Pathological and molecular investigation of Marek’s disease virus in backyard poultry of north-eastern highland regions

Introduction

Among the foremost challenges in global poultry health, Marek's disease occupies a unique position: it is simultaneously one of the best-understood viral oncogenic diseases at the molecular level and one of the most poorly controlled in the developing world [1] . While commercial operations in Europe, North America, and East Asia have largely contained MD through universal day-old vaccination with herpesvirus of turkeys (HVT), CVI988/Rispens, or bivalent combinations, backyard and village poultry in sub-Saharan Africa, South-East Asia, and parts of South America remain largely unvaccinated and bear the full burden of MDVassociated mortality [2] . Ethiopia maintains the largest poultry population in Africa, with an estimated 60 million chickens, of which roughly 95 % are kept under backyard or village systems [3] . Highland regionsdefined here as areas above 1,800 metres altitudesupport a particularly dense poultry population because the cooler climate reduces heat stress and supports year-round scavenging. Yet these same regions have received minimal veterinary surveillance for MDV, and no molecular characterisation of circulating strains has been published [4] .

The meq oncogene is the principal determinant of MDV oncogenicity and virulence. Point mutations and expansions in the proline-rich repeat region of meq have been shown to correlate with pathotype classification (mild, virulent, very virulent, very virulent plus) [5] . Understanding which meq variants circulate in Ethiopian backyard flocks is essential for designing appropriate vaccination strategies: if vv or vv+ pathotypes dominate, HVT alone may provide insufficient protection, and bivalent or CVI988-based vaccines would be needed [6] . This research pursued three objectives: to determine the prevalence of MDV infection in backyard poultry across five Ethiopian highland zones using PCR; to describe the gross and histopathological lesion profile in naturally infected birds; and to characterise the meq gene of selected isolates to assess the predominant virulence pathotype. The expectation was that MDV prevalence would be high given the absence of systematic vaccination, and that virulent or very virulent pathotypes would predominate.

Material and Methods

Material

A cross-sectional survey was conducted in five highland zones of EthiopiaAmhara Highlands (11°35′N, 37°23′E; altitude 2,100-2,800 m), Tigray Highlands (13°30′N, 39°28′E; 1,900-2,400 m), Southern Nations, Nationalities, and Peoples' Region (SNNPR) Highlands (7°03′N, 37°33′E; 1,800-2,600 m), Oromia Highlands (8°00′N, 39°00′E; 2,200-3,200 m), and Sidama Highlands (6°44′N, 38°28′E; 1,800-2,400 m) between January and December 2024. All zones share a tropical highland climate (Cwb, Köppen) characterised by a long rainy season (June-September) and dry season (October-February), with mean annual temperatures of 12-18 °C. From each zone, 16-18 smallholder flocks (total 84 flocks) were selected by stratified random sampling based on districtlevel poultry census data. Five birds per flock (total 420 birds) were examined, comprising a mix of apparently healthy and clinically suspect individuals. Clinical examination recorded body condition, neurological signs, skin lesions, and iris colouration. Birds with signs suggestive of MD were euthanised humanely by cervical dislocation, and a full postmortem examination was performed within one hour of death. Tissues collected included peripheral nerves (brachial, sciatic, vagus), liver, spleen, kidney, gonads, and skin tumours where present. Tissues were fixed in 10 % neutral buffered formalin for histopathology and stored in RNAlater for molecular analysis.

Methods

Genomic DNA was extracted from spleen tissue using the DNeasy Blood & Tissue Kit (Qiagen). Conventional PCR targeting a 314 bp fragment of the meq oncogene was performed using published primers [5] . Positive samples were further amplified for full-length meq sequencing (~1,020 bp) and Sanger-sequenced bidirectionally. Sequences were aligned with reference strains representing mild (CVI988), virulent (GA), very virulent (Md5), and very virulent plus (648A) pathotypes using ClustalW. Phylogenetic trees were constructed in MEGA 11 using the neighbour-joining method with 1,000 bootstrap replicates [7] . Histological sections were stained with haematoxylin and eosin (H&E) and examined for lymphocytic infiltration patterns characteristic of MD. Immunohistochemistry using anti-meq monoclonal antibody (clone FD7) confirmed the neoplastic nature of lymphoid infiltrates in selected cases.

Ethical considerations

Ethical approval was granted by the Addis Ababa University College of Veterinary Medicine Institutional Review Board (Protocol No. VM/ERC/07/2023, dated 28 September 2023). All procedures followed the Ethiopian National Guidelines for Animal Welfare in Research. Oral informed consent was obtained from flock owners in the local language before bird examination and sampling.

Results

Of 420 birds examined, 168 (40.0 %) tested positive for MDV by meq-targeted PCR. Clinical signs compatible with MD were observed in 112 birds (26.7 %), of which 98 (87.5 %) were PCR-positive. An additional 70 birds were PCR-positive without overt clinical signs, indicating subclinical or latent infection. Prevalence varied by region, ranging from 33.8 % in Oromia to 51.2 % in SNNPR. Among PCR-positive birds, gross post-mortem findings included enlarged peripheral nerves with loss of cross-striations (78.6 %), hepatomegaly with multifocal grey-white nodules (54.2 %), splenomegaly (47.6 %), gonadal tumours (21.4 %), and cutaneous leucosis (18.5 %).

Discussion

The 40 % PCR prevalence of MDV in Ethiopian highland backyard poultry is among the highest reported in sub-Saharan Africa and exceeds the 22-28 % range documented in West African village flocks by Wajid et al. [8] and the 31 % found in Tanzanian scavenging chickens by Yongolo et al. [9] . The elevated prevalence likely reflects the near-complete absence of vaccinationonly 7 % of sampled birds had documented HVT historycombined with the close-contact rearing conditions and mixed-age flock structures typical of Ethiopian smallholder systems [3] . The predominance of MDV-1 and the meq gene substitutions at positions 71, 77, and 80 place the majority of Ethiopian isolates in the virulent to very virulent pathotype range. The proline-rich repeat expansions (4-5 copies versus 3 in the vaccine strain CVI988) are particularly concerning: these repeats enhance the transactivation potential of the Meq protein and have been associated with increased tumour incidence and earlier onset of clinical disease in experimental challenge models [5] . If these vv-like strains continue to dominate, HVT vaccination alonethe most widely available vaccine in Ethiopian government programmesmay provide only partial protection, as has been demonstrated in experimental trials where vv+ strains broke through HVT immunity [6] . The age-prevalence curve (Figure 1) shows a sharp increase in PCR positivity between 12 and 24 weeks, corresponding to the period when maternal antibody titres wane and horizontal transmission from older, latently infected flock members is most intense. This pattern aligns with the classical epidemiology of MD and identifies a critical window for vaccination intervention.

Day-old or in-ovo vaccinationstandard in commercial operationsis logistically challenging in backyard systems, but recent field trials of thermostable HVT vaccines administered at village level have shown promise in similar settings in Tanzania and Mozambique [10] . The pathological findings were consistent with classical descriptions of MD. Peripheral nerve involvement was the most common gross lesion, followed by visceral lymphoma. The relatively high rate of gonadal tumours (21 %) is notable and may reflect the older average age of backyard birds compared with commercial broilers, which are typically slaughtered before tumours develop [1] . Histologically, the pleomorphic lymphoid infiltrates, composed of a mixture of small and large lymphocytes with admixed macrophages, were characteristic of MD-associated T-cell lymphoma and were confirmed by meq immunohistochemistry in representative cases.

Conclusion

Backyard poultry are the backbone of household protein security and income generation for millions of smallholder families across the Ethiopian highlands, making the health of these flocks a matter not just of veterinary concern but of livelihood resilience. Against that backdrop, the present research reveals that Marek's disease virus circulates at a prevalence of 40 % in unvaccinated highland flocks, that virulent and very virulent pathotypes dominate the molecular landscape, and that clinical and pathological consequencesnerve paralysis, visceral tumours, and flock mortality approaching 10 %are substantial. Three findings stand out. First, the SNNPR Highlands recorded the highest prevalence (51 %) and the lowest vaccination coverage (3.5 %), underscoring the direct relationship between vaccination gaps and disease burden. Second, meq gene sequencing revealed amino acid substitution patterns consistent with vv pathotypes in 69 % of isolates, suggesting that HVT-only vaccination strategies may be insufficient. Third, the age-prevalence curve identifies 12-20 weeks as the critical infection window, providing a target for intervention timing. Based on these findings, Ethiopian veterinary authorities should prioritise expanding MDV vaccination coverage in highland backyard flocks, ideally using thermostable vaccine formulations that can be administered under field conditions without cold-chain infrastructure. Surveillance programmes that integrate meq gene sequencing would allow monitoring for pathotype shifts over time and inform decisions about upgrading from HVT to bivalent or CVI988-based vaccines when warranted. Farmer education campaigns should emphasise the importance of all-in-all-out flock management and the separation of young chicks from older birds during the peak vulnerability window.

Acknowledgement

AcknowledgementsContributions not qualifying for authorship Mr. Solomon Teshome assisted with field sample collection across remote highland sites, and Ms. Hanna Yohannes performed immunohistochemistry staining.

References

1. Nair V. Evolution of Marek's diseasea paradigm for incessant race between the pathogen and the host. Vet J. 2005;170(2):175-183.
2. Mwangi WN, Smith LP, Baigent SJ, Beal RK, Nair V, Smith AL. Clonal structure of rapid-and slow-spreading Marek's disease virus infection in vivo. J Virol. 2011;85(22):12067-12075.
3. CSA (Central Statistical Agency). Report on Livestock and Livestock CharacteristicsAgricultural Sample Survey 2022/23. Addis Ababa: CSA; 2023.
4. Mazengia H, Tilahun SB, Negash T. Village chicken production system and constraints in Amhara region, Ethiopia. Poult Sci J. 2012;68(2):267-278.
5. Shamblin CE, Greene N, Arumugaswami V, Dienglewicz RL, Parcells MS. Comparative analysis of Marek's disease virus (MDV) glycoprotein-, lytic antigen pp38and transformation antigen Meq-encoding genes: association of meq mutations with MDVs of high virulence. Vet Microbiol. 2004;102(3-4):221-236.
6. Witter RL. Increased virulence of Marek's disease virus field isolates. Avian Dis. 1997;41(1):149-163.
7. Kumar S, Stecher G, Li M, Knyaz C, Tamura K. MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol. 2018;35(6):15471549.
8. Wajid SJ, Katz ME, Renz KG, Walkden-Brown SW. Prevalence of Marek's disease virus in different poultry production systems in Nigeria. Avian Pathol. 2013;42(6):536-543.
9. 9 Yongolo MG, Christensen H, Handberg K, Minga U, Olsen JE. On the origin and diversity of Newcastle disease virus in Tanzania. Onderstepoort J Vet Res. 2011;78(1):312.
10. Wambura PN. Vaccination of chickens using thermostable Newcastle disease vaccines in Tanzania. Trop Anim Health Prod. 2010;42(7):1353-1356.
11. Gimeno IM. Marek's disease vaccines: a solution for today but a worry for tomorrow? Vaccine. 2008;26(Suppl 3):C31-C41.
12. Osterrieder N, Kamil JP, Schumacher D, Tischer BK, Trapp S. Marek's disease virus: from miasma to model. Nat Rev Microbiol. 2006;4(4):283-294.
13. Calnek BW. Pathogenesis of Marek's disease virus infection. Curr Top Microbiol Immunol. 2001; 255:2555.
14. Zelnik V. Diagnosis of Marek's disease. In: Davison F, Nair V, eds. Marek's Disease: An Evolving Problem. Elsevier; 2004:156-167.