Avian leukosis virus subgroup J associated with the outbreak of erythroblastosis in chickens in China

This study was carried out in strict adherence to the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Healthy. The protocol was approved by the Committee on the Ethics of Animal of Shandong (Permit Number: 20100326).

From the year 2010 to 2011, our laboratory (The Molecular Pathology Laboratory, College of Veterinary Medicine, Shandong Agricultural University) received six sick representatives of the 20-day-old commercial layer chickens (flock 1), three 90-day-old (flock 2) and three 110-day-old (flock 3) Chinese local breed chickens for the diagnostic purpose. The birds of flock 1 presented depression, recumbency and pale cockscomb started from 15 days of age, and the mortality in the population was 18%. The birds of flock 2 showed symptoms of nerve system disorders such as depression and ataxia started from 85-day-old, and the mortality was 12%. The birds of flock 3 anorexia, lethargy and emaciation started from 90-day-old, and the mortality reached 20% at 100-day-old.

The samples of the liver, spleen, kidney, heart, lung, proventriculus, sciatic nerve, brain, and bone marrow were collected and fixed in 10% buffered neutral formalin. The fixed tissues were embedded in paraffin, sectioned at 4 μm thick, and stained with haematoxylin and eosin. The sample slides were observed under light microscopy.

DF-1 cells were seeded in 6-well plate at a density of approximately 1×10⁶ cells per well. Tissue extracts from ill chickens were inoculated onto DF-1 and incubated at 37°C for 2 h. Then the cells were cultured with fresh medium contained 1% fetal bovine serum (FBS, Invitrogen, CA, USA). Observed daily, on the seven days of the post-inoculation, provirus DNA were extracted from infected DF-1 cells using DNA extraction kit (TaKaRa, Bio, Inc., Beijing, China). The PCR amplifications using provirus DNA as templates with the primers (Table 1) specific for the avian erythroblastosis virus (AEV) specific primers (Genbank number : K02006.1), ALV-A, ALV-B [7], ALV-J [8], REV [9] and MDV [10] respectively were performed. The amplification of the target gene was set up in a 25 μL reaction containing 1 μL of DNA, 2.5 μL of 10×Taq buffer (TaKaRa, Bio, Inc., Beijing, China), 2.5 μL of dNTP (2.5 mmol/ L), 1 μL of each primer (10 mmol/ L), and 17 μL of ddH₂O. The PCR products were detected by 0.8% agarose gel electrophoresis with ErBr staining.

To detect the presence of ALV-J and AEV antigen, tissues were fixed with 10% buffered neutral formalin, paraffin-embedded, sectioned with the thickness of 4 μm, and mounted on poly-l-lysine-coated slides. The tissue sections were stained with a routine streptavidin biotin/horseradish peroxidase (HRP)-conjugated immunohistochemical technique as described by [11]. Briefly, the sections were pre-treated with 3% hydrogen peroxide in methanol, and blocked with 5% bovine serum albumin in PBS for 10 min. Then the slides were incubated with primary antibody (a rabbit anti-ALV-J and anti-AEV surface protein prepared by our lab) at a dilution of 1: 400 for 1 h, washed three times with PBS, and incubated with the secondary antibody (biotinylated goat anti-rabbit IgG, Santa Cruz, CA, USA) at a dilution of 1:5000 for 30 min. After three washes, the tertiary conjugate streptavidin/HRP was applied for 30 min. Chromogen (AEC) was applied and developed microscopically for positive straining. The reaction was stopped by water and the slides were then counterstained with hematoxylin. Finally, the slides examined microscopically with light microscopy. In negative immunostaining controls, the primary antibody was replaced with non-immune rabbit IgG.

The birds examined were characterized with pale pectoral muscles, myocardium hemorrhage (Figure 1A-B), and the enlarged visceral organs especial liver with multifocal, white raised foci throughout (Figure 1C), spleen (Figure 1D) and kidney with piebald (Figure 1E) which were brittle fragile. The bone marrow became jelly like with lighter colour. The diffuse, multifocal white raised foci were observed on the lung surface (Figure 1F).

There were numerous erythroblasts at different growth stages replaced the normal erythrocytes in the blood smears as shown in Figure 2A. The erythroblasts had polymorphism (spherical, ellipse and irregular shape), greater cellularity, loosen chromatin than normal erythrocytes. Their cytoplasm was basophilic and contained vacuolus surrounded the spherical or ellipse nuclei. In the bone marrow smears, the amount of erythroblasts was significantly increased. The erythroblasts had bigger volume, round shape and irregular edge. The features of the cytoplasm, nuclei and chromatin of the bone marrow erythroblasts were similar to that of the blood erythroblasts (Figure 2B).

There were some similar pathological changes in various tissues observed under light microscope. Severe hemorrhage and congestion were found in all tissue sections of the examined birds and the normal architecture of all tissues were damaged with different degrees. The Disse’s space of livers (Figure 3A) and the blood capillary in spleens (Figure 3B), myocardium (Figure 3C), lungs (Figure 3D) and spinal cords (Figure 3E) were dilated, in which massive erythroblasts were accumulated as observed in blood smears. The parenchyma hyperplasia and increased erythroblast were seen in bone marrow (Figure 3F). At high magnification, some of erythroblasts in all tissue sections had mitotic figures.

Provirus DNAs extracted from livers of the chickens of all the three flocks were assayed with PCR using AEV, ALV-A, ALV-B, ALV-J, REV and MDV specific primers. The results showed that all the samples tested were negative for AEV, ALV-A, ALV-B, REV and MDV (data not shown). Ten of the twelve samples were positive for ALV-J with a PCR product of 924 bp as expected, one sample from flock 1 and one from flock 2 were PCR negative (Figure 4).

In order to further detect the distribution of ALV-J antigen in different organs, immunohistochemistry using the anti-ALV-J specific antibody was performed to detect the ALV-J positive signals. The ALV-J positive signals were indicated by the brown staining of the erythroblast cytoplasm in the immunohistochemical stain assays. The results showed that the ALV-J positive signals were mainly presented in spleen (Figure 5B-C), lung (Figure 5E-F) and other tissues especially rich in blood. However, AEV was negative label (data not shown).