Immunopathological investigation and genetic evolution of Avian leukosis virus Subgroup-J associated with myelocytomatosis in broiler flocks in Egypt

Avian leucosis virus (ALV) is an RNA virus belonging to the genus Alpharetrovirus, family Retroviridae, and induces a lot of neoplastic diseases with other reproduction troubles in different poultry species worldwide [1, 2]. Avian leucosis virus type J (ALV-J), strain HPRS-103, was first isolated from commercial meat-type chickens in the United Kingdom in the late 1980s [1, 3]. Moreover, in China, ALV-J infection was first detected in 1999 then followed by devastation to layers, of native breeds of chickens and ducks, causing catastrophic economic losses [4]. To date, ALVs have been classified into 11 viral subgroups, based on their host range, as well as viral envelope interference and cross-neutralization patterns [5]. Subgroups A-D, K, and J are exogenous viruses; that mainly infect chickens and turkeys, whereas subgroup E belongs to endogenous viruses. Subgroups ALV-A and ALV-B are common pathogens inducing lymphoid leukosis tumors with high incidence, while subgroups ALV-C and ALV-D have rarely affected the chicken [6]. Recently, subgroup ALV-K induces fowl glioma [7].

Importantly, chickens infected with ALV-J usually exhibit tumor development, depressed immunity, growth retardation, reduced egg productivity, and a considerable increase in the morbidity and mortality rates that are most apparent in broiler breeder hens. ALV-J infection in breeder flocks is associated with the occurrence of myeloid leucosis (myelocytomatosis) [8]. Myelocytomatosis was first observed in broiler breeder birds between 25 and 55 weeks. Furthermore, myelocytomatosis tumors are now being reported to appear in the field as early as 17 weeks [5, 9]. Numerous further cases of myelocytomatosis among broiler breeding flocks have also been reported in several European countries [10]. America, Asia [4], Africa (Egypt) [11], and Australia [12].

In recent years, numerous strains of ALV Subgroup-J have been isolated from white meat-type chickens; causing a serious impact on the growth performance of the poultry industry [3]. Due to horizontal and vertical transmission, ALV-J has caused increasingly severe damage to the poultry industry worldwide as infected broilers exhibit various tumor phenotypes, such as hemangioma, myeloid leukosis, and myelocytoma with decreased weight gain [13, 14]. Moreover, in other parts of the world as China, the mixed structure of the chicken breeding industry through crossbreeding Chinese local breeds with other western breeds may potentially be of concern in increasing the high frequency of ALV-J infection, especially in the case of vertical transmission to their progeny [15]. In Egypt, ALV-J-induced myelocytomatosis was reported in broilers − 28 days old-depending on histopathological lesions and antibody detection as the positive samples were (26%) with a mortality rate was (3.5%) [11, 16]. Taken into account, myelocytomatosis diagnosis is based on gross pathological lesions and antibody titer in a 27-week-old broiler breeder. Additionally, in laying hens, ALV-J particles are presented abundantly in the ovarian stroma, bud from cells in direct contact with oogonia, and oocytes with the highest concentration in the albumen-secreting glands of the magnum. This early ovarian and oviduct exposure may cause an early and diffuse infection [17]. Moreover, monoclonal antibodies against ALV-J envelop glycoproteins have been produced with broad reactivity for most ALV-J isolates. These antibodies have been used to determine tissue tropism of ALV-J naturally infected chickens [18].

Currently, there is no vaccination strategy or specific treatment available for ALV-J. Thus, control measures depended on the elimination of positive cases, management approaches and biosecurity programs in the poultry farms to decrease ALV spreading and clinical symptoms [19]. Therefore, rapid and confirmatory diagnosis is necessary to eradicate ALV from breeding flocks [20]. The ELISA is a useful serological diagnosis for the detection of ALV-J antibodies as it is a sensitive, easy, safe, and rapid diagnostic tool [11]. ELISA diagnostic method of ALV-J is reported to have sensitivity (99.2%), and specificity (100%), and can be used clinically for screening purposes [21].

Recently, molecular-based diagnostic techniques including insitu hybridization, PCR, and sequencing analysis have been developed for ALV detection [22]. Particularly, the proviral DNA arrangement of the ALV genome involves three important structural proteins (gag, pol, and env) which have been translated into the specific group antigen and envelop glycoproteins. The gp85 envelop glycoprotein, is closely associated with the viral entry, and host range, inducing host-neutralizing antibodies, tissue tropism, and virulence. Moreover, it is the major subgrouping determinant responsible for host infection and tumor formation [23‐25]. In addition, the gp85 gene is the most variable region of the envelope which evolves more rapidly in ALV-J compared to the other subgroups causing serious economic losses. Thus, it is crucial to monitor the gp85 gene evolution continuously to update any new strains and mutations [19, 26, 27].

Taken together, the present study targets the molecular characterization, serological assay, and sequencing analysis of ALV-J isolates that are circulating in broiler flocks in El-Sharqia, El-Dakahliya, and Al-Qalyubiyya Egyptian governorates through PCR technique, ELISA, and molecular sequencing approaches. Our study also involved the myelocytomatosis diagnosis in naturally infected broiler chickens with a complete pathological and immunohistochemical picture of different infected organs.

ALV-J was a great threat causing huge economic losses in the poultry industry worldwide. Notably, ALV-J spread rapidly through poultry populations, and the emergence viral new strains has been spread rapidly among different countries thus, viral eradication is very necessary for commercial breeding flocks. ALV-J Infection is challenging to control without a vaccination program, thus the only obtainable control method is flock condemnation; certainly, China has managed and controlled ALV-J infection by cautiously selecting non-infected breeders for broiler and layer industry [25, 38, 39]. Interestingly, in Egypt, ALV-J spreads quickly during 2014 throughout Egyptian poultry flocks including native and foreign breeds of layers and ducks, with high mortality rates [16, 40]. Recently, sporadic cases of ALV-J were detected in Egypt in poultry and ducks with various genetic backgrounds [19, 41].

In this study, broiler flocks in highly poultry production three governorates (El-Sharqia, El-Dakahliya, and Al-Qalyubiyya) have shown general clinical symptoms with suspicion of viral natural infections. Also, the mortality rate was recorded at about 7% while the morbidity was about 20%; with non-specific necropsy except for off-white masses on the sternum and pelvis in some investigated birds. To the best of our knowledge, no research or report has been achieved mainly on ALV-J associated with myelocytomatosis in broiler flocks, in Egypt.

According to our findings, ALV-J CPE was observed after 72 h in inoculated CER cell culture as aggregation, rounding, ballooning, degeneration, and enormous detachments of cells. These results were in line with the CPE findings of [16, 28]. Also, [42] mentioned that cytopathic ALV strains inoculated in chicken embryo fibroblast cells have given CPE and cell detachment 3 days after infection. Concerning egg inoculation, ALV-J pathological lesions on SPF-ECE were mainly stunting, curling, anomalies, and enlarged liver. These lesions may be due to the ALV-J direct effect. Embryo mortality increased as the virus passing increased until the third passage. ALV-J has been reported to cause severe hemorrhage and embryo death within 4–5 days following embryo infection. These results came in accordance with [29] At the same time, results analysis obtained from the serological survey showed that 14 farms were positive for ALV-J (77.7%). The virus identification using antigen capture ELISA revealed that only 79 serum samples were positive for ALV-J (67.52%) based on the S/P ratio. These subsequent results are in agreement with [43, 44]. In addition, these findings are nearly in agreement with [16] who reported that the positive ELISA results of collected serum samples reach to 74.2% in broiler chickens. Moreover, [11] stated that ALV-J antibody titer was significantly (P < 0.01) increased in experimentally infected SPF chicks of one-day-old from the 3rd-month post-infection (mpi) till the 5th mpi (experiment end). Interestingly, our promising results confirm the high incidence of ALV-J in the currently examined farms indicating a vertical transmission of the causative virus. We first followed up on the ALV-J infected breeder hens and then confirmed by histopathology examination (unpublished data), after that all clinical specimens were collected from affected broiler flocks. However, [45] suggested that the hatching of one-day-old egg-type chicks with ALV-J-infected meat-type chicks in the same hatchery had contributed to horizontal infection.

Despite evidence of virus growth in SPF-ECE embryo and tissue culture, PCR is the most appropriate and rapid method to detect ALV-J; providing epidemiological data of various isolates prevalent in infected flocks periodically. Analysis of PCR assay with ALV-J specific primers from (the liver, spleen, and kidney) revealed that only 43 samples were positive with a percentage of 75%. Currently, the liver and spleen show very high tropism for ALV-J according to PCR results at 91.1% and 89.4%; respectively. These results are similar to those [46] who recorded the ALV-J high tropism in the different visceral organs, especially the liver and spleen. This result indicated that these samples were collected from viremic-tolerant chickens. Parallel to our results, [11, 16] reported that all examined samples obtained from the liver, spleen, and kidney indicated a positive reaction with ALV-J at 545 bp. Also, [19] mentioned that ALV-J infection was detected in the Lower Egypt layer farms in El-Qalyubia, El-Monofia, El-Gharbia, El-Behera, and El-Daqhlia governorates. Regarding our results, [41] reported also different rates of ALV-J infections in the different governorates (Gharbia, Damietta, Sharkia, and Dakahlia) based on qRT-PCR in collected breeder chickens and duck samples. Our results indicated that ALV-J is the main etiology of viral tumors in broilers at these three El-Sharqia, El-Dakahliya, and Al-Qalyubiya governorates. Regarding previous studies, the gp85 encoding protein is highly evolved and capable of receptor-binding site, which plays a critical role in viral entry that determines the host ranges and tumor types [24, 25]. To investigate the genetic evolution of the gp85 gene in our ALV-J isolates, they were sequenced, systematically analyzed then compared to other ALV-J reference sequences. The genetic characteristics of the reported Egyptian strains (ALV-J Dakahlia-2 and ALV-J Sharqia-1) were highly similar to other ALVs. Phylogenetic analysis indicated that the ALV-J Dakahlia-2 isolate has the highest genetically related to Egyptian isolates as ALV-EGY/YA 2021.3, ALV-EGY/YA 2021.4, ALV-EGY/YA 2021.14, and ALV-EGY/YA 2021.9 with nucleotide identity percentage 100%, 97%, 96%, 96%; respectively, and on the amino acid level were with 96%, 97%; 96%, 96%; respectively. Furthermore, ALV-J Sharqia-1 isolate is highly similar to other Egyptian strains like ALV-EGY/YA 2021.14, ALV-EGY/YA 2021.9, ALV-J isolate QL1, ALV-J isolate QL4, ALV-J isolate QL3, ALV-EGY/YA 2021.4 with nucleotide identity percentage 98%, 98%, 98%, 98%, 97%; respectively, and on the amino acid level were with 97%, 97%; 98%, 97%,97%, 95%; respectively. In particular, our previous results may suggest that these prevalent ALV-J isolates might be of the same sources or have similar ancestors. This reasonably elevated detection rate might be attributed to the vertical transmission of the. ALV-J infection. Meanwhile, ALV-J Dakahlia-2 and ALV-J Sharqia-1 isolates shared 73–75% homology with the American strain (ALJ-10022-2). Our findings are not in agreement with [47] who reported that the gp85 of PK19SA01 shares 95.5% identity with the American strain. Also, ALV-J Dakahlia-2 and ALV-J Sharqia-1 isolates shared 91% -93% similarity with the American reference strain (ALJ-ADOL-7501). These subsequent results are not in agreement with [19] who mentioned that the Egyptian ALV-J isolates were similar to HPRS-1003 (prototype strain) with an identity percentage of 91.2–91.8%. Importantly, we can speculate that our Egyptian ALV-J strains might be introduced from America through chicken breeding. On the other hand, ALV-J isolates were distinctly apparent from Chinse isolates with 71% and 73% identity. These results are not parallel to [19] who stated that the nucleotide identity percentage of their Egyptian isolates was within the range of 88–94% when compared to Chinese reference strains.

The core region of the gp85 gene contains five variable regions (hr1, hr2, vr1, vr2, and vr3) [48]. These regions (hr1, hr2, and vr3) are ALV-J receptor interaction determinants [49]. Previous studies revealed that the gp85 gene tends to mutate as a result of immune pressure, causing a lot of changes in antigenic properties and virulence [20]. In the present study, no evidence of variations or amino acid mutations of our isolates in the gp85 gene were detected in the putative variable regions, vr2. These current findings are similar to those [19] who recorded that no changes existed in the vr2 domain of the gp85 gene of their isolates at the layer farms. In contrast, these results are not in agreement with [41] who reported 25 true SNPs among the five strains of chicken and duck breeders, in which only 5 SNPs lead to amino acid mutation. These amino acid substitutions might cause variations in the pathogenicity, oncogenicity, and ALV-J host range. Finally, our findings act as a warning that the ALV-J eradication is not disposable, so continuous monitoring is essential.

The histopathological picture besides the molecular characterization gave an accurate diagnosis for ALV-J. ALV-J could induce malignant or benign tumorigenic diseases and immunosuppressive responses in poultry such as hemangiomas, myelomas, and myelocytomatosis. Tumor development is a multi-step process representing the abnormal expression of an apoptotic gene, inactivation of tumor suppressor genes, or activation of proto-oncogenes [50]. Myelocytomatosis causes high economic losses in white meat-type breeder farms [51]. Furthermore, myelocytomatosis is a tumor disease in which tumor progress is a process that is complicated and related to many factors as genetic background, immune competence, and viral infection factors. The process of promoting carcinogenesis is still unknown, but the integration of the myelocytomatosis provirus may interfere with the function of the host endogenous gene [52, 53].

The immune suppression due to myelocytomatosis may involve atrophy of lymphoid organs, decreased mitogen-induced blastogenesis, and decreased antibody response [54]. Interestingly, the immune system alteration occurs as a result of cessation of B cell maturation in addition to a blockage in the development of T-suppressor cells, probably due to hindrance with functional IL-2 synthesis [55, 56] showed that ALV-J could induce lymphocyte apoptosis in immune organs, particularly in young chickens. Lymphocyte death increases susceptibility to other diseases.

Myelocytomas have a characteristic gross appearance in which myelocytes proliferate and soon overgrow the bone marrow. Tumors are formed by the expansion of marrow growth and may crowd through the bone and periosteum. They occur frequently on the surface of bone such as the costochondral junctions of the ribs and on the sternum and pelvis, these aforementioned lesions agree with [46, 57].

Histopathological evaluation of the liver concluded the presence of myeloid and lymphoid. The features of the liver lesions were aggregations of mature granulated myeloid cells. The neoplastic cells replaced the hepatocytes with relative atrophy of the surrounding cells. The same findings were recorded by [11, 37]. Meanwhile, focal aggregations of lymphoblastic cells were detected in the hepatocyte. Furthermore, In the liver, the brown granules resembled a positive reaction for specific viral particles present in the Kupffer cells and lymphocytes as well as erythroblasts; this supports the previous findings which were also confirmed by detecting the virus from liver tissues using PCR. These results agree with [5, 41, 53, 58, 59].

In contrast, the microscopic picture of spleen sections showed that ererea a pleomorphic lymphoid population surrounding arteriole. By Giemsa stain, myeloid cells appear obviously with their eosinophilic granules. Viral antigen was greatest in the splenic trabeculae, subcapsular sinuses, and lymphoid follicles by IHC. These results were supported by [37] who recorded ALV-J positive signals in the erythroblast cytoplasm, spleen, lung, and other tissues especially rich in blood.

The microscopic examination of the kidney revealed multifocal neoplastic aggregations of granular myeloid cells. The tumor cells were aggregated between the degenerated renal tubules and around the congested blood vessels. Granulated myeloid cells were seen infiltrating the interstitial tissue causing pressure atrophy and loss of some renal tubules when stained with Giemsa stain, that result was confirmed by [41, 58, 60, 61].

The proliferation of granulated myelocytes was detected in the bone marrow and the periosteum of the sternum. Proliferation begins in the bone marrow of epiphysis. The Myelocytes invaded from the bone marrow to periosteal areas through Haversian and Volkmann’s canals. Myelocyte proliferation was also detected in the bone marrow. Additionally, there is a wall thickening and lumen narrowing of the sternal bone together with dentation in the periosteum which is considered as osteopetrosis. Finally, ALV-J tropism for chicken bone marrow cells, and induces their neoplastic transformation [62]. This finding agrees with that recorded by [11, 63, 64].

We first isolated two ALV-J strains associated with myelocytomatosis from broiler flocks, in Egypt. In summary, the circulating ALV-J infection associated with myelocytomatosis during 2023 in broiler flocks at different localities of Egypt was diagnosed through PCR technique, serological assay, molecular sequencing approaches, pathological, and immunohistochemical examinations. ALV-J causes neoplastic diseases in broiler flocks, with the highest rate of infection presented in these governorates as El-Sharqia, El-Dakahliya, and Al-Qalyubiyya. In addition, the ALV-J gp85 gene evolution of our isolate (Dakahlia-2, identified as subgroup II) have the highest genetically related to ALV-EGY/YA 2021.3, ALV-EGY/YA 2021.4, and ALV-EGY/YA 2021.14 with nucleotide identity percentage 100%, 97%, 96%; respectively, and on the amino acid level were with 96%, 97%; 96%; respectively. Moreover, ALV-J Sharqia-1 isolate is highly similar to ALV-EGY/YA 2021.14, ALV-EGY/YA 2021.9, and ALV-J isolate QL1 with nucleotide identity percentage of 98%, and on the amino acid level were with 97%, 97%; 98%; respectively. Our Egyptian ALV-J isolates (ALV-J Dakahlia-2 and ALV-J Sharqia-1) were submitted to Genbank with accession numbers (OR509852–OR509853). The phylogenetic analysis based on the nucleotide and deduced amino acid sequences of the gp85 gene showed no evidence of variations or amino acid mutations in the putative variable domains, vr2. Currently, no vaccinations or treatments for ALV are presented and such ALV still threatens the local poultry industry. This reminds us to eradicate the positive cases, strengthen the breeder introduction detection, and apply periodic molecular monitoring for all recent Egyptian strains. Furthermore, the whole genome sequencing of these isolates is recommended to detect both the pathogenicity and antigenicity of these circulating ALV-J strains.