Cytological Changes in Chicken Infectious Anemia Virus Infected Bone Marrow in Broilers

This study involved farms in Lahore district and its surroundings. We included this particular area because poultry is too dense here and occurrence of multiple infection across the area is not very uncommon. During a period of one year (2017–2018), we screened a total of fifteen broiler flocks having the capacity of 5000–30000 birds each. Each of the farm was suspected of having CIAV on the basis of clinical symptoms that included poor growth, anemia, anorexia and depression. Bone marrow samples were obtained from each of the flock and transferred to University Diagnostic Laboratory, University of Veterinary and Animal Sciences, Lahore. The negativity and positivity of each of the flock was confirmed through PCR as described previously (Herman et al., 2012). The samples were processed as pool of samples from each of the individual flock. Finally, after PCR confirmation, a total of five CIAV positive along with an equal number of negative broiler flocks were selected for bone marrow examination. From each flock, bone marrow (n=5) were collected from tibiotarsus and femur of chickens during necropsy and smears were made. The smear slides were fixed with methanol and stained by Giemsa stain (Al-Sadi and Hussein, 2010). The slides were then examined by microscope to evaluate cellularity of the bone marrow, to identify different stages of erythroid, myeloid and thrombocyte precursors maturation. Myeloid Erythroid Ratio (M:E) was also noted in each slide. Five hundred cells parameter was used for differential counting of all marrow cells precursors. Developmental stages of erythroid maturation including pro-erythroblast, basophilic normoblast, polychromatic normoblast and orthochromatic erythroblast were identified and calculated. Likewise, the developmental stages of myeloid progenitors (promyelocytes, myelocytes, meta myelocytes, stab form and mature lobulated granulocytes) were also calculated. The M/E ratio was calculated by dividing total number of nucleated cells of granulocytic group with total number of nucleated erythroid cells.

Complete concerned data was analyzed by using SPSS (23.0) and final results were declared as mean ± SEM.

In CIAV perspective, the bone marrow-based immunosuppression studies are limited and a much of detailed analysis has previously been documented for other avian pathogens such as infectious bursal disease (IBD) and Newcastle disease (ND). This was the reason that results interpretation along with evidences are documented or referenced while comparing other immunosuppressive agents available in the literature so far.

Immune system in chicken is constituted by lymphoid organs which provide a strong protective mechanism against all bacterial, viral and fungal infections. These lymphoid organs are labelled as primary or secondary lymphoid organs. Thymus, bone marrow and bursa of Fabricius are grouped under the heading of primary lymphoid organs whereas spleen, collections of mucosae related lymphoid tissues as secondary lymphoid organs (Riddel, 2001). In chicken development of erythrocytes and production of platelets takes place within the dilated vascular channels whereas production of granulocytic cells occurs outside the vascular sinuses (Bounous et al., 2000). Our study highlighted the depression of bone marrow activity in CIAV infected birds as compared to healthy birds. A depletion in mean percentage of erythroid precursors in bone marrow of CIAV infected bird (17.9±0.30) compared to healthy ones (39.95±0.55) was observed (Table I). Similarly, depletion in mean percentage of myeloid series precursors in infected bone marrow was observed (21.98±0.30) than in healthy ones (57.28±0.35). Decreased in M/E ratio of 1.22±0.15 was observed in infected chicken as compared to normal chicken (Table I). Our study results agree with the findings of a research conducted on bone marrow cytological changes in IBD, ND and Colibacillosis infected birds in comparison with healthy birds. In his study, deletion in erythroid and myeloid progenitor series were reported in infected birds bone marrow cytology (Wani et al., 2016); however, the depletion in the percentage of both progenitor series noted was non-significant when compared to control group results. In our study the difference in infected and control group cytology is significant. This might be due to the differences in the viruses used in their study, managemental practices, bird maternal immunity, use of intoxicated feed etc. In our study, a decrease in lymphoid progenitors were evidenced in infected bone marrow than in the control group. This finding is in line with the results of study conducted previously on IBD, ND and colibacillosis (Wani et al., 2016). In this study, it was noticed that among the cells of erythroid series, highest percentage of polychromatic erythroblast was found in both infected and healthy birds however the percentage of subject cells were less (8.70 ± 0.38) in infected than in control group (20.50 ± 0.58) (Table I). The morphology of cells was also examined in infected and control group. It was interesting to note that no differences in the morphology of cells were seen in both study groups. These polychromatic erythroblasts were circular in shape with a basophilic cytoplasm. In polychromatic erythroblast nucleus was small as compared to cytoplasm and was having dense clumped chromatin whereas orthochromatic erythroblasts were oval in shape with irregularly condensed chromatin material. Similar findings were noted in ducks and quails by Campbell and Coles (1986) and Tadjalli et al. (2002).

Among the granulocytic cells, myelocytes with eccentric nucleus and secondary granules were predominant cells which could be labelled as heterophils, basophils and eosinophils considering the characteristics of secondary granules. Metamyelocytes possess bean shaped nucleus along with secondary granules. Stab cells or band cells showed band shaped nucleus without lobulation. Monoblast and monocytes were having irregular shaped nucleus with transparent basophilic cytoplasm containing only few granules. Megakaryocytes were the largest cells in the bone marrow smear having large multi lobulated nucleus with granular cytoplasm. Lymphoblast and prolymphocytes were relatively small in size having high nuclear cytoplasmic ratio and scanty cytoplasm without granules. These mentioned morphological findings were found in concordance with the findings of earlier researchers (Tadjalli et al., 1997).

The bone marrow examination showed a significant difference in the bone marrow cellularity of normal and CAV infected bird. There was marked depression in erythroid, myeloid and megakaryocyte precursors along with developing cells, which ultimately leads to anemia, leucopenia and thrombocytopenia. Hence, bone marrow examination is easy, rapid and cost-effective analysis which may aid in the presumptive diagnosis of Chicken Infectious Anemia Virus in field contidions.

Authors declared no conflict of interest.