Cellular and cytokine responses in feathers of chickens vaccinated against Marek's disease

In Marek's disease virus infection, feather follicle epithelium (FFE) constitutes the site of formation of infectious virus particles and virus shedding. The objective of this study was to characterize cellular and cytokine responses as indicators of cell-mediated immune response in FFE and associated feather pulp following immunization against Marek's disease. Analysis of feather tips collected between 4 and 28 days post-immunization (d.p.i.) from chickens vaccinated post-hatch with either CVI988/Rispens or herpesvirus of turkeys revealed that replication of these vaccine viruses started at 7d.p.i., peaked by 21d.p.i., and subsequently, showed a declining trend. This pattern of viral replication, which led to viral genome accumulation in feather tips, was associated with infiltration of T cell subsets particularly CD8+ T cells into the feather pulp area and the expression of cytokine genes such as interferon-gamma, which is an indication of elicitation of cell-mediated immune responses at the site of virus shedding.     

Delayed-type hypersensitivity skin responses associated with feline infectious peritonitis in two cats

Two cats previously challenge-exposed and seropositive to feline infectious peritonitis virus (FIPV) were evaluated for delayed-type hypersensitivity (DTH) skin responses to intradermal FIPV. Before testing, cat 1 (FIP-resistant) had survived a severe experimental FIPV challenge-exposure and had remained asymptomatic, whereas cat 2 (FIP-susceptible) developed acute fulminant FIP after a considerably smaller virus challenge-exposure. Cat 1 developed a focal thickened plaque at the FIPV-injected skin site at 48 hours after injection. Histological examinations of serial punch biopsies from virus-inoculated skin revealed perivascular and diffuse dermal infiltrations of macrophages, lymphocytes and polymorphonuclear leucocytes which were maximal at 48 to 72 hours after injection. In contrast, cat 2 did not react grossly and showed only very mild dermal infiltrates at 72 hours after injection. The present findings of strong DTH responses to FIPV in a resistant cat and minimal responses in a cat with acute fulminant FIP suggest that certain in vivo cellular immune reactions may be associated with disease resistance.     

Differential expression of inducible nitric oxide synthase and cytokine mRNA in chicken lines divergent for cutaneous hypersensitivity response

Phytohemagglutinin (PHA)-induced delayed-type hypersensitivity is an immunocompetent trait considered an indicator of cell-mediated immune or T-cell responses. Divergent selection was performed to generate high and low lines for response to PHA-P. Extreme-responder birds of the F2 generation in each line were used to study possible differences in macrophage activity and the associated functional genes. To evaluate macrophage activity, nitric oxide (NO) was estimated both systemically in serum and in in vitro monocyte culture. Semi-quantitative RT-PCR was used to detect the differential mRNA expression patterns of iNOS and MIP-1beta in monocyte culture, whereas T(H)1 cytokines (IL-2 and IFN-gamma) were studied in peripheral blood mononuclear cells (PBMC) at different time intervals after lipopolysaccharide (LPS) induction. The high line showed strong systemic, as well as in vitro NO production, compared to the low line, upon stimulation with NDV and LPS, similar to early and high iNOS mRNA expression. Following the pattern of iNOS gene expression, an early strong expression of cytokines with powerful iNOS-inducing action, such as IFN-gamma and the chemokine MIP-1beta, was observed in the high line. In contrast, for response to PHA-P, low expression of IL-2 was observed in the high compared to the low line. In conclusion, the study revealed that divergent selection for response to PHA-P resulted in a divergent effect on T(H)1 cell activity, resulting in altered macrophage function in chickens. Selection, based on response to PHA-P, could lead to more resistant birds or birds with an enhanced immune response.     

Effects of the lipopolysaccharide-protein complex and crude capsular antigens of Pasteurella multocida serotype A on antibody responses and delayed type hypersensitivity responses in the chicken.

The effects of the lipopolysaccharide-protein complex (LPS) and crude capsular antigen (CCA) prepared from Pasteurella multocida serotype A isolated from a duck in the Philippines, on antibody responses to sheep red blood cells (SRBC) and Brucella abortus (BA) and delayed type hypersensitivity (DTH) responses to bovine serum albumin (BSA) in the chickens were studied. Chickens injected subcutaneously with LPS and CCA at 1 and 2 weeks of age and immunized intravenously with the mixed antigens of SRBC and BA, at 3 and 4 weeks of age showed significantly increased antibody responses against both SRBC and BA, when evaluated at 7 days after each immunization. In addition, these chickens sensitized intramuscularly with the emulsion of BSA in complete Freund's adjuvant at 5 weeks of age, and then injected into the wattle with BSA at 7 weeks of age also showed significantly increased DTH responses against BSA, when evaluated at 24 and 48 hr after challenge. These results indicate that LPS and CCA of P. multocida serotype A have a property enhancing humoral and cell-mediated immune responses.     

Lymphocyte blastogenic responses to inciting food allergens in dogs with food hypersensitivity

Lymphocyte blastogenic responses against food allergens in dogs with food hypersensitivity were evaluated in this study. Eleven dogs with food hypersensitivity, based on food elimination and oral food provocation tests and allergic responses to food allergens, were examined by various tests such as intradermal testing, antigen-specific IgE testing, and lymphocyte blastogenic responses. The number and kinds of food allergens identified as positive by these tests were compared with the offending food allergens that were found in an oral food provocation test. In 9 (82%) of the 11 dogs with food hypersensitivity, there was close agreement for positive allergens between the results of lymphocyte blastogenic responses and oral food provocation test; however, there was little agreement for intradermal and IgE testing of the positive allergens with those of the oral food provocation test (11% and 31%, respectively). In the 9 dogs, the stimulation indices of lymphocyte blastogenic responses increased to 2.0-10.1 upon food provocation but decreased significantly to 0.7-1.4 upon feeding the elimination diet until clinical signs disappeared. These results indicate that lymphocyte blastogenic responses may fluctuate because of exposure to offending food allergens in dogs with food hypersensitivity. Lymphocytes reactive to food allergens may play an important role in the pathogenesis of food hypersensitivity in dogs.     

Clinical findings in 40 dogs with hypersensitivity associated with administration of potentiated sulfonamides

The purpose of this study was to summarize the clinical findings in 40 dogs with systemic hypersensitivity reactions associated with the administration of potentiated sulfonamides. Dogs ranged from 6 months to 14 years of age, with a mean of 5.7 +/- 3.2 years. Spayed female dogs were overrepresented (24 of 40, or 60% of the dogs), as were Samoyeds (3 of 40; 8%) and Miniature Schnauzers (5 of 40; 13%). Mean dosages of potentiated sulfonamides were 47.0 +/- 14.9 mg/kg/d (range, 23.4-81.4 mg/kg/d). The time from the 1st administration of the drug to the onset of the clinical signs of hypersensitivity ranged from 5 to 36 days, with a mean of 12.1 +/- 5.9 days. There was no relationship between either the dosage or type of sulfonamide given and the time to the onset of the clinical signs. Fever was the most common clinical sign observed (55% of the dogs); thrombocytopenia was 2nd (54%), and hepatopathy (28%) was 3rd. Neutropenia, keratoconjunctivitis sicca (KCS), hemolytic anemia. arthropathy, uveitis, skin and mucocutaneous lesions, proteinuria, facial palsy, suspected meningitis, hypothyroidism, pancreatitis, facial edema, and pneumonitis were also observed in some patients. Of 39 dogs with adequate follow-up, 30 (77%) recovered, whereas 8 (21%) either died or were euthanized, and 1 recovered clinically but had persistent increases in alanine aminotransferase (ALT) activity. Dogs with hepatopathy generally had a poorer prognosis (46% recovery) than dogs without hepatopathy (89% recovery; P = .0035). Sixty-three percent of the dogs with thrombocytopenia recovered, compared to 90% of the dogs without thrombocytopenia (P = .042). Recovery was not associated with sex, age, breed, or type of sulfonamide administered.     

Modulation of the cytokine responses in equine macrophages following TACE-inhibition

The detrimental effects of the pro-inflammatory cytokine TNF-alpha during equine acute abdominal disease are well known. Its pivotal role in many human diseases has led to various in-depth studies regarding its release mechanism, in particular by TNF-alpha converting enzyme (TACE). In this study we investigated the inhibitory effect of a TACE-inhibitor on cytokine release (TNF-alpha, IL-1alpha and IL-6) in three different cell models, including U937 cells, a recently established equine macrophage cell line, known as eCAS, and primary equine PBMC. The aim was to show the similarity of TNF-alpha release through the TACE mechanism in human and equine cells after stimulation with LPS. Results indicate that, by using a TACE-inhibitor, TNF-alpha, IL-1alpha and IL-6 release can be reduced in both equine cell models and achieved comparable results in the human U937 cells. These results suggest that equine TNF-alpha, like its human homologue, is released from its membrane-bound position by TACE.     

Cellular immune responses of the rat to experimental infection with Dermatophilus congolensis

The host cell-mediated immune response was examined following experimentally-induced infection of rats with Dermatophilus congolensis, the causal agent of the skin disease dermatophilosis. Mononuclear cells (MC) isolated from Wistar rats 10 days following the induction of a third infection underwent a strong and specific proliferative response, as assessed by a [3H]thymidine incorporation assay, when cultured with various concentrations of inactivated D. congolensis cocci. Using specific monoclonal antibodies in an indirect fluorescent antibody test, this in vitro response was found to be characterised by a large expansion of the W3/25 (T-helper phenotype) population to form 56% of the total. Finally, the primed and stimulated MC were assessed for their ability to produce factors capable of inhibiting macrophage migration. The culture supernatants of D. congolensis-stimulated MC from infected rats caused significant migration inhibition of normal rat peritoneal exudate cells, whilst the supernatants of similarly-stimulated MC from naive rats failed to cause significant inhibition. The results show that a MC subpopulation becomes primed following experimentally-induced infection with D. congolensis and becomes activated after subsequent, in vitro, exposure.     

Attenuated cytokine responses in porcine lymph node cells stimulated with CpG DNA are associated with low frequency of IFNalpha-producing cells and TLR9 mRNA expression

The immune stimulatory effects of synthetic CpG DNA, on porcine peripheral blood mononuclear cells (PBMC) have been reported, but little is known about CpG-induced responses in other lymphoid tissues of pigs. We investigated innate immune responses induced by CpG DNA in cells from blood, lymph nodes (LN) and spleens of pigs. Porcine PBMC and lymph node cells (LNC) were stimulated in vitro with three classes (A-, B- and C-class) of CpG oligodeoxynucleotides (ODNs), and a non-CpG control ODN. All three classes of CpG ODNs induced significant production of IFNalpha, TNFalpha, IL-1, IL-6 and IL-12 in PBMC. In contrast, in LNC, only IL-12 was stimulated by all three classes of CpG ODNs, while IFNalpha, and IL-6 were induced by A- and C-class ODNs. No TNFalpha was induced in LNC by any of the ODNs. Significant lymphocyte proliferation was induced in PBMC by all three classes of CpG ODNs and non-CpG control. However, in LNC, B- and C-class ODNs induced significant proliferation, while no proliferation was seen with A-class and non-CpG control ODN. All three classes of ODNs induced NK-like cytotoxicity in PBMC and spleen cells, but were less effective in inducing NK cytotoxicity in LNC. We then investigated the reasons for the relatively poor CpG-induced responses in LNC. Our investigations revealed that LNC had a lower frequency of IFNalpha-secreting cells and expressed low levels of TLR9 mRNA compared to PBMC. We conclude that the lower number of IFNalpha-secreting cells and receptor expression may contribute to the attenuated responses in LNC following stimulation with CpG ODN.     

Immunologic responses against hydrolyzed soy protein in dogs with experimentally induced soy hypersensitivity

OBJECTIVE: To assess whether dogs with experimentally induced type I hypersensitivity against soy protein would respond to soy hydrolysate and develop cutaneous or gastrointestinal tract reactions after intradermal and oral challenge exposure. ANIMALS: 12 na?ve Beagle pups (9 sensitized and 3 control dogs). PROCEDURE: 9 dogs were sensitized against soy protein by administration of allergens during a 90-day period. After the sensitization period, serum concentrations of soy-specific IgE were determined and an intradermal test was performed to confirm the dogs were sensitized against soy protein. An intradermal challenge test and an oral challenge test with native and hydrolyzed soy protein were conducted on 6 sensitized and 2 control dogs. RESULTS: High serum concentrations of soy-specific IgE and positive results for the intradermal test were observed for the 9 sensitized dogs after completion of the sesitization process. Sensitized dogs challenge exposed with hydrolyzed soy protein had a reduced inflammatory response after intradermal injection and no clinical response after an oral challenge exposure, compared with responses after intradermal and oral challenge exposure with native soy protein. CONCLUSIONS AND CLINICAL RELEVANCE: Soy-sensitized dogs did not respond to oral administration of hydrolyzed soy protein. Thus, hydrolyzed soy protein may be useful in diets formulated for the management of dogs with adverse reactions to food.     

Changes in blastogenic responses of lymphocytes and delayed type hypersensitivity responses after vaccination in dogs.

To clarify the immunologic effects of vaccination in dogs, we monitored total leukocyte and lymphocyte counts, humoral antibody responses, blastogenic responses of lymphocyte, and delayed type hypersensitivity (DTH) responses after vaccination. Mixed vaccines were administered on day 0 except for canine parvovirus (CPV) vaccine which was readministered on day 21. The puppy and adult dogs had a significant decrease in leukocyte and lymphocyte counts on day 7. The puppies showed a significant increase in the blastogenesis of lymphocytes after each vaccination, whereas the adult dogs had no significant changes. However, the adult dogs were divided into two groups, high responders and low responders in blastogenesis of lymphocytes. The dogs with higher or lower response in SI values on day 0 tended to show decrease or increase after the first vaccination, respectively. Since almost all dogs developed high titers of humoral antibody, it is considered that vaccination acts in an immunomodulative fashion. DTH responses to phytohemagglutinin (PHA) and CPV vaccine monitored at 0, 3, and 8 weeks after the first vaccination produced strong reactions, in particular those to CPV vaccine rose significantly after vaccination and maintained the higher responses for at least 2 months. These results suggest that DTH responses to PHA and CPV vaccine are helpful to monitoring non-specific and specific immune functions in vivo, therefore, DTH could be used as simple and rapid immunologic tests in canine practice.     

Cellular responses in the skin of merino sheep to repeated inoculation with Dermatophilus congolensis

The cellular response in the skin of Merino sheep was examined after three successive inoculations with Dermatophilus congolensis. There was a massive neutrophil influx into the infected epidermis and underlying dermis at 4-10 days after the first inoculation. A lymphocyte-macrophage response occurred at 10-12 days, followed by a plasma cell response at 14-38 days. Resolution of skin lesions after the first inoculation corresponded to the time when the plasma cell response in the skin was most intense. A second inoculation with D. congolensis, 70 days after the first, failed to produce skin lesions typical of dermatophilosis. Typical lesions of dermatophilosis did develop after a third inoculation of the same sheep 140 days after the first inoculation, but the lesions resolved in most sheep within 13 days. Dermatophilosis did not develop in some of these sheep at sites inoculated with 100-1000-fold lower infective doses of D. congolensis, whereas control sheep did develop lesions.     

Proinflammatory cytokine responses of cultured equine keratinocytes to bacterial pathogen‐associated molecular pattern motifs

Reasons for performing study: Further knowledge of equine keratinocyte physiology and keratinocyte response to various stimuli is important in developing a better understanding of disease states involving the epidermis. Objectives: To assess the inflammatory cytokine response of cultured equine keratinocytes to various pathogen‐associated molecular pattern molecules (PAMPs) from both Gram‐negative and positive bacteria likely to be present in equine sepsis. Methods: Keratinocytes were isolated from skin of 2 horses and primary cultures performed. Keratinocytes were harvested for RNA extraction after exposure to lipopolysaccharide (LPS), lipoteichoic acid (LTA), peptidoglycan (PGN), bacterial DNA (CpG), flagellin or maintained in medium (controls) for 4 or 24 h. Real time‐quantitative PCR was used to quantify interleukin‐1β (IL‐1β), interleukin‐6 (IL‐6) and CXCL8 mRNA concentrations. Results: Increases (P<0.05) in IL‐1β, IL‐6 and CXCL8 mRNA concentrations were induced by LPS exposure compared to controls. Increased mRNA concentrations of both IL‐6 and CXCL8 were also noted (vs. controls) upon exposure to flagellin. Overall, responses were greater at 4 h. No increases (P>0.05) in cytokine expression by keratinocytes were present after LTA, PGN or CpG exposure. Conclusions: Increased proinflammatory cytokine expression in response to LPS and flagellin indicate that equine keratinocytes have functional TLR4 and TLR5 receptor signalling. However, the lack of keratinocyte stimulation by PGN, LTA or CpG provides no evidence for functional TLR2, TLR9 or NOD receptor signalling. These results suggest that equine keratinocytes are more responsive to PAMPs usually associated with Gram‐negative sepsis and unresponsive to PAMPs most commonly associated with Gram‐positive sepsis. Potential relevance: The increased incidence of injury of epidermal structures in clinical cases of Gram‐negative (vs. Gram‐positive) sepsis in the horse may be due to a lack of functional TLR signalling for Gram‐positive PAMPs in the equine keratinocyte.     

A comparison of the clinical manifestations of feeding whole and hydrolysed chicken to dogs with hypersensitivity to the native protein

Twenty‐six dogs with known adverse food reactions were fed whole chicken for 14 days. From this group, 12 dogs with cutaneous manifestations following exposure to chicken meat were selected and randomly divided into two groups (n = 6). Each group was then fed hydrolysed chicken or hydrolysed soy for 14 days in a blinded crossover design with a 17‐day washout period between each diet. Assessments of a CADESI (Canine Atopic Dermatitis Extent and Severity Index) score and pruritus were performed throughout the entire study, and combined in a global score (GS). Serum was collected weekly for the measurement of chicken‐ and soy‐specific IgG and IgE. Dogs displayed the most severe clinical response when eating whole chicken compared to baseline (P < 0.001). The GS was significantly reduced in 11 of the 12 dogs when fed hydrolysed chicken were compared to those fed whole chicken (3.58 ± 2.81 versus 20.38 ± 14.65, P < 0.01). Serum immunoglobulin G and E responses were variable and did not show relationship with specific dietary exposure.     

鸡体内减毒鼠伤寒沙门氏菌的繁殖和免疫反应

初步研究了cya和crp双基因突变减毒鼠伤寒沙门氏菌(Salmonella typhimurium)在鸡体内的繁殖和免疫反应。1、4或7日龄肉鸡分别经口感染10^8或10^9CFU减毒S．typhimurium X4550，接种后3—4周鸡体内特异性细胞和体液免疫达到最高峰。4日龄口服10^9CFu组免疫效果最佳，但1日龄高剂量组表现增重降低及免疫抑制。28日龄每鸡口服攻击10^10CFU强毒S．typhimurium 50333，各免疫组保护率为100％。同时免疫鸡还能全部或部分阻止沙门氏菌在肝脏和脾脏的繁殖。接种后病毒S．typhimurium在泄殖腔的检出时间为4周左右。