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MHC-B haplotypes impact susceptibility and resistance to RSV-A infection

Vishwa M. Khare1,*,Vishesh K. Saxena2,Alka Tomar3,K.P. Singh4,Kunwar B. Singh5,Amit K. Tiwari6
1
Cell and Developmental Biology, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
2
Disease Genetics and Biotechnology Lab, CARI, Izatnagar, UP-243 122, India
3
Tumor Immunology Lab, IVRI, Izatnagar, UP-243 122, India
4
CADRAD, IVRI, Izatnagar, UP-243 122, India
5
Animal Science Department, Rohilkhand University, Bareilly (UP), India
6
Department of Pharmacology and Experimental Therapeutics, The University of Toledo, Toledo, OH 43614, USA
DOI: 10.2741/E837 Volume 10 Issue 3, pp.506-519
Published: 01 June 2018
(This article belongs to the Special Issue Cell death mechanisms)
*Corresponding Author(s):  
Vishwa M. Khare
E-mail:  
khare_vm@yahoo.com
Abstract

We investigated the impact of haplotype of major histocompatibility complex (MHC)-B on the outcome of infection of Synthetic Dam Line (SDL) broiler strain with Rous Sarcoma Virus (RSV). Genomic analysis of MHC-B haplotypes, revealed a total of 12 known standard haplotypes that constituted to twenty-five different genotypes and one new haplotype of 217 bp size, designated BX. The inoculation of RSV-A in SDL chicks resulted in the development of tumors of progressive or regressive phenotypes with varying tumor profile index (TPI). Haplotypes B2, B21 and B22 had low TPI scores (1 or 2) with less mortality and were resistant to RSV-A tumor. The haplotypes B13, B13.1., B15, B15.1. and B15.2. had significantly higher TPI scores (5 or 6), indicating a susceptibility to RSV-A. The genotype, Bx /Bx, had a mean TPI score of 3.67 ± 1.33, which was closer to the resistant haplotype. Sequence analysis of the new haplotype (BX) revealed 99.5% similarity with B2 haplotype. Metastases was observed in 44% of chicks and comprised of mixed fibrosarcoma and myxosarcoma.

Key words

Major histocompatibility complex, Rous sarcoma virus–A, synthetic dam line, tumors, susceptibility, resistance, metastasis

2. Introduction

Avian leucosis or lymphoid leucosis is the most common form of neoplastic diseases observed in chicken and other avian species (1). Most of the neoplastic diseases have a viral etiology and are caused by avian retroviruses of genus Alpharetrovirus of Retroviridae family (2). These diseases cause significant economic losses in commercial layers and breeder flocks due to tumor mortality and other reproductive problems such as delayed sexual maturity, low fertility and hatchability and drop in egg production (3). Moreover, avian leucosis virus (ALV) is present in commercial chickens and eggs, thus exposing human beings on a consistent basis. Till date, since no commercial vaccines are available for control of ALV infection, improving the genetic attribute to disease resistance is the best approach for sustainable control of ALV infectious diseases in poultry.

Genetic resistance to diseases is a multigenic trait controlled primarily by the immune system and their interactions with physiological and environmental factors (4). In order to develop genetically resistant breeds, it is essential to recognize the immunological factors that are responsible for differences in natural resistance (5). The diversity of the major histocompatibility complex (MHC), T cell receptors (TcR) and immunoglobulins (B cell receptors) constitute the primary immunological basis for variations in disease resistance (4). In chickens, the association between MHC polymorphisms and susceptibility to infectious pathogens is well established (6, 7). Indeed, MHC protein polymorphisms have been shown to have a significant positive correlation with resistance or susceptibility to numerous autoimmune, viral, bacterial, and parasitic diseases, including avian leucosis (8) and Rous sarcoma virus (RSV) tumors (9, 10).

Disease resistance studies in Rous sarcoma virus have allowed new findings on related mechanisms and the genes involved (10). Analysis of inbred lines, their crosses, congenic lines, and noninbred populations have revealed the anti-RSV response of many B complex haplotypes (10). Particular MHC genotypes determine RSV tumor regression or progression and based upon degree of tumor growth, a tumor profile index (TPI) was assigned (11).

MHC haplotypes are being determined from genomic DNA by PCR using a polymorphic microsatellite marker LEI0258 and MCW0371 (12), which is located within the MHC region on chromosome 16 (13) between BG and BF region or sequence-based typing (SBT) of the MHC-B locus class I α chain 2 (BF2) allele (14). Recently, Suzuki (15) investigated the relationship between MHC-B haplotypes and response to RSV-J in an F2 commercial chicken family by microsatellite marker LEI0258, BF2 SBT and an additional 35 SNP to discriminate more precisely between regression and progression phenotypes.

The variation in the resistance/ susceptibility of high yielding synthetic dam line (SDL) a broiler strain of India to RSV-A has not been studied so far. Therefore, this study was designed to explore the prevailing MHC haplotypes/ genotypes in the Synthetic Dam Line (SDL) broiler strain and their association with resistance and susceptibility to RSV-A. The MHC haplotypes of the SDL chicks were determined using genomic DNA as a template before challenging the chicks with RSV-A. Genotyping was accomplished using the PCR microsatellite markers LEI0258 and MCW0371 located within the MHC region.

The information on MHC haplotypes of the Indian native SDL broiler to Rous sarcoma virus will help in identifying chickens with susceptibility and resistance phenotypes and in their conservation through targeted strategies for control of tumor outcome.

3. Materials and methods

3.1. Virus

Bryan Standard strain of Rous sarcoma virus (Rous associated virus-1) (BS-RSV (RAV-1)), henceforth to be referred as RSV-A, was used for the present study.

Freeze dried ampoules of RSV-A (Rous sarcoma virus) obtained from Tumor Immunology Lab, IVRI, Izatnagar were used for the fresh preparation of RSV. Each freeze dried ampoule was reconstituted in 0.5 ml of Normal saline solution (NSS). 9 to 11 day old embryonated chick eggs procured from Experimental Broiler Farm, CARI, Izatnagar were utilized to determine titer of Rous Sarcoma Virus (RSV) by chorioallantoic membrane (CAM) assay as per the method of Groupe et al. (16). Infectivity titer of the virus was found to be 1x103 pock forming units (p.f.u.) / ml. The virus was handled under biosafety level 2 facilities.

3.2.Chicks and management practices

The protocols involving the care and use of animals for these experiments were in accordance with the with the rules of the Animal Ethics Monitoring Committee of the Institute, Government of India.

One day old chicks, belonging to white-plumaged Synthetic Broiler Dam Line (SDL), were obtained from the Experimental Broiler Farm, Central Avian Research Institute (CARI), Izatnagar Uttar Pradesh (UP), India. The chicks were shifted to challenge sheds at the Indian Veterinary Research Institute (IVRI), Izatnagar, UP, India, where they were infected with Rous sarcoma virus (RSV-A). The birds were housed in battery brooders and were maintained under standard management and nutrition. The chicks were brooded under controlled warm conditions and were fed ad lib starter ration (upto 3 week) and finisher ration thereafter ( upto 5 week). They had free access to feed and water. Individual chicks were wing banded for identification. A group of uninfected control chicks was simultaneously maintained in the same facility.

3.3. MHC haplotyping

We determined the MHC haplotypes of 80 SDL chicks using PCR-based genotyping of microsatellite sequences within the MHC region, using the method of Fulton et al. (12). We used genomic DNA from pre-challenged chicks with RSV-A, as a template and LEI0258 as a microsatellite marker. MHC haplotypes that could not be distinguished using the LEI0258 microsatellite alone were distinguished by the MCW0371 marker. The details of primer sequences used for haplotyping are given in Table 1.

Table 1. Primers For MHC-B Haplotyping
S. No.PrimersSequencesAccession No.Allele size range
1LEI0258(F) CAC GCA GCA GAA CTT GGT AAGG
(R) AGC TGT GCT CAG TCC TCA GTGC
AL023516182-552 bp
2MCW0371(F) CTG CTC CGA GCT GTA ATC CTG
(R) TTT CAT GGC ATC CTA AGA TG
AL023516200-209 bp
The PCR products of individual DNA samples were resolved on a 4% MetaPhor agarose gel.

3.4. Tumor induction

One week old SDL broiler chicks were subcutaneously infected with Rous Sarcoma Virus (2000 p.f.u/0.2 mL of RSV-A suspension per chick) in the left wing-web and reared in the Challenge Sheds, IVRI, Izatnagar under contained facilities. The droppings, dead and necropsied birds, and other discards were disposed through incineration. Chicks were observed regularly for the appearance of tumors at the primary site, as well as in other organs. Uninfected control birds were maintained separately, observed daily, and sacrificed by cervical dislocation at the end of the experiment.

3.5. Categorization of chicks

The growth pattern of the primary tumors induced by Rous sarcoma virus was observed every day, and chicks were categorized according to the magnitude of the growth of the tumor. The scoring was done to assign a tumor profile index (TPI) (17) using the following: TPI-1= no palpable tumor; TPI-2= complete regression by 42 or 56 days; TPI-3= complete regression by 70 days, a decreasing slope, or complete regression by 56 days followed by recurrence; TPI-4= tumor covering > half the wing web area, but < entire wing web; TPI-5= tumor filling the entire wing web; or TPI-6 = massive tumor extending beyond the wing web and metastasizing to other organs. Chicks with a TPI score of 1 were deemed non-responders, 2 or 3 were regressors and 4, 5 and 6 were progressors.

3.6. Gross pathological examinations

The growth pattern of the primary tumors at the wing web that was induced by Rous sarcoma virus was assessed by measuring the volume of primary tumor with the help of vernier calipers every day. Dead chicks were necropsied for gross pathological findings in different organs of the body and the site of gross lesions were recorded.

3.7. Histopathological examinations

3.7.1. Collection of samples for histopathology

The experimental chicks were sacrificed by cervical dislocation and tissue samples viz., primary tumor tissue, lung, liver, spleen, heart, kidney, neck and leg muscle were collected from progressor birds and fixed in 10% neutral buffered formalin solution for histopathological studies.

3.7.2. Histopathological procedure

Tissue pieces were microsectioned into 2-3 mm and washed overnight with running water. The samples were then dehydrated with ascending grades of ethanol starting from 70% ethanol, to 80%, 90% and 95%, absolute ethanol for 1 hour at each grade. Following dehydration, the tissues were cleared in three changes of xylene for 1 hour for each change. The tissue pieces were embedded in paraffin and sections were cut at 4 microns. These microsections were fixed on slides and the sections were stained with hematoxylin and eosin (H&E).

3.7.3. Hematoxylin and Eosin (H & E) staining procedure

Sections were deparafinized in three changes of Xylene for 10 min. for each change. The sections were rehydrated in descending grades of ethanol (90%, 80%, 70%) and finally brought to water. The sections were stained with H & E stains and finally mounted in DPX (Distyrene Plasticizer Xylene). Sections were examined under compound microscope.

3.8. Statistical analysis

3.8.1. Effects of MHC-genotypes on tumor profile index (TPI)

The fixed effect of MHC genotypes on TPI was evaluated using SPSS version 16.0. The MHC genotypes were coded and analyzed using following model:

Yij = µ + Gi + eij

Where,

Yij=TPI under ith genotype

µ =Overall mean

Gi=Effect of ith MHC-genotype

eij=Random error distributed with mean 0 and variance σ2

4. Results

4.1. Determination of various MHC haplotypes and genotypes

Numerous studies have been conducted globally to identify different MHC haplotypes present in different chicken breeds (12, 18, 19). However, there are scanty reports on Indian chicken germplasm and this investigation is the first report on indigenously developed broiler parent stock in India. Therefore, the present investigation was conducted on SDL chicks using PCR- based genotyping of the genomic DNA by LEI0258 and MCW0371 microsatellite markers (12), which are located within MHC region on chromosome 16 (13) between BG and BF region. The band sizes of different haplotypes were detected using a 4% MetaPhor agarose gel and categorized into different B haplotypes based on their LEI0258 polymorphic microsatellite marker sizes (Figure 1A). Allele size variations for LEI0258 ranged from 182 bp to 552 bp and ranged from 200 bp to 209 bp for MCW0371. The MHC haplotypes that shared a common LEI0258 allele size were distinguished by the MCW0371 marker (Figure 1B). It was observed that the B2 and B15 haplotypes had an identical allele size of 261bp for LEI0258. However, their MCW0371 allele differed (B2=206, B15=203) such that these two haplotypes could be distinguished with this marker.

Figure 1. Amplification of MHC-B Haplotypes using (A). LEI0258 microsatellite (B). MCW0371 microsatellite.

MHC-B haplotypes results revealed the following twelve haplotypes: B2, B8, B12.3, B13, B13.1, B15, B15.1, B15.2, B21, B22 , B72, and Bx, a newly identified haplotype that was 217 bp in length (Table 2).

Table 2. Different MHC haplotypes and their sizes with LEI0258 and MCW0371 microsatellite marker
S.No.HaplotypeLEI0258 size (bp)MCW0371 size (bp)
1B2261206
2B8405206
3B12.3513205
4B13205202
5B13.1381206
6B15261203
7B15.1193200
8B15.2249206
9B21357205
10B22249206
11B72307208
12BX217206

Further confirmation of identified B-haplotypes was done by cloning, sequencing and sequence alignment. A comparison of individual B-haplotypes sequences, using a BLAST algorithm, indicated that all the sequences belonged to Gallus gallus chromosome 16 genomic contig. The sequence of each haplotype was aligned with the reported sequence of each haplotype. The new sequence of 217 bp size (Figure 2A) showed high resemblance (99.5%) with the B2 haplotype (261), but had a deletion of 24 bp (Figure 2B). Further confirmation is therefore required to find out whether this is a new haplotype or a variant of the B2 haplotype.

The above 12 haplotypes were present in different combinations, constituting 25 different genotypes. The genotypes identified were: B2/ B2, B2/ B13, B2/ B15.1, B8/ B12.3, B8/ B13, B13/ B13, B13/ B13.1, B13/ B15, B13/ B15.2, B13/ B21, B13/ B22, B13/ Bx, B13.1/ B22, B15/ B15, B15/ B15.1, B15/ B15.2, B15/ B72, B15.1/ B15.2, B15.1/ B21, B15.1/ B22, B15.2/ B15.2, B15.2/ B21, B15.2/ Bx, B21/ Bx and Bx/ Bx. Also, it was observed that most of the genotypes were present in the heterozygous condition and a few were in the homozygous condition. Only four genotypes viz. B2/B2, B13/B13, B15 /B15 and Bx/Bx were present in the homozygous condition. The occurrence of a higher proportion of heterozygotes might be due to selective advantage to heterozygotes for survival as well as the line has never bred as per MHC haplotypes (Table 3).

Table 3. Frequency and Mean tumor profile index (TPI) scores of different MHC Genotypes in SDL chicken population
S. No.GenotypeFrequencyTPI (Mean ± SE)
1B2/ B20.0251.00 ± 0.00
2B2/B130.0132.01 ± 0.11
3B2/B15.10.0252.00 ± 0.00
4B8/ B12.30.0134.30 ± 0.45
5B8/B130.0134.83 ± 0.34
6B13/ B130.0635.50 ± 0.29
7B13/ B13.10.0256.00 ± 0.00
8B13/ B150.0385.00 ± 0.58
9B13/ B15.20.1634.92 ± 0.08
10B13/ B210.0252.00 ± 0.00
11B13/ B220.1132.11 ± 0.11
12B13/ Bx0.0384.33 ± 0.67
13B13.1/B220.0132.14 ± 0.13
14B15/ B150.0386.00 ± 0.11
15B15/ B15.10.0135.45 ± 0.18
16B15/ B15.20.0385.33 ± 0.33
17B15/ 720.0135.77 ± 0.25
18B15.1/ B15.20.054.50 ± 0.50
19B15.1/ B210.055.25 ± 0.25
20B15.1/ B220.0252.00 ± 0.00
21B15.2/ B15.20.0255.00 ± 0.00
22B15.2/ B210.054.75 ± 0.25
23B15.2/Bx0.0755.67 ± 0.21
24B21/Bx0.0252.00 ± 0.00
25Bx/Bx0.0383.67 ± 1.33

Figure 2. Sequence of (A). BX, New B-haplotype (217 bp); (B). Alignment of 217 bp pair sequences with B2 haplotype.

4.2. Frequency of different genotypes

The genotype frequencies of the SDL chicks ranged from 0.013 to 0.163. Genotype B13/ B15.2 was present at highest frequency (0.163), followed by B13/ B22 (0.113), B15.2/Bx (0.075) and B13/ B13 (0.063). Genotypes B15.1/ B15.2, B15.1/ B21, B15.2/ B21 were at same frequency (0.050). Genotypes B2/B13, B8/ B12.3, B8/B13, B13.1/B22, B15/ B15.1, B15/ B72 were present at a very low frequency 0.013 (Table 3).

4.3. Genetic manifestation of different B genotypes

Chicks of the genotype B2/B2 did not develop any tumors during the entire period of the experiment; therefore, they were categorized as non-responders. The mean TPI score of 1.00±0.00 was assigned to this genotype, indicating that this genotype conferred resistance, resulting in the complete absence of tumors in these chicks. The tumors with the B2/B15.1., B13/B21, B13/B22, B15.1/B22, and B21/Bx genotypes chicks regressed and had a very low mean TPI (2.00±0.00 to 2.11±0.11). Thus, they were categorized as regressors. Chicks with the genotypes B13/B15, B13/B15.2, B13/Bx, B15/B15.1, B15/B15.2, B15.1/B15.2, B15.1/B21, B15.2/B15.2, B15.2/B21, and B15.2/Bx had progressive tumors, with mean TPI scores from 4.33 ±0.67 to 5.67±0.21. They were therefore categorized as progressors. Genotypes B13/B13, B13/B13.1, and B15/B15 had the highest TPI scores (5.50±0.29 to 6.00±0.11). These tumors extended beyond the wing web and metastasized to other organs.

Genotype Bx/Bx had mean TPI score of 3.67±1.33 which indicated resistance of this haplotype towards Rous sarcoma virus. It was observed that if this haplotype was present in homozygous condition it made the bird resistant towards Rous sarcoma and if present in heterozygous condition in combination with resistant haplotype it became resistant genotype as indicated in genotype B21/Bx with TPI 2.00±0.00 and if present with susceptible haplotype as in genotype B15.2/Bx the TPI score was increased to 5.67±0.21 indicating its susceptibility towards Rous sarcoma virus. Thus, the haplotype Bx might be resistant recessive (Table 3).

4.4. Gross pathology

The inoculation of SDL chicks with RSV-A resulted in the appearance of a primary tumor at the site of inoculation between 2–10 days post infection. The tumor appeared as either a single pin head sized nodule or as multiple beads that coalesced later on to form one tumor mass (Figure 3A & B.). The tumor grew rapidly and eventually involved the whole wing web, which restricted the movement of the bird (Figure 3C). Tumors varied in consistency from a soft palpable to hard, solid nodular mass (Figure 3D). Soft and palpable tumors contained reddish – brown, slimy tenacious material with foul smelling fluid. The solid tumor mass had dirty, white - colored cheesy material and in some tumor masses, concentric rings were observed upon cross-section.

Figure 3. Primary solid tumor (A). as a single nodule on wing web (B). coalesced (C). covering whole wing web (D). transverse section.

In addition to the primary tumor, some chicks developed metastatic tumors in different organs of the body and on the skin (Figure 4A). Post-mortem reports revealed metastasis in the mandibular region, covering the jaw, tongue (Figure 4B), neck and thymus (Figure 4C), the area between the rib bones and chest muscle (Figure 4D) and leg muscle (Figure 4E). Pearl - like white nodules were seen on the surface of the lung, liver, spleen, heart and kidney (Figure 4E. - Figure 4F). The liver and spleen had multiple, white colored pearl - like lesions that were friable and soft and were 1mm to 5mm in diameter. The lung, heart and kidney had small, pin-point white foci. The chest, neck and leg muscles were thickened due to the proliferation of cells. The proventriculus, gizzard and caecum were also affected and showed micro-foci on their surfaces.

Figure 4. Representative tumor lesions metastasized to (A). Skin (B). Jaws (C). Neck (D). Ribs and chest (E). Leg (F). Lung (G). Liver (H). Spleen (I). Heart (J). Kidney.

The percentage of chicks showing metastasis in different organs are shown in Table 4. In the present study, the incidence of metastasis was mostly observed in the lung, liver and heart (24%), followed by the spleen (10%) and kidneys (8%). The least affected organs were the proventriculus, gizzard, caecum, intestine and serosa (2%). Splenomegaly and hepatomegaly was also observed but the bursa was not affected in any of the chicks.

Table 4. Percentage of chicks showing metastasis in different organs
S. No.Organ% of Chicks
1Lung24
2Liver24
3Heart24
4Spleen10
5Chest10
6Kidney8
7Leg8
8Neck6
9Skin6
10Jaw & Tongue2
11Proventriculus2
12Gizzard2
13Caecum2
14Bursa0
15Splenomegaly10
16Hepatomegaly12

4.5. Histopathology

Histopathological analysis typically revealed two types of tumors which were fibrosarcoma and myxosarcoma. Fibrosarcoma consisted of immature fibroblasts, loosely arranged in irregular interwoven bundles, with a moderate amount of collagen. Myxosarcoma was characterized by stellate – like or fusiform - shaped tumor cells, with extended cytoplasm. The tumor cells were homogenous, with slightly basophilic mucinous contents. However, in few birds, myxofibrosarcoma was also observed.

4.5.1. Progressor birds

4.5.1.1 Primary tumor

At the site of inoculation, the tumors appeared as fibrosarcoma. In most cases, a typical fibrosarcoma had degeneration of tissues in the center and pronounced infiltration of mesenchymal cells at the periphery. The cancer cells were spindle - shaped and multidirectional, with no set pattern. At some locations, , the cancer cells were compact and at other locations, they appeared myxomatous. The cancer invaded muscle tissue, causing atrophy. The primary tumors were highly vascularized, containing numerous and highly engorged blood vessels, as well as extensive hemorrhages. There was degeneration and necrosis of cells. The tumor stroma was infiltrated with neutrophils, mononuclear cells and plasma cells. There was proliferation of cells around hair follicles but the epidermis and skin were intact. Neoplastic cells were found in bundles (Figure 5).

Figure 5. Primary tumor showing severe hemorrhages of RBCs of tumor tissue and presence of dense fibro-sarcoma (H&E; 25 x 6.3.).

4.5.1.2. Leg muscle

There was extensive proliferation of neoplastic cells, causing replacement and atrophy of muscle fibers. Fibrosarcoma caused extensive damage to the underlying muscle, such that the muscle fibers were ruptured and separated due to presence of erythrocyte-rich, edematous fluid between the fibers. There were different patterns of cancerous cells. They were found in compact, long spindle - shaped cells or loosely arranged with different orientations. There were whorl formations with no specific direction. The cancerous cells were highly vascularized, extensively myxomatous, with only remnants of muscle fibers and at a few places, the shape of the cells was oval in shape. The necrosis of the muscle cells, along with infiltration of inflammatory cells, was evident at certain places (Figure 6A).

4.5.1.3. Chest muscle

The chest muscles were completely replaced by neoplastic cells. Cells were oval - shaped and loosely arranged with myxomatous tissues in between the cells. Neoplastic cells were rarely of the fibrosarcomatous type. Groups of secretory cells were present in myxomatous tissues. The nature of the cells suggested the presence of an advanced stage of cancer (Figure 6B).

4.5.1.4. Lung

Histopathologically, both fibrosarcoma and myxosarcoma was observed, in combination with proliferation of lymphoid cells. The tumor cells had loosely arranged, elongated cutaneous cells with loss of orientation characterized by a whirling pattern. In some cases with myxosarcomatous tissues, cells were conglomerated to form a mass or clump of nuclei (Figure 6C).

4.5.1.5. Liver

Moderate to extensive tumor growths of varying size were present and replaced the normal hepatic parenchyma. Degenerative tissues, vacuolations, lymphocytic infiltration and hemorrhages were seen in the liver parenchyma. The perivascular area had mononuclear cell and fatty cell infiltration. There was proliferation of cutaneous cells, which were loosely arranged. Fibrosarcoma could be seen as well (Figure 6D).

4.5.1.6. Spleen

The spleen was infiltrated by loosely arranged lymphoid cells. Concentrated sarcomatous tissues were visible and the compact mass of cells that could be seen was fibrosarcoma (Figure 6E).

4.5.1.7. Heart

The myocardial fibers were separated and showed degenerative changes due to the infiltrating tumor. A mixed type of cellular infiltration, with mononuclear cells and heterophils, were present (tissue slide not shown).

4.5.1.8. Kidney

There was degeneration of the kidney tubules and glomeruli. Ruptured RBC and debris were deposited in the center of glomeruli (Figure 6F).

Figure 6. (A) Leg muscle showing proliferation of neoplastic cells causing atrophy of muscle fiber and presence of myxomatous cancerous cells that are loosely arranged with different orientation (H&E; 16 x 6.3.) (B). Presence of myxomatous cancerous cells with oval shape and loose arrangement in chest muscle (H&E; 25 x 6.3.) (C). Lung showing presence of both fibro-sarcoma and myxosarcoma, proliferation of lymphoid cells and elongated cutaneous cells that are loosely attached with loss of orientation in lungs (H&E; 25 x 6.3.) (D). Liver showing fibro sarcoma with degenerative changes, vacuolations lymphocytic infiltration and hemorrhages in parenchyma and proliferation of loosely arranged cutaneous cells (H&E; 25 x 6.3.) (E). Spleen showing compact cells of fibro-sarcoma and proliferation of loosely arranged lymphoid cells (H&E; 25 x 6.3.) (F). Kidney showing degeneration of kidney tubules and glomerulli and rupturing of RBCs with deposition of debris at the center (H&E; 25 x 6.3.).

4.5.2. Regressor birds

Lung, liver, spleen, heart, kidney and the site of inoculation were carefully examined histopathologically and there was no tumor formation was observed.

4.5.3. Non-responder and Control birds

Non-Responder and uninfected control birds also did not show any tumor formation in different the organs viz. Lung, liver, spleen, heart, kidney and the site of inoculation.

5. Discussion

The present investigation was designed to determine the MHC-B haplotype status of the SDL broiler strain and the association of MHC haplotypes with cancer induction and progression. First, we determined the MHC haplotypes present in SDL chicks using LEI0258 microsatellite marker. We also used the MCW0371 marker when the haplotypes could not be differentiated by LEI0258 alone. Our study revealed twelve different haplotypes, constituting twenty-five different genotypes in combination. Apart from the standard haplotypes, a new, 217 bp haplotype was found and designated haplotype as Bx. The majority of the genotypes were heterozygous, with the frequency ranging from 0.013 to 0.163. The current study demonstrated a wide variety of MHC haplotype, which was in accordance with the findings of previous studies in domestic chickens (12, 18-20). Fulton et al. (12), identified 51 different genotypes with 26 distinctive alleles, using LEI0258 marker in chickens. Similarly, in Brazilian local chickens, using the LEI0258 microsatellite (19), 53 different genotypes, with 15 different alleles, were reported. Schou et al. (18) also used this marker to identify two different chicken breeds in Vietnam. In one breed, 19 different alleles were found and in another breed, 24 distinctive alleles were identified. Lwelamira et al. (20) reported the presence of 22 and 23 alleles of LEI0258 in the Tanzania Kuchi and Medium ecotypes, respectively. The number of alleles in these studies, including our work, were much higher. However, a lower number of alleles were reported for commercial breeds such as the Lohman Silver Line (3 alleles) and the Lohman Brown line (5 alleles) (18). The presence of a higher number of alleles in the SDL chickens suggested greater variability at their MHC locus.

To elucidate the genetic manifestation of the different B genotypes, the overall tumor growth pattern was observed and a tumor profile index (TPI) score was determined. A higher TPI indicated greater progression of tumor growth, and thus greater susceptibility to the Rous sarcoma virus. Conversely, a lower TPI indicated resistance towards the virus, as demonstrated by either no induction or regression of the tumor. Based on the TPI scores, it was noted that haplotypes B2, B21, and B22 were resistant, as these SDL chicks had a lower TPI score (1 or 2). Haplotypes B13, B13.1., B15, B15.1., and B15.2., however, were deemed more susceptible as their TPI scores were from 4 to 6. We also observed that chickens with the haplotypes B2, B22, and B21 showed reduced mortality and increased survival, further corroborating our hypothesis that these haplotypes confer resistance to RSV-A induction. Similar results were obtained by Collins et al. (11), who obtained TPI scores in two highly inbred lines homozygous for B2 and B5. They reported that the most resistant genotype, B2B2, had a 5% mortality rate and a mean TPI of 2.94, whereas the most susceptible genotype, B5B5, had a 93% mortality rate and a mean TPI of 4.93, and the heterozygotes had TPI values similar to the resistant genotype compared to those that were deemed susceptible to infection.

Furthermore in our study the haplotypes B2 and B22 appeared to be resistant and dominant, as their presence in both homozygous and heterozygous conditions led to the reduction in TPI scores. The presence of these haplotypes in the homozygous condition (B2/B2) produced chicks that were non-responders. These chicks demonstrated no tumor development and their TPI scores were 1.00±0.00, indicating complete resistance against the Rous sarcoma virus. In the heterozygous condition, such as in genotype B2/B15.1, the presence of the B2 allele reduced the TPI score (2.00), leading to tumor regression hence, indicating an incomplete dominance effect.

Various studies have reported that MHC complementation results in better outcomes compared to the homozygous condition. The susceptible homozygous genotypes B13/B13, B13.1/B13.1, and B15/ B15 had high TPI scores, ranging from 5.50±0.29 to 6.00±0.11, indicating their high susceptibility to RSV. Heterozygous genotypes had intermediate TPI scores between the susceptible and resistant homozygous genotypes. Senseney et al., (21) also found MHC complementation between BQ and B17 haplotypes in a Line UCD 001 (BQBQ) × UCD 003 (B17B17) cross. In addition, heterozygote animals had lower TPI scores than the homozygote animals. Genotype B23/B26 had a lower TPI compared with either the B23/B23 or B26/B26 homozygotes (22, 23) and the B22/B26 genotype had a TPI lower than the B22/B22 or B26/B26 homozygotes (24). However, Taylor et al., (25) suggested negative complementation between haplotypes B24 and B30. Heterozygous B24B30 chickens had a higher TPI than B24/B24 and B30/B30 chickens but only the B24/B30 and B30/B30 types were significantly different.

We observed that the resistant haplotype B21 was dominant over B13 and Bx, but recessive to B15.1 and B15.2.. When haplotype B21 was present, along with B13 or Bx, the tumor regressed; however, when it was present with B15.1. or B15.2., the tumor progressed (TPI score of 5 to 6). Furthermore, it is worthwhile to emphasize that the newly identified Bx /Bx genotype had a mean TPI score of 3.67±1.33, which was closer to that of the resistant haplotype. Complementation of this haplotype, with a resistant haplotype, such as B21, reduced the TPI score and conferred resistance. However, when Bx was combined with a susceptible haplotype, the TPI score was increased, indicating susceptibility to RSV. Similar results were reported by Taylor et al., (25) and Lukacs et al., (24), while studying the anti-tumor response of the B30 haplotype. Their results indicated that in the homozygous condition, B30/B30 was more regressive; however, in the heterozygous state, it had a variable anti-tumor response. The combination with a progressive haplotype such as B24 resulted in a more progressive state, whereas coupling with a regressive haplotype such as B22 resulted in more regressive state. Thus, we concluded that in our study, haplotype Bx is likely to be resistant but recessive.

Based on our results, the order from dominant to recessive, was: B2 > B22 >B15 > B15.1 >B15.2 > B21>B13 > Bx for RSV-A tumor resistance. We examined the gross pathology of the tumors and noted that primary tumors first appeared as a single pin - head sized nodule that grew and became extensive, covering the whole wing-web. Halpern et al., (26) also found RSV - induced tumors highly enlarged, which reflected both tumor cell proliferation and viral replication generating new tumor cells. In the present study, the lung, liver and heart were the most affected organs, followed by the spleen and kidney and the least affected organs were proventriculus, gizzard, and intestine. Similar findings were reported by Collins et al. (27),where the incidence of metastatic tumors in the heart and pericardial sac was 27%, followed by the liver and pancreas (18%), gastrointestinal tract (15%) and the least affected organ was the kidney (2%).

Comparing the histopathological lesions in different organs of chicks, two types of sarcomas were encountered in progressor birds; however the regressor, non-responder and uninfected control did not show tumour in any of the organs studied. Mostly the tumors found in progressor birds were fibrosarcomas and to a lesser extent the myxosarcomas were found. Primary tumors had extensive fibrosarcomas, with compact to lose architecture in the proliferating neoplastic cells. Metastatic organs indicated a variable degree of infiltration of fibro and myxo sarcomatous tissues, causing atrophy of adjoining areas and replacing the original parenchyma, leaving only remnants of normal structure. Halpern et al., (26) also found mostly fibrosarcomas, which consisted primarily of immature fibroblasts loosely arranged in irregular bundles. Histiocytic sarcomas as described by Arshad et al and Sastri et al. (28, 29) were not seen in this study which might be possibly due to a difference in the subgroup viral strain that was used.

6. Conclusion

The present study demonstrated a higher number of alleles in SDL chickens, suggesting greater variability at MHC locus. The role of MHC haplotypes in resistance/susceptibility was confirmed and the halpotypes B2, B21 and B22 , exhibited resistance with dominance compared to the most susceptible haplotypes B13, B13.1, B15, B15.1 and B15.2. Furthermore, in the homozygous condition, resistant haplotypes became more resistant to RSV, as indicated by the absence of tumor induction, while homozygosity in the susceptible haplotypes increased susceptibility. Genotypes in the heterozygous condition showed intermediate tumor growth between susceptible and resistant homozygous genotypes. Thus, we conclude that the heterozygous condition has better outcomes in tumor regression compared to the homozygous condition, except for B2, B21 and B22. Genetic complementation increased the response of heterozygotes compared to their component homozygotes. Although the exact mechanism of genetic complementation is not known, Brown et al., (21) described the anti-tumor response as a complex reaction to various viral and tumor antigens. Heterozygotes may recognize a greater spectrum of antigenic determinants from RSV compared to their component homozygotes. Alternatively, heterozygotes may recognize the same antigenic determinants more efficiently (21, 23). Thus, the understanding of the mechanisms of genetic complementation may help in the development of genetically resistant breeds. The majority of the haplotypes in the SDL population were present in the heterozygous condition. The higher variability at the MHC proteins in the heterozygous condition may produce a survival advantage for this SDL stock in their native environment. Moreover, the results of the present study indicate that the breeding of chicks with haplotypes such as B2, B21 and B22 may help generating RSV-A resistant flocks.

7. Acknowledgments

The facilities provided by Director CARI, Izatnagar and Director IVRI, Izatnagar for carrying out the investigation are thankfully acknowledged. We thank Dr. Charles R. Ashby, Jr. (St. John’s University, Queens, NY) for editorial assistance.

References

    1. Witter RL, Bacon LD, Hunt HD, Silva RE, Fadly AM: Avian leukosis virus subgroup J infection profiles in broiler breeder chickens: association with virus transmission to progeny. Avian Dis, 44:913-931 (2000)
    DOI: 10.2307/1593066
    PMid:11195648

    2. van Regenmortel MH, Mayo MA, Fauquet CM, Maniloff J: Virus nomenclature: consensus versus chaos. Arch Virol, 145:2227-2232 (2000)
    DOI: 10.1007/s007050070053
    PMid:11087105

    3. Gao Y, Yun B, Qin L, Pan W, Qu Y, Liu Z, Wang Y, Qi X, Gao H, Wang X: Molecular epidemiology of avian leukosis virus subgroup J in layer flocks in China. J Clin Microbiol, 50:953-960 (2012)
    DOI: 10.1128/JCM.00956-12
    DOI: 10.1128/JCM.06179-11
    PMid:22205787 PMCid:PMC3295186

    4. Zekarias B, Ter Huurne AA, Landman WJ, Rebel JM, Pol JM, Gruys E: Immunological basis of differences in disease resistance in the chicken. Vet Res, 33:109-125 (2002)
    DOI: 10.1051/vetres:2002001
    PMid:11944802

    5. Praharaj NK, Gross WB, Dunnington EA, Siegel PB: Feeding regimen by sire family interactions on growth, immunocompetence, and disease resistance in chickens. Poult Sci, 75:821-827 (1996)
    DOI: 10.3382/ps.0750821
    PMid:8805197

    6. Briles WE, Stone HA, Cole RK: Marek’s disease: effects of B histocompatibility alloalleles in resistant and susceptible chicken lines. Science, 195:193-195 (1977)
    DOI: 10.1126/science.831269
    PMid:831269

    7. Lamont SJ: The chicken major histocompatibility complex in disease resistance and poultry breeding. J Dairy Sci, 72:1328-1333 (1989)
    DOI: 10.3168/jds.S0022-0302(89)79240-7

    8. Yoo BH, Sheldon BL: Association of the major histocompatibility complex with avian leukosis virus infection in chickens. Br Poult Sci, 33:613-620 (1992)
    DOI: 10.1080/00071669208417500
    PMid:1322760

    9. Bacon LD, Witter RL, Crittenden LB, Fadly A, Motta J: B-haplotype influence on Marek’s disease, Rous sarcoma, and lymphoid leukosis virus-induced tumors in chickens. Poult Sci, 60:1132-1139 (1981)
    DOI: 10.3382/ps.0601132
    PMid:6267578

    10. Taylor RL, Jr.: Major histocompatibility (B) complex control of responses against Rous sarcomas. Poult Sci, 83:638-649 (2004)
    DOI: 10.1093/ps/83.4.638
    PMid:15109061

    11. Collins WM, Briles, W.E. and Zsigray, R.M. : The B locus (MHC) in the chicken: association with the fate of RSV-induced tumors. Immunogenetics, 5:333-343 (1977)
    DOI: 10.1007/BF01570490

    12. Fulton JE, Juul-Madsen HR, Ashwell CM, McCarron AM, Arthur JA, O’Sullivan NP, Taylor RL, Jr.: Molecular genotype identification of the Gallus gallus major histocompatibility complex. Immunogenetics, 58:407-421 (2006)
    DOI: 10.1007/s00251-006-0119-0
    PMid:16738938

    13. McConnell SK, Dawson DA, Wardle A, Burke T: The isolation and mapping of 19 tetranucleotide microsatellite markers in the chicken. Anim Genet, 30:183-189 (1999)
    DOI: 10.1046/j.1365-2052.1999.00454.x
    PMid:10442979

    14. Livant EJ, Brigati JR, Ewald SJ: Diversity and locus specificity of chicken MHC B class I sequences. Anim Genet, 35:18-27 (2004)
    DOI: 10.1111/j.1365-2052.2003.01078.x
    PMid:14731225

    15. Suzuki K, Kobayashi E, Yamashita H, Uenishi H, Churkina I, Plastow G, Hamasima N, Mitsuhashi T: Structural analysis of MHC alleles in an RSV tumour regression chicken using a BAC library. Anim Genet, 43:348-351 (2012)
    DOI: 10.1111/j.1365-2052.2011.02247.x
    PMid:22486511

    16. Groupé, V., Dunkel, V. C., and Manake, R. A. Improved pock counting method for the titration of rous sarcoma virus in embryonated eggs. J Bacteriol. Sep; 74(3): 409–410 (1957).

    17. Collins WM, Zervas, N.P., Urban, W.E. Jr., Briles, W.E., Aeed, P.A.: Response of B complex haplotypes B22, B24, and B26 to Rous sarcomas. Poultry Science, 64:2017-2019 (1985)
    DOI: 10.3382/ps.0642017
    PMid:2999742

    18. Schou TW, Permin A, Juul-Madsen HR, Sorensen P, Labouriau R, Nguyen TL, Fink M, Pham SL: Gastrointestinal helminths in indigenous and exotic chickens in Vietnam: association of the intensity of infection with the Major Histocompatibility Complex. Parasitology, 134:561-573 (2007)
    DOI: 10.1017/S0031182006002046
    PMid:17166322

    19. Lima-Rosa CA, Canal CW, Streck AF, Freitas LB, Delgado-Canedo A, Bonatto SL, Salzano FM: B-F DNA sequence variability in Brazilian (blue-egg Caipira) chickens. Anim Genet, 35:278-284 (2004)
    DOI: 10.1111/j.1365-2052.2004.01160.x
    PMid:15265066

    20. Lwelamira J, Kifaro GC, Gwakisa PS: Genetic parameters for body weights, egg traits and antibody response against Newcastle Disease Virus (NDV) vaccine among two Tanzania chicken ecotypes. Trop Anim Health Prod, 41:51-59 (2009)
    DOI: 10.1007/s11250-008-9153-2
    PMid:19052902

    21. Senseney HL, Briles WE, Abplanalp H, Taylor RL, Jr.: Allelic complementation between MHC haplotypes B (Q) and B17 increases regression of Rous sarcomas. Poult Sci, 79:1736-1740 (2000)
    DOI: 10.1093/ps/79.12.1736
    PMid:11194035

    22. Brown DW, Collins WM, Ward PH, Briles WE: Complementation of major histocompatibility haplotypes in regression of Rous sarcoma virus-induced tumors in noninbred chickens. Poult Sci, 61:409-413 (1982)
    DOI: 10.3382/ps.0610409
    PMid:6283511

    23. Collins WM, Briles, W.E., Corbett, A.C., Clark, K.K., Zsigray, R.M. and Dunlop, W.R. : B locus (MHC) effect upon regression of RSV-induced tumors in noninbred chickens. Immunogenetics, 9:97–100 (1979)
    DOI: 10.1007/BF01570396

    24. Lukacs NW, Briles WE, Briles RW, Taylor RL, Jr.: Response of major histocompatibility (B) complex haplotypes B22, B26, and B30 to Rous sarcomas. Poult Sci, 68:233-237 (1989)
    DOI: 10.3382/ps.0680233
    PMid:2539589

    25. Taylor RL, Jr., Clare RA, Ward PH, Briles RW, Briles WE: Anti-Rous sarcoma response of major histocompatibility (B) complex haplotypes B23, B24 and B30. Anim Genet, 19:277-284 (1988)
    DOI: 10.1111/j.1365-2052.1988.tb00816.x
    PMid:2849889

    26. Halpern MS, Ewert DL, Flores LJ, Fujita DJ, Aldrich CE, Mason WS: Sarcoma growth in 15I5 x 7 (2) chickens infected with avian sarcoma viruses of subgroup B or G. Virology, 134:472-476 (1984)
    DOI: 10.1016/0042-6822(84)90315-5

    27. Collins WM, Dunlop, W.R., Zsigray, R.M., Briles, R.W., Fite, R.W. : Metastasis of Rous sarcoma tumors in chickens is influenced by the major histocompatibility (B) complex and sex. Poult Sciences, 65:1642-1648 (1986)
    DOI: 10.3382/ps.0651642
    PMid:3022275

    28. Sastri GA, Rao SP, Narayana JV, Rao PR, Christopher J: Histiocytic-Sarcomas in Domestic Fowls. (a Report of Five Cases) Indian J Pathol Bacteriol, 52:277-281 (1964)
    PMid:14247636

    29. Arshad SS, Howes K, Barron GS, Smith LM, Russell PH, Payne LN: Tissue tropism of the HPRS-103 strain of J subgroup avian leukosis virus and of a derivative acutely transforming virus. Vet Pathol, 34:127-137 (1997)
    DOI: 10.1177/030098589703400205
    PMid:9066079

5. Discussion

The present investigation was designed to determine the MHC-B haplotype status of the SDL broiler strain and the association of MHC haplotypes with cancer induction and progression. First, we determined the MHC haplotypes present in SDL chicks using LEI0258 microsatellite marker. We also used the MCW0371 marker when the haplotypes could not be differentiated by LEI0258 alone. Our study revealed twelve different haplotypes, constituting twenty-five different genotypes in combination. Apart from the standard haplotypes, a new, 217 bp haplotype was found and designated haplotype as Bx. The majority of the genotypes were heterozygous, with the frequency ranging from 0.013 to 0.163. The current study demonstrated a wide variety of MHC haplotype, which was in accordance with the findings of previous studies in domestic chickens (12, 18-20). Fulton et al. (12), identified 51 different genotypes with 26 distinctive alleles, using LEI0258 marker in chickens. Similarly, in Brazilian local chickens, using the LEI0258 microsatellite (19), 53 different genotypes, with 15 different alleles, were reported. Schou et al. (18) also used this marker to identify two different chicken breeds in Vietnam. In one breed, 19 different alleles were found and in another breed, 24 distinctive alleles were identified. Lwelamira et al. (20) reported the presence of 22 and 23 alleles of LEI0258 in the Tanzania Kuchi and Medium ecotypes, respectively. The number of alleles in these studies, including our work, were much higher. However, a lower number of alleles were reported for commercial breeds such as the Lohman Silver Line (3 alleles) and the Lohman Brown line (5 alleles) (18). The presence of a higher number of alleles in the SDL chickens suggested greater variability at their MHC locus.

To elucidate the genetic manifestation of the different B genotypes, the overall tumor growth pattern was observed and a tumor profile index (TPI) score was determined. A higher TPI indicated greater progression of tumor growth, and thus greater susceptibility to the Rous sarcoma virus. Conversely, a lower TPI indicated resistance towards the virus, as demonstrated by either no induction or regression of the tumor. Based on the TPI scores, it was noted that haplotypes B2, B21, and B22 were resistant, as these SDL chicks had a lower TPI score (1 or 2). Haplotypes B13, B13.1., B15, B15.1., and B15.2., however, were deemed more susceptible as their TPI scores were from 4 to 6. We also observed that chickens with the haplotypes B2, B22, and B21 showed reduced mortality and increased survival, further corroborating our hypothesis that these haplotypes confer resistance to RSV-A induction. Similar results were obtained by Collins et al. (11), who obtained TPI scores in two highly inbred lines homozygous for B2 and B5. They reported that the most resistant genotype, B2B2, had a 5% mortality rate and a mean TPI of 2.94, whereas the most susceptible genotype, B5B5, had a 93% mortality rate and a mean TPI of 4.93, and the heterozygotes had TPI values similar to the resistant genotype compared to those that were deemed susceptible to infection.

Furthermore in our study the haplotypes B2 and B22 appeared to be resistant and dominant, as their presence in both homozygous and heterozygous conditions led to the reduction in TPI scores. The presence of these haplotypes in the homozygous condition (B2/B2) produced chicks that were non-responders. These chicks demonstrated no tumor development and their TPI scores were 1.00±0.00, indicating complete resistance against the Rous sarcoma virus. In the heterozygous condition, such as in genotype B2/B15.1, the presence of the B2 allele reduced the TPI score (2.00), leading to tumor regression hence, indicating an incomplete dominance effect.

Various studies have reported that MHC complementation results in better outcomes compared to the homozygous condition. The susceptible homozygous genotypes B13/B13, B13.1/B13.1, and B15/ B15 had high TPI scores, ranging from 5.50±0.29 to 6.00±0.11, indicating their high susceptibility to RSV. Heterozygous genotypes had intermediate TPI scores between the susceptible and resistant homozygous genotypes. Senseney et al., (21) also found MHC complementation between BQ and B17 haplotypes in a Line UCD 001 (BQBQ) × UCD 003 (B17B17) cross. In addition, heterozygote animals had lower TPI scores than the homozygote animals. Genotype B23/B26 had a lower TPI compared with either the B23/B23 or B26/B26 homozygotes (22, 23) and the B22/B26 genotype had a TPI lower than the B22/B22 or B26/B26 homozygotes (24). However, Taylor et al., (25) suggested negative complementation between haplotypes B24 and B30. Heterozygous B24B30 chickens had a higher TPI than B24/B24 and B30/B30 chickens but only the B24/B30 and B30/B30 types were significantly different.

We observed that the resistant haplotype B21 was dominant over B13 and Bx, but recessive to B15.1 and B15.2.. When haplotype B21 was present, along with B13 or Bx, the tumor regressed; however, when it was present with B15.1. or B15.2., the tumor progressed (TPI score of 5 to 6). Furthermore, it is worthwhile to emphasize that the newly identified Bx /Bx genotype had a mean TPI score of 3.67±1.33, which was closer to that of the resistant haplotype. Complementation of this haplotype, with a resistant haplotype, such as B21, reduced the TPI score and conferred resistance. However, when Bx was combined with a susceptible haplotype, the TPI score was increased, indicating susceptibility to RSV. Similar results were reported by Taylor et al., (25) and Lukacs et al., (24), while studying the anti-tumor response of the B30 haplotype. Their results indicated that in the homozygous condition, B30/B30 was more regressive; however, in the heterozygous state, it had a variable anti-tumor response. The combination with a progressive haplotype such as B24 resulted in a more progressive state, whereas coupling with a regressive haplotype such as B22 resulted in more regressive state. Thus, we concluded that in our study, haplotype Bx is likely to be resistant but recessive.

Based on our results, the order from dominant to recessive, was: B2 > B22 >B15 > B15.1 >B15.2 > B21>B13 > Bx for RSV-A tumor resistance. We examined the gross pathology of the tumors and noted that primary tumors first appeared as a single pin - head sized nodule that grew and became extensive, covering the whole wing-web. Halpern et al., (26) also found RSV - induced tumors highly enlarged, which reflected both tumor cell proliferation and viral replication generating new tumor cells. In the present study, the lung, liver and heart were the most affected organs, followed by the spleen and kidney and the least affected organs were proventriculus, gizzard, and intestine. Similar findings were reported by Collins et al. (27),where the incidence of metastatic tumors in the heart and pericardial sac was 27%, followed by the liver and pancreas (18%), gastrointestinal tract (15%) and the least affected organ was the kidney (2%).

Comparing the histopathological lesions in different organs of chicks, two types of sarcomas were encountered in progressor birds; however the regressor, non-responder and uninfected control did not show tumour in any of the organs studied. Mostly the tumors found in progressor birds were fibrosarcomas and to a lesser extent the myxosarcomas were found. Primary tumors had extensive fibrosarcomas, with compact to lose architecture in the proliferating neoplastic cells. Metastatic organs indicated a variable degree of infiltration of fibro and myxo sarcomatous tissues, causing atrophy of adjoining areas and replacing the original parenchyma, leaving only remnants of normal structure. Halpern et al., (26) also found mostly fibrosarcomas, which consisted primarily of immature fibroblasts loosely arranged in irregular bundles. Histiocytic sarcomas as described by Arshad et al and Sastri et al. (28, 29) were not seen in this study which might be possibly due to a difference in the subgroup viral strain that was used.

6. Conclusion

The present study demonstrated a higher number of alleles in SDL chickens, suggesting greater variability at MHC locus. The role of MHC haplotypes in resistance/susceptibility was confirmed and the halpotypes B2, B21 and B22 , exhibited resistance with dominance compared to the most susceptible haplotypes B13, B13.1, B15, B15.1 and B15.2. Furthermore, in the homozygous condition, resistant haplotypes became more resistant to RSV, as indicated by the absence of tumor induction, while homozygosity in the susceptible haplotypes increased susceptibility. Genotypes in the heterozygous condition showed intermediate tumor growth between susceptible and resistant homozygous genotypes. Thus, we conclude that the heterozygous condition has better outcomes in tumor regression compared to the homozygous condition, except for B2, B21 and B22. Genetic complementation increased the response of heterozygotes compared to their component homozygotes. Although the exact mechanism of genetic complementation is not known, Brown et al., (21) described the anti-tumor response as a complex reaction to various viral and tumor antigens. Heterozygotes may recognize a greater spectrum of antigenic determinants from RSV compared to their component homozygotes. Alternatively, heterozygotes may recognize the same antigenic determinants more efficiently (21, 23). Thus, the understanding of the mechanisms of genetic complementation may help in the development of genetically resistant breeds. The majority of the haplotypes in the SDL population were present in the heterozygous condition. The higher variability at the MHC proteins in the heterozygous condition may produce a survival advantage for this SDL stock in their native environment. Moreover, the results of the present study indicate that the breeding of chicks with haplotypes such as B2, B21 and B22 may help generating RSV-A resistant flocks.

7. Acknowledgments

The facilities provided by Director CARI, Izatnagar and Director IVRI, Izatnagar for carrying out the investigation are thankfully acknowledged. We thank Dr. Charles R. Ashby, Jr. (St. John’s University, Queens, NY) for editorial assistance.

References

    1. Witter RL, Bacon LD, Hunt HD, Silva RE, Fadly AM: Avian leukosis virus subgroup J infection profiles in broiler breeder chickens: association with virus transmission to progeny. Avian Dis, 44:913-931 (2000)
    DOI: 10.2307/1593066
    PMid:11195648

    2. van Regenmortel MH, Mayo MA, Fauquet CM, Maniloff J: Virus nomenclature: consensus versus chaos. Arch Virol, 145:2227-2232 (2000)
    DOI: 10.1007/s007050070053
    PMid:11087105

    3. Gao Y, Yun B, Qin L, Pan W, Qu Y, Liu Z, Wang Y, Qi X, Gao H, Wang X: Molecular epidemiology of avian leukosis virus subgroup J in layer flocks in China. J Clin Microbiol, 50:953-960 (2012)
    DOI: 10.1128/JCM.00956-12
    DOI: 10.1128/JCM.06179-11
    PMid:22205787 PMCid:PMC3295186

    4. Zekarias B, Ter Huurne AA, Landman WJ, Rebel JM, Pol JM, Gruys E: Immunological basis of differences in disease resistance in the chicken. Vet Res, 33:109-125 (2002)
    DOI: 10.1051/vetres:2002001
    PMid:11944802

    5. Praharaj NK, Gross WB, Dunnington EA, Siegel PB: Feeding regimen by sire family interactions on growth, immunocompetence, and disease resistance in chickens. Poult Sci, 75:821-827 (1996)
    DOI: 10.3382/ps.0750821
    PMid:8805197

    6. Briles WE, Stone HA, Cole RK: Marek’s disease: effects of B histocompatibility alloalleles in resistant and susceptible chicken lines. Science, 195:193-195 (1977)
    DOI: 10.1126/science.831269
    PMid:831269

    7. Lamont SJ: The chicken major histocompatibility complex in disease resistance and poultry breeding. J Dairy Sci, 72:1328-1333 (1989)
    DOI: 10.3168/jds.S0022-0302(89)79240-7

    8. Yoo BH, Sheldon BL: Association of the major histocompatibility complex with avian leukosis virus infection in chickens. Br Poult Sci, 33:613-620 (1992)
    DOI: 10.1080/00071669208417500
    PMid:1322760

    9. Bacon LD, Witter RL, Crittenden LB, Fadly A, Motta J: B-haplotype influence on Marek’s disease, Rous sarcoma, and lymphoid leukosis virus-induced tumors in chickens. Poult Sci, 60:1132-1139 (1981)
    DOI: 10.3382/ps.0601132
    PMid:6267578

    10. Taylor RL, Jr.: Major histocompatibility (B) complex control of responses against Rous sarcomas. Poult Sci, 83:638-649 (2004)
    DOI: 10.1093/ps/83.4.638
    PMid:15109061

    11. Collins WM, Briles, W.E. and Zsigray, R.M. : The B locus (MHC) in the chicken: association with the fate of RSV-induced tumors. Immunogenetics, 5:333-343 (1977)
    DOI: 10.1007/BF01570490

    12. Fulton JE, Juul-Madsen HR, Ashwell CM, McCarron AM, Arthur JA, O’Sullivan NP, Taylor RL, Jr.: Molecular genotype identification of the Gallus gallus major histocompatibility complex. Immunogenetics, 58:407-421 (2006)
    DOI: 10.1007/s00251-006-0119-0
    PMid:16738938

    13. McConnell SK, Dawson DA, Wardle A, Burke T: The isolation and mapping of 19 tetranucleotide microsatellite markers in the chicken. Anim Genet, 30:183-189 (1999)
    DOI: 10.1046/j.1365-2052.1999.00454.x
    PMid:10442979

    14. Livant EJ, Brigati JR, Ewald SJ: Diversity and locus specificity of chicken MHC B class I sequences. Anim Genet, 35:18-27 (2004)
    DOI: 10.1111/j.1365-2052.2003.01078.x
    PMid:14731225

    15. Suzuki K, Kobayashi E, Yamashita H, Uenishi H, Churkina I, Plastow G, Hamasima N, Mitsuhashi T: Structural analysis of MHC alleles in an RSV tumour regression chicken using a BAC library. Anim Genet, 43:348-351 (2012)
    DOI: 10.1111/j.1365-2052.2011.02247.x
    PMid:22486511

    16. Groupé, V., Dunkel, V. C., and Manake, R. A. Improved pock counting method for the titration of rous sarcoma virus in embryonated eggs. J Bacteriol. Sep; 74(3): 409–410 (1957).

    17. Collins WM, Zervas, N.P., Urban, W.E. Jr., Briles, W.E., Aeed, P.A.: Response of B complex haplotypes B22, B24, and B26 to Rous sarcomas. Poultry Science, 64:2017-2019 (1985)
    DOI: 10.3382/ps.0642017
    PMid:2999742

    18. Schou TW, Permin A, Juul-Madsen HR, Sorensen P, Labouriau R, Nguyen TL, Fink M, Pham SL: Gastrointestinal helminths in indigenous and exotic chickens in Vietnam: association of the intensity of infection with the Major Histocompatibility Complex. Parasitology, 134:561-573 (2007)
    DOI: 10.1017/S0031182006002046
    PMid:17166322

    19. Lima-Rosa CA, Canal CW, Streck AF, Freitas LB, Delgado-Canedo A, Bonatto SL, Salzano FM: B-F DNA sequence variability in Brazilian (blue-egg Caipira) chickens. Anim Genet, 35:278-284 (2004)
    DOI: 10.1111/j.1365-2052.2004.01160.x
    PMid:15265066

    20. Lwelamira J, Kifaro GC, Gwakisa PS: Genetic parameters for body weights, egg traits and antibody response against Newcastle Disease Virus (NDV) vaccine among two Tanzania chicken ecotypes. Trop Anim Health Prod, 41:51-59 (2009)
    DOI: 10.1007/s11250-008-9153-2
    PMid:19052902

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Vishwa M. Khare, Vishesh K. Saxena, Alka Tomar, K.P. Singh, Kunwar B. Singh, Amit K. Tiwari. MHC-B haplotypes impact susceptibility and resistance to RSV-A infection. Frontiers in Bioscience-Elite. 2018. 10(3); 506-519.