Early cell-mediated immune responses to Marek's disease virus in two chicken lines with defined major histocompatibility complex antigens

N2a and P2a chickens, resistant and susceptible to Marek's disease (MD), respectively, were used to examine relationships between major histocompatibility complex (MHC)-restricted cytotoxic T lymphocytes (CTL) and natural killer (NK)-like cell activity with resistance to infection with Marek's disease virus (MDV). Ten-day-old chickens were infected with MDV and euthanatized at selected times to evaluate for NK cell and MHC-restricted cytotoxicity. The N2a MDV-infected chickens had an early cell-mediated immune response characterized by a sustained NK-like cytotoxicity that coincided with a measurable MHC-cytotoxicity that was lower than controls. Although MHC-restricted and NK cell cytotoxicity was demonstrated in P2a MDV-infected chickens at 8 dpi, both abruptly decreased and remained low for the remainder of the 20-day experiment. The critical time point that may determine the resistance to MD appears to be within the first 2 weeks post-infection. Improvement of the chicken NK cell activity may be a good candidate for both selection and immunomodulation MD control programs.     

The efficacy of recombinant fowlpox vaccine protection against Marek's disease: its dependence on chicken line and B haplotype

Earlier studies have shown that the B haplotype has a significant influence on the protective efficacy of vaccines against Marek's disease (MD) and that the level of protection varies dependent on the serotype of MD virus (MDV) used in the vaccine. To determine if the protective glycoprotein gene gB is a basis for this association, we compared recombinant fowlpox virus (rFPV) containing a single gB gene from three serotypes of MDV. The rFPV were used to vaccinate 15.B congenic lines. Nonvaccinated chickens from all three haplotypes had 84%-97% MD after challenge. The rFPV containing gB1 provides better protection than rFPV containing gB2 or gB3 in all three B genotypes. Moreover, the gB proteins were critical, since the B*21/*21 chickens had better protection than chickens with B*13/*13 or B*5/*5 using rFPV with gB1, gB2, or gB3. A newly described combined rFPV/gB1gEgIUL32 + HVT vaccine was analyzed in chickens of lines 15 x 7 (B*2/*15) and N (B*21/*21) challenged with two vv+ strains of MDV. There were line differences in protection by the vaccines and line N had better protection with the rFPV/gB1gEgIUL32 + HVT vaccines (92%-100%) following either MDV challenge, but protection was significantly lower in 15 X 7 chickens (35%) when compared with the vaccine CVI988/Rispens (94%) and 301B1 + HVT (65%). Another experiment used four lines of chickens receiving the new rFPV + HVT vaccine or CVI988/Rispens and challenge with 648A MDV. The CVI 988/Rispens generally provided better protection in lines P and 15 X 7 and in one replicate with line TK. The combined rFPV/gB1gEgIUL32 + HVT vaccines protected line N chickens (90%) better than did CVI988/Rispens (73%). These data indicate that rFPV + HVT vaccines may provide protection against MD that is equivalent to or superior to CVI988/ Rispens in some chicken strains. It is not clear whether the rFPV/gB1gEgIUL32 + HVT vaccine will offer high levels of protection to commercial strains, but this vaccine, when used in line N chickens, may be a useful model to study interactions between vaccines and chicken genotypes and may thereby improve future MD vaccines.     

Natural killer cell activity in chickens exposed to Marek's disease virus: inhibition of activity in susceptible chickens and enhancement of activity in resistant and vaccinated chickens

Chickens of 2 genetic lines (lines P and N) were inoculated with a pathogenic strain of Marek's disease (MD) virus (MDV) and chronologically examined for disease response and natural killer (NK) cell expression. The NK cell reactivity was assayed in an in vitro cytotoxicity assay in which effector cells from the spleen of test chickens were reacted with 51Cr-labeled LSCC-RP9 target cells. Chickens of line P developed progressive debilitating disease and a high incidence of gross tumors and death. The NK cell reactivity of line-P chickens infected with MDV was significantly lower than that of uninfected control hatchmates. In contrast, NK cell levels were significantly elevated in MDV-inoculated line-N chickens that were resistant to MD and in chickens of lines P or N that had been inoculated with herpesvirus of turkeys (HVT). NK cell levels were also elevated in line P if chickens were vaccinated with HVT before infection with MDV. Inhibition of NK reactivity in susceptible chickens and elevation of reactivity in naturally resistant or vaccinated chickens may indicate a role for the NK cell system in regulating resistance to MD.     

Marek's disease virus infection in the brain: virus replication, cellular infiltration, and major histocompatibility complex antigen expression

Marek's disease virus (MDV) infection in the brain was studied chronologically after inoculating 3-week-old chickens of two genetic lines with two strains of serotype I MDV representing two pathotypes (v and vv+). Viral replication in the brain was strongly associated with the development of lesions. Three viral antigens (pp38, gB, and meq) were detected in the brain of infected chickens. Marked differences between v and vv+ pathotypes of MDV were identified for level of virus replication, time course of brain lesions, and expression of major histocompatibility complex (MHC) antigens. Two pathologic phenomena (inflammatory and proliferative) were detected in the brain of chickens inoculated with vv+MDV, but only inflammatory lesions were observed in those inoculated with vMDV. Inflammatory lesions, mainly composed of macrophages, CD4+ T cells, and CD8+ T cells, started at 6-10 days postinoculation (dpi) and were transient. Proliferative lesions, characterized by severe infiltrates of CD4+CD8- T cells (blasts), started at 19-26 dpi and persisted. Expression of MHC antigens in endothelial cells and infiltrating cells within the brain was influenced by MDV infection. Upregulation of MHC class II antigen occurred in all treatment groups, although it was more severe in those inoculated with vv+MDV. MHC class I antigen was downregulated only in those groups inoculated with vv+MDV. These results enhance our understanding of the nature and pattern of MDV infection in the brain and help to explain the neurovirulence associated with highly virulent MDV.     

A comparative evaluation of the protective efficacy of rMd5deltaMeq and CVI988/ Rispens against a vv+ strain of Marek's disease virus infection in a series of recombinant congenic strains of White Leghorn chickens

Marek's disease (MD) is a lymphoproliferative disease of domestic chickens caused by a highly infectious, oncogenic alpha-herpesvirus known as Marek's disease virus (MDV). MD is presently controlled by vaccination. Current MD vaccines include attenuated serotype 1 strains (e.g., CVI988/Rispens), avirulent serotype 2 (SB-1), and serotype 3 (HVT) MDV strains. In addition, recombinant MDV strains have been developed as potential new and more efficient vaccines to sustain the success of MD control in poultry. One of the candidate recombinant MDV strains, named rMd5deltaMeq, was derived from Md5, a very virulent strain of MDV lacking the MDV oncogene Meq. Our earlier reports suggest that rMd5deltaMeq provided protection equally well or better than commonly used MD vaccines in experimental and commercial lines of chickens challenged with very virulent plus (vv+) strains of MDV. In this study, maternal antibody-positive (trial 1) and negative (trial 2) chickens from a series of relatively MD resistant lines were either vaccinated with the rMd5deltaMeq or CVI988/Rispens followed by infection of a vv+ strain of MDV, 648A, passage 10. This report presents experimental evidence that the rMd5deltaMeq protected significantly better than the CVI988/Rispens (P < 0.01) in the relatively resistant experimental lines of chickens challenged with the vv+ strain of MDV. Together with early reports, the rMd5deltaMeq appeared to provide better protection, comparing with the most efficacious commercially available vaccine, CVI988/Rispens, for control of MD in lines of chickens regardless of their genetic background.     

Effects of Compound Chinese Herbal Medicinal Polysaccharides on Immune Signal Molecules Expression in Different MHC B-LβⅡ Genotypes Chickens Lymphocyte

This study was aimed to explore the effect of compound Chinese herbal medicinal polysaccharides (cCHMPS) on immunomodulatory in different MHC B-LβⅡ genotype chickens.MHC B-LβⅡ genotype was analyzed by PCR-SSCP method in 500 chickens.Collected the blood from different MHC B-LβⅡ genotype chickens,isolated peripheral blood lymphocytes,and added cCHMPS with the final concentration of 200,100,75,50,25 and 0 μg/mL cocultured for 24 h.Then the lymphocyte supernatant cAMP,cGMP,Ca²⁺,NO and iNOS content were detected by ELISA.The results showed that cCHMPS could increase cAMP,cGMP,Ca²⁺,NO and iNOS levels in chicken lymphocyte supernatant compared with control group in each MHC B-LβⅡ genotype chickens.And when the concentration of cCHMPS was 50 μg/mL,cAMP,Ca²⁺,NO and iNOS levels of AA and BC genotypes chicken lymphocyte supernatant were higher than that of the same genotype chicken,cGMP level of BC genotype chicken lymphocyte supernatant was higher than that of the same genotype chicken;When cCHMPS concentration was 75 μg/mL,cGMP level of AA genotype chicken lymphocyte supernatant was higher than that of the same genotype chicken;When cCHMPS concentration was 100 μg/mL,cAMP,cGMP,Ca²⁺,NO and iNOS levels of BB genotype chicken lymphocyte of the supernatant were higher than that of the same genotype chicken.cCHMPS could increase cAMP,cGMP,Ca²⁺,NO and iNOS levels in different MHC B-LβⅡ genotypes chickens,and the cCHMPS optimum immunomodulatory doses were different in each MHC B-LβⅡ genotypes chickens.     

Expression kinetics of chicken β2-microglobulin and Class I MHC in vitro and in vivo during Marek’s disease viral infections

Marek’s disease virus (MDV) is a highly cell-associated herpesvirus that causes a disease in chickens characterized by tumor formation and immunosuppression. The changes of major histocompatibility complex (MHC) expression in different MDV-infected cells are not completely understood. In this study, we investigated the expression of the Class I MHC and β2-microglobulin (β2m) genes in response to MDV infection at different time points by real-time PCR. In both in vitro and in vivo, the expression levels of Class I MHC and β2m genes were upregulated during early MDV infections in comparison to control cells; We also found that the expression of Class I MHC gene was downregulated in BudR (5-bromo-2′-deoxyuridine)-treated MSB1 cells at 48 h and MDV-infected chicken embryo fibroblast cells (CEF) at 120 and 168 h post infection (hpi); Furthermore, compared to control groups, Class I MHC and β2m expression levels were downregulated in peripheral blood lymphocytes (PBLC) from MDV-infected chickens at 14 and 28 days post infection (dpi); Interestingly, both Class I MHC and β2m gene expression levels increased again in PBLC from MDV RB1B-infected chickens at 35 dpi, in which MDV was in the latent or transformed infection stages. In addition, Class I MHC expression was clearly decreased in MDV-infected CEF at 120 hpi although β2m expression was significantly increased. These changes in Class I MHC and β2m gene expression might provide more insights into host-virus interaction.     

2018年中国部分地区猪瘟病毒2.1d亚型新流行株的基因组特征分析

为了解中国猪瘟病毒（classical swine fever virus，CSFV）的分子流行病学及遗传变异情况，本研究应用RT-PCR方法对2018年采集自河南、河北、山东、黑龙江和辽宁5个省份的350份疑似CSFV感染的病料进行E2和NS5B基因扩增，并对PCR产物进行测序和序列分析。结果显示，350份样品中21份为CSFV阳性，共获得14株CSFV的E2基因序列和7株CSFV的部分NS5B基因序列。E2全基因、NS5B部分基因序列分析表明，21份阳性样品均属于近年来在中国流行的CSFV 2.1d亚型，且新发现的2.1d亚型CSFV与中国较早2.1d亚型CSFV毒株间同源性差异不大，新发现的2.1d亚型CSFV分离株在E2基因的6个氨基酸（R³¹、S³⁴、W¹⁸²、K²⁰⁵、K³⁰³、A³³¹）上具有相同的分子特征，E2蛋白中15个位点上的半胱氨酸均未发生变异。韩国2.1d亚型CSFV在E2蛋白上具有3个独特的氨基酸（N⁹⁷、K¹⁵⁹、R²⁰⁵）特征，并且发现了韩国毒株YC11WB可能作为2.1b和2.1d亚型CSFV过渡毒株的证据，流行于中国和韩国的2.1d亚型CSFV可能分别来自于本国早期2.1b亚型CSFV的衍化。本研究证实，2018年中国及周边国家CSFV较为活跃，且流行毒株依然以2.1d亚型为主，为中国科学防控CSFV提供了依据。     

Effects of a second mutant allele (V199I) at the PRKAG3 (RN) locus on carcass composition in pigs

The effect of a second mutant allele (V199I, here denoted rn*) at the PRKAG3 (RN) locus on carcass composition was determined in 334 pigs, entire males and females, from crosses between Swedish Hampshire (H) and Finnish Landrace (L) (H × LH; LH × H; LH × LH). Pigs were classified according to DNA test into the following PRKAG3 genotypes: RN⁻/RN⁻ (23%), RN⁻/rn⁺ (24%), RN⁻/rn* (33%), rn⁺/rn⁺ (8%), rn⁺/rn* (9%) and rn*/rn* (2%). The pigs were slaughtered at a commercial slaughterhouse and assessed 24 h postmortem. Right sides were fabricated into primary wholesale cuts, then further processed into defatted hams and loins, and a subset of hams (n = 122) was dissected into the five major individual muscles. The genotype frequencies for the subsample were RN⁻/RN⁻ (27%), RN⁻/rn⁺ (20%), RN⁻/rn* (35%), rn⁺/rn⁺ (9%), rn⁺/rn* (8%) and rn*/rn* (1%). Weights were recorded for meat and bone in ham and loin, fat in ham, back and shoulder and the individual dissected muscles. The genotype effect was significant (P < 0.05) for estimated lean meat content and the proportions of meat and bone and fat in ham and loin (of carcass weight). Also, the content of meat and bone in ham and loin, in proportion of whole ham and loin, respectively, differed significantly (P < 0.01) between genotypes. Estimated lean meat content was highest for RN⁻/RN⁻ (63.0%) and RN⁻/rn⁺ (63.1%) and lowest in the combined group rn*/⁻ (rn⁺/rn* and rn*/rn*, 61.7%); RN⁻/rn* (62.5%) and rn⁺/rn⁺(62.1%) were intermediate. The same results were found for meat and bone in ham and loin, as a proportion of whole ham and loin, respectively. RN⁻/RN⁻ and RN⁻/rn⁺ did not differ in any trait; however, they produced carcasses with the lowest proportions of fat within loin and the major wholesale cuts (ham, loin and shoulder). The carcass percentage of meat and bone in ham was higher in the three RN⁻/ genotypes (RN⁻/RN⁻, RN⁻/rn⁺ and RN⁻/rn*, P < 0.05) than in the rn*/⁻ group, whereas rn⁺/rn⁺ did not (P > 0.05) differ from any of the other genotypes. RN⁻/rn⁺ and RN⁻/rn* had higher (P < 0.05) proportion of meat and bone in loin compared to the rn*/⁻ group. We conclude that the second mutant allele found at the PRKAG3 (RN) locus, rn*, decreased the lean meat content compared with the two other alleles (RN⁻, rn⁺). The RN⁻/RN⁻ and RN⁻/rn⁺ genotypes were leanest, followed by RN⁻/rn* and rn⁺/rn⁺, and rn⁺/rn* and rn*/rn* were the fattest.     

重组鸡白细胞介素18(rChIL-18)对MDV感染SPF鸡细胞免疫的影响

用马立克氏病病毒（MDV）感染5日龄SPF鸡后,取21日龄和35日龄鸡的淋巴细胞,运用3H-TdR掺入法检测T淋巴细胞对ConA和重组鸡白细胞介素18（rChIL-18）的反应,并用MTT法检测NK细胞和CTL对MD肿瘤细胞系CU147的杀伤活性及rChIL-18和IFN-γ对它们的作用。结果显示,SPF鸡感染MDV后淋转水平显著下降,NK细胞、CTL杀伤活性在感染后21日龄时升高,而在35日龄时NK细胞杀伤活性显著下降。rChIL-18对对照组和感染组SPF鸡的淋转水平和杀伤活性均有提高作用,同样IFN-γ也具有提高NK细胞和CTL杀伤活性的作用。     

通过雏鸡MDV攻毒试验选择抗马立克氏病亲本

对F0、F1、F2亲本经继代No.614血型基因选择后繁殖的F2、F3雏鸡,10日龄以马立克氏病强毒（MDV）攻击至60日龄的死亡率,其父母亲对No.614抗血清呈阳性反应的试验组显著低于其父母亲对此抗血清呈阴性反应的对照组。试验组25个家系中,有14个家系的F2雏鸡死亡率显著低于对照组;F3雏鸡有21个家系的死亡率低于对照组,其中8个家系具有统计意义。肿瘤发生率,以肾脏和腺胃最高,肺脏和神经组织最低。综合血型抗原检测和MDV攻毒结果,从试验组25个家系中选留强MDV抗性家系留种繁殖,继代选育。     

Serological Screening for MHC (B)-Polymorphism in Indigenous Chickens

As part of a series of studies to characterize innate and specific immune responses of indigenous chicken lines, birds from Bolivia and India were screened serologically for MHC class IV (BG) polymorphism by direct haemagglutination using haplotype-specific antisera (B2, B4, B12, B13, B14, B15, B19, B21). The sample consisted of 95 Bolivian indigenous chickens and 119 hens from the four most common North Indian 'back-yard' chicken lines: Yellow Aseel (AP), Kadaknath (KN), frizzled typed (Ff-) and naked neck (NN). Of all chickens tested, the majority were haplotyped as B2, B15, B19 and B21. Of the Bolivian chickens, 89.5% could be haplotyped: 54.9% were homozygous (including 43.3% B15), and 34.6% were heterozygous (including 15.7% B15). B2-like haplotypes were not found among the Bolivian hens, and only 3.2% of these birds showed homozygous B21-like proteins. Of the Indian hens, MHC (BG)-like proteins could be detected in 60.0% of the AP birds, 6.7% of the KN birds; 40.0% of the Ff- birds; and 10.3% of the NN birds. In these lines, a total of 40.1% (AP), 6.7% (KN), 30.1% (Ff-) and 10.3% (NN) were homozygous for the B-haplotype. Only in the AP line (19.9%), and the Ff- line (9.9%) were heterozygous B-haplotypes types found. The B2 haplotype was found in all Indian chicken lines. Most Indian birds have completely unknown haplotypes, indicating a potentially interesting genetic pool. Subgrouping the Bolivian and Indian indigenous hens into monomorphic BG populations revealed individual differences based on the B-types.     

Humoral immune responses to chicken infectious anemia virus in three strains of chickens in a closed flock

This is a comparative study on seroconversion to chicken infectious anemia virus (CIAV) in a closed flock of specific-pathogen-free chickens undergoing a natural outbreak and after vaccination of some of these flocks with a commercial, live vaccine. The N2a strain (B21B21 haplotype) had the highest seroconversion after natural infection (94%) or vaccination (100%), followed by the P2a strain (B19B19) at 75%-82% seroconversion after natural infection and 85% seroconversion after vaccination. The S13 (B13B13) chickens were 26% seropositive after natural infection and 75% seropositive after vaccination. N2a chickens with polymerase chain reaction (PCR)-positive tissues were 97% seropositive compared to 80%-83% PCR-positive and seropositive for the P2a chickens and only 8% seropositive and PCR-positive for the S13 chickens. Seroconversion occurred at or near sexual maturity after natural infection in seven flocks studied.     

Cell-mediated cytolysis of lymphoblastoid cells expressing Marek's disease virus-specific phosphorylated polypeptides.

Cell-mediated immune responses against Marek's disease virus (MDV)-antigens were examined using reticuloendotheliosis virus (REV)-transformed lymphoblastoid cell line CU91 and three cell lines derived from CU91. CU210 was established by establishing a latent MDV infection in CU91. Transfection of CU210 with pNL1, a selectable plasmid or with pNL1 and the cloned BamHI A fragment of MDV DNA resulted in the establishment of CU212 and CU211, respectively. CU211 expressed a MDV-specific phosphorylated polypeptide, while CU210 and CU212 were negative for MDV antigens. Only CU211 was lysed by MDV-specific effector cells. All cell lines were lysed by syngeneic REV-specific effector cells, although high levels of expression of the phosphorylated protein reduced the level of REV-specific lysis.     

Mortality of one-week-old chickens during naturally occurring Marek's disease virus infection

Marek's disease (MD) is a disease of chickens that occurs worldwide and has serious economic consequences. MD can present as one of several forms, with the most commonly occurring forms being the lymphoproliferative diseases. Under experimental conditions, an early mortality syndrome has been recognized following infection by some but not all strains of MD virus (MDV). This is the first report of a confirmed case of mortality due to naturally occurring MDV infection in 1-week-old, nonvaccinated, chickens. Necrotizing lesions were observed in the bursa of Fabricius, lung, duodenum, jejunum, and proventriculus, and large intranuclear inclusion bodies were a striking feature in tissues with lesions in all birds. Immunohistochemical staining for the pp38 protein of MDV revealed abundant pp38 antigen in the affected tissues, confirming the presence of MDV within the lesions. PCR yielded an amplicon with 97% homology to the meq gene of MDV. No evidence of co-infection by either of the immunosuppressive agents chicken anemia virus and infectious bursal disease virus was detected.     

氮、磷添加对高寒草原群落多样性和生物量的影响

为探讨环青海湖区域轻度退化高寒草原适宜的氮（N）、磷（P）添加量,筛选区域N,P合施的优化方案,本试验选用尿素与磷酸二铵,采用随机区组设计,设置3个氮元素水平（35,70,140 kg·hm^-2）和3个磷元素水平（26,52,65 kg·hm^-2）,氮元素和磷元素水平组合为：N1P1（35,26 kg·hm^-2）,N1P2（35,52 kg·hm^-2）,N1P3（35,65 kg·hm^-2）,N2P1（70,26 kg·hm^-2）,N2P2（70,52 kg·hm^-2）,N2P3（70,65 kg·hm^-2）,N3P1（140,26 kg·hm^-2）,N3P2（140,52 kg·hm^-2）,N3P3（140,65 kg·hm^-2）9个施肥梯度,以不施肥为对照,研究N、P合施当年和次年对该草原群落特征的影响。结果表明：N、P合施显著（P<0.05）增加禾本科、豆科植物的盖度、高度和重要值。施肥不同程度降低了高寒草原植物群落多样性,但差异性不显著（P>0.05）。施肥当年与次年地上生物量分别增产59.22%~205.57%,73.41%~180.10%,次年比当年增产2.72%~17.34%。该区域最适宜植物生长的N、P添加量分别为70~140 kg·hm^-2,26~52 kg·hm^-2。综上所述,人工施肥是天然高寒草原轻度退化草地改良一种快速有效的措施,能够有效的提高草地生物量,同时也有利于草地植被的恢复。     

Relationship between tumour development and detection of Marek's disease virus in the feather follicular epithelium of older chickens

To demonstrate the relationship between tumour development and virus replication, eight specific-pathogen-free pullets of line P2 (Group P; 14 weeks old) and five adult chickens (Group A; 96 weeks old) were inoculated with virulent Marek's disease virus (vMDV). Five chickens of Group P died or were euthanised due to moribund condition following the development of neoplastic lesions between days 53 and 91. On histopathological examination, these lesions were characterised by the proliferation of lymphoid cells of variable size. On analysis by polymerase chain reaction (PCR), the MDV meq gene was detected in Group P from day 21, and it was continuously identified in five chickens until they died or were euthanised. Abnormal signs and histopathological changes were not observed in chickens of Group A. The MDV meq gene was temporarily detected in some chickens of Group A, but it remained almost undetectable throughout the experimental period. In older chickens inoculated with vMDV, the onset of MD lymphoma development tended to be delayed as compared with the young chicks. The relationship between MD lymphoma development and virus replication in older chickens has been suggested. Our data might indicate the underlying existence of an age-related resistance to vMDV challenge.     

Immune responses associated with progression and control of infection in calves experimentally challenged with Mycobacterium avium subsp. paratuberculosis

Cells expressing CD4, CD8, major histocompatibility complex (MHC) Class II, and macrophage biomarkers in lungs of chickens were quantified by measuring total area of antigen expressed using imageJ, a software program developed at the National Institutes of Health and available at no cost. The procedures reported here were rapid, and reproducible. Total area of antigen expressed had positive correlation with manual counts of cells expressing CD4 and CD8 biomarkers after inoculation with serotype 1 Marek's disease virus (MDV) vaccines. Visual inspection and overlays prepared from outlines of cells counted by imageJ confirmed agreement between antigen expression and area measured. Total area measured was not dependent on time of image acquisition from randomly selected fields from the same slides. Total area values were not computer specific, but acquisition of the original images required standardization of microscope used and camera setup. All steps in the process from sample collection through sectioning, staining, and image acquisition must be standardized as much as possible. Chickens infected with a very virulent+ (vv(+)) isolate of MDV (648A) had increased CD4, CD8, MHC Class II, and macrophage biomarker expression compared to noninfected control chickens at 10 days post infection, but variable responses depending on the specific biomarker measured at 3 and 5 days post infection. The procedure described here is faster and more reproducible than manual counting in cases (CD4 and CD8) where the number of positive cells is low enough for manual counts. Manual counting is not possible with MHC Class II and macrophage antigens nor when CD4(+) cells are present in large numbers following proliferation to tumors, thus subjective systems are used for scoring in these conditions. Using imageJ as described eliminates the need for subjective and less reproducible methods for measuring expression of these antigens.     

Early posthatch protection against Marek's disease in chickens vaccinated in ovo with a CVI988 serotype 1 vaccine

CVI988, a serotype 1 Marek's disease virus (MDV), was used as an in ovo vaccine in specific-pathogen-free chickens to determine if this virus induces early posthatch protection against Marek's disease as has been shown previously for turkey herpesvirus. MDV CVI988 was injected at embryonation day (ED) 17 (group 1) or at hatch (group 2). A third group (group 3) was left unvaccinated. At 1, 2, 3, 4, 5, and 7 days of age, chickens from each group were sampled and examined as follows: a) single-cell suspensions of spleen were inoculated onto chicken embryo fibroblast monolayers to isolate the virus; b) sections of bursal tissues were stained by indirect immunofluorescence assays with anti-pp38 monoclonal antibody to identify viral antigen expression; and c) chickens were exposed intra-abdominally to MDV RB1B, a virulent serotype 1 MDV. Results revealed that in chickens given MDV CVI988 at ED 17, virus and virus-encoded protein were not detected until chickens were 3 and 2 days old after hatching, respectively. Results also indicated that during the first 4 days after hatch, the chickens given MDV CVI988 at ED 17 were better protected against virulent MDV than those given MDV CVI988 at hatch (P < or = 0.001). These results suggested that MDV CVI988 proteins were adequately expressed in the embryo to initiate prehatch immunologic response. Additional efforts with more sensitive techniques than used in this study are needed to identify the nature of viral expression in embryos.