Brazilian avian metapneumovirus subtypes A and B: experimental infection of broilers and evaluation of vaccine efficacy

Santos M.B., Martini M.C., Ferreira H.L., Silva L.H.A., Fellipe P.A., Spilki F.R. & Arns C.W. 2012. Brazilian avian metapneumovirus subtypes A and B: experimental infection of broilers and evaluation of vaccine efficacy. Pesquisa Veterinaria Brasileira 32(12):1257-1262. Laboratorio de Virologia, Instituto de Biologia, Universidade Estadual de Campinas, Rua Monteiro Lobato s/n, Cx. Postal 6109, Campinas, SP 13083-970, Brazil. E-mail: arns@unicamp.br Avian metapneumovirus (aMPV) is a respiratory pathogen associated with the swollen head syndrome (SHS) in chickens. In Brazil, live aMPV vaccines are currently used, but subtypes A and, mainly subtype B (aMPV/A and aMPV/B) are still circulating. This study was conducted to characterize two Brazilian aMPV isolates (A and B subtypes) of chicken origin. A challenge trial to explore the replication ability of the Brazilian subtypes A and B in chickens was performed. Subsequently, virological protection provided from an aMPV/B vaccine against the same isolates was analyzed. Upon challenge experiment, it was shown by virus isolation and real time PCR that aMPV/B could be detected longer and in higher amounts than aMPV/A. For the protection study, 18 one-day-old chicks were vaccinated and challenged at 21 days of age. Using virus isolation and real time PCR, no aMPV/A was detected in the vaccinated chickens, whereas one vaccinated chicken challenged with the aMPV/B isolate was positive. The results showed that aMPV/B vaccine provided a complete heterologous virological protection, although homologous protection was not complete in one chicken. Although only one aMPV/B positive chicken was detected after homologous vaccination, replication in vaccinated animals might allow the emergence of escape mutants.

Rapid sample preparation procedure for As speciation in food samples by LC-ICP-MS

This paper describes a rapid method for arsenic (As) speciation by LC-ICP-MS in several types of food samples. Prior to analysis, samples were milled and the As species extracted from biological tissues by sonication in only 2 min with a solution containing MeOH (10%, v/v) plus HNO3 (2%, v/v). As species were separated by LC using an anion exchange column. Method detection limits for AsB, As3+,DMA, MMA and As5+ were 1.3, 0.9, 0.6, 0.7 and 0.8 ng g(-1), respectively. Method accuracy and precision were traceable to Certified Reference Materials SRM1577 bovine liver from the National Institute of Standards and Technology, CE278 mussel tissue from the Institute of Reference Materials and Measurements and DOLT-3 dogfish liver tissue and DORM-3 fish protein from the National Research Council of Canada. Finally, the method was applied to speciate As in food samples (egg, fish muscle, beef and chicken) purchased in Brazilian markets.

Serologic and molecular diagnostic and bioassay in mice for detection of Toxoplasma gondii in free ranges chickens from Pantanal of Mato Grosso do Sul

Holsback L., Pena H.F.J., Ragozo A., Lopes E. G., Gennari S. M. & Soares R. M. 2012. Serologic and molecular diagnostic and bioassay in mice for detection of Toxoplasma gondii in free range chickens from Pantanal of Mato Grosso do Sul. Pesquisa Veterinaria Brasileira 32(8): 721-726. Setor de Veterinaria e Producao Animal, Universidade Estadual do Norte do Parana, Campus Luiz Meneghel, Rodovia BR 369 Km 54, Bandeirantes, PR 86360-000, Brazil. E-mail: lhsfertonani@uenp.edu.br The aim of this study was to investigate the occurrence of Toxoplasma gondii and compare the results obtained in the Modified Agglutination Test (MAT), Polimerase Chain Reaction (PCR) and bioassay in mice. In order to accomplish this, 40 free-range chickens from eight farms in neighboring areas to the Pantanal in Nhecolandia, Mato Grosso do Sul, were euthanized and blood samples, brain and heart were collected. The occurrence of anti-T. gondii antibodies found in chickens was 67.5% (27 samples), considering as a cutoff point the dilution 1:5. Among the samples analyzed, 7 (25.9%) were positive in the dilution 1: 5, 3 (11.1%) in 1: 10, 2 (7.4%) in 1: 20, 3 (11.1%) in 1: 320, 1 (3.7%) in 1: 640, 3 (11.1%) in 1: 1280, 2 (7.4%) in 1: 2560, 4 (14.8%) in 1: 5120 and 2 (7.4%) in 1: 10.240. From the mixture of tissue samples (brain and heart) from the chickens analyzed, 16 (40%) presented electrophoretic bands compatible with T. gondii by PCR (gene B1). In the comparison of techniques, 59.26% positivity in PCR was revealed among animals that were seropositive in MAT (cutoff 1: 5). From 141 inoculated mice, six (4.44%) died of acute toxoplasmosis between 15 and 23 days after inoculation. Surviving mice were sacrificed at 74 days after inoculation, and a total of 28 cysts were found in the brains of 10 distinct groups. From the seropositive hens, 27 bioassays were performed and 11 (40.7%) isolates were obtained. A greater number of isolations happened in mice that were inoculated with tissues from chickens that had high titers for anti-T. gondii antibodies. Chronic infection in mice was observed in nine groups (33.3%) from five different properties. Among the surviving mice, 25.6% were positive for T. gondii in MAT (1: 25). From mice positive in PCR, 87.5% were also positive in MAT. Among the PCR-negative mice, 5.2% were positive for T. gondii in MAT. It can be concluded through this study that the occurrence of infecton by T. gondii in the rural properties studied was high, that PCR directed to gene B1 does not confirm the viability of the parasite, but it can be used as a screening method for the selection of chickens infected by T. gondii, that the animals with titer greater than 10 must be prioritized for the selection of animals for bioassay, since for them, the chances of isolating the parasite are greater and that seroconversion in experimentally infected mice is not a good indicator for isolating the agent.

Acute heat stress impairs performance parameters and induces mild intestinal enteritis in broiler chickens: Role of acute hypothalamic-pituitary-adrenal axis activation

Studies on the environmental consequences of stress are relevant for economic and animal welfare reasons. We recently reported that long-term heat stressors (31 +/- 1 degrees C and 36 +/- 1 degrees C for 10 h/d) applied to broiler chickens (Gallus gallus domesticus) from d 35 to 42 of life increased serum corticosterone concentrations, decreased performance variables and the macrophage oxidative burst, and produced mild, multifocal acute enteritis. Being cognizant of the relevance of acute heat stress on tropical and subtropical poultry production, we designed the current experiment to analyze, from a neuroimmune perspective, the effects of an acute heat stress (31 +/- 1 degrees C for 10 h on d 35 of life) on serum corticosterone, performance variables, intestinal histology, and peritoneal macrophage activity in chickens. We demonstrated that the acute heat stress increased serum corticosterone concentrations and mortality and decreased food intake, BW gain, and feed conversion (P < 0.05). We did not find changes in the relative weights of the spleen, thymus, and bursa of Fabricius (P > 0.05). Increases in the basal and the Staphylococcus aureus-induced macrophage oxidative bursts and a decrease in the percentage of macrophages performing phagocytosis were also observed. Finally, mild, multifocal acute enteritis, characterized by the increased presence of lymphocytes and plasmocytes within the lamina propria of the jejunum, was also observed. We found that the stress-induced hypothalamic-pituitary-adrenal axis activation was responsible for the negative effects observed on chicken performance and immune function as well as for the changes in the intestinal mucosa. The data presented here corroborate with those presented in other studies in the field of neuroimmunomodulation and open new avenues for the improvement of broiler chicken welfare and production performance.

No evidence of selenosis from a selenium-rich diet in the Brazilian Amazon

Selenium (Se) is an essential element and a well-known anti-oxidant. In the Lower Tapajos River region of the Brazilian Amazon, biomarkers of Se range from normal to very high. The local traditional diet includes important Se sources such as Brazil nuts, chicken, game meat and certain fish species. Some studies have reported alterations in keratin structure, gastrointestinal problems and paresthesia in populations with high Se intake. The objective of the present study was to evaluate cutaneous and garlic odor of the breath signs and sentinel symptoms of Se toxicity (selenosis) in relation to Se status in communities along the Tapajos River. Participants (N = 448), aged 15-87 years, were recruited from 12 communities. Se concentrations were measured in blood (B-Se) and plasma (P-Se) by ICP-MS. A nurse performed an examination of the hair, nails, skin and breath for signs of Se toxicity. Interview-administered questionnaires were used to collect information on socio-demographics, medical history and possible symptoms of Se toxicity. In this population, the median levels of B-Se and P-Se were 228.4 mu g/L (range 103.3-1500.2 mu g/L) and 134.8 mu g/L (range 53.6-913.2 mu g/L) respectively. Although B-Se and P-Se surpassed concentrations considered toxic (B-Se: 1000 mu g/L (U.S. EPA, 2002)), no dermal or breath signs or symptoms of Se toxicity were associated with the biomarkers of Se status. In the present study population, where Se intake is mostly from traditional diet, there is no evidence of selenosis. These findings support the need to re-assess Se toxicity considering factors such as the chemical form of Se exposure, route of exposure (inhaled versus ingested), co-exposures to toxic elements such as mercury. Considering the current food transition towards a western diet in the Amazon, further studies should address the possible association between high Se status and cardiometabolic health in this study population. (C) 2011 Elsevier Ltd. All rights reserved.

Description of the female and new records of Triatoma baratai Carcavallo & Jurberg, 2000 (Hemiptera: Heteroptera: Reduviidae: Triatominae) from Mato Grosso do Sul, Brazil, with a key to the species of the Triatoma matogrossensis subcomplex

Triatoma baratai Carcavallo & Jurberg, 2000, a species similar to Triatoma williami Galvao, Souza & Lima, 1967 and belonging to the T. matogrossensis subcomplex, was described based on a male specimen collected in a sylvatic environment, near a cave, in Bonito county, Bodoquena mountain range, state of Mato Grosso do Sul, Brazil. In the present work we describe the female of T. baratai, captured in a chicken house, in Nioaque county, state of Mato Grosso do Sul, Brazil. Furthermore, we recorded the occurrence of T. baratai in domiciles and peridomestic environment in another four municipalities (Bodoquena, Bela Vista, Corumba, and Miranda), extending its geographical distribution. Finally, we present a key to the species of the Triatoma matogrossensis subcomplex.

Seroprevalence and isolation of Toxoplasma gondii from free-range chickens from Espirito Santo state, southeastern Brazil

Prevalence of Toxoplasma gondii infection in 510 free-range (FR) chickens (380 from 33 small farms, and 130 from a slaughter house for FR chickens) from Espirito Santo state, southeastern Brazil, was investigated. Antibodies to T. gondii were sought using commercial indirect haemagglutination (IHAT, Imuno-HAI Toxo (R), Wama Diagnostica, Sao Paulo, Brazil, cut-off 1:16) and the modified agglutination test (MAT, cut-off 1:25) tests. Attempts were made to isolate viable T. gondii from seropositive chickens by bioassay in mice. Pooled samples of brain, heart and quadriceps muscle of one thigh (total 40 g) from 64 chickens with IHAT titers of >= 1:16 were minced, digested in pepsin and bioassayed in mice. Antibodies to T. gondii were found in 40.4% (206/510) FR chickens by IHAT (titer >= 1:16) and 38.8% (198/510) by MAT (titer >= 1:25); concordance between IHAT and MAT was 81.6% (kappa index = 0.614). Viable T. gondii was isolated (designated TgCkBr234-281) from 48 of 64(75%) seropositive (IHAT titers >= 1:32) FR chickens. Most isolates of T. gondii were virulent for mice; 100% of mice inoculated with 44 of 48 isolates died of toxoplasmosis within 30 days post inoculation (p.i). An epidemiological investigation revealed that people living in rural areas have little knowledge about the parasite and about the risk of acquiring it from raw meat. Results indicated that the locally available IHAT was useful for screening of chicken sera for T. gondii antibodies. (C) 2012 Elsevier B.V. All rights reserved.

Avian coronavirus Spike Glycoprotein Ectodomain Shows a Low Codon Adaptation to Gallus gallus with Virus-Exclusive Codons in Strategic Amino Acids Positions

This is a study on the Avian coronavirus IBV and chicken host-relationship from the codon usage point of view based on fifty-nine non-redundant IBV S1 sequences (nt 1-507) from strains detected worldwide and chicken tissue-specific protein genes sequences from IBV-replicating sites. The effective number of codons (ENC) values ranged from 36 to 47.8, indicating a high-to-moderate codon usage bias. The highest IBV codon adaptation index (CAI) value was 0.7, indicating a distant virus versus host synonymous codons usage. The ENC x GC3 % curve indicates that both mutational pressure and natural selection are the driving forces on codon usage pattern in S1. The low CAI values agree with a low S protein expression and considering that S protein is a determinant for attachment and neutralization, this could be a further mechanism besides mRNA transcription attenuation for a low expression of this protein leading to an immune camouflage.

Current perspectives in avian salmonellosis: Vaccines and immune mechanisms of protection

Salmonellosis is one of the most prevalent foodborne diseases worldwide. Food animals have been identified as reservoirs for nontyphoid Salmonella infections. in poultry, host-specific Salmonella infections cause fowl typhoid and pullorum diseases that produce economic losses in different parts of the world. Several measures have been used to prevent and control Salmonella infections in poultry, and vaccination is the most practical measure because it avoids contamination of poultry products and by-products and prevents disease in humans. Salmonella vaccines can decrease public health risk by reducing colonization and organ invasion, including invasion of reproductive tissues, and by diminishing fecal shedding and environmental contamination. We review available information on the host-specific and non-host-specific Salmonella serotypes found in poultry and the improved understanding of the pathogenesis of and immune responses to infection. We also include some approaches based on updated publications regarding killed and live attenuated vaccines and their immune mechanisms of protection.

Molecular surveillance of the Newcastle disease virus in domestic and wild birds on the North Eastern Coast and Amazon biome of Brazil

Brazil is one of the world's largest countries with a rich diversity of wildlife, including resident and migratory wild birds, which may be natural reservoirs of the Newcastle disease virus (NDV). Because Brazil is a major global exporter of chicken meat, the emergence of such a disease may have a huge negative impact not only on the economy due to trade restrictions and embargoes, but also on the quality of life of the population. Samples were collected from 1,022 asymptomatic domestic and wild birds from the Brazilian coast and the Amazon region using tracheal/cloacal swabs and tested by RT-qPCR. The results showed 7 (0.7%) birds were positive for NDV. The positive samples were then isolated in embryonated chicken eggs and their matrix protein genes were partially sequenced, revealing a low-pathogenicity NDV. This study confirms the maintenance of the velogenic-NDV free status of Brazil.

Emergence of enteric viruses in production chickens is a concern for avian health

Several viruses have been identified in recent years in the intestinal contents of chickens and turkeys with enteric problems, which have been observed in commercial farms worldwide, including Brazil. Molecular detection of these viruses in Brazil can transform to a big threat for poultry production due to risk for intestinal integrity. This disease is characterized by severely delayed growth, low uniformity, lethargy, watery diarrhea, delayed feed consumption, and a decreased conversion rate. Chicken astrovirus (CAstV), rotavirus, reovirus, chicken parvovirus (ChPV), fowl adenovirus of subgroup I (FAdV-1), and avian nephritis virus (ANV) were investigated using the conventional polymerase chain reaction (PCR) and the reverse transcription polymerase chain reaction (RT-PCR). In addition, the infectious bronchitis virus (IBV), which may play a role in enteric disease, was included. The viruses most frequently detected, either alone or in concomitance with other viruses, were IBV, ANV, rotavirus, and CAstV followed by parvovirus, reovirus, and adenovirus. This study demonstrates the diversity of viruses in Brazilian chicken flocks presenting enteric problems characterized by diarrhea, growth retard, loss weight, and mortality, which reflects the multicausal etiology of this disease

Application of chromatographic and spectroscopic techniques in the evaluation of the lipid fraction of animal products

Lipolysis and oxidation of lipids in foods are the major biochemical and chemical processes that cause food quality deterioration, leading to the characteristic, unpalatable odour and flavour called rancidity. In addition to unpalatability, rancidity may give rise to toxic levels of certain compounds like aldehydes, hydroperoxides, epoxides and cholesterol oxidation products. In this PhD study chromatographic and spectroscopic techniques were employed to determine the degree of rancidity in different animal products and its relationship with technological parameters like feeding fat sources, packaging, processing and storage conditions. To achieve this goal capillary gas chromatography (CGC) was employed not only to determine the fatty acids profile but also, after solid phase extraction, the amount of free fatty acids (FFA), diglycerides (DG), sterols (cholesterol and phytosterols) and cholesterol oxidation products (COPs). To determine hydroperoxides, primary products of oxidation and quantify secondary products UV/VIS absorbance spectroscopy was applied. Most of the foods analysed in this study were meat products. In actual fact, lipid oxidation is a major deterioration reaction in meat and meat products and results in adverse changes in the colour, flavour and texture of meat. The development of rancidity has long recognized as a serious problem during meat handling, storage and processing. On a dairy product, a vegetal cream, a study of lipid fraction and development of rancidity during storage was carried out to evaluate its shelf-life and some nutritional features life saturated/unsaturated fatty acids ratio and phytosterols content. Then, according to the interest that has been growing around functional food in the last years, a new electrophoretic method was optimized and compared with HPLC to check the quality of a beehive product like royal jelly. This manuscript reports the main results obtained in the five activities briefly summarized as follows: 1) comparison between HPLC and a new electrophoretic method in the evaluation of authenticity of royal jelly; 2) study of the lipid fraction of a vegetal cream under different storage conditions; 3) study of lipid oxidation in minced beef during storage under a modified atmosphere packaging, before and after cooking; 4) evaluation of the influence of dietary fat and processing on the lipid fraction of chicken patties; 5) study of the lipid fraction of typical Italian and Spanish pork dry sausages and cured hams.

Quality and safety of oils and fats obtained as co-products or by-products of the food chain and destined to animal production

The aim of the first part of this thesis was to evaluate the effect of trans fatty acid- (TFA), contaminant, polycyclic aromatic hydrocarbon (PAH)- and oxidation productenriched diets on the content of TFA and conjugated linoleic acid (CLA) isomers in meat and liver of both poultry and rabbit. The enriched feedings were prepared with preselected fatty co-and by-products that contained low and high levels of TFA (low, palm fatty acid distillate; high, hydrogenated palm fatty acid distillate), environmental contaminants (dioxins and PCBs) (two different fish oils), PAH (olive oil acid oils and pomace olive oil from chemical refining, for low and high levels) and oxidation products (sunflower-olive oil blend before and after frying), so as to obtain single feedings with three enrichment degrees (high, medium and low) of the compound of interest. This experimental set-up is a part of a large, collaborative European project (http://www.ub.edu/feedfat/), where other chemical and health parameters are assessed. Lipids were extracted, methylated with diazomethane, then transmethylated with 2N KOH/methanol and analyzed by GC and silver-ion TLC-GC. TFA and CLA were determined in the fats, the feedings, meat and liver of both poultry and rabbit. In general, the level of TFA and CLA in meat and liver mainly varied according to those originally found in the feeding fats. It must be pointed out, though, that TFA and CLA accumulation was different for the two animal species, as well as for the two types of tissues. The TFA composition of meat and liver changes according to the composition of the oils added to the feeds with some differences between species. Chicken meat with skin shows higher TFA content (2.6–5.4 fold) than rabbit meat, except for the “PAH” trial. Chicken liver shows higher TFA content (1.2–2.1 fold) than rabbit liver, except for the “TRANS” and “PAH” trials. In both chicken and rabbit meats, the TFA content was higher for the “TRANS” trial, followed by the “DIOXIN” trial. Slight differences were found on the “OXIDATION” and “PAH” trends in both types of meats. In both chicken and rabbit livers, the TFA content was higher for the “TRANS” trial, followed by those of the “PAH”, “DIOXIN” and “OXIDATION” trials. This trend, however, was not identical to that of feeds, where the TFA content varied as follows: “TRANS” > “DIOXIN” >“PAH” > “OXIDATION”. In chicken and rabbit meat samples, C18:1 TFA were the most abundant, followed by C18:2 TFA and C18:3 TFA, except for the “DIOXIN” trial where C18:3 TFA > C18:2 TFA. In chicken and rabbit liver samples of the “TRANS” and “OXIDATION” trials, C18:1 TFA were the most abundant, followed by C18:2 TFA and C18:3 TFA, whereas C18:3 TFA > C18:2 in the “DIOXIN” trial. Slight differences were found on the “PAH” trend in livers from both species. The second part of the thesis dealt with the study of lipid oxidation in washed turkey muscle added with different antioxidants. The evaluation on the oxidative stability of muscle foods found that oxidation could be measured by headspace solid phase microestraction (SPME) of hexanal and propanal. To make this method effective, an antioxidant system was added to stored muscle to stop the oxidative processes. An increase in ionic strength of the sample was also implemented to increase the concentration of aldehydes in the headspace. This method was found to be more sensitive than the commonly used thiobarbituric acid reactive substances (TBARs) method. However, after antioxidants were added and oxidation was stopped, the concentration of aldehydes decreased. It was found that the decrease in aldehyde concentration was due to the binding of the aldehydes to muscle proteins, thus decreasing the volatility and making them less detectable.

Effect of rearing techniques and feed composition on productive traits, bird welfare and quality of poultry products

The PhD project was focused on the study of the poultry welfare conditions and improvements. The project work was divided into 3 main research activities. A) Field evaluation of chicken meat rearing conditions kept in intensive farms. Considering the lack of published reports concerning the overall Italian rearing conditions of broiler chickens, a survey was carried out to assess the welfare conditions of broiler reared in the most important poultry companies in Italy to verify if they are in accordance with the advices given in the European proposal COM (2005) 221 final. Chicken farm conditions, carcass lesions and meat quality were investigated. 1. The densities currently used in Italy are in accordance with the European proposal COM 221 final (2005) which suggests to keep broilers at a density lower than 30-32 kg live weight/m2 and to not exceed 38-40 kg live weight/m2. 2. The mortality rates in summer and winter agree with the mortality score calculated following the formula reported in the EU Proposal COM 221 final (2005). 3. The incidence of damaged carcasses was very low and did not seem related to the stocking density. 4. The FPD scores were generally above the maximum limit advised by the EU proposal COM 221 final (2005), although the stocking densities were lower than 30-32 kg live weight per m2. 5. It can be stated that the control of the environmental conditions, particularly litter quality, appears a key issue to control the onset of foot dermatitis. B) Manipulation of several farm parameters, such litter material and depth, stocking density and light regimen to improve the chicken welfare conditions, in winter season. 1. Even though 2 different stocking densities were established in this study, the performances achieved from the chickens were almost identical among groups. 2. The FCR was significantly better in Standard conditions contrarily to birds reared in Welfare conditions with lower stocking density, more litter material and with a light program of 16 hours light and 8 hours dark. 3. In our trial, in Standard groups we observed a higher content of moisture, nitrogen and ammonia released from the litter. Therefore it can be assumed that the environmental characteristics have been positively changed by the improvements of the rearing conditions adopted for Welfare groups. 4. In Welfare groups the exhausted litters of the pens were dryer and broilers showed a lower occurrence of FPD. 5. The prevalence of hock burn lesions, like FPD, is high with poor litter quality conditions. 6. The combined effect of a lower stocking density, a greater amount of litter material and a photoperiod similar to the natural one, have positively influenced the chickens welfare status, as a matter of fact the occurrence of FPD in Welfare groups was the lowest keeping the score under the European threshold of the proposal COM 221 final(2005). C) The purpose of the third research was to study the effect of high or low stocking density of broiler chickens, different types of litter and the adoption of short or long lighting regimen on broiler welfare through the evaluation of their productivity and incidence of foot pad dermatitis during the hot season. 1. The feed efficiency was better for the Low Density than for High Density broilers. 2. The appearance of FPD was not influenced by stocking density. 3. The foot examination revealed that the lesions occurred more in birds maintained on chopped wheat straw than on wood shaving. 4. In conclusion, the adoptions of a short light regimen similar to that occurring in nature during summer reduces the feed intake without modify the growth rate thus improving the feed efficiency. Foot pad lesion were not affected neither by stocking densities nor by light regimens whereas wood shavings exerted a favourable effect in preserving foot pad in good condition. D) A study was carried out to investigate more widely the possible role of 25-hydroxycholecalciferol supplemented in the diet of a laying hen commercial strain (Lohmann brown) in comparison of diets supplemented with D3 or with D3 + 25- hydroxycholecalciferol. Egg traits during a productive cycle as well as the bone characteristics of the layers have been as well evaluated to determine if there the vitamin D3 may enhance the welfare status of the birds. 1. The weight of the egg and of its components is often greater in hens fed a diet enriched with 25-hydroxycholecalciferol. 2. Since eggs of treated groups are heavier and a larger amount of shell is needed, a direct effect on shell strength is observed. 3. At 30 and at 50 wk of age hens fed 25 hydroxycholecalciferol exhibited greater values of bone breaking force. 4. Radiographic density values obtained in the trial are always higher in hens fed with 25-hydroxycholecalciferol of both treatments: supplemented for the whole laying cycle (25D3) or from 40 weeks of age onward (D3+25D3).

Analysis of the influence of epitope flanking regions on MHC class I restricted antigen presentation

Peptides presented by MHC class I molecules for CTL recognition are derived mainly from cytosolic proteins. For antigen presentation on the cell surface, epitopes require correct processing by cytosolic and ER proteases, efficient TAP transport and MHC class I binding affinity. The efficiency of epitope generation depends not only on the epitope itself, but also on its flanking regions. In this project, the influence of the C-terminal region of the model epitope SIINFEKL (S8L) from chicken ovalbumin (aa 257-264) on antigen processing has been investigated. S8L is a well characterized epitope presented on the murine MHC class I molecule, H-2Kb. The Flp-In 293Kb cell line was transfected with different constructs each enabling the expression of the S8L sequence with different defined C-terminal flanking regions. The constructs differed at the two first C-terminal positions after the S8L epitope, so called P1’ and P2’. At these sites, all 20 amino acids were exchanged consecutively and tested for their influence on H-2Kb/S8L presentation on the cell surface of the Flp-In 293Kb cells. The detection of this complex was performed by immunostaining and flow cytometry. The prevailing assumption is that proteasomal cleavages are exclusively responsible for the generation of the final C-termini of CTL epitopes. Nevertheless, recent publications showed that TPPII (tripeptidyl peptidase II) is required for the generation of the correct C-terminus of the HLA-A3-restricted HIV epitope Nef(73-82). With this background, the dependence of the S8L generation on proteasomal cleavage of the designed constructs was characterized using proteasomal inhibitors. The results obtained indicate that it is crucial for proteasomal cleavage, which amino acid is flanking the C-terminus of an epitope. Furthermore, partially proteasome independent S8L generation from specific S8L-precursor peptides was observed. Hence, the possibility of other existing endo- or carboxy-peptidases in the cytosol that could be involved in the correct trimming of the C-terminus of antigenic peptides for MHC class I presentation was investigated, performing specific knockdowns and using inhibitors against the target peptidases. In parallel, a purification strategy to identify the novel peptidase was established. The purified peaks showing an endopeptidase activity were further analyzed by mass spectrometry and some potential peptidases (like e.g. Lon) were identified, which have to be further characterized.