Immune response to Cystoisospora suis in piglets: local and systemic changes in T-cell subsets and selected mRNA transcripts in the small intestine.

Infections of neonatal piglets with Cystoisospora suis are responsible for substantial economic losses in pig production. To investigate kinetics of T-cell populations, which are possibly involved in this infection, lymphocytes from blood, spleen, mesenteric lymph nodes and the jejunal mucosa of infected and noninfected piglets were investigated by flow cytometry and immunohistochemistry at five time points during the acute phase of primary infection. Additionally, mRNA expression levels of pattern recognition receptors and immunomodulatory cytokines in the jejunum were investigated. T-cell receptor-γδ(+) T cells were found to be increased in the gut mucosa 4 days after infection and were most likely involved in the primary local immune response. Five to eleven days later, cytotoxic T cells peaked in this location, which was preceded by an expansion of this lymphocyte population in the mesenteric lymph nodes. In intestines of infected piglets, mRNA expressions of TLR-2, NOD2 and TNF-α were significantly upregulated, suggesting an involvement in parasite recognition, immune response and possibly also in immunopathology. Taken together, this study identifies cellular and molecular players involved in the early immune responses against C. suis, but their precise role in the pathogenesis and control of this neonatal disease requires further investigation.

Bone and cellular immune system of multiparous sows are insensitive to ovariectomy and nutritive calcium shortage.

Research in osteoporosis, which is a complex systemic disease, demands suitable large animal models. In pigs, most research has been done in growing minipigs, which probably are not ideal models for postmenopausal osteoporosis. Therefore, our aim was to analyze the effects of ovariectomy (OVX) and nutritive calcium shortage on multiparous Large White sows. 32 animals were randomly assigned to 4 groups in a cross design with OVX vs. sham and physiological calcium supplementation (0.75% calcium) vs. dietary calcium shortage (0.3% calcium). The observation period was 10 months with blood sampling every 2 months for hematological, immunological, and biochemical bone marker measurements. At the termination of the experiment, animals were sacrificed. Samples of trabecular bone of distal radius, proximal tibia, and sixth lumbar vertebra were subjected to micro-computed tomography imaging and ashed afterwards. Dual X-ray absorptiometry scans of the proximal femora were performed with prepared bones being placed in a water bath for mimicking soft tissue. Analyses of bone marker and cytokine profile kinetics, distribution of leukocyte subpopulations, and morphometrical and densitometrical analyses showed no evidence of any impact of OVX or calcium shortage. In conclusion, the skeleton of adult sows of a conventional breed is seemingly protected from effects of OVX and calcium shortage.

Faeces, FACS, and functional assays--preparation of Isospora suis oocyst antigen and representative controls for immunoassays.

SUMMARY: Highly purified antigen and appropriate controls are essential for antigen-specific immunoassays. In the case of Isospora suis, the causative agent of neonatal porcine coccidiosis, the only current source of antigen is oocysts isolated from faeces. The aim of this study was to develop a procedure for high-grade purification of I. suis oocysts from piglet faeces to obtain both antigen and representative controls suitable for in vitro re-stimulation of lymphocytes. This was achieved by use of filtration, density-gradient centrifugation and fluorescence-activated cell sorting (FACS). The feasibility for immunological studies was demonstrated with IFN-gamma ELISPOT assays after in vitro re-stimulation of lymphocytes from previously infected swine using the obtained antigen. The developed method allowed the production of highly purified antigen and representative controls from faeces with an oocyst recovery rate of 14%. Regarding the application of the obtained material it could be shown that lymphocytes from I. suis-infected pigs react in an antigen-specific manner in terms of an in vitro recall response by the production of IFN-gamma. This demonstrates the suitability of the developed method for the production of antigen and controls for sensitive immunological readout systems. Moreover, the detected specific IFN-gamma response encourages further functional studies on the cellular immune response to I. suis.

Changes in lymphocyte populations in suckling piglets during primary infections with Isospora suis.

Isospora suis, a common intestinal parasite of piglets, causes neonatal porcine coccidiosis, which results in reduced and uneven weaning weights and economic losses in pig production. Nevertheless, there are no detailed studies available on the immune response to I. suis. The aim of this study was to carry out phenotypical characterization of lymphocytes during primary infections on day 3 after birth. Infected and noninfected piglets were investigated between days 7 and 16 after birth. Lymphocytes from the blood, spleen and mesenteric lymph nodes (flow cytometry) and of the jejunal mucosa (immunohistochemistry) were analysed. A decrease in T cells, especially with the phenotype of resting T-helper cells, T-cell receptor-gammadelta-T cells, and regulatory T cells in the blood, spleen and mesenteric lymph nodes was noticeable. An increase in cells with the phenotype of natural killer cells in the spleen of infected animals was found, and the subset of TcR-gammadelta-T cells was strongly increased in the gut mucosa. Our findings suggest an accelerated migration of those cells into the gut. This study provides a strong indication for the involvement of adaptive and innate immune response mechanisms in the primary immune response to I. suis, especially of TcR-gammadelta-T cells as a linkage between innate and adaptive immunity.

Ornithinibacillus gen. nov., with the species Ornithinibacillus bavariensis sp. nov. and Ornithinibacillus californiensis sp. nov.

A Gram-positive, aerobic, rod-shaped, motile, endospore-forming bacterium was isolated from pasteurized milk from Bavaria, Germany. 16S rRNA gene sequence similarities indicated that strain WSBC 24001(T) was most closely related to Virgibacillus species (95.3-96.1 %), Oceanobacillus species (95.6-95.7 %), Bacillus firmus IAM 12464(T) (95.5 %) and Bacillus niacini IFO 15566(T) (95.2 %). However, strain WSBC 24001(T) showed the highest level of sequence similarity to an unnamed strain, MB-9(T) (97.6 %), which was isolated from coastal surface sediments in California. Hence, this strain was included in our study. The genomic DNA G + C contents of strains WSBC 24001(T) and MB-9(T) were 36.4 mol and 40.8 mol%, respectively. The major respiratory quinone of both strains was menaquinone MK-7 and the peptidoglycan type was A4beta (L-orn<--D-Asp). The polar lipid profiles of these strains contained a predominance of diphosphatidylglycerol and moderate to minor amounts of phosphatidylglycerol, an unknown phospholipid and an unknown aminophospholipid. However, strain WSBC 24001(T) could be distinguished from strain MB-9(T) by the presence of an unknown lipid. The fatty acid profiles of the two strains comprised mainly iso- and anteiso-branched acids, but showed some significant quantitative differences in the amounts of certain acids. The DNA-DNA relatedness value (15.5 %) clearly demonstrated that strains WSBC 24001(T) and MB-9(T) are representatives of two different species. On the basis of their phylogenetic position and morphological, physiological and chemotaxonomic properties, a novel genus is proposed, Ornithinibacillus gen. nov., with two novel species, the type species Ornithinibacillus bavariensis sp. nov. (type strain WSBC 24001(T) = DSM 15681(T) = CCM 7096(T)) and Ornithinibacillus californiensis sp. nov. (type strain MB-9(T) = DSM 16628(T) = CCM 7237(T)).