Research Note: Reticulocytes and related erythroid atypia of ducks-indicators of stress or pathology?

Two kinds of reticulocytes, and atypical erythroid cells, were found in the blood of apparently healthy ducks with Wright-Giemsa (W-G) stain. The reticulin of network-type examples (nRtc) is of a large complex (3-D) form. The punctate reticulocyte (pRtc) contains small pin-point cytoplasmic granulations; both cells are distinct from a polychromatic RBC (pRBC). Atypical erythroid cells with oval or irregular shapes reminiscent of primary or yolk-sac RBCs (ysRBC) accompanied the Rtc. Rare binuclear cells (bi) were either polychromatic (pRBC and pRtc) or full hemoglobin (Hb) types (RBC). Some bi with equal-sized daughter nuclei were presumed mitotic products. Conversely, unequal daughter nuclei were amitotic products, a nuclear division without chromosomes or a spindle. Erythrocytes formed tight aggregations with thrombocytes or other cells called "toroids"; further indicating a reactive hemogram. Erythroplastids (ep) anuclear erythroid cells, found along with other atypia, were either pRBC or full Hb types. The total white blood counts (TWBC) of the study set ranged from 5 K/µL (embryo E [d24]) to >100 K/µL (older ducks) with heterophil ratios (H/L) ranging from 0.5 to >4.0. Atypical erythroid cells, like atypical leukocytes, indicate a transition from homeostasis to a reactive state. Recognition of nRtc, pRtc, and toroids as reactive forms, expands the utility of hematologic data in assessing stress levels, indicating pathology, and exploring welfare questions. The present observations support earlier work showing some reticulocytes are detected by W-G alone and do not require vital dyes. Cells and behaviors described here demonstrate the benefit that a description of overall cytology adds to H/L ratios in evaluating a hemogram.

Atypical hemograms of the commercial duck.

A description of standard and atypical heterophils, lymphocytes, and 2 types of giant cells found in the circulation of 17 wk commercial ducks (N = 24) in apparent good health is the subject. Heterophils were sorted as either "classic" (HC) having red rod-shaped cytoplasmic granules, "typical" (HT) having weakly stained granules providing a reticular cytoplasmic appearance, or rarely as "variant" types (HV) having orange spherical granules. Atypical HT's and HC's were in 14 of 24 (58%) of the ducks. Small lymphocytes (Ls), reactive lymphocytes and plasmacytes (Lm) were routinely found. Giant cells, also present, were placed with Lm or monocytes (Mn) depending on cytology. Two counts of 200 leukocytes gave the total white count (TWBC) and 2 heterophil/lymphocyte ratios. H/L 1 = (HT + HC +HV) / Ls; and H/L 2 = (HT + HC + HV) / (Ls + Lm). The results showed that TWBC were normal (~ 23,000 /µL) but both H/L ratios were highly variable. HT were differentiated from HC on nuclear and cytoplasmic criteria. Many HT and HC exhibited signs of deterioration. Some giant cells likely represented developmental stages. Multiple nucleoli were evident in others suggesting polyploidy. The more common lymphoid giants were usually round whereas monocyte types were irregular. Mn types were actively phagocytic often consuming thrombocytes or rarely erythrocytes (RBC). Giant cells of either type were in 13 of 24 (54%) of the duck hemograms. Conidiospores were detected in the blood smears of 4 ducks and bacteria in 2 with 1 duck having both. As all ducks were in apparent good health the blood born microorganisms likely represented low grade infections. Presumably the atypical cells were a response to the presence of toxins of bacterial and fungal origin. The presence of atypical heterophils and lymphocytes complicates interpretation of H/L ratios traditionally used to establish stress. As atypical cells can be found in the context of normal TWBC or nonstress H/L values cytological observations attain additional importance. Moreover, giant cells may be useful indicators of infection even without direct microscopic observation or isolation of the offending organisms.

A comparison of Mott cell morphology of three avian species. II. - Bad behavior by plasmacytes?

Mott cells are atypical plasmacytes recognized microscopically by endoplasmic reticulum (ER) distensions (Russell bodies) a result of retained secretory product (antibody). Originally associated with parasitism, they are observed in a broad spectrum of immunopathology, sometimes involving hypergammaglobulinemia. Few descriptions of Mott cells appear in avian literature. The purpose of the manuscript is to provide examples identified by light microscopy in three poultry species. Transmission electron micrographs (TEM) of plasmacytes from the turkey oviduct mucosa are included for comparison with Mott cell light microscopic images. Wright's stained blood and bone marrow from commercial and specific pathogen free (SPF) chickens, ducks, and turkeys are the sources. Mott cell positive samples commonly occurred with leukocytosis or leukemoid reactions, polymicrobial bacteremia, and fungemia. Atypical granulocytes and leukocytes regularly accompanied Mott cells. It is proposed that circulating Mott cells are "sentinels" indicative of stress, dyscrasia, and pathology. Moreover, Mott cells, like other atypia, complicate the interpretation of simple heterophil/lymphocyte (H/L) ratios. As Mott cells are defective plasmacytes these observations address hematology, immunology, pathology, and welfare issues.

Atypical lymphocytes and leukocytes in the peripheral circulation of caged hens.

Lymphocytes comprise a family of cells descended from bursa and thymus progenitors whose differentiation is not possible by standard hematology. However, if they are small with a nuclear/cytoplasmic ratio near 1, they are "resting" at least in the microscopic sense. Activation, increases their size, and decreases the nuclear:cytoplasmic (N:C) ratio. Reactive cells are infrequent in healthy animal blood. Their presence indicates an immune response in progress, inflammation, stress, or other pathology. Here the purpose is to describe unusual leukocytes and lymphocytes found in the periphery of commercial hens. Samples of Wright stained blood films obtained from commercial hens housed in modern cages are the data source. Photomicroscopy used an Olympus CX41 light microscope equipped with an Infinity-2 1.4 megapixel charge-coupled device (CCD) Universal Serial Bus (USB) 2.0 camera, at 100× (oil) magnification. Collectively these cells illustrate a continuum between mildly "reactive" to grossly "atypical" states. The description begins with normal resting cells, proceeds to mildly atypical, and concludes with grossly abnormal cells. Bone marrow cells, a source of plasmacytes, are included for comparison. Examples of circulating plasmacytes, large plasmacytoid lymphocytes (LPL), foam cells, and cells expressing properties of more than one lineage are included. The importance of these observations lies in their contribution to cytology, hematology, and immunology. Last, because of the wide use of heterophil:lymphocyte ratios (H:L) as a stress measure they bear directly the welfare issues of caged animals. When cells similar to the types described here are in blood, they indicate stress independent of H:L or other standard measures.

Are peripheral Mott cells an indication of stress or inefficient immunity?

Atypical plasmacytes having distinctive cytoplasmic vacuoles (Mott cells) were detected in 77/1,000 (7.7%) of commercial hens housed conventionally, in aviaries, or in enriched environments. The earliest Mott positive peripheral blood samples were at placement (18 wk) from 2 consecutive commercial flocks. Additional samples obtained at 32, 48, 56, and 77 wk were positive. Most Mott cells came from hens with high total white blood cell counts as a component of leukocytosis. However, Mott cells were found in hens with low total white blood cell counts, and low heterophil/lymphocyte ratios. Phagocytosis of bacteria by some Mott cells was a remarkable feature. Many of the Mott positive hens had polymicrobial bacteremia and a few had fungemia likely accounting for the leukocytosis. In other cases, free-swimming bacteria were located near to a Mott cell. These atypical cells were in the peripheral blood samples from other poultry; a tom at slaughter (17 wk), experimental toms (10 wk), and experimental ducklings. Examples are included.As descriptions of avian Mott cells are few, the purpose of describing these cells is their contribution to hematology, immunology, and cytology. Mott cells like other atypia are sentinels, frank cytological indicators of an unusual hemogram, and consequently infer stress. Therefore, they bear directly on welfare issues.

An examination of the utility of heterophil-lymphocyte ratios in assessing stress of caged hens.

To measure stress in caged hens, differential counts of their wing vein blood were used to determine heterophil/lymphocyte (H/L) ratios and total white blood cell counts (TWBC). The H/L values of 18-wk samples from conventionally caged hens (CC) were not statistically different from hens raised in aviaries (AV) when calculated by either of 2 methods (H/L 1 and H/L 2). However, there was a high degree of variation among samples within each cage type. The TWBC data and hematology indicated leukocytosis, leukemoid reactions, and a high frequency of atypia. Reactive lymphocytes, large plasmacytoid lymphocytes, cyanophils, coccinocytes, and atypical heterophils were common. Analysis of 77-wk data indicated significant differences among 3 cage types. The H/L 1 of enriched caged (EN) hens was twice (0.91) that of either AV (0.33) or CC (0.44) hens (P<0.01); the H/L 2 values were also highest for EN (0.46) versus AV (0.29) and CC (0.34; P<0.01). As was the case with 18-wk samples, TWBC distributions and hematological data indicated leukocytosis, leukemoid reactions, and a high frequency of atypia. Among the likely reasons for the hematological observations was the occurrence of polymicrobial bacteremia and fungemia, both of which could account for high TWBC and atypical cells. Collectively, these observations challenge the general application of the H/L ratio method when applied alone as an indicator of stress and welfare of hens caged in modern systems.