Development of γδ T cell subset responses in gnotobiotic pigs infected with human rotaviruses and colonized with probiotic lactobacilli

γδ T cell responses are induced by various viral and bacterial infections. Different γδ T cells contribute to activation and regulation of the inflammatory response and to epithelial repair. How γδ T cells respond to rotavirus infection and how the colonization of probiotics influences the γδ T cell response were unknown. In this study, we evaluated by multicolor flow cytometry the frequencies and distribution of total γδ T cells and three major subsets (CD2-CD8-, CD2+CD8- and CD2+CD8+) in ileum, spleen and blood of gnotobiotic (Gn) pigs at early (3-5 days) and late phases (28 days) after rotavirus infection. The Gn pigs were inoculated with the virulent human rotavirus Wa strain and colonized with a mixture of two strains of probiotics Lactobacillus acidophilus and Lactobacillus reuteri. In na?ve pigs, the highest frequency of total γδ T cells was found in blood, followed by spleen and ileum at the early age (8-10 days old) whereas in older pigs (32 days of age) the highest frequency of total γδ T cells was found in ileum and spleen followed by blood. Rotavirus infection significantly increased frequencies of intestinal total γδ T cells and the putatively regulatory CD2+CD8+ γδ T cell subset and decreased frequencies of the putatively proinflammatory CD8- subsets in ileum, spleen and blood at post-infection days (PID) 3 or 5. The three γδ T cell subsets distributed and responded differently after rotavirus infection and/or lactobacilli colonization. The CD2+CD8+ subset contributed the most to the expansion of total γδ T cells after rotavirus infection in ileum because more than 77% of the total γδ T cells there were CD2+CD8+ cells. There was an additive effect between lactobacilli and rotavirus in inducing total γδ T cell expansion in ileum at PID 5. The overall effect of lactobacilli colonization versus rotavirus infection on frequencies of the CD2+CD8+ γδ T cell subset in ileum was similar; however, rotavirus-infected pigs maintained significantly higher frequencies of CD8- subsets in ileum than lactobacilli-colonized pigs. The dynamic γδ T cell responses suggest that γδ T cell subsets may play important roles in different stages of immune responses after rotavirus infection and probiotic colonization. The knowledge on the kinetics and distribution patterns of γδ T cell subsets in na?ve pigs and after rotavirus infection or lactobacilli colonization provides the foundation for further mechanistic studies of their functions.     

Broiler responses to supplementation of phytase and admixture of carbohydrases and protease in maize–soyabean meal diets with or without maize Distillers’ Dried Grain with Solubles

1. This experiment investigated growth performance and nutrient utilisation responses of broilers to partial replacement of maize and soyabean meal in broiler diets with 100 g kg^?1 maize Distillers’ Dried Grain with Solubles (mDDGS) as well as responses to supplementation of an admixture of carbohydrases and protease (XAP) or phytase individually or in combination in the diets.

2. A total of 288 one-day-old broilers were allocated to 8 treatments in a randomised complete block design and a 2 × 2 × 2 factorial arrangement of treatments. The three factors were two levels each of mDDGS (0 or 100 g kg^?1), phytase (0 or 1000 FTU kg^?1), and XAP (0 or 500 mg kg^?1).

3. Each treatment had 6 replicate cages with 6 birds per replicate cage. The control diets were formulated to meet all nutrient requirements of broilers according to National Research Council recommendations of 1994, but were marginally deficient in non-phytate P and ME.

4. Weight gain and gain:food were higher in broilers receiving diets containing mDDGS. The coefficient of apparent ileal N digestibility was lower in diets with mDDGS. Phytase increased the coefficient of apparent ileal DM digestibility in all diets.

5. Phytase improved the coefficient of the apparent total tract DM retention independently of mDDGS and tended to improve the coefficient of apparent P retention in the diets without mDDGS. The enzymes were additive in their effects in the diets with mDDGS. Overall, the results showed that adding 100 g kg^?1 mDDGS to a maize–soyabean meal diet had no negative effect on growth when energy and nutrient concentrations were similar to the maize–soyabean meal diet, and that phytase or an admixture of carbohydrases and protease individually or in combination modestly improved nutrient utilisation independently of mDDGS addition. Combination of the enzymes did not produce greater benefit than the use of phytase alone.     

Treatment of cattle with DNA-encoded Flt3L and GM-CSF prior to immunization with Theileria parva candidate vaccine antigens induces CD4 and CD8 T cell IFN-γ responses but not CTL responses

Theileria parva antigens recognized by cytotoxic T lymphocytes (CTLs) are prime vaccine candidates against East Coast fever in cattle. A strategy for enhancing induction of parasite-specific T cell responses by increasing recruitment and activation of dendritic cells (DCs) at the immunization site by administration of bovine Flt3L and GM-CSF prior to inoculation with DNA vaccine constructs and MVA boost was evaluated. Analysis of immune responses showed induction of significant T. parva-specific proliferation, and IFN-γ-secreting CD4(+) and CD8(+) T cell responses in immunized cattle. However, antigen-specific CTLs were not detected. Following lethal challenge, 5/12 immunized cattle survived by day 21, whereas all the negative controls had to be euthanized due to severe disease, indicating a protective effect of the vaccine (p<0.05). The study demonstrated the potential of this technology to elicit significant MHC class II and class I restricted IFN-γ-secreting CD4(+) and CD8(+) T cells to defined vaccine candidate antigens in a natural host, but also underscores the need to improve strategies for eliciting protective CTL responses.     

Co-incubation with IL-18 potentiates antigen-specific IFN-γ response in a whole-blood stimulation assay for measurement of cell-mediated immune responses in pigs experimentally infected with Lawsonia intracellularis

The whole-blood interferon-gamma (IFN-γ) assay is a quantitative in vitro assay for a direct read-out of Ag-specific cell-mediated immune (CMI) responses to infectious diseases. The IFN-γ assay is robust in severe intracellular infections like Brucella or mycobacteria, but more difficult to evaluate for less severe or immunocompromising infections. Here we investigated the performance of the assay when recombinant co-stimulatory cytokines IL-12 and/or IL-18 were added along with Ag or PBS to cultures of whole-blood from pigs infected with Lawsonia intracellularis. In pigs recovering from a natural infection, addition of rIL-12 or rIL-18 alone increased the Ag-specific IFN-γ release while addition of both cytokines resulted in increased IFN-γ release also in PBS cultures. In analyses after experimental infections with L. intracellularis, significant increased levels of Ag-specific IFN-γ production were measured in Ag+rIL-18 cultures from infected pigs compared to the background response in PBS+rIL-18 control samples (p<0.01) or to Ag+rIL-18 cultures from non-inoculated control pigs (p<0.05). Flow cytometry identified two lymphocyte subsets as the Ag-specific IFN-γ producers. The highest IFN-γ production was by CD4(+)CD8(+) cells while a more numerous population of CD4(-)CD8(+) cells produced lower amounts of IFN-γ in response to rIL-18 and L. intracellularis Ag.     

Is meat quality from Longissimus lumborum samples correlated with other cuts in horse meat?

The present work aims to investigate if the variation of each parameter in Longissimus lumborum muscle could correspond to the same or to a similar variation of the parameter in the other muscles. The work presents results of Pearson's correlations between Longissimus lumborum samples and other muscle samples, such as Biceps femoris, Rectus femoris, Semimembranosus, Supraspinatus and Semitendinosus in horse meat. A total of 27 male IHDH (Italian Heavy Draught Horse) breed foals were employed. They were slaughtered at 11 months of age and the above‐mentioned muscles were sampled. The Longissimus lumborum muscle showed to be representative of other muscles and of the whole carcass for some chemical parameters (moisture, protein and ash) and for some fatty acids profile patterns such as C12:0, C14:0, total monounsaturated fatty acid and polyunsaturated fatty acid, but poor correlations were recorded for intramuscular fat concentration, rheological and colorimetric parameters. Although almost all the qualitative parameters in meat are affected by the anatomical site and by the muscle, the Longissimus lumborum is often not representative in horse meat with regard to modifications of this parameters.     

Modulating immune responses with dendritic cells: an attainable goal in veterinary medicine?

Dendritic cells (DCs) are antigen presenting cells that potently modulate immune responses with varying outcomes depending on the DC sub-population involved. To understand how DC sub-types arise, it is necessary to determine which factors influence their differentiation. At least three major sub-populations of DCs have been described in mice: CD4+/CD8- "myeloid" DCs, CD4-/CD8+ "lymphoid" DCs and Langerhans cell-derived DCs. Whilst somewhat comparable populations have been described in man, in most other species very little is known. The identification of cytokines which stimulate proliferation of DC precursors, and the observation that the cytokine environment influences the phenotype and the function of the DCs that subsequently develop, has provided a useful tool for evaluating these rare cells. We describe the influence of cytokines on the phenotype of DCs generated in the rat. Using bone marrow cells as the source of precursors we generated "myeloid-type" DCs from the adherent population using granulocyte-macrophage colony stimulating factor (GM-CSF), IL-4 and Flt-3L or "lymphoid-type" DCs from the non-adherent population using cytokines which included IL-7, IL-3, SCF and TNFalpha. In order to facilitate similar approaches to the study of equine DCs we have identified the nucleotide sequence encoding GM-CSF from the m-RNA of equine PBMC stimulated with Concanavalin A, amplified the cDNA by PCR and cloned it in eukaryotic and prokaryotic expression vectors. We report on the structure and function of this molecule.     

Serotypes of Streptococcus agalactiae cultured from dairy milk samples in Québec

Streptococcus agalactiae remains an important pathogen of dairy herds in Québec, but data about antigenic characteristics of this microorganism are sparse. This study was conducted to determine the variety of S. agalactiae serotypes in dairy herds in Québec. Two hundred and ninety-five isolates cultured from the milk of individual cows from 7 regions of Québec were serotyped. Sixty-two percent of the isolates were untypable. Among the 38% of typeable isolates, serotype III was found most frequently. In conclusion, the heterogeneity found among antigenic determinants of isolates from bovine milk suggests that an immunological method for the detection of S. agalactiae performed directly on bovine milk would not be a practical approach.     

Pro-inflammatory and pro-apoptotic responses of TNF-α stimulated bovine mammary endothelial cells

Coliform mastitis may be severe in periparturient cows due to enhanced expression of pro-inflammatory cytokines that contribute to disease pathogenesis. Tumor necrosis factor (TNF)-α is implicated with the severity of coliform mastitis by provoking inflammatory responses in affected tissues. The endothelium is an integral organ in regulating inflammatory responses and loss of endothelial integrity may be fatal. Studies in humans suggest that endothelial cell apoptosis may be a consequence of TNF-α exposure and contributes to the development of sepsis, however, its impact on bovine mammary endothelial cells (BMEC) is unknown. We sought to determine the inflammatory and apoptotic responses of primary BMEC exposed to TNF-α in vitro. Stimulation of endothelial monolayers with TNF-α resulted in significant increase of toll-like receptor 4, interleukin-6 and -8, and intercellular adhesion molecule-1 and vascular cellular adhesion molecule-1 gene expression in a time-dependent manner. Caspase-8 and caspase-3 mRNA expression, as well as caspase enzyme activity, also increased significantly following TNF-α stimulation. Cell viability assessed by ATP activity and BMEC apoptosis determined by flow cytometry revealed no significant changes across time with TNF-α stimulation. Results suggest that TNF-α stimulation, at the dose used in this study, can elicit a pro-inflammatory response in BMEC, but not induce apoptosis. The impact of TNF-α on mammary vascular function and the subsequent impact on the pathophysiology of severe coliform mastitis warrant further investigation.     

Growth and meat yield responses of Ross × Ross 708 male broilers fed diets formulated with distillers dried grains with solubles varying in ether extract content and inclusion rate from 1 to 49 days of age

An experiment was conducted to evaluate the effects of diets containing low-, moderate-, or high-oil dried distillers dried grains with solubles (DDGS) included at conventional- or increased-inclusion rates fed to 1,500 Ross × Ross 708 male broilers that were assigned to 60 floor pens from 1 to 49 d of age. Three sources of DDGS had ether extract composition of 6.06, 8.80, or 11.59%, on dry matter (DM) basis, representing low-oil, moderate-oil, or high-oil DDGS, respectively. Diets were formulated to contain corn, soybean meal, animal protein meal as the primary ingredients, and 1 of the 3 DDGS sources at either 5, 7, 9, or 11% (conventional-inclusion rate) and 8, 10, 12, or 14% (increased-inclusion rate) in the starter (1 to 14 d), grower (15 to 24 d), finisher 1 (25 to 34 d), and finisher 2 (35 to 49 d) periods, respectively. Apparent ME_n (low-oil:1,975, moderate-oil: 2,644, and high-oil: 3,137 kcal/kg) and digestible amino acid (AA) values of the 3 DDGS sources were determined from previous research. No differences were detected for cumulative BW gain and feed conversion 1 to 49 d of age or meat yields at 50 d of age. Feeding broilers diets containing the low-oil DDGS source increased feed cost per BW gain and breast meat weight of $0.025/kg and $0.004/kg compared with birds fed diets containing high-oil DDGS or moderate-oil and high-oil DDGS sources, respectively. These data indicated that DDGS source and inclusion rate did not affect cumulative growth and carcass characteristics of broilers from 1 to 50 d of age but demonstrate differences in feed cost/BW gain and feed cost/breast meat weight.     

Detection of antibiotic residues and association of cefquinome residues with the occurrence of Extended-Spectrum β-Lactamase (ESBL)-producing bacteria in waste milk samples from dairy farms in England and Wales in 2011

Waste milk samples from 103 farms in England and Wales were examined for the presence of β-lactam antibiotics and ESBL-producing Enterobacteriaceae. Approximately 10 months after the initial sampling, further waste milk, environmental and faecal samples from farms shown to be positive for CTX-M Escherichia coli were investigated further. Isolates with an ESBL phenotype were tested by PCR for the presence of bla_CTX-M, bla_OXA, bla_SHV and bla_TEM genes. Isolates positive for bla_CTX-M were sequenced to determine CTX-M type. Representative isolates were further examined by PFGE, plasmid replicon typing and serotyping. Of particular interest, 21.4% of waste milk samples contained residues of the cephalosporin cefquinome, which was significantly associated with CTX-M bacteria. Such bacteria occurred in 5.8% of the waste milk samples (including 3.9% CTX-M E. coli). CTX-M types identified were 1, 14, 14b and 15, but none of the E. coli were serotype O25, the serotype of the human pandemic strain.     

Comparative responses of genetically lean and fat chickens to lysine,arginine and non‐essential amino acid supply. II. plasma amino acid responses

1. Three experiments performed to study the effects of amino acid imbalances on the growth of genetically lean (LL) and fat (FL) male chickens from 28 to 42 d of age were described by Leclercq et al. (1994). The plasma amino acid concentrations of birds on selected treatments from that paper are reported here. In experiment 1, three dietary concentrations of digestible lysine were compared (4.75, 6.75 and 7.75 g/kg). In experiment 2, two dietary concentrations of digestible arginine were compared (6.53 and 10.00 g/kg). In experiment 3, three diets were compared: a high‐protein diet (189 g CP/kg), a low‐protein diet containing added essential amino acids (144 g CP/kg), and this low‐protein diet supplemented with 40 g/kg of non‐essential amino acids (NEAA; glutamic and aspartic acids).

2. The present results are compared with two earlier reports on the same genotypes. The LL consistently had lower plasma concentrations of me‐thionine, cystine, phenlyalanine, isoleucine and valine, and higher concentrations of histidine, than the FL chickens. In 4 of 5 experiments, LL leucine concentrations were lower, and glutamic acid, tyrosine, glutamine and alanine were higher, than in the FL. The other amino acids measured; arginine, lysine, aspartic acid, glycine and serine, exhibited variable responses among the experiments.

3. When the limiting essential amino acids, lysine and arginine, were added to a deficient diet, the plasma concentration of the supplemented amino acid increased while the others remained constant or decreased.

4. When glutamic and aspartic acids were added to the low protein diet, plasma amino acid responses were similar to those of adding a limiting amino acid to a deficient diet, except that alanine exhibited a dramatic increase.

5. Although there were genotype by diet interactions for several amino acids, the interactions were caused by differences in the degree of the responses, not in their direction.

6. These results suggest that the FL and LL genotypes do not utilise various amino acids with the same efficiency and, as a consequence, the ideal profile of dietary amino acids should not be the same for both lines. The results support the hypothesis that selection for fatness and leanness changed the amino acid requirements independently of the: effects of food intake.     

Molecular identification of Cryptosporidium spp. from fecal samples of felines, canines and bovines in the state of São Paulo, Brazil

The objective of this study was to obtain information of epidemiological nature through genotypic characterization of Cryptosporidium isolates from dogs, cats and bovines from the state of São Paulo, Brazil. The extraction of DNA from oocysts was carried out and polymerase chain reaction was accomplished using specific primers to 18S rRNA gene. The amplicons were directed sequenced. Seven cat samples, nine dog samples and nine bovine samples were analysed. From the seven cat samples the genotypic analyses revealed Cryptosporidium felis in all. These were the first genotypic characterization of Cryptosporidium from domestic felines in Brazil. In nine sequenced samples from dogs, genotypic identities compatible with Cryptosporidium canis were revealed in all samples. The genotypic analyses in bovines revealed Cryptosporidium parvum in eight samples and Cryptosporidium bovis in another sample, the last one being a non-zoonotic species, not related to clinical symptoms and described for the first time in Brazil.     

A comparison of larval development and mucosal mast cell responses in worm-naïve goat yearlings,kids and lambs undergoing primary and secondary challenge with Teladorsagia circumcincta

Larval development, mucosal mast cell (MMC) and eosinophil responses in worm-nai;ve lambs, yearling goats and goat kids were compared using two different experimental challenge regimes involving oral administration of infective Teladorsagia circumcincta L(3). Experimental challenge regimes enabled primary and secondary immune responses in the two species to be compared. Goats carried higher worm burdens than lambs and there were significant differences in the stages of development attained by the larval challenge that established in the two species. Possible physiological reasons for these differences are discussed. There were also differences in the establishment and development of larvae in individual yearlings which may indicate the development of a weak age-related immune response. Quantitative analysis of MMC and globule leukocyte (GL) recruitment and functional activity in the form of mast cell-specific proteinase (MCP) production demonstrated differences between the species with goat tissues containing significantly higher numbers of GL and lower concentrations of MCP than the lambs. Quantitative analysis of blood and tissue eosinophil responses failed to demonstrate any significant differences in either species under the two challenge regimes.     

A live attenuated mutant of Salmonella Montevideo triggers IL-6, IFN-γ and IL-12 cytokines that co-related with humoral and cellular immune responses required for reduction of challenge bacterial load in experimental chickens

A live attenuated Salmonella enterica serovar Montevideo (SM) mutant JOL1599 was constructed by deletion of virulence-associated genes. The protective efficacy and immune response profiles of chickens immunized with JOL1599 were investigated. Immunized chickens demonstrated significant increases in plasma IgG and lymphocyte proliferative responses (P ≤ 0.05). Increased levels of IL-6, INF-γ, and IL-12 were also observed. Immunized birds strongly responded to infection by rapid stimulation of a CD4+ subset of T cells. Organ bacterial recovery assay revealed a significant reduction in the challenge strain among immunized birds. Multiple doses of JOL1599 enhanced the immune responses of the birds as revealed by ascending trends of the immunological profiles. These findings indicate that immunization of chickens with JOL1599 may provide protection against Salmonella Montevideo infection via induction of IL-6, INF-γ, and IL-12 protective cytokines, which in turn triggers conducive humoral and cell-mediated immune responses.     

Does examination of fecal samples 24 hours after cestocide treatment increase the sensitivity of Anoplocephala spp. detection in naturally infected horses?

Fecal samples were examined immediately before and 24 to 48 h after cestocide treatment for a comparative detection of tapeworm-positive horses. In early winter, 17 weanlings, 20 yearlings, 15 2-year-old horses, 24 breeding mares, and 2 stallions were treated with praziquantel in combination with a macrocyclic lactone. The horses were presumed to be naturally infected with tapeworms after pasture grazing. Fecal samples were collected before treatment (Day 0), at 24 or 48 h after treatment (Day 1-2), and 16 to 21 d after treatment (Day 16-21). A Wisconsin test was done on all fecal samples. Odds of detection of infection for all age groups increased by a factor of 2.04 [95% confidence interval (CI): 1.30 to 3.20] from Day 0 to Day 1-2 (P = 0.002).     

Impact of genotype 1 and 2 of porcine reproductive and respiratory syndrome viruses on interferon-α responses by plasmacytoid dendritic cells

Porcine reproductive and respiratory syndrome (PRRS) virus (PRRSV) infections are characterized by prolonged viremia and viral shedding consistent with incomplete immunity. Type I interferons (IFN) are essential for mounting efficient antiviral innate and adaptive immune responses, but in a recent study, North American PRRSV genotype 2 isolates did not induce, or even strongly inhibited, IFN-α in plasmacytoid dendritic cells (pDC), representing “professional IFN-α-producing cells”. Since inhibition of IFN-α expression might initiate PRRSV pathogenesis, we further characterized PRRSV effects and host modifying factors on IFN-α responses of pDC. Surprisingly, a variety of type 1 and type 2 PRRSV directly stimulated IFN-α secretion by pDC. The effect did not require live virus and was mediated through the TLR7 pathway. Furthermore, both IFN-γ and IL-4 significantly enhanced the pDC production of IFN-α in response to PRRSV exposure. PRRSV inhibition of IFN-α responses from enriched pDC stimulated by CpG oligodeoxynucleotides was weak or absent. VR-2332, the prototype genotype 2 PRRSV, only suppressed the responses by 34%, and the highest level of suppression (51%) was induced by a Chinese highly pathogenic PRRSV isolate. Taken together, these findings demonstrate that pDC respond to PRRSV and suggest that suppressive activities on pDC, if any, are moderate and strain-dependent. Thus, pDC may be a source of systemic IFN-α responses reported in PRRSV-infected animals, further contributing to the puzzling immunopathogenesis of PRRS.     

Effects of single or trickle Haemonchus contortus experimental infection on digestibility and host responses of naïve Creole kids reared indoor

The objective of this study was to compare the effects of the type of Haemonchus contortus experimental infection (trickle infection, TI versus single infection, SI) on feed intake, nutrients digestibility, parasitological and haematological measures, and plasma leptin in Creole kids. The animals were infected over 2 periods (challenge 1 and challenge 2) of 6 weeks each, corresponding respectively to the primary and the secondary infection. Periods prior infection (1 week each) were considered as controls. The primary infection was realized with 35 Creole kids (18.40 ± 3.76 kg BW) housed in individual boxes and fed a hay-based diet. The secondary infection continued with 29 kids (21.90 ± 3.40 kg BW) from the initial 35. A total of 6 kids and 8 kids were slaughtered for measuring nematode burden at the end of the primary and the secondary infection, respectively. Measurements of nutrients digestibility were made at 0, 3 and 5 weeks post-infection for both challenges. Faecal egg count (FEC), blood eosinophilia and packed cell volume (PCV) were monitored weekly. Feed intake (dry matter intake, DMI) and nutrients digestibility were negatively affected by H. contortus infection only during the primary infection. Plasma leptin changed significantly over time (P = 0.0002) but was not affected by the infection type. Effect of infection type was observed only on crude protein digestibility during the primary infection, which was lower in the TI group (P < 0.01). The overall level of blood eosinophilia was significantly higher in the TI group (P < 0.0001) during both challenges. The overall FEC mean was significantly higher in the SI compared with the TI groups, during both challenges (P < 0.02). These results were related to the mean female length significantly higher in the SI group compared with the TI group during challenge 1 (P = 0.004), and the number of adult nematode significantly lower in the TI group compared with the SI group during the challenge 2 (P = 0.05). The results showed that the response of Creole kids to H. contortus experimental infection was in part dependent on the type of experimental infection. Our data suggest that plasma leptin would not be involved in the response of Creole kids against H. contortus infection, as no relationship between its plasma level and the transient reduction in voluntary feed intake observed in both groups during the primary infection was observed.