Cytochemical studies
Acid ferrocyanide staining in this study revealed positive results in mature erythrocytes (Table 2), with occasional cells exhibiting blue siderocytic granules in their cytoplasm (Fig 1A), a finding consistent with previous reports by
Gupta and Singh (2012) in guinea fowl,
Yadav (2012) in Kadaknath fowl and Mohd
et al. (2015) in Uttara fowl. Similarly,
Doley et al., (2024) observed a sparse presence of these granules in erythrocytes of Zoar. Most of the acid ferrocyanide-positive erythrocytes in this study exhibited nuclear deformities, potentially indicating disrupted erythropoiesis, which aligns with earlier findings by
Cartwright et al., (1975) who reported siderocytic granules as a typical feature during normoblast maturation in humans and pigs. Conversely, all leukocytes and thrombocytes were negative for acid ferrocyanide, consistent with findings by
Doley et al., (2024) and
Valenciano et al., (2010), who noted the rarity of siderocytic leukocytes, typically observed only in cases of feline haemolytic anaemia.
Barger (2022) associated siderocytes with conditions like myeloproliferative disorders, lead toxicity and haemolytic anaemia, while
Meguro et al., (2007) emphasized the role of non-heme iron histochemistry as a critical tool for studying degenerative diseases and pathological processes.
In this study, basophils exhibited intense positivity for acid mucopolysaccharides, while erythrocytes, thrombocytes, heterophils, eosinophils, monocytes and lymphocytes showed no reaction (Table 2). These findings align with earlier reports by
Gupta et al., (2010) in Guinea fowls,
Yadav and Singh (2012) in Kadaknath fowl,
Mohd et al. (2015) in Uttara fowl and
Shalini (2015), who noted strong reactions in fowl, moderate responses in ducks and weak to negative reactions in quail.
Kumar (2019) observed weak positivity in the basophils of domestic fowl. Under toluidine blue staining, basophils appeared as round cells filled with intensely violet metachromatic granules throughout the cytoplasm (Fig 1B), similar to observations by
Gupta et al., (2010), Yadav and Singh (2012),
Mohd et al., (2015) and
Doley et al., (2024). Shalini (2015) similarly reported strong granule positivity in fowl, moderate in ducks and weak to negative in quail.
Raskin (2010) explained that toluidine blue, a basic dye, forms metachromatic complexes with acid mucopolysaccharides, resulting in red-purple staining.
Kiehl et al., (1994) highlighted that toluidine blue staining could be metachromatic under acidic conditions but not at neutral pH.
Ribatti (2018) attributed the metachromasia to dye polymerisation, causing a shift from blue to violet, red, or orange hues.
Shao et al., (2013) identified glycosaminoglycans in leukocytes as primarily chondroitin sulfate with minor heparin sulfate, while
Fong and Cranes (2023) linked basophil and mast cell metachromasia to highly acidic heparin molecules in their granules.
In this study, heterophils exhibited strong periodic acid-Schiff (PAS) positivity (Fig 1D), consistent with
Shalini (2015), who reported similar results in ducks and quail but moderate positivity in fowl. However andreasen and
Latimer (1990) and
Thrall et al., (2004) observed PAS negativity in chicken heterophils, while
Salakij et al., (2019) reported species-specific differences, with PAS positivity in Black kites but negativity in Black-shouldered and Brahminy kites. Eosinophils displayed moderate PAS positivity (Fig 1E), aligning with findings by
Mohd et al., (2015) in Uttara fowl, though weaker reactions were noted by
Yadav (2011) in Kadaknath fowl and
Raskin (2010) in chickens. Lymphocytes showed weak PAS positivity (Fig 1F), while other cells were non-reactive (Table 2). Interestingly, thrombocytes exhibited moderate PAS positivity in fowl, ducks and quail, corroborating
Mohd et al., (2015). Salakij et al., (2019) reported PAS-negative erythrocytes in Black-shouldered and Brahminy kites, contrasting with
Chen et al., (2019), who found PAS-positive erythrocytes in domestic pigeons.
Raskin (2010) linked PAS positivity in blood cells to glycoproteins, mucoproteins, glycolipids and glycogen, highlighting its diagnostic value for various leukemias in animals.
In this study, eosinophils showed strong positivity for Sudan Black B, characterized by black, round cytoplasmic granules (Fig 1C), consistent with findings in guinea fowl
(Gupta et al., 2010), Kadaknath fowl (
Yadav and Singh, 2012), Uttara fowl (
Mohd et al., 2015), Crested Serpent Eagles
(Salakij et al., 2015), domestic pigeons
(Chen et al., 2019) and Zoar
(Doley et al., 2024). However,
Kumar (2019) reported only moderate positivity in domestic fowl eosinophils. Heterophils also displayed strong positivity with black elongated granules (Fig 1C), aligning with
Shalini (2015) in ducks and quails but contrasting with weak positivity reported in Uttara fowl
(Mohd et al., 2015), painted storks
(Salakij et al., 2003) and the negative results observed by
Bounous and Stedman (2000) and
Salakij et al., (2004) in other avian species. In contrast, erythrocytes, basophils, lymphocytes, monocytes and thrombocytes showed no reaction (Table 2), consistent with findings in domestic fowl, ducks, quails (
Shalini, 2015) and domestic pigeons
(Chen et al., 2019). Exceptions include positivity in painted stork erythrocytes
(Salakij et al., 2003) and occasional positivity in avian monocytes (
Raskin, 2010). Sudan Black B, a fat-soluble dye that stains lipid particles in myelocytic and monocytic granules (
Jamal, 2020), is also useful in diagnosing various leukemias, including myeloblastic, eosinophilic, monocytic, myelomonocytic and erythroleukemia in several species (
Raskin, 2010).
Cytoenzymatic studies
In this study, basophils displayed strong positivity for acid phosphatase (Fig 2A), contrasting with findings in guinea fowl (
Gupta and Singh, 2008) and domestic pigeons
(Chen et al., 2019), where basophils were negative for this enzyme. Heterophils showed moderate positivity (Fig 2B), aligning with findings in rock partridges (
Dönmez and Sur, 2008), avian heterophils (
Claver and Quaglia, 2009) and Zoar
(Doley et al., 2024), although previous studies in chickens (
Andreasen and Latimer, 1990) and other birds reported negative or varying reactivity. Weak positivity in eosinophils was reported in guinea fowl (
Gupta and Singh, 2008), Uttara fowl
(Mohd et al., 2018) and domestic pigeons
(Chen et al., 2019). Eosinophils and lymphocytes in this study exhibited weak positivity (Fig 2C), consistent with
Andreasen and Latimer (1990) in chickens,
Yadav et al., (2015) in Kadaknath fowl and other studies highlighting acid phosphatase in avian eosinophils (
Bounous and Stedman, 2000;
Raskin and Valenciano, 2007). Other leukocytes, including thrombocytes and erythrocytes, were non-reactive (Table 3), consistent with observations in chickens and fowl, although acid phosphatase activity has been noted in monocytes (
Raskin and Valenciano, 2007), lymphocytes (
Shalini, 2015) and thrombocytes (
Dönmez and Sur, 2008).
Raskin (2010) highlighted the diagnostic utility of acid phosphatase activity in identifying blast cells and aiding in the diagnosis of acute myeloblastic leukemia and its subtypes, such as myelomonocytic, erythroleukemia and megakaryoblastic leukemia.
In this study, eosinophils showed moderate positivity (Fig 2D) and heterophils weak positivity for alkaline phosphatase (Fig 2E), while other blood cells were non-reactive (Table 3). Similar findings were reported by
Gupta and Singh (2008a) in guinea fowl,
Yadav (2011) in Kadaknath fowl and
Mohd et al., (2018) in Uttara fowl, as well as by
Doley et al., (2024) in Zoar. In contrast
andreasen and Latimer (1990) and
Raskin and Valenciano (2007) observed no alkaline phosphatase activity in chickens’ heterophils and eosinophils.
Chen et al., (2019) found alkaline phosphatase positivity in the erythrocytes of domestic pigeons, while
Genovese et al., (2013) reported negative reactivity in avian heterophils. Previous studies by
Nanba et al., (1977) and
Elghetany et al., (1990) demonstrated alkaline phosphatase activity in granulocytes and specific lymphocyte subsets, with
Raskin (2010) noting its potential as a diagnostic marker for myelogenous leukemia, although late-stage neutrophils in dogs and cats lacked activity.
In this study, thrombocytes showed strong positivity for peroxidase (Fig 2F), consistent with Santos
et al.
(2003) in roadside hawks, but differing from
Salakij et al., (2004) in Greater and Lesser Adjutants,
Chen et al., (2019) in domestic pigeons and
Salakij et al., (2019) in Black-shouldered Kites, Brahminy Kites and Black Kites. Eosinophils and erythrocytes in White Pekin ducks showed weak peroxidase positivity, similar to
Bounous and Stedman (2000),
Gupta and Singh (2008),
Bonadiman et al., (2009) and
Mohd et al., (2018), but contrasting with
Chen et al., (2019) and
Salakij et al., (2015), who found erythrocytes in pigeons and Crested Serpent Eagles negative for peroxidase. All other blood cells in this study were non-reactive, aligning with
Bounous and Stedman (2000),
Salakij et al., (2004) and
Gupta and Singh (2008a), who reported no peroxidase activity in avian heterophils, lymphocytes, basophils and monocytes.
Raskin (2010) noted peroxidase activity in the matrix of eosinophilic granules but not the crystalloid core, emphasizing its diagnostic value for myeloblastic and myelomonocytic leukaemia in various animal species.
In this study, eosinophils and lymphocytes showed weak positivity for b-glucuronidase (Fig 3B, 3C), consistent with
Yadav and Singh (2015) in Kadaknath fowl, but contrasting with
Salakij et al., (2004), who found no reactivity in the eosinophils of Greater and Lesser Adjutants. Other blood cells were non-reactive for this enzyme (Table 3), aligning with
Salakij et al., (2004), who also reported no reactivity in erythrocytes, heterophils and thrombocytes, while basophils were positive and monocytes weakly positive.
Yadav and Singh (2015) noted that basophils, lymphocytes and monocytes in Kadaknath fowl were negative, differing from
Salakij et al., (2015), who observed weak positivity in erythrocytes of Crested Serpent Eagles and Shikras.
Salakij et al., (2003) found weak or no staining in heterophils of Painted Storks, while thrombocytes were positive.
Genovese et al., (2013) reported positivity in avian heterophils and
Salakij et al., (2015) noted positivity in heterophils, basophils, lymphocytes and thrombocytes of Crested Serpent Eagles, with weak positivity in monocytes. Furthermore,
Salakij et al., (2019) observed b-glucuronidase positivity in lymphocytes and thrombocytes of Black-shouldered Kites, with variability in other kite species.
Haskins et al., (1984) associated β-glucuronidase deficiency with mucopolysaccharidosis VII (Sly syndrome), leading to glycosaminoglycan accumulation in tissues.
In this study, eosinophils and heterophils exhibited strong positivity for α-naphthol acetate esterase (ANAE) (Fig 3E, 3F), consistent with
Salakij et al., (2015) in Crested Serpent Eagles and Shikras, while
Shalini (2015) reported weak positivity in fowl eosinophils and weak to negative reactions in ducks. Lymphocytes showed weak positivity (Fig 3D), in line with
Ergün et al. (2004) in ostriches,
Oznurlu et al., (2012) in pigeons and
Shalini (2015) in quails. All other cell types were non-reactive (Table 3), aligning with
Dönmez and Sur (2008), who found negative results in erythrocytes and positive reactions in heterophils of Rock Partridges, as well as
Salakij et al., (2004), who reported negative results in heterophils of Lesser and Greater Adjutants. These findings contrast with
Salakij et al., (2003), who observed strong ANAE positivity in heterophils of Painted Storks, highlighting interspecies variability. According to
Pinkus et al., (1979), ANAE activity is mainly associated with T lymphocytes and is a crucial marker in diagnosing lymphoproliferative disorders such as leukemia and lymphomas, helping differentiate T and B cell populations and providing insights into disease progression and the rapeutic responses.
