorg101177104063870902100109

109 Brown HM Modaresi SM Cook JL Latimer KS Peterson DS 2009Genetic variability of archived Cytauxzoon felis histologic specimensfrom domestic cats in Georgia 1995ndash2007 J Vet Diagn Invest 21493ndash 498 httpsdoiorg101177104063870902100410

110 Shock BC Birkenheuer AJ Patton LL Olfenbuttel C Beringer J GroveDM Peek M Butfiloski JW Hughes DW Lockhart JM Cunningham MWBrown HM Peterson DS Yabsley MJ 2012 Variation in the ITS-1 andITS-2 rRNA genomic regions of Cytauxzoon felis from bobcats andpumas in the eastern United States and comparison with sequencesfrom domestic cats Vet Parasitol 19029 ndash35 httpsdoiorg101016jvetpar201206010

111 Schreeg ME Marr HS Griffith EH Tarigo JL Bird DM Reichard MV CohnLA Levy MG Birkenheuer AJ 2016 PCR amplification of a multi-copymitochondrial gene (cox3) improves detection of Cytauxzoon felis in-fection as compared to a ribosomal gene (18S) Vet Parasitol 225123ndash130 httpsdoiorg101016jvetpar201606013

112 Lewis KM Cohn LA Marr HS Birkenheuer AJ 2012 Diminazene diace-turate for treatment of chronic Cytauxzoon felis parasitemia in naturallyinfected cats J Vet Intern Med 261490 ndash1493 httpsdoiorg101111j1939-1676201201003x

113 Lewis KM Cohn LA Marr HS Birkenheuer AJ 2014 Failure of efficacyand adverse events associated with dose-intense diminazene diacetu-rate treatment of chronic Cytauxzoon felis infection in five cats J FelineMed Surg 16157ndash163 httpsdoiorg1011771098612X13502974

114 Cohn LA Birkenheuer AJ Brunker JD Ratcliff ER Craig AW 2011Efficacy of atovaquone and azithromycin or imidocarb dipropionate incats with acute cytauxzoonosis J Vet Intern Med 2555ndash 60 httpsdoiorg101111j1939-167620100646x

115 Brown HM Latimer KS Erikson LE Cashwell ME Britt JO Peterson DS2008 Detection of persistent Cytauxzoon felis infection by polymerasechain reaction in three asymptomatic domestic cats J Vet Diagn Invest20485ndash 488 httpsdoiorg101177104063870802000411

116 Schreeg ME Marr HS Tarigo J Cohn LA Levy MG Birkenheuer AJ 2013Pharmacogenomics of Cytauxzoon felis cytochrome b implications foratovaquone and azithromycin therapy in domestic cats with cytaux-zoonosis J Clin Microbiol 513066 ndash3069 httpsdoiorg101128JCM01407-13

117 Schreeg ME Marr HS Tarigo JL Cohn LA Levy MG Birkenheuer AJ2015 Rapid high-resolution melt analysis of Cytauxzoon felis cyto-chrome b to aid in the prognosis of cytauxzoonosis J Clin Microbiol532517ndash2524 httpsdoiorg101128JCM00635-15

118 Reine NJ 2004 Infection and blood transfusion a guide to donorscreening Clin Tech Small Anim Pract 1968 ndash74 httpsdoiorg101053jctsap200401002

119 Wardrop KJ Reine N Birkenheuer A Hale A Hohenhaus A Crawford CLappin MR 2005 Canine and feline blood donor screening for infec-tious disease J Vet Intern Med 19135ndash142 httpsdoiorg101111j1939-16762005tb02672x

120 Hillis DM Dixon MT 1991 Ribosomal DNA molecular evolution andphylogenetic inference Q Rev Biol 66411ndash 453 httpsdoiorg101086417338

121 Zieman EA Jimenez FA Nielsen CK 29 March 2017 Concurrent exam-ination of bobcats and ticks reveals high prevalence of Cytauxzoon felisin southern Illinois J Parasitol httpsdoiorg10164516-133

122 Diacuteaz-Regantildeoacuten D Villaescusa A Aylloacuten T Rodriacuteguez-Franco F Baneth GCalleja-Bueno L Garciacutea-Sancho M Agulla B Sainz Aacute 2017 Moleculardetection of Hepatozoon spp and Cytauxzoon sp in domestic and straycats from Madrid Spain Parasit Vectors 10112 httpsdoiorg101186s13071-017-2056-1

Jin-Lei Wang received a Bachelor of Veter-inary Science (BVSc) degree from Shang-dong Agricultural University Taian Chinaand a PhD in parasitology from the Grad-uate School of Chinese Academy of Agri-cultural Sciences Beijing China Dr Wangis now a research scientist at the Depart-ment of Parasitology Lanzhou VeterinaryResearch Institute Chinese Academy ofAgricultural Sciences China He is inter-ested in epidemiology molecular phylog-eny and gene functions of parasitic protozoa as well as controlstrategies for parasitic infections in animals and humans

Ting-Ting Li received a Bachelor of Veteri-nary Science (BVSc) degree from Shang-dong Agricultural University Taian Chinaand a Master of Veterinary Science (MVSc)degree from Lanzhou Veterinary ResearchInstitute Chinese Academy of AgriculturalSciences Lanzhou Gansu Province ChinaShe is most interested in epidemiology andcontrol of parasitic infections and the innateimmunity of hosts to parasitic infections

Guo-Hua Liu received a Master of VeterinaryScience (MVSc) degree and a PhD in par-asitology from Hunan Agricultural Univer-sity Changsha China and had his postdoc-toral training in molecular parasitology atthe Lanzhou Veterinary Research InstituteChinese Academy of Agricultural SciencesChina Dr Liu is currently a Professor of Par-asitology at the Department of ParasitologyCollege of Veterinary Medicine Hunan Agri-cultural University China His current re-search interests focus on the genetics genomics transcriptomics sys-tematics epidemiology and control of parasitic infections He wasawarded the Odile Bain Memorial Prize in 2016 He has published over50 original papers in well-regarded international journals His researchhas been well supported by research grants from the National NaturalScience Foundation of China the China Postdoctoral Science Founda-tion and Hunan Agricultural University




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Xing-Quan Zhu obtained a Bachelor of Vet-erinary Science (BVSc) degree from the Si-chuan Institute of Animal Sciences and Vet-erinary Medicine a Master of VeterinaryScience (MVSc) degree from the GraduateSchool of the Chinese Academy of Agricul-tural Sciences China and a PhD in parasi-tology from the Department of VeterinaryScience The University of Melbourne Aus-tralia He acquired postdoctoral training inmolecular parasitology at the Department ofVeterinary Science The University of Melbourne He is currently Headand Professor at the Department of Parasitology Lanzhou VeterinaryResearch Institute Chinese Academy of Agricultural Sciences China Hiscurrent research interests focus on the genetics genomics transcrip-tomics proteomics and metabolomics of parasites parasite-host inter-actions and molecular vaccines and the epidemiology and control ofparasitic infections He serves as a subject editor for the journal Parasitesamp Vectors and on the editorial boards of several international parasitol-ogy journals He has published more than 300 papers in well-regardedinternational journals

Chaoqun Yao received Bachelor of Medicineand Master of Medicine degrees from theHuazhong University of Science and Tech-nology in Wuhan China and a PhD in vet-erinary parasitology from the University ofGeorgia in Athens GA He was a researchfaculty member at the University of Iowawith a concomitant appointment as a Re-search Health Science Specialist at the IowaCity VA Medical Center following postdoc-toral fellowships at Washington State Uni-versity and the University of Iowa He held the position of tenure-trackAssistant Professor at the University of Wyoming and was Head of theParasitology Lab of the Wyoming State Veterinary Laboratory He wasan adjunct professor of the University of Washington at the same timeHe is currently at the Ross University School of Veterinary Medicine DrYaorsquos research interests since graduate school have been the molecularpathogenesis and host-parasite interactions of parasites of medicalveterinary importance




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INTRODUCTION

Cytauxzoonosis an emerging tick-borne disease of domestic cats and wild felids iscaused by the protozoa belonging to the genus Cytauxzoon Cytauxzoon felis the

agent of cytauxzoonosis of major concern belongs to the phylum Apicomplexa classSporozoasida order Piroplasmorida and family Theileriidae (1ndash8) For many years itwas endemic exclusively to North America ie to the southern southeastern andmid-Atlantic states of the United States (9ndash16) Only recently has it been reported inSouth America ie in Brazil and a few other identified species have been reported inEurope and other geographic regions (17ndash28) The most common natural host for Cfelis is the bobcat (Lynx rufus) Upon infection bobcats generally experience a shortcourse of non-life-threatening illness followed by a full recovery They serve as a naturalreservoir for the pathogen (10 29 30) In contrast infected domestic cats (Felis catus)usually succumb to infections within 9 to 15 days postinfection (pi) Infected domesticcats show clinical signs such as pyrexia anorexia dehydration depression icterus andhepatosplenomegaly They usually die within 24 to 48 h upon presentation to veteri-narians in the presence or absence of supportive therapy and experimental therapeuticregimens against this protozoan (2ndash6 11ndash13 15 31 32) However recent research hasindicated that this scenario can also present the other way around That is somebobcats suffer from severe acute cytauxzoonosis sometimes even leading to deathand in contrast the domestic cat can be clinically normal or carry the parasite for a longperiod upon recovery from acute cytauxzoonosis (14 17 33ndash37) It appears that themore we learn about the parasite and the disease the more questions are being raisedand need to be answered

Due to the severity of feline cytauxzoonosis and its geographic expansion it iscrucially important to raise awareness of the disease among veterinarians pet ownersand the general public Thus the current article reviews the parasite and its life cyclegeographic distribution genetic variability and pathogenesis as well as host immu-nology and the diagnosis treatment and prevention of infection in the domestic cat

THE PARASITES AND THEIR LIFE CYCLECytauxzoon Species

Feline cytauxzoonosis was first reported in 1976 in Missouri with four fatal cases (1)The disease has been endemic mainly in the domestic cat in the southern andsoutheastern United States which is considered ground zero of the disease (2ndash6) Thedisease has also been reported from other geographic regions such as South AmericaEurope Africa and Asia (17ndash28) The species and nomenclature of the causativeprotozoa have been a topic of debate for over a decade due to the indistinguishablemorphological features of the intraerythrocytic merozoites commonly called piro-plasms In general intraerythrocytic piroplasms have a diameter of 1 to 2 m and mayappear in several different forms (2ndash6 38) The most common form is a round ldquosignetringrdquo form (Fig 1A) (16) Bipolar oval ldquosafety pinrdquo and round anaplasmoid bodies are lesscommon whereas tetrads are occasionally discovered in Giemsa-stained blood smears(2ndash6 38) However genomic data (httppiroplasmadborgpiro) and DNA sequenceshave revealed differences that are sufficient enough to differentiate the causativeprotozoan agents at the species level and finally to allow for species classificationwithin the genus (7 8 39ndash43)

The genus Cytauxzoon is closely related to Theileria (43) The piroplasm stages in theerythrocytes of mammalian hosts are morphologically similar making it very difficult ifnot impossible to distinguish them The two were originally thought to differ from oneanother by the location of schizogony within the host cells Schizogony of Cytauxzoonspp occurs in macrophages whereas that of Theileria spp takes place in lymphocytes(2ndash6 38 44) However Theileria spp were later found to also infect macrophages (4546) Macrophages containing mature Cytauxzoon schizonts are greatly increased in sizeto up to 250 m in diameter and are often associated with endothelial cells ofcapillaries of many internal organs (Fig 1B) (16) which contributes to capillary occlu-sion resulting in tissue damage and organ failure (2ndash6 15 32 44)




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Parasite Life Cycle






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two tales of cytauxzoon felis infections in domestic cats · cytauxzoon species harbored by the...

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