Small Mammal Hematology: Leukocyte Identification in Rabbits and Guinea Pigs

Vanessa K. Lester, DVM; Heather L. Tarpley, DVM; Kenneth S. Latimer, DVM, PhD

Class of 2005 (Lester) and Department of Pathology (Tarpley, Latimer) College of Veterinary Medicine, University of Georgia, Athens, GA 30602-7388

General Information

Rabbits and guinea pigs are common companion and laboratory animals. This web page is designed as a descriptive and visual guide to help veterinarians recognize the different leukocyte types in Romanowsky-stained blood smears. Also presented is basic information concerning leukogram characteristics in these animals during health and disease.

Most of the leukocytes in rabbits and guinea pigs appear very similar to those of other mammals. Novices may easily mistake heterophils (the lapine and cavian equivalent of neutrophils) for eosinophils. Heterophils have the same function as other mammalian neutrophils, but they have acidophilic or eosinophilic granules in their cytoplasm. They are sometimes referred to as "pseudoeosinophils" in the literature. Heterophils are present in a number of animal species including birds, reptiles, amphibians, some fish, rabbits, guinea pigs, and hamsters. The functions of the leukocytes in rabbits and guinea pigs are similar to those of other mammals.

Foa-Kurloff cells are a leukocyte type that is unique to guinea pigs. These are specialized mononuclear cells that contain an intracytoplasmic inclusion body of mucopolysaccharide. Although Foa-Kurloff cells can be present in the blood of both males and females, they are most commonly observed in blood smears from pregnant guinea pigs. These cells may be more prominent during pregnancy because they shift from the lungs and spleen to the thymus and placenta under estrogen stimulation.¹ Foa-Kurloff cells possibly function as natural killer cells².

Leukocytes of Rabbits

Lymphocyte - The morphology of lapine lymphocytes is similar to that of other species. Lymphocytes have a large nucleus, that may be slightly indented, and a small amount of light blue cytoplasm. Although small lymphocytes predominate, large lymphocytes may be present. These cells are similar in size to heterophils (or neutrophils of other mammals). Large lymphocytes may occasionally contain azurophilic granules near the nuclear indentation. Reactive lymphocytes (immunocytes) are antigenically-stimulated lymphocytes that are larger cells with a more intensely blue cytoplasm (Fig. 1).

Figure 1. A normal, small, well differentiated lymphocyte is on the left, while a larger, reactive lymphocyte with dark blue cytoplasm is on the right.

Heterophil - Inexperienced microscopists often misidentify rabbit heterophils as eosinophils. Heterophils range from 10 to15 µm in diameter. They have a light purple, lobulated nucleus surrounded by cytoplasm containing diffuse, variably-sized reddish granules. Heterophilic granules are generally smaller than those of eosinophils and may not occupy all of the cytoplasm. Although the nucleus is usually segmented, there may be infrequent band heterophils in the blood of healthy rabbits. Minor heterophil degranulation may accompany the use of rapid Romanowsky-type stains such as Diff-Quick. Stain-induced degraulation is presumed to be a sequel of short fixation time (5 to 10 seconds) during staining. Degranulated cells will resemble heterophils, but vacuoles will be present where the granules previously were located. Stain-induced degranulation should not be confused with toxic change in which the cytoplasm has a blue cast.

Figure 2. Normal rabbit heterophils have a lobulated nucleus and small, diffuse, red, cytoplasmic granules (left). Stain-induced degranulation of heterophils may be observed in some blood smears following Diff-Quik staining (right).

Eosinophil - Eosinophils are slightly larger than heterophils and are 12 to 16 µm in diameter. The nucleus stains purple and often appears bilobed. Intensely acidophilic, round, cytoplasmic granules are present that are larger and more numerous than the granules in heterophils.

Figure 3. Normal rabbit eosinophils have a lobulated nucleus and numerous, round, intensely red, cytoplasmic granules.

Monocyte - Monocytes are the largest circulating leukocytes in health and measure 15 to 18 µm in diameter. Monocytes have a large, variably-shaped nucleus with chromatin that appears less condensed than that of heterophils. The cytoplasm is abundant and stains gray to blue-gray. A few cytoplasmic vacuoles may be observed. Large, dark red granules have been described in the cytoplasm of some monocytes in association with nonspecific toxicity.³

Figure 4. A rabbit monocyte with a nonlobated nucleus and abundant blue-gray cytoplasm.

Basophil - Basophils have a light purple, lobulated nucleus and dark purple to purple-black cytoplasmic granules. They are approximately the same size as heterophils.

Figure 5. A rabbit basophil with a lubulated nucleus and chunky purple granules that partially obscure nuclear morphology.

Comments Concerning the Hematology of Rabbits

In contrast to some other mammals, 2 to 4% polychromasia may be a normal observation in stained blood smears of healthy rabbits. An occasional nucleated RBC or Howell Jolly body also may be present. The estimated lifespan of lapine RBCs is 57-67 days.⁴ This relatively short erythrocytic lifespan is associated with increased polychromasia to replace senescent erythrocytes.

Leukocyte counts are variable both between animals and for different samples from the same animal. The total leukocyte count is lowest in newborns and has dual peaks at 3 months and 12 months of age, with a decline between those time periods.⁴ There is also diurnal variation in the leukocyte count with the nadir occurring in the late afternoon to evening.⁵ Stress may increase total leukocyte count by 15 to 30%.⁴

The relative distribution of rabbit leukocyte subtypes is also variable. The lymphocyte is the most common leukocyte in the blood of young animals that are < 12 months of age. After 13 months of age, the heterophils and lymphocytes may be present in approximately equal numbers.⁴ In contrast to many other mammals, healthy rabbits may have basophils ranging from 5 to 30% of the leukocyte differential count.⁴ The following published reference ranges⁴ provide the expected frequency of the total and differential leukocyte counts in rabbits: WBC = 6,300 - 10,060 cells /µl; segmented heterophils = 1,490 - 3,210 cells /µl; band heterophils = 0 cells /µl; lymphocytes = 3,360 to 7,000 cells /µl; monocytes = 50-450 cells /µl; eosinophils = 100-150 cells /µl; and basophils = 60-360 cells /µl.

Aberrations in the rabbit leukogram may be more difficult to interpret than those in most companion animals. Rabbits do not commonly develop a leukocytosis with bacterial infections, but may display an inverse heterophil:lymphocyte (H:L) ratio. Leukogram interpretation is complicated by the commonality of an inverse H:L ratio which occurs secondary to any source of stress (cortisol), including stress from transport or any chronic disease. Stressful events, such as that related to transport, may last for 24 to 48 hrs.⁵ The processes of venipuncture and blood collection do not appear to have these effects.⁵ Stress (endogenous cortisol release) should not be confused with an excitement (epinephrine release). Excitement should actually cause lymphocytosis, while stress may result in lymphopenia. The presence of other systemic signs of illness, such as fever or toxic changes, may help determine if leukogram changes are due to infectious causes.

Due to the similarity in cell function across species, other changes in the leukogram may have the same general etiologies. Leukocytosis may occur with lymphosarcoma, especially if abnormal lymphocytes are present in the stained blood smear. Leukopenia, especially lymphopenia, may indicate chronic disease. Chronic parasitism may cause an eosinophilia.³ Monocytosis, if present, suggests chronic inflammation.

Rabbits may have a rare autosomal dominant genetic condition called Pelger-Huët anomaly, which also has been described in people, dogs, and cats. This anomaly is characterized by granulocytic nuclear hyposegmentation with the retention of a coarse, mature chromatin pattern. Affected animals are typically heterozygotes. The homozygous state of Pelger-Huët anomaly usually is lethal in utero; however, the rare surviving rabbits have granulocytes with round to oval nuclei and an extremely coarse chromatin pattern, severe skeletal deformities including dyschondroplasia, and an increased neonatal mortality rate.

Leukocytes of Guinea Pigs

Lymphocyte - The appearance of lymphocytes in blood smears from guinea pigs is similar to that in other species. Small, well differentiated lymphocytes predominate and are slightly larger than RBCs. The larger lymphocytes are almost twice as large and may have azurophilic granules, as in the rabbit (Fig. 6).

Figure 6. Appearance of large and small lymphocytes in a stained blood smear from a guinea pig. The large lymphocyte at left has several metachromatic granules in the area of nuclear indentation.

Heterophil - Heterophils of guinea pigs are 10 to 12 µm in diameter. The nucleus of individual cells usually is purple, segmented, and has a dense chromatin pattern. The nuclei of some heterophils in females have a "drumstick" sex chromatin lobe. The cytoplasm has scattered acidophilic granules that are smaller than those of eosinophils. More of the cytoplasm is visible than in eosinophils.

Figure 7. The heterophil (upper right) has small, widely scattered, eosinophilic, cytoplasmic granules compared to the eosinophil (lower left) that has numerous, large, round, brightly eosinophilic, cytoplasmic granules.

Eosinophil - Eosinophils are slightly larger than heterophils. The nucleus is less segmented and the cytoplasmic granules are larger, round, and bright red compared to heterophils of this species. Granules usually completely fill the cytoplasm (Fig. 7).

Monocyte - Monocytes are the largest leukocyte in circulation. These cells have a variably-shaped nucleus, less condensed chromatin pattern, and moderately abundant blue-gray cytoplasm. Compared to lymphocytes, monocytes are larger and have darker, more abundant (Fig. 8).

Figure 8. Image of a monocyte to be added

Basophil - Basophils are the same size as the other granulocytes. They have a purple, lobated nucleus and variably-sized, purple granules in the cytoplasm.

Figure 9. Image of a basophil to be added.

Foa-Kurloff cells - Foa-Kurloff cells are unique to guinea pigs and capabaras. These mononuclear cells approximate the size of a large lymphocyte. They have a large, round, purple nucleus that is often eccentrically located and blue cytoplasm. The distinguishing characteristic of this cell is a very large, slightly granular, magenta, cytoplasmic inclusion body (Fig. 10).

Figure 10. Foa-Kurloff cell in the blood smear of a guinea pig. Notice the characteristic large, slightly granular, magenta, cytoplasmic inclusion.

Comments Concerning the Hematology of Guinea Pigs

The predominant circulating leukocyte in healthy guinea pigs is the lymphocyte. In contrast to the rabbit, basophils are rarely observed. Foa-Kurloff cells may comprise 3 to 4% of leukocyte differential count.¹ The published reference intervals for the total and differential leukocyte counts are as follows:⁴ WBC = 8,220 - 14,000 cells /µl; segmented heterophils = 1,350 - 3,650 cells /µl; band heterophils = 0 – 10 cells /µl; lymphocytes = 5,470 - 10,550 cells /µl; monocytes = 60-560 cells /µl; and basophils = 0 – 20 cells /µl.

There are few publications on leukogram changes of guinea pigs with naturally occurring diseases. Guinea pigs experimentally infected with Trixacarus caviae (guinea pig mange mites) developed a heterophilia, monocytosis, eosinophilia, and basophilia.⁶ Although guinea pigs are considered a tick-resistant species, they may develop an eosinophilia and basophilia in response to infestation with Amblyomma americanum⁷ (lone star tick) and a significant basophilia in response to Rhipicephalus sanguineus (brown dog tick) .⁸ Guinea pigs also have developed eosinophilia in response to Treponema pallidum (syphilis) infections.⁹

References

1. Moore DM: Hematology of Rabbits and Hematology of the Guinea Pig. In: Feldman BF, Zinkl JG, Jain NC (eds): Schlam’s Veterinary Hematology, 5th ed, Lippincott Williams & Wilkins, 2000, pp.1100-1110.

2. Pouliot N, Maghni K, Blanchette F, et al: Natural killer and lectin-dependent cytotoxic activities of Kurloff cells: Target cell selectivity, conjugate formation, and Ca++ dependency. Inflammation 20:647-671, 1996.

3. Benson KG, Paul-Murphy J: Clinical pathology of the domestic rabbit: Acquisition and interpretation of samples. Vet Clin N Am Exotic Anim Pract 2:539-552, 1999.

4. Campbell TW: Mammalian hematology: Laboratory animals and miscellaneous species. In: Thrall MA: Veterinary Hematology and Clinical Chemistry, 1st ed, Lippincott Williams and Wilkins, 2004, pp. 211-224.

5. Harcourt-Brown F. Textbook of Rabbit Medicine, 1st ed, Elsevier Science Limited, 2002, pp.142-147.

6. Rothwell TLW, Pope SE, Rajczyk ZK, Collins GH: Haematological and pathological responses to experimental Trixacarus caviae infection in guinea pigs. J Comp Pathol 104:179-185, 1991.

7. Brown SJ, Askenase PW: Blood eosinophil and basophil responses in guinea pigs parasitized by Amblyomma americanum ticks. Am J Trop Med Hygiene 31:593-598, 1982.

8. Szabo MPJ, Aoki VL, Sanches FPS, et al: Antibody and blood leukocyte response in Rhipicephalus sanguineus tick-infested dogs and guinea pigs. Vet Parasitol 115:49-59, 2003.

9. Wicher V, Scarozza AM, Ramsingh AI, et al: Cytokine gene expression in skin of susceptible guinea-pig infected with Treponema pallidum. Immunology 95:242-247, 1998.

Acknowledgment

"Ranch Rabbit", an acrylic painting by Malcolm Furlow, is from the Creative Expressions Gallery website and permission to use has been requested.

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