FIGURE 1 Blood smear from a dog with Babesia gibsoni, a classic, small-form Babesia spp with a thin, blue cell membrane (A-D; solid arrows) and appears as small, oval to pleomorphic structures with an eccentric dotlike nucleus (C, D; dashed arrows). B gibsoni has worldwide distribution, can cause severe disease in dogs, and has been associated with secondary immune-mediated hemolytic anemia.⁷ In studies of samples submitted to infectious disease and international diagnostic laboratories, B gibsoni was the most commonly identified species affecting dogs.^2,8 Quick Romanowsky stains are commonly used in the clinic and highlight visualization of organisms better than traditional Romanowsky stains. Wright Giemsa stain, a traditional Romanowsky stain, was used in A and B, and the cytoplasmic membrane is not highly visible. Quick Romanowsky stain was used in C and D, and the nucleus and cytoplasmic membrane are highly visible. This effect is enhanced when slides are slightly overstained with a dark-blue stain solution (solution II). Original magnification, 100× objective

FIGURE 2 Blood smear from a dog with Babesia vulpes, a classic, small-form Babesia spp that appears as a small ring with a dotlike nucleus (arrows). B vulpes is also known as Babesia annae, Theileria annae, Babesia microti-like, and Babesia ‘Spanish isolate’.⁹ In dogs, B vulpes has been shown to cause significant clinical disease, including hemolytic anemia and thrombocytopenia.⁸ In a study of samples submitted to an infectious disease laboratory, B vulpes was the second most commonly identified organism infecting dogs.⁸ A high level of coinfection with B gibsoni and a high prevalence in American Staffordshire terriers and American pit bull terriers was also found, suggesting that bite wounds may play an important role in transmission.^8,10 Quick Romanowsky stain. Original magnification, 100× objective. Images courtesy of Barbara Qurollo, DVM, MS

FIGURE 3 Blood smear from a dog with Babesia canis, a classic, large-form Babesia spp easily identified due to its large size and robust staining. B canis typically appears as a pear- or piriform-shaped structure with a purple–red, dotlike, round-to-flattened nucleus (A-D; dashed arrows), a relatively thick basophilic cell membrane (A-D; solid arrows), and light-blue to nearly clear cytoplasm. B canis often appears in pairs with the thin ends touching; this is sometimes described as a double piriform (A). B canis can occasionally be found extracellularly on blood smears (B). A cell with multiple organisms is visible (C), but individual organisms can be artifactually rounded on the feathered edge of the slide (D). There is controversy as to whether there are 3 different subspecies of B canis (ie, B canis canis, B canis rossi, B canis vogeli) or whether these represent 3 distinct species; however, consensus appears to be moving toward acceptance as different species.¹¹ B canis appears to be uncommon in the United States but is relatively common in Europe, where it was characterized by a large commercial laboratory as the second most commonly identified species after B gibsoni.^8,10 Disease caused by B canis is milder in immunocompetent animals as compared with B gibsoni. Thrombocytopenia is the most common finding, but dogs can also develop mild to occasionally moderate anemia.¹² Wright-Giemsa stain. Original magnification, 100× objective

FIGURE 4 Blood smear from a dog with Babesia sp (coco), a novel, large-form Babesia spp first isolated from an immunosuppressed dog in North Carolina (designated coco after this first index patient).¹³ A small case series suggested this infection in dogs may be generally associated with immunosuppression or an immunocompromised state (eg, splenectomy, chemotherapy treatment)¹⁴; moderate to marked thrombocytopenia and mild anemia were the most common diagnostic findings. A recent study found low prevalence in samples submitted to an infectious disease testing laboratory.⁸ This organism is more pleomorphic than B canis and has a thinner cell membrane. Although it can form classic, paired piriform-shaped structures (A; arrow), highly pleomorphic ameboid-shaped structures are more common (B-D; arrows).¹³ Wright-Giemsa stain. Original magnification, 100× objective

FIGURE 5 Blood smear from a 2.5-year-old maned wolf (Chrysocyon brachyurus) in a zoologic park with a novel, small-form Babesia. The wolf was severely anemic with thrombocytopenia, icteric plasma, 3+ hematuria, 3+ bilirubinuria, and a positive blood saline agglutination test. Genetic analysis characterized the Babesia species as being distinct from known organisms reported in dogs and most closely resembling a species identified in raccoons in North America.^15,16 The patient’s presentation was similar to a previously reported case of a maned wolf from the same park.¹⁷ Many small, oval to elongated organisms with a thin, delicate basophilic cell membrane (solid arrows) and a dotlike nucleus (dashed arrows), features that are characteristic of small-form organisms, are visible. The inset shows a single, red cell with 3 organisms (arrowheads). Romanowsky stain. Original magnification, 100× objective. Case from American Society of Veterinary Clinical Pathology, Mystery Slide Conference 2015, Case 8, submitted by Dr. Adi Wasserkrug Naor

FIGURE 6 Blood smear from a juvenile male raccoon (Procyon lotor) found to be nonambulatory near a road. Routine blood work revealed several hematologic and serum chemistry abnormalities, including moderate to marked anemia and hypoproteinemia. Blood smear examination showed low numbers of small-form, round, oval, and ameboid Babesia organisms (arrows) with thin, blue cell membranes and a dotlike nucleus (features that are characteristic of small-form species). Organisms were detected periodically on the blood smear up to the time of release of the raccoon. Whether the infection was incidental or contributing to illness was unknown. Genetic analysis confirmed Babesia, but one that was distinct from a previously reported small form identified in raccoons as Babesia microti-like.¹⁶ A more recent study of wild trapped raccoons in North Carolina showed a high prevalence of both Babesia spp, suggesting they are unlikely to be highly pathogenic in raccoons.⁵ Wright-Giemsa stain. Original magnification, 100× objective

FIGURE 7 Blood smear from a 3-year-old North American elk (Cervus elaphus) with Babesia odocoitei. The elk was presented for a 2-day history of hemoglobinuria, lethargy, and anorexia and was from a herd that had experienced several deaths in the preceding 2 months. On routine blood work, the patient was found to be mildly anemic, and blood smear examination showed several piriform-shaped organisms. Genetic analysis confirmed B odocoitei, a largely nonpathogenic hemoparasite of white-tailed deer. Screening of a selection of elk from the herd revealed a high incidence of infection. Although this patient had acute hemolytic anemia, the authors indicated that infections are typically subclinical unless additional stressors are present.¹⁸ Most clinical case reports of elk with B odocoitei have been in the United States, but a recent report suggests possible emergence in Canada that could be related to spread of the host tick’s range.¹⁹ B odocoitei have a flattened appearance, are typically on the periphery of the cells (solid arrows), and are often arranged in double piriforms (dashed arrows). The inset shows a double piriform with distinct eccentric nuclei. The eccentric position, flattening, and multiple double-piriform formations are classic for this organism, but they are not unique to it.⁶ Wright’s stain. Original magnification, 100× objective. Case from American Society of Veterinary Clinical Pathology, Mystery Slide Conference 2003, Case 3, submitted by Dr. Armando R. Irizarry-Rovira

FIGURE 8 Blood smear from a reindeer (Rangifer tarandus tarandus) with Babesia odocoilei (A), which are piriform-shaped, often flattened, and typically eccentrically positioned (sometimes referred to as the accolé position).²⁰ Occasional rounded forms (B), a rare tetrad (ie, Maltese cross formation; C), and a classic double piriform (D) can be seen. Fatal B odocoilei infections in reindeer have been reported and appear to be capable of infecting a wide range of hosts with varying degrees of pathology²⁰; this may relate to additional stressors or introduction of the parasite to a naive population. Wright-Giemsa stain. Original magnification, 100× objective. Case from American Society of Veterinary Clinical Pathology, Mystery Slide Conference 2015, Case 12, submitted by Dr. Kimberly Pattullo

FIGURE 9 Blood smear from a North American river otter (Lontra canadensis) with a novel Babesia species detected during a routine health check. Subsequent analysis of samples obtained from wild-trapped animals showed a high prevalence in the group. The index case showed no clinical, hematologic, or serum chemistry abnormalities. Genetic analysis showed the organisms to be unique but closely related to Babesia microti-like organisms found in other species.^9,21 Pathogenicity in otters is unknown, but the lack of clinical or hematologic signs combined with a high prevalence in wild-caught animals suggests pathogenicity is unlikely to be significant.²¹ Organisms can be present as small pleomorphic or angular forms (A-C; dashed arrows) or can have a classic, small, round to oval appearance with dotlike nuclei and a thin cell membrane (D; solid arrow). Modified Wright-Giemsa stain. Original magnification, 100× objective

FIGURE 10 Blood smear from a 4- to 6-month-old female red fox (Vulpes vulpes) found in a semirural region with Babesia vulpes. She was thin and weak with mild to moderate regenerative anemia. Blood smear examination showed occasional very small ring- and oval-shaped organisms with dotlike nuclei and thin cytoplasmic membranes. Genetic analysis showed that the organism was identical to Babesia vulpes.²² Wright-Giemsa stain. Original magnification, 100× objective. Case from American Society of Veterinary Clinical Pathology, Mystery Slide Conference 2009, Case 20, submitted by Dr. Noel Clancey

FIGURE 11 Blood smear from a 2-month-old female golden (Syrian) hamster (Mesocricetus auratus) with Babesia duncani (ie, Babesia WA1) that was part of an experimental study; the hamster was euthanized after developing clinical signs. Blood collected antemortem demonstrated marked anemia with gross hemolysis. On blood smear examination, many small ring-shaped organisms were found with classic small-form morphology. In addition, there were large numbers of ghost cells (empty red cell membranes from intravascular lysis of red cells) and free organisms in the background of the slide. The platelet count from the automated hematology analyzer was increased, but only low numbers of platelets were seen on the smear.²³ It was speculated that the free organisms were falsely interfering with the platelet count. Hamsters are known to be susceptible to both B microti and B duncani, and although both can cause severe disease, B duncani appears to be more pathogenic.²⁴ Multiple ghost cells and free organisms with occasional intracellular organisms (A) and an intact cell with a small, eccentrically placed Babesia trophozoite and a pale ghost cell indicating acute intravascular hemolysis (B) can be seen. A large platelet with prominent purple granules (C; solid arrow) is to the lower right of a polychromatic red cell with an intracellular organism (dashed arrow), whereas to the left, there is a small free organism with a single purple–red, eccentric, dotlike nucleus and a small amount of light basophilic cytoplasm (arrowhead). An oval-shaped organism in an intact red cell with free organisms in the surrounding background (D) is also visible. Modified Wright stain. Original magnification, 100× objective. Case from American Society of Veterinary Clinical Pathology, Mystery Slide Conference 2006, Case B1, submitted by Dr. Casey M. Wood