Introduction
The quest to satisfy the ever-rising demand for protein food has led to the intensification and industrialization of poultry farming. Despite improving productivity of the sector, this also lend to a situation highly conducive to pathogen evolution as a result of cramped living conditions and shorter rearing periods. A typical example of this is marek’s disease virus (MDV), which is a pathogen of poultry that has evolved from a relatively harmless paralytic syndrome into a highly virulent pathogen [
1] as a result of industrialization [
2,
3].
Marek’s disease (MD) is a highly contagious, immunosuppressive [
4], and lymphoproliferative disease of chickens [
5] with a mortality rate reaching 100% [
1,
6]. The causative agent, Gallid alphaherpes virus 2 (GaHV-2), is a dsDNA virus belonging to the genus
Mardivirus, subfamily
Alphaherpesvirinae, and family Herpesviridae [
7]. Within the genus
Mardivirus, are three closely related but distinct virus species. Gallid alphaherpes virus 2 (formerly serotype 1 MDV, MDV-1), consists of all known pathogenic strains, which vary in their pathogenic and oncogenic potential and being classified as mild (m), virulent (v), very virulent (vv), and very virulent plus (vv +) GaHV-2 [
8,
9]. Gallid alphaherpes virus 3 (GaHV-3, formerly serotype 2 MDV), and Meleagrid alphaherpes virus 1 (MeHV-1, also called herpes virus of turkey (HVT) and formerly serotype 3 MDV) are avirulent strains isolated from chickens and turkey respectively [
7].
The disease is characterized by general inflammation of peripheral nerves (polyneuritis) and development of solid tumors in multiple organs that originate from transformed T lymphocytes [
10‐
12]. Two forms of the disease are well recognized, neural and visceral types [
13], with 10–25% and above 70% mortality, respectively. In neural MD, the main clinical symptoms include a complete or partial paralysis of the neck, wings, and limbs. Such paralyses are mainly induced by lesions of the vagus, brachial, and sciatic plexuses that show enlargement and yellowish color on the surface. In visceral MD, the gross tumors can be observed in the gonads, liver, kidney, lung, heart, spleen, and proventriculus in larger sizes and higher numbers [
14]. At necropsy, MD gross lesions are characterized by diffuse enlargement of the liver and the spleen, presence of lymphomas in liver, kidney, ovary, proventriculus, spleen, lungs, nerves, heart, skin, and atrophy of the bursa of Fabricius and thymus [
15].
Diagnosis is based on isolation and identification of MDV from infected tissues. Virus isolation is usually by virus propagation in cell culture and identification/quantification by cytopathic changes (plaque formation) or identification of the infected cells by immunostaining [
16]. Several PCR and real-time-based techniques have been developed for detection, as well as quantification, of the MDV genome of field and vaccine strains from blood, organ samples, and feather tips [
17]. In addition, loop-mediated isothermal amplification (LAMP), has been presented as attractive alternative to the PCR-based methods. LAMP is a rapid technique that can be performed at a single temperature (60 °C to 65 °C) in a laboratory water bath or a dry heat block, and the results can be read with the naked eye [
18].
Vaccination against MD denotes one of the most successful examples of protection against virally induced tumor. All the currently used vaccines are live vaccines derived from the three viral strains: the HVT FC126 strain [
19], the GaHV-3 SB-1 strain [
20], and the GaHV-2 CVI988/Rispens strain [
21]. HVT and SB-1 vaccines are considered heterologous vaccines as they are derived from a different viral species than the target virus, while the Rispens vaccine is considered homologous because it is from the same viral species as the targeted virus.
Currently, MD has a global distribution with increasing reports of vaccination breaks and the emergence of more virulent pathotypes [
22,
23] and is responsible for a massive economic burden [
24]. Outbreaks of MD have been reported in different parts of Ethiopia. Lobago and Woldemeskel (2004) [
25]have reported MD in a commercial poultry farm in Central Ethiopia on clinico-pathological criteria. Similarly, Duguma et al. (2005) [
26] conducted serological and clinico-pathological investigation reporting a higher (97.9%) mortality of chickens due to MD. However, the first genetic confirmation of MDV-1 was reported by Demeke et al. (2017) [
27]from commercial farms in Central Ethiopia. Furthermore, MDV has been isolated from outbreaks in different zones of Southwestern Ethiopia [
28]. It is becoming clear that farms in the country need to consider MD prevention and control plans to avoid the drastic consequences of the disease. Despite the occurrences of disease outbreaks suggestive of MD in chickens in Northwest Ethiopia, there are no genetic evidences and official reports on the presence and circulation of MDV in the region. In this study we conducted a clinico-pathological and molecular investigation in MD-suspected outbreaks in commercial poultry farms in Northwest Ethiopia. Thus, this study provides the first molecular evidence of MDV-1 in chicken flocks in Northwest Ethiopia.
Discussion
In the present, outbreak-based investigation, gross pathological examination was conducted on chickens suspected of being morbid and dead of MDV infection. marek’s disease virus was isolated from tissue samples, PCR confirmed, and representative samples sequenced. This is the first confirmatory report of MDV in chicken farms located at different agro-ecological zones and production systems in Northwestern Ethiopia. Thus, the results presented here contribute to the accumulating knowledge of MD in country.
In this study, the clinical signs shown by affected chickens such as depression, shrunken combs, and paralysis of leg, wing, and neck, are consistent with literature description of MD in chickens reported elsewhere [
13]. Upon postmortem examination of dead and sacrificed chickens, tumor-like nodular lesions of various size were widely observed in visceral organs. In addition, gross abnormalities such as splenomegaly, hepatomegaly, renomegaly, and sciatic nerve enlargement were recorded which were concordant with reports of other studies [
14,
15].
Diagnosis of MD is usually based on isolation and identification of MDV in cell culture and identification by cytopathic changes (plaque formation) [
16]. In this study, pooled spleen and feather follicle samples were targeted for virus isolation. Of the 22 samples considered positive for MDV isolation, 17 (85%) were from pooled feather samples. In fact, it has been reported that the epithelium of feather follicles is the tissue most commonly found positive in infected chickens, compared to other tissues [
35,
36].
Characteristic CPEs such as rounding of cells, formation of foci area and syncytia that detached later from the wall of the cell culture flask were observed on CEF cell cultures. In line with this, other authors [
27,
28] reported similar cytopathic changes on CEF upon inoculation with MD suspected tissue samples. However, CEF is permissible for other poultry herpesviruses [
37,
38] that can induce similar or comparable cytopathic changes. Therefore, the identity of MDV was confirmed using a conventional PCR, amplifying 318 bp of the
ICP4 gene of MDV-1. Of the 22 pooled samples, 9 (33.33%) were found PCR-positive. Similarly, several other authors have targeted the
ICP4 gene for genetic confirmation of MDV-1 [
17,
27,
39,
40]. Amplification of the conserved
ICP4 GaHV-2 gene is expected to be more reliable as the gene is specific for pathogenic MDV, MDV-1.
In order to have a better understanding of the epidemiologic situation,
ICP4 genes from 5 samples that vary by farm and study site were partially sequenced. Phylogenetic tree was re-constructed using
ICP4 partial gene sequences of the current study and homologous sequences retrieved from GenBank. Two of the isolates from the same site, Metema (GenBank accession numbers: OP485106 and OP485107) seem to be clonal complexes forming distinct cluster (bootstrap value: 73.2). The other three isolates, two from Merawi (GenBank accession numbers: OP485108 and OP485109) and one isolate from Debretabor (GenBank accession number: OP485110) seem to represent distinct genotypes although the isolate from Debretabor is closer to the Metema clonal complex (bootstrap value: 86.8). On the other hand, the isolates from Merawi appeared genetically far related to the rest of the 3 isolates and clustered with Indian MDV strains included in the analysis (bootstrap value: 59.4). Moreover, none of the isolates from the current study clustered with strains from central Ethiopia, indicating the high diversity of the virus in the country. This is consistent with previous reports of the continuous evolution of field MDV strains [
30].
As mentioned before, there are 4 pathotypes of the serotype 1 MDV, mild (m), virulent (v), very virulent (vv), and very virulent + (vv +) [
1]. In addition to their association with distinct virulence and subsequent losses in commercial chicken flocks, each pathotype appeared to encode various level of resistance against defined MD vaccines. For example, v pathotype strains induced high levels of disease in non-vaccinated chickens, but little disease in chickens vaccinated with HVT. In contrast, vv pathotype strains induced high levels of disease in HVT-vaccinated chickens, but little disease in chickens vaccinated with bivalent vaccines composed of HVT and selected serotype 2 strains such as SB-1 or 301B/1 [
9]. Therefore, pathotyping would have a practical significance when it comes to vaccination-based control of MD. However, as a limitation, in our study, we did not conduct molecular analysis of genes that have profound impact on virulence and enable pathotyping of MDV, such as marek’s EcoRI-Q (
MEQ), phosphoprotein-38 (
pp38) and viral interleukin 8 (
vIL-8) [
41,
42], neither did we perform pathotypic classification of the isolates using the Avian Disease and Oncology Laboratory (ADOL) method.
Based on the comparative phylogenetic analysis, it can be speculated that the virus is introduced to the country through importation of vaccines, chickens’ eggs or live chickens. Despite the true sources of the virus, it has become clear that MDV is circulating in chicken farms in Northwest Ethiopia. The current live chicken market and the extensive movement of chickens within and across the region coupled with lack of MD preventive measures, is expected to favor the spread of the virus which will result in a severe consequence.
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