Genetic susceptibility of chicken macrophages to in vitro infection with infectious laryngotracheitis virus

Chicken macrophages are susceptible to infection with infectious laryngotracheitis virus (ILTV), the level of infection being dependent, in part, on the genotype of the host cell. Following infection in vitro a greater proportion of macrophages from the ILTV-resistant J1(B113/113) and N1(B114/114) inbred lines of chickens were found to be positive for ILTV antigens, than macrophages from the ILTV-susceptible M1(B15/15) chickens. The proportion of ILTV-positive macrophages was found to be genetically regulated, in part by the chicken major histocompatibility complex (MHC), although alloantisera to class I and class II MHC antigens did not reduce the number of macrophages infected. Similarly, the phagocytic activity of the cultured macrophages from chickens of different MHC genotype did not correlate with their susceptibility to infection with ILTV.     

The major histocompatibility complex and genetic control of antibody response to sheep red blood cells in chickens

The genetic association of the B-F/B-L region of the chicken major histocompatibility complex (MHC) with the antibody response to sheep red blood cells (SRBC) was investigated using three inbred lines of White Leghorn chickens. Two out of the three inbred lines of chickens. J1 and N1, were found to be a low responder to SRBC. Planned matings between the three inbred lines of chickens showed that the genetic regulation of the magnitude of the antibody response to SRBC was associated with the MHC. The low antibody response to SRBC was inherited as a recessive trait linked to the B15 MHC phenotype of the M1 inbred line.     

The major histocompatibility complex and genetic control of antibody response to synthetic antigens in chickens

The genetic association of the B-F/B-L region of the chicken major histocompatibility complex (MHC) with the antibody responses to the hapten 2,4-dinitrophenol (DNP) conjugated to human gamma globulin and to the synthetic antigens, GAT and (T,G)-A-L were investigated using five inbred lines of White Leghorn chickens. Two of the five inbred lines of chickens J1 and M1, were found to be high responders to DNP and (T,G)-A-L, whilst L1, M4 and N1 chickens were found to be relatively low responders to both antigens. Chickens from all five inbred lines were low responders to GAT. Matings between the inbred lines. J1, M1 and N(1), showed that the magnitude of the antibody response to (T,G)-A-L in backcross chickens was regulated by a dominant gene or set of genes linked to the MHC. High antibody responsiveness to (T,G)-A-L was inherited as a dominant trait linked to the B113 and the B15 MHC alleles of the J1 and M1 inbred lines respectively. The similarity of the anti-(T,G)-A-L responses in chickens from the three B113 homozygous inbred lines L1, M4 and N1, also suggests that control of this response was linked to the MHC. The magnitude of the antibody response to DNP in backcross chickens from matings between the inbred lines N1 and J1 or M1 was also found to be regulated by a gene or set of genes linked to the MHC. Again, high antibody responsiveness to DNP was inherited as a dominant trait linked to the B113 and the B15 MHC alleles of the J1 and M1 inbred lines respectively.     

Complementing MHC- and non-MHC-linked genes and resistance to avian sarcoma virus-induced tumours in inbred lines of chickens

Schmidt-Ruppin Rous sarcoma virus, subgroup B (SR-RSV-B), was inoculated into the wingwebs of chickens from three partially congenic inbred lines, G-B1, G-B2 and G-B3, homozygous for different MHC (B region) haplotypes (genotypes = B1/B1, B2/B2 and B3/B3 respectively). All birds developed tumours but only the G-B2 line resisted progressive tumour growth. Birds from lines G-B1 and G-B3 approached 100% susceptibility to progressive tumour growth, whereas most (G-B1 X G-B3) F1 hybrids were resistant to tumours induced by SR-RSV-B. The association of the resistance trait in F1 hybrids with genes of the B region was investigated by testing progeny of (G-B1 X G-B3) X B-B1 F1 and (G-B1 X G-B2) X G-B3 F1 backcross matings. Approximately 27% of the backcross population was resistant to SR-RSV-B-induced tumours and these resistant offspring were predominantly of the B1/B3 phenotype. We interpret these results to mean that resistance to progressive tumour growth involves complementation between genes (allelic or at separate loci) linked to or within the B region and that resistance is effective only when the complementing B region genes act in concert with complementing genes which assort independently of the MHC. We suggest that complementing B region-linked genes are homologues of complementing murine H-2-linked Ir genes. The function of the B region in determining growth of sarcomas may therefore be analogous to that of Ir genes.     

A strong antigen-specific T-cell response is associated with age and genetically dependent resistance to avian enteric salmonellosis

Chicken genetics and age affect resistance to enteric infection with Salmonella enterica serovar Typhimurium and were used to identify the immune responses that may contribute to rapid clearance. When birds were infected at 40 days of age, line 6(1) chickens cleared the infection more effectively than line N chickens, whereas when birds were infected at 10 days of age, both chicken lines were highly susceptible to infection. Antibody levels, T-cell responsiveness, and cytokine mRNA levels were all elevated during infection. A negative correlation between resistance and antigen-specific antibody production was observed in older chickens. However, this finding was not replicated for age-related resistance; we found that older chickens exhibited a stronger and more rapid antibody response than younger chickens. The levels of interleukin-1beta (IL-1beta) and gamma interferon (IFN-gamma) mRNA were similar in the spleens and cecal tonsils of both line 6(1) and line N chickens, except for higher levels of IL-1beta in the spleens of line 6(1) chickens at 6 days postinfection. Differences in the levels of IFN-gamma and IL-1beta 1beta mRNA between the lines were more apparent in younger chickens, but while the increases were greater than those observed in the older chickens, the clearance of enteric S. enterica serovar Typhimurium was much slower. The level of antigen-specific proliferation of splenocytes was associated with increased resistance in both experimental systems, and the strongest responses were observed in older and genetically resistant chickens. The data presented here implicate T-cell responses in the clearance of S. enterica serovar Typhimurium from the intestine of infected chickens.     

Methods for evaluating and developing commercial chicken strains free of endogenous subgroup E avian leukosis virus.

The genome of nearly all chickens contains various DNA proviral insertions of retroviruses of subgroup E avian leukosis virus (ALVE). However, the elimination or control of ALVE gene expression is desirable to improve productivity, to improve resistance to avian leukosis virus (ALV)-induced tumours, and to develop safer live virus vaccines in chick embryos and cultured chick cells. Restriction fragment length polymorphism and polymerase chain reaction methods are used to define the presence of ALVE genes; and the expression of ALVE in chicken plasma or on cells, and the susceptibility of cells to ALVE is determined by flow cytometry using a specific (R2) antibody. ADOL line 0 chickens have been selected to be free of ALVE genes, while being resistant (i.e. lack receptors to ALVE), but susceptible to exogenous ALV (i.e. ALVA, ALVB, ALVC and ALVJ). To develop improved line 0-type chickens, ADOL line 0 was outcrossed to a commercial line that had one ALVE gene and evidence for ALVE resistance. Rous sarcoma virus (RSV) challenge was used to confirm resistance of F1 chickens to ALVE, and susceptibility of F2 breeders to ALVA and ALVB using test chicks produced by matings to line 7(2). Selected F2 breeders were resistant to ALVE, but susceptible to exogenous ALVA, ALVB, ALVC and ALVJ, based on challenge tests of progeny chick cells using an enzyme-linked immunosorbent assay. The new line, 0(1), has evidence for improved egg size, productivity, fertility and hatchability. Similar procedures may be used for development of productive ALVE free chicken lines with preferred ALV susceptibility traits.     

A Th1 cell line (3E9.1) from resistant A/J mice inhibits induction of macrophage procoagulant activity in vitro and protects against MHV-3 mortality in vivo.

Induction of immune coagulants has been implicated in the pathogenesis of murine hepatitis virus strain 3 (MHV-3)-induced fulminant hepatic necrosis. Previous work from our laboratory has shown that the induction of procoagulant activity (PCA) correlates with the resistance/susceptibility to disease in inbred and recombinant inbred (RI) strains of mice. Macrophages from susceptible, but not resistant, strains of mice expressed increased levels of PCA in response to MHV-3 stimulation. T lymphocytes, however, had a marked regulatory role in the final expression of macrophage PCA. CD3+ CD4+ CD8- lymphocytes from RI H-2 compatible susceptible mice were able to instruct macrophages from susceptible mice to express significantly augmented levels of PCA, whereas CD3+ lymphocytes from RI H-2 compatible MHV-3-immunized resistant mice were able to suppress induction of PCA. In this present study, T-cell lines were derived from draining popliteal lymph nodes from resistant A/J mice, which had been immunized with MHV-3. All T-cell lines showed marked proliferation to MHV-3 and MHV-JHM which was major histocompatibility complex (MHC) restricted. All cell lines were CD3+, four of these were CD4+ and one was CD8+. All of the CD4+ cell lines produced IL-2 and two produced interferon-gamma (IFN-gamma), consistent with the Th1 cytokine profile. One cell line (3E9.1) was able to inhibit the induction of macrophage PCA through production of a soluble factor although cell-to-cell contact could not be excluded. This CD4+ T-cell line conferred protection to infected and susceptible AXB8 mice. These results demonstrate that the existence of a Th1 subpopulation of cells with a regulatory effect on macrophage PCA induction in MHV-3-infected mice contributes to the resistance of the A/J strain of mice to MHV-3 infection.     

Genetic differences in the mitogenic response of peripheral blood lymphocytes in the chicken.

The mitogenic responses of chicken peripheral blood lymphocytes were compared in two partially inbred chicken lines and in their hybrids. The lymphocytes of line P chickens showed a higher phytohaemagglutinin (PHA) response than those of line V chickens and (P X V)F1 hybrids. The Con A responses were of the same magnitude in all chicken lines. The higher PHA response in line P chickens was not sex-linked, apparently was not associated with the major histocompatibility complex and it did not correlate with immunoglobulin G allotypes.     

GENETIC DIFFERENCES IN THE MITOGENIC RESPONSE OF PERIPHERAL BLOOD LYMPHOCYTES IN THE CHICKEN

The mitogenic responses of chicken peripheral blood lymphocytes were compared in two partially inbred chicken lines and in their hybrids. The lymphocytes of line P chickens showed a higher phytohaemagglutinin (PHA) response than those of line V chickens and (P x V)F₁ hybrids. The Con A responses were of the same magnitude in all chicken lines. The higher PHA response in line P chickens was not sex-linked, apparently was not associated with the major histocompatibility complex and it did not correlate with immunoglobulin G allotypes.     

Inflammatory response of different chicken lines and B haplotypes to infection with infectious bursal disease virus.

Chickens representing two different inbred lines (layer and meat-type) and three different B haplotypes (BW1, B19 and B131) were infected with infectious bursal disease virus (IBDV) at 21 days of age. Mortality was recorded, and surviving chickens were killed and examined either 3 or 17 days post-infection. Non-infected control chickens were run in parallel. The variables recorded were all significantly changed by the virus infection, including the serum concentration of mannan-binding lectin that was increased 2-fold 3 days post-infection. A significantly increased mortality, liver to body weight ratio and erythrocyte sedimentation rate (ESR) was seen among chickens from the layer type line compared with the meat-type line. In addition, the haplotype of the chickens influenced the atrophy and hypertrophy of the thymus. It was concluded that the meat-type chicken line was more resistant to IBDV infection than the layer-type line, and that mortality rate, liver to body weight ratio and ESR were valuable variables for evaluation of the level of IBDV infection-induced inflammation and disease.     

Retrospective evidence that the MHC (B haplotype) of chickens influences genetic resistance to attenuated infectious bronchitis vaccine strains in chickens.

Infectious bronchitis is a respiratory disease of chickens that is caused by the coronavirus infectious bronchitis virus (IBV). Virtually all broiler and layer breeder flocks are routinely vaccinated against IBV. Two hatches of 1-day-old chicks from four lines were mistakenly vaccinated for infectious bronchitis using a moderately attenuated vaccine designed for chicks of an older age. The vaccination resulted in high mortality, and chicks from three of four lines died with signs typical of infectious bronchitis. The mortality that occurred using this less-attenuated vaccine was significantly influenced by the genetic line, and the MHC (B) haplotype in chickens of three B congenic lines. B congenic chickens possessing the B*15 haplotype were resistant in contrast to chickens possessing the B*13 or B*21 haplotypes. Chicks from two further hatches of the four lines were vaccinated appropriately with a more attenuated IBV vaccine, and only limited chick mortality was seen. These retrospective data from two repeated hatches confirm earlier data indicating chicken genes influence resistance to IBV, and indicate for the first time that genes tightly linked to the B haplotype are relevant in resistance to IBV. Due to extenuating circumstances it was not possible to verify results with chicks from F2 matings. Factors that may enhance definition of the role of the B haplotype in immune response to IBV, and the desirability for further analysis of a B haplotype-linked influence on immunity to IBV are discussed.     

Heterologous tumor growth patterns induced in related MHC-defined chicken lines by separate isolates of an avian sarcoma virus strain

Different patterns of tumour growth resulted from inoculation of separate isolates of the Subgroup C Bratislava 77 strain (B77) of Avian Sarcoma Virus (ASV) into three closely-related inbred lines of chickens. The major genetic difference between these chicken lines is that each is homozygous for a different MHC haplotype. Since for one of the viral isolates, resistance to progressive tumour growth has been shown to be controlled by MHC-linked genes, the data presented here suggest that MHC-controlled tumour rejection operates on viral or cellular determinants different from those which define classical viral group of subgroup specificity.     

Role of B cells in the expression of genetic resistance to growth of Rous sarcoma in the chicken.

Resistance to the development of progressively growing tumors induced by Rous sarcoma virus is a dominant trait controlled by a gene linked to the major histocompatibility complex (MHC). The effect of bursectomy (Bx) on the expression of this trait was studied in two inbred lines of chickens homozygous for different MHC alleles, and which differ with respect to the gene controlling resistance to Rous tumors. The results show that Bx alters the expression of the trait, since genetically resistant birds were rendered highly susceptible to progressive tumor growth. The bursa of Fabricius thus makes an important contribution to resistance. The results do not indicate whether genetic resistance is mediated exclusively by B cells or by another bursa-dependent population.     

Very Virulent Plus Strains of MDV Induce an Acute Form of Transient Paralysis in Both Susceptible and Resistant Chicken Lines

Abstract Marek's disease (MD) is a lymphoproliferative disease of domestic chickens caused by a highly cell-associated alpha herpesvirus, Marek's disease virus (MDV). Clinical signs of MD include depression, crippling, weight loss, and transient paralysis (TP). TP is a disease of the central nervous system that affects MD-susceptible chickens 8-11 days post-infection (dpi), normally resulting in recovery 1-3?d after the onset of clinical signs. In this study we inoculated chickens from lines 7(2) (MD-susceptible) and 6(3) (MD-resistant) with a very virulent plus strain of MDV at 2?wk of age, and collected brain samples from birds with and without TP at 5, 11, and 21?dpi for gene expression profiling and histological analysis. Data revealed that chickens inoculated with MDV had higher levels of IL-6, IL-10, IL-18, IFN-α, IFN-β, IFN-γ, MHC I, and CD18 in their brains at 11?dpi compared to the uninfected control birds. In addition, the expression levels of IL-6, IL-10, IFN-α, IFN-β, and IFN-γ were significantly higher in the brains of the birds showing clinical signs of TP than in asymptomatic chickens. Comparative analysis between the two chicken lines showed that the expression levels of IL-6, IL-10, IFN-β, IFN-γ, IL-18, CD18, and MHC I were significantly higher in the brains of the birds from line 6(3) with TP than those of line 7(2) exhibiting neurological disorders. A differential expression pattern was observed for some of the tested genes at different time points post-inoculation. Histological analysis showed lymphocytic meningitis, perivascular cuffing, and neuronal degeneration within the brains of birds from both susceptible and resistant lines exhibiting TP at 11?dpi. Vaccination prevented development of TP and other MD-associated clinical symptoms. These observations are suggestive of an underlying immunological mechanism for viral-induced neurological dysfunction, and the differential responses of the two chicken lines to MDV infection.     

Major histocompatibility complex effect on cellulitis among different chicken lines

The chicken major histocompatibility complex (MHC) has been implicated in conferring resistance/susceptibility to several bacterial, parasitic, and viral diseases. Investigators have shown that the chicken MHC plays a major role in determining the outcome of a Marek's disease infection, in that standard B 13 is susceptible to the virus while B 21 confers resistance to the virus. Previous work with a broiler line has shown that B 21 is susceptible to an Escherichia coli -induced cellulitis infection and that B 13 conferred resistance to the infection. For this experiment, a broiler and a Leghorn chicken line shown to contain standard B 13 and B 21 were examined in a challenge model for cellulitis. The birds were challenged with a cellulitis-causing E. coli isolate. Homozygous B 21 had the highest incidence of cellulitis development compared with either homozygous B 13 or the heterozygous B 13 / B 21 for both the broiler and Leghorn lines. Additionally, cellulitis lesion severity was measured in both lines and shown to be independent of MHC type.     

Development of an alloantiserum (R2) that detects susceptibility of chickens to subgroup E endogenous avian leukosis virus

An alloantiserum, termed R2, specifically agglutinates red blood cells (RBC) from line 100B chickens that are susceptible to avian leukosis viruses (ALV) belonging to subgroups B and E, but does not agglutinate RBC from congenic inbred line 7(2 )chickens that are resistant to ALV B and E. The R2 antigen was also detected on lymphocytes and thrombocytes. Using chickens from a special cross, it was found that R2 reactivity requires that the chickens must: (1) be susceptible to infection by ALV-E; and (2) express a viral envelope gene with subgroup E specificity. With R2 antiserum, a nearly perfect association was observed between agglutination and susceptibility to ALV-B in F(2) chickens containing endogenous viral genes ev2 and/or ev3. These results support earlier evidence that ALV-B and ALV-E share receptors. Moreover, the R2 antiserum was shown to neutralize ALV-E. The R2 antigen showed Mendelian segregation in chickens of a commercial White Leghorn strain-cross containing ev3, ev6 and ev9. However, commercial chickens with or without the R2 antigen did not differ in susceptibility to lymphoid leukosis induction or immune response on infection with ALV of subgroup A for complex reasons we discuss.     

Effects of reticuloendotheliosis virus on the response of chickens to infectious laryngotracheitis virus

Effects of reticuloendotheliosis virus (REV) on the response to infectious laryngotracheitis virus (ILTV) were investigated in young chickens with and without maternally derived antibody (MAb) to REV. In the first experiment a group of 1-day-old chickens without REV MAb were inoculated at 1 day of age with REV whilst another group of similar chickens were left uninoculated. All chickens were vaccinated with ILTV at 7 days of age. There was a significantly higher proportion of infectious laryngotracheitis (ILT) post-vaccinal ophthalmia (p.v.o.) in the group inoculated with REV. In the second experiment chickens with and without MAb to REV were inoculated at 1 day old with REV. These chickens, together with others not inoculated with REV, were vaccinated with ILTV isolate SA-2 8 days later. A virulent ILTV isolate, G, was used to challenge all the chickens 20 days after vaccination. Again the chickens without MAb to REV inoculated with REV showed a higher proportion of ILT p.v.o. and a significantly higher mortality rate due to ILT following vaccination. In the chickens inoculated with REV at 1 day of age and not vaccinated but challenged with ILTV there was a significantly higher mortality and rate of clinical signs due to ILT in those birds without than in those with REV MAb. In both experiments chickens from REV negative parents were found to be free of REV neutralising MAb. However, only 30% of chickens originating from a flock known to be infected with REV had a titre of 1/40 or higher. In spite of this, this group was significantly more resistant than the group without REV MAb to the immunosuppressive effect of inoculation at 1 day old with REV. This was demonstrated by their lower susceptibility (i.e. less p.v.o. and mortality) to the vaccination and challenge with ILTV. Chickens without REV MAb developed neutralising antibodies within 2 weeks of inoculation with REV. Irrespective of the REV MAb status 1-day-old chickens inoculated with REV were viraemic within a week.     

Host defenses in experimental rickettsialpox: genetics of natural resistance to infection.

The genetic basis for natural resistance to lethal infection with Rickettsia akari was studied in over 25 inbred strains, inbred hybrids, and outbred stocks of mice. Inbred mice infected intraperitoneally with the Kaplan strain of R. akari demonstrated three levels of response, susceptible (C3H/HeJ), intermediate (A/HeJ, A/J, A/WySn, BALB/cDub, BALB/cJ, and SJL/J), and resistant (AKR/J, AL/N, BALB/cAnN, BALB/cNCr1BR, C3H/HeN, C57BL/6J, C57L/J, CBA/J, DBA/2J, and SWR/J). No correlation was evident between the six H-2 haplo-types tested and susceptibility to Kaplan infection. Four outbred mouse stocks, Dub: (ICR), Wrc:(ICR), Caw:(CF1), and Mai:(S) were all resistant. The F1 inbred hybrids of resistant X resistant (AKD2F1/J), resistant X intermediate (CB6F1/U), intermediate X intermediate (CAF1/J), and resistant X susceptible (C3D2F1/J) parents were all resistant. The F2 and parental backcross generations of C3H/HeJ and DBA/2J hybrids yielded ratios of resistant to susceptible mice that suggested resistance was under multigeneic control. Susceptible mice (C3H/HeJ) were capable of mounting an immune response, since prior infection with the avirulent Hartford strain of R. akari rendered them resistant to subsequent lethal challenge with the Kaplan strains.     

A high capacity in vitro assay for measuring the cytoadherence of Plasmodium falciparum-infected erythrocytes

A novel flow cytometric technique was developed to determine the absolute numbers of leukocytes of specific phenotypes in whole blood from two lines of inbred chickens (line 7(2) and line 6(1)). This single step method is rapid, accurate, repeatable, can be used in the presence of nucleated erythrocytes and addresses the problems encountered when electronically counting the numbers of leukocytes in specific subpopulations in the blood of non-mammalian species. It is superior to previous methods in that (1) peripheral blood leukocytes (PBL) do not need to be separated by density gradient centrifugation, (2) erythrocyte lysis is not necessary and (3) absolute numbers of specific phenotypes of cells are determined directly. A standard volume of diluted whole blood was added to a standard number of fluorescent beads before incubation with fluorescently-conjugated monoclonal antibodies recognising specific PBL surface antigens. Samples were analysed by flow cytometry and electronic gates were set to count a standard number of beads and the concomitant fluorescently-labelled cells. Absolute numbers of B, CD4+ and CD8+ PBL were determined. Since the bead fluorescence is constant, it was also possible to measure relative MHC class I expression using fluorescence intensity. In both lines of chickens absolute numbers of all of the phenotypes of PBL measured increased with age. Although line 7(2) chickens had greater numbers of B, CD4+, and CD8+ PBL than line 6(1) chickens, there was no significant difference in the CD4+:CD8+ PBL ratios, the T:B PBL ratios or relative MHC class I expression between the two lines. Relative MHC class I expression increased with age in both lines.     

The major histocompatibility complex of outbred chickens: II, Analysis of the typing response in mixed lymphocyte culture stimulated by homozygous typing cells

MLR phenotypes of outbred and inbred chickens typing B13 of the major histocompatibility complex (MHC) of the chicken were compared. F1 hybrids of outbred and inbred B13 positive chickens were analysed in mixed lymphocyte culture (MLC). The intermediate strength responses of cells from B13 heterozygous outbred chickens stimulated by inbred B13 homozygous chicken cells were not due to minor variations of B encoded lymphocyte activating determinants (Lads). Nor were Lads encoded by genes unlinked to the B complex responsible for these reactions. In contrast, F1 anti-parental type reactions were observed, and these alone are probably responsible for the intermediate strength reactions so often seen in typing of heterozygous outbreds with homozygous typing cells.