PROCEEDINGS OF THE NORTH AMERICAN VETERINARY CONFERENCE VOLUME 20

JANUARY 7-11, 2006 ORLANDO, FLORIDA

SMALL ANIMAL EDITION

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Small Animal Hematology ______________________________________________________________________________________________

INFECTIOUS DISEASE OF FELINE BLOOD

John W. Harvey, DVM, PhD, Diplomate ACVP College of Veterinary Medicine University of Florida Gainesville, FL HEMOTROPHIC MYCOPLASMOSIS (HAEMOBARTONELLOSIS) Hemotrophic mycoplasmas are gram-negative, nonacid-fast, bacteria that attach to the external surface of erythrocytes. Damage caused by parasite attachment and immune-response by the host result in increased erythrocyte destruction and anemia. Hemotrophic mycoplasmas were classified as rickettsia in the genus Haemobartonella or the genus Eperythrozoon for many years; however, results from sequencing of the 16S rRNA gene indicate that these parasites are closely related to mycoplasmas. Consequently the Haemobartonella and Eperythrozoon genera have been discarded and organisms in these genera were moved to the genus Mycoplasma. Hemoplasmas has been proposed as a trivial name for these hemotrophic mycoplasmas. Although preweaning cats have been recognized with hemotrophic mycoplasmosis, most cases occur in adult animals. Risk factors reported to be associated with the visual identification of hemoplasma organisms in blood include anemia, clinical signs of illness, cat-bite abscesses, FeLV-positive status, roaming outdoors, and age <3 years. Hemotrophic mycoplasmosis is reported more commonly in male than in female cats. The severity of disease varies from unapparent infection in some animals to marked depression and death in other animals. Two distinctly different hemoplasmas have been identified in cats based on 16S rRNA gene sequences, Mycoplasma haemofelis (formerly called the large form of H. felis) and Mycoplasma haemominutum (formerly called the small form of H. felis). Reports to date indicate that M. haemofelis generally produces anemia and clinical signs of disease, while M. haemominutum generally results in unapparent infections and minimal change in hematocrit, unless complicated by other disorders such as FeLV, FIV infections, and neoplasia. A new hemoplasma isolate has been identified from a cat with hemolytic anemia that is most closely related to rodent hemoplasmas. Clinical Signs The most common clinical signs of acute hemotrophic mycoplasmosis are weakness, depression, anorexia, paleness of mucous membranes, and variable weight loss. Animals are often, but not consistently, febrile. Icterus is sometimes observed after rapid decreases in hematocrit. Splenomegaly is commonly noted in cats and dyspnea may be observed late in the disease. The mortality of acutely-affected cats with uncomplicated hemotrophic mycoplasmosis may be as high as 30%.

Clinical signs are mild or absent in cats experimentally infected only with M. haemominutum. Mild fever may occur at times. M. haemominutum infection has been demonstrated more often in sick cats than in healthy cats, but it is unclear if the clinical signs present were related to the M. haemominutum infection or to other accompanying disease processes. Transmission Transmission of infection by bloodsucking arthropods, especially fleas, is considered by many to be the primary mode of transmission in cats, although some cats with hemotrophic mycoplasmosis have never had flea infestations. Hemoplasmas have been detected in fleas using PCR, and transient transmission of M. haemofelis from infected fleas to one cat has been reported. M. haemofelis can be transmitted from female cats with clinical disease to their newborn offspring in the absence of arthropod vectors. It is not known whether transmission occurs in utero, during parturition, or via nursing. Iatrogenic transmission of hemoplasmas can occur by the transfusion of blood from normal-appearing carrier cats. In order to prevent this transmission, it is suggested that prospective blood donors be tested using PCR-based tests discussed later. Routine Laboratory Findings Parasites may not be visualized in blood even when animals are markedly anemic. Organisms tend to appear in blood during discrete parasitemic episodes of one or more days duration, and they can disappear from blood in a matter of two hours or less in infected cats. In many instances, a rapid decrease followed by a rapid increase in hematocrit occurs in association with the appearance and disappearance of organisms from the blood. These fluctuations in hematocrit in cats appear to be associated with splenic sequestration of parasitized erythrocytes and with later release of nonparasitized erythrocytes. In other instances, the hematocrit remains decreased or continues to decline for one or more days after a parasitemic episode, probably as a result of erythrocyte destruction. Cats often have multiple parasitemic episodes before severe anemia develops. In uncomplicated (primary) hemotrophic mycoplasmosis, hematocrits are generally reduced to one-half normal or less when clinical signs of illness are apparent, and a reticulocytosis is present in most cases by the time affected animals are presented to a veterinarian. The MCV is generally increased and the MCHC may be decreased concomitant with the regenerative response of the bone marrow to the anemia. The anemia may appear nonregenerative if a precipitous decrease in hematocrit has occurred early in the disease or if other concurrent disorders (e.g., FeLV or FIV infections in cats) are present. Persistent infections with hemoplasmas may promote myeloproliferative disorders in FeLV-infected cats. A bone marrow biopsy and evaluation may be helpful to determine if other disorders (e.g., myeloproliferative disease) are also present. Hematocrits are normal or

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The North American Veterinary Conference 2006 ______________________________________________________________________________________________ only slightly low in animals with unapparent infections (such as is reported for M. haemominutum infections in cats) and in carriers animals that have recovered from clinical hemotrophic mycoplasmosis. Slight hemoglobinemia is rarely observed, but hemoglobinuria has not been reported. Autoagglutination may be seen in blood samples after they cool below body temperature and the direct Coombs' test may be positive at 37oC. Total and differential leukocyte counts are variable and of little diagnostic assistance. Bizarre monocytes, sometimes demonstrating erythrophagocytosis, may be seen in acute feline hemotrophic mycoplasmosis. Clinical chemistry profiles may demonstrate abnormalities related to anemic hypoxia, but profiles can be normal. Plasma protein concentrations are usually normal but may be increased. Hypoglycemia may occur in moribund cats. Hyperbilirubinemia may be measured at times following an abrupt decrease in hematocrit, but it is seldom severe.1 Diagnosis For years, documentation of infection with hemoplasmas required the identification of organisms in stained blood films. Organisms are more readily visualized when stained with routine Wright or WrightGiemsa stains than they are when stained with Diff-Quik stains. Blood films must be examined for organisms before therapy is begun. Reticulocyte stains cannot be used to identify organisms, because punctate reticulocytes in cats appear similar to the parasites. M. haemofelis organisms appear as small, blue-staining cocci, rings, or rods on erythrocytes. M. haemominutum organisms are rarely observed in stained blood films from infected cats. When seen, M. haemominutum organisms appear to stain less densely and be about half the size of M. haemofelis organims. Hemoplasmas must be differentiated from precipitated stain, retractile drying or fixation artifacts, poorly staining Howell-Jolly bodies, basophilic stippling, iron-positive inclusions, Cytauxzoon felis organisms, and small Babesia organisms. Highly sensitive feline PCR-based assays have been developed that can detect hemotrophic mycoplasmas in blood when there are fewer organisms present than would be required to make a diagnosis using stained blood films. Using competitive, quantitative PCR, it has been determined that as few as about 50 organisms can be detected by PCR. Genotype-specific PCR primers have been developed to differentiate between M. haemofelis and M. haemominutum infections in cats. Using these primers, investigators in the United States reported finding about 30% of anemic cats suspected of having hemotrophic mycoplasmosis to be positive for hemotrophic mycoplasmas by PCR, with a majority of cats being positive for M. haemofelis alone or in combination with M. haemominutum. Additionally, about 14% of healthy control cats in the United States study were PCR positive for hemotrophic mycoplasmas. Of these PCR-positive healthy cats, 19 were infected only with M. haemominutum and 1 was infected with both variants. A survey of feral cats in northern Florida revealed that 8% were PCR-positive for M. haemofelis and 12% were PCR-positive for M. haemominutum. As in other reports, males were more commonly infected than females, and there was a positive correlation between hemoplasma infections and FeLV and FIV infections. Whether diagnosed by identification of parasites in blood films or PCR-based assays, the significance of hemoplasma infections must be interpreted in light of hematologic and clinical findings. Therapy Doxycycline (5 mg/kg PO q12h) should be given for 3 weeks. Unfortunately, tetracycline antibiotics may produce fever and/or evidence of gastrointestinal disease in cats. Recent findings indicate that enrofloxacin (10 mg/kg PO q24h) may be an efficacious alternative for cats that do not tolerate tetracycline antibiotics. Glucocorticoids such as prednisolone (1-2 mg/kg PO BID) may be given to severely anemic animals in an effort to decrease erythrophagocytosis, with the dosage gradually decreased as the hematocrit increases. Blood transfusions are required when the anemia is considered life-threatening. IV fluid containing glucose is recommended in moribund animals. CYTAUXZOONOSIS Cytauxzoon felis is a protozoal parasite that occurs in a genus in the family Theileriidae. Cytauxzoon differs from the genus Theileria in that schizogony occurs in macrophages rather than lymphocytes, but intraerythrocytic piroplasms of both genera are morphologically indistinguishable. Cytauxzoonosis was first recognized as a fatal infection of domestic cats in southwestern Missouri, but has now been observed in many states in the Southeast. Reservoir and Transmission The disease occurs most frequently in rural, heavily wooded areas. Since domestic cats generally die of acute illness in a matter of days, they are believed to be dead end hosts. Bobcats, however, usually do not die when infected with C. felis. They have been shown to be a reservoir of infection by experimental transmission to domestic cats with ticks infected from carrier bobcats. Florida panthers are sometimes infected with C. felis, but like bobcats they usually do not die. Fatal cytauxzoonosis has occurred in a white tiger in Florida. Clinical Signs The first clinical signs observed in infected animals are depression and anorexia. The rectal temperature rises gradually, at times to 41oC (106oF) or more, during the 3 to 4 days after illness first becomes clinically apparent. Icterus, dehydration, and a moderate anemia develop rapidly during the febrile period. Thereafter, the body temperature begins to drop, becoming subnormal

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Small Animal Hematology ______________________________________________________________________________________________ in 2 to 3 days. Dyspnea generally develops late in the course of the disease. Splenomegaly may be palpable. Experimental animals inoculated with infected blood or tissues rarely live more than 3 weeks after inoculation, and cats are usually dead within a week after clinical signs of illness are observed. Laboratory Findings In the terminal stages of the disease, most cats will have icteric plasma. The PCV may be in the low 30's but is usually in the 20's or upper teens. Reticulocyte counts are not increased in response to the anemia. Cats become thrombocytopenic during late stages of disease. Coagulation tests may be prolonged or remain normal. Elevations in ALT, BUN, and glucose are frequently measured and serum protein is variably decreased. White blood cell counts are variable, but leukopenia generally develops terminally. Parasitemia occurs late in the disease. When routinely-stained blood films are examined, organisms appear in erythrocytes either as rounded "signet ring" bodies 1 to 1.5 microns in diameter or as bipolar, oval or "safety pin" bodies 1 by 2 microns. The cytoplasm of the protozoan stains a light blue, while the nucleus appears red to purple. In the late stages of the disease, up to 25% of erythrocytes may contain protozoa, but parasites may be absent. If parasites are absent or their identification is equivocal, schizonts may be identified in bone marrow aspirate smears. A PCR-based test for the nuclear small subunit (NSS) of the Cytauxzoon rRNA gene has been developed, but it is of limited use in the diagnosis of acutely ill animals, because the test results would generally not be available in time to effectively treat the animal. Similarly, a serologic assay for antibodies using a microfluorometric immunoassay can identify animals that have survived the infection, but may not identify acutely infected animals. Therapy and Prognosis Nearly all untreated cases in domestic cats are fatal, although a less virulent strain has been reported in a restricted area of northwestern Arkansas and northeastern Oklahoma. In this area, cats have survived without specific treatment and 4 asymptomatic cats were discovered by bleeding healthy cats in households with ill cats. Treatment for cytauxzoonosis may be efficacious if infected animals are recognized early. Therapy should include imidocarb dipropionate (Imizol) or diminazene aceturate (2 mg/kg IM given twice with a 3 to 7 day interval), atropine (0.05 mg/kg SC) given prior to each imidocarb treatment, heparin (150 U/kg SC q8h) with tapered decrease in dosage after the second imidocarb treatment, fluid therapy, and a blood transfusion if needed. References 1. Harvey JW. Hemotrophic mycoplasmosis. In: Greene CE, 3rd ed. Infectious Diseases of the Dog and Cat. Philadelphia: W.B. Saunders; 2005:in press. Meinkoth JH, Kocan AA. Feline cytauxzoonosis. Vet Clin North Am Small Anim Pract. 2005;35:89-101. Messick JB. Hemotrophic mycoplasmas (hemoplasmas): a review and new insights into pathogenic potential. Vet Clin Pathol. 2004;33:2-13. Willi B, Boretti FS, Cattori V et al. Identification, molecular characterization, and experimental transmission of a new hemoplasma isolate from a cat with hemolytic anemia in Switzerland. J Clin Microbiol. 2005;43:2581-2585. Woods JE, Brewer MM, Hawley JR, Wisnewski N, Lappin MR. Evaluation of experimental transmission of Candidatus Mycoplasma haemominutum and Mycoplasma haemofelis by Ctenocephalides felis to cats. Am J Vet Res. 2005;66:1008-1012.

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