Recognizing infectious organisms in the blood film (Proceedings)

Common RBC parasites of dogs and cats

Hemotropic parasites are often associated with hemolytic anemia.

• Red blood cell parasites that cause extravascular hemolysis include Mycoplasma, Cytauxzoon, and Babesia. Clinical signs include a regenerative anemia, leukocytosis, splenomegaly, and hyperbilirubinemia.

• RBC parasites that cause intravascular hemolysis include Babesia and occasionally Cyauszoon. Clinical signs are similar to those for extravascular hemolysis, but also include hemoglobinemia and hemoglobinuria.

Mycoplasma

Mycoplasma haemofelis is an epicellular organism that loosely adhere to the RBC membrane. Transmission is via blood?sucking arthropods such as fleas and ticks, saliva from infected cats, in utero, via nursing, or by blood transfusion. Three strains have been described

• Mycoplasma haemofelis – large form, may be the most pathogenic, found in ~ 5% of anemic cats, ~50% of cats with M. haemofelis infection are FeLV +

• Mcyoplasma turicensis – recently recognized, found in ~ 7% of anemic cats

• Mycoplasma hemominutum – small form, ‘California variant', least pathogenic, minimal disease unless another disease is present, found in ~ 25% of anemic cats

Organisms are 0.1?1.5m dark blue cocci, rods, or rings attached to the RBC membrane. Organisms may dissociate from RBCs in EDTA and can resemble stain precipitate, HJ bodies, or debris. Parasitemia is cyclical: 1?5 days of parasitemia with 3?11 days in between each cycle. Organisms can disappear from blood within 2 hours. Inability to detect organisms on blood film does not eliminate Mycoplasma spp as organisms may not be present in sufficient numbers to be recognized. Fresh, non-EDTA samples may be useful for examination. Examination of epeated blood samples and PCR are recommended diagnostics.

Mycoplasma haemofelis  is an epicellular organism similar in size to M. haemofelis that forms chains (sometimes branching) across the RBC surface. Transmission is likely via ticks, but may also occur in utero, via nursing, or by blood transfusion. Clinical signs (e.g. extravascular hemolysis) are rarely present unless the dog has been splenectomized, is being treated with immunosuppressive drugs, or has other significantly immunosuppressive diseases. Organisms are rarely present on blood smear. PCR available.       

Cytauzoon

Cytauzoon is often a fatal disease in domestic cats although newer reports suggest a second lesser pathogenic disease process. The organism is transmitted by ticks (Dermacentor variabilis) from bobcats, the natural reservoir. Bobcats remain persistent carriers after mild or subclincal infection. Cytauxzoon felis is an intracellular protozoan that has a two stages: macrophage stage and an RBC stage. In RBCs, the the organisms appear as oval signet ring bodies 0.5-1.5 um with a small peripherally located magenta nucleoid structure. Parasitemia is a late stage of the disease; in about 50% of the cats the parasite cannot be found in the red cells and in the other cats often only 1-2% of the red cells are parasitized. Histologiclly, schizonts can be observed in macrophages associated with endothelium lining venous channels in major organs such as lung, spleen, lymph nodes, and bone marrow.  Occasionally, these will be seen on the feather edge of blood films. Infection primarily results in extravascular hemolysis, but intravascular hemolysis may also be present.  

Babesia

Canine babesiosis occurs mainly in the SW and SE regions of the US. Transmission by ticks requires 2-3 days of feeding; transmission also occurs across the placenta or by blood transfusion. Concurrent infections with other hemoparasties are not uncommon. B. canis and B. gibsoni are intracellular piroform protozoan parasites that induce intravascular (more important in acute infection) and extravascular hemolysis (especially in chronic infection). Cyclical parasitemia may occur and only low numbers of parasites may be seen on the blood film. Using capillary blood and examining cells at the periphery of the blood film may facilitate the identification of parasites. There may be no parasitemia in chronic cases. A high percentage of cases are also Coomb's positive. Detection of chronic carriers by serology is not reliable. PCR assays can differentiate subspeicies and detect low levels of parasitemia

• B. canis appear as single or paired (2-5µ), basophilic, tear-drop shaped trophozoites with an eccentric nucleus. There are three subtypes of B. canis: B. canis canis, B. canis vogeli, and B. canis rossi. They appear identical but differ in virulence, geographic location, and tick vector

• B. gibsoni appear as small (1-3µ), paler, round to oval or ring-shaped organisms.

• Feline babesiosis is rarely diagnosed in the US - Babesia felis (S Africa, Sudan) and Babesia cati (India)

Bartonella

Bartonellosis is caused by small, curved, gram-negative bacteria that live within RBCs. These cannot be readily detected by light microscopy. Transmission between cats by fleas and between dogs, other mammals, and humans by ticks. Clinical signs vary between species and stage of disease. Serology and PCR are available for dogs. Culture is may be used for definitive diagnosis.

Common WBC parasites of dogs and cats

Ehrlichiae

Ehrlichiae are obligate intracellular, small gram-negative pleomorphic cocci that replicate within host cells. There are three Ehrlichia genogroups based on 16S rRNA nucleotide sequence:

• Group 1: genus Ehrlichia

• Group 2: genus Anaplasma

• Group 3: genus Neorickettsia

Ehrlichia canis infects dogs, wolves, and jackals and is the causative agent of Canine Monocytic Ehrlichiosis. E. canis is transmitted by ticks. German Shepherds, young and aged dogs, and immunocompromised dogs are more susceptible to infection. Clinical signs include fever, anorexia, lymphadenopathy, splenomegaly, hepatomegaly, petecchiae, ecchymoses. Thrombocytopenia is the most common laboratory abnormality; hypergammaglobulinemia and anemia may also be present. E. canis is primarily found in mononuclear cells as clusters called morulae (2-6μ blue to purple inclusions) during the acute stage of infection. SNAP 4Dx, serology, and PCR  are available diagnostics.

Ehrlichia ewingii infects dogs, humans, and white tailed deer and is the causative agent of Canine Granulocytic Ehrlichiosis. E. ewingii is transmitted by ticks. Clinical disease tends to be milder than with E. canis. Polyarthritis may also be seen. Thrombocytopenia is common. E. ewingii is primarily found in granulocytic cells (neutrophils and eosinophils) in blood, joint fluid, and other sites as clusters called morulae (2-6μ blue to purple inclusions) during the acute stage of infection. Serology, and PCR are available diagnostics.

Ehrlichia chaffeensis infects humans and white tailed deer and has also been identified in dogs. It is the causative agent of Human Monocytic Ehrlichiosis. E. chaffeensis is transmitted by ticks. The role of dogs as a reservoir for humans is not clear. Clinical disease appears similar to E. canis. Thrombocytopenia is common; also seen is lymphocytosis, lymphadenopathy, uveitis, epistaxis, GI disease. E. chaffeensis is primarily found in mononuclear cells as clusters called morulae (2-6μ blue to purple inclusions) during the acute stage of infection. Serology, and PCR are available diagnostics.

Feline mononuclear Ehrlichiosis has been reported but a definitive organism has not been identified. Healthy cats may be seropositive for a variety of Ehrlichial agents (e.g. E. canis and N. risticii), cats appear to be naturally infected with E. canis or closely related organism (by PCR) with morulae seen in mononuclear cells and rarely in neutrophils. Cats experimentally infected with E. canis and N. risticii develop clinical signs and morulae can be identified in mononuclear cells.  Similar to dogs, cats develop thrombocytopenia, hyperglobulinemia, anemia, and possibly arthritis. Diagnosis is made by detection of morulae, serology (although not sufficient for diagnosis as positive results in healthy cats), and PCR.

Anaplasma phagocytophilum now includes 3 agents that were previously considered to be 3 different species and are now recognized as different strains of the same species.  These were:

•  HGE agent - Human Granulocytic Ehrlichiosis

•  E. equi - agent of Equine Granulocytic Ehrlichiosis 

•  E. phagocytophilum - agent of Ruminant Ehrlichiosis

A. phagocytophilum is transmitted by ticks and infects a variety of species including dogs, cats, humans, horses, cattle, sheep, llamas, deer, elk and rodents. Lyme's disease and A. phagocytophilum share vectors and reservoirs, thus ticks are often co-infected with both organisms.  Clinical signs vary by host species and organism strain. In dogs and cats, thrombocytopenia is common; in dogs, polyarthritis is a prominent feature. Morulae may be found in neutrophils in blood and joint fluid (less commonly in eosinophils in blood). For dogs, SNAP 4Dx (although A. platys also cross reacts), serology, and PCR are available diagnostics. For cats, serology and PCR.

Salmon disease occurs when dogs ingest raw salmon type fish infected with the parasite Nanophyetus  salmincola that itself is infected with the parasite Neorickettsia helminthoeca. This disease is most typically seen in the northwest. Irregular inclusions and morulae are not seen in the blood, but instead are  diagnosed cytologically by examining lymph node aspirates. The organism will be seen in macrophages as small basophilic to dark granules of variable size.

Protozoal agents

Leismaniasis is a protozoal disease of dogs and cats. At least two species of Leishmania have been detected in animals in the United States. Transmission occurs primarily through sandflies, but can also be transmitted by blood transfusion and to nursing pups. There appears to be an increased risk in Foxhounds. Skin, bone marrow, and lymph nodes are common sites of involvement. Thrombocytopenia, anemia, and hypergammaglobulinemia are common. Amistigotes have a characteristic appearance, are 1-5 um in size, contain a magenta nucleus and a bar shaped kinetoplast placed perpendicularly to the nucleus. Serology and PCR are other diagnostics.

Canine hepatozoonosis is a protozoon disease primarily of dogs, but can also infect domestic cats, coyotes, bobcats, and ocelots. H. americanum is recognized in the US and appears to be a more aggressive and progressive disease; H. canis is seen in other parts of the world. Transmission occurs by ingestion of infected ticks. A marked neutrophilic leukocytosis, with or without an eosinophilia is common. Gamonts are detected in neutrophils and monocytes as large (5x11u), variably basophilic inclusion with occasional granular internal structure. In H. americanum, the number of infected cells is low.

Infectious agents in platlets

Anaplasma platys is a rickettsial agent that  infects canine platelets and causes infectious thrombocytopenia. The organism is similar to E. canis but does not cross?reactivity serologically. Tick transmission is likely. Thrombocytopenia may be present, and may be cyclical, but some dogs have minimal clinical signs. Morula may rarely be detected in blood. An indirect IFA is available.

Infectious agents in plasma

Trypanosoma cruzi is a flagellated protozoanresponsible for Chagas diease in people.  T. cruzi also infects dogs, cats, and many other domestic and wild animals. Transmission is by reduviid  “assassin”. When seen in blood, T. cruzi is 16?20 um long with a large, subterminal kinetoplast, a midsection nucleus, an undulating membrane, and a free flagellum. Detection may be facilitated by examination of a buffy coat smear. Serology is also available.

Microfilaria found in blood include both Dirofilaria immitis (heartworm) and Dipetalonema reconditum.

Miscellaneous agents

Rickettsia rickettsi is the etiologic agents of Rocky Mountain Spotted Fever. It is an obligate intracellular bacterium similar to gram negative bacteria that infects endothelial cells. Clinical and laboratory signs are similar to Ehrlichia and similar agents, however, the organisms are not visualized on the CBC being 0.3-0.5 um in size and localized in endothelial cells.

Histoplasma capsulatum is occasionally seen on the blood film, usually within neutrophils. The small yeast-like organisms are round to oval and 1 to 4 µm in diameter with a purple nucleus and lightly basophilic protoplasm surrounded by a thin, clear halo.