Evidence for the existence of a new genus Chlamydiifrater gen. nov. inside the family Chlamydiaceae with two new species isolated from flamingo (Phoenicopterus roseus): Chlamydiifrater phoenicopteri sp. nov. and Chlamydiifrater volucris sp. nov.

The family Chlamydiaceae currently comprises a single genus Chlamydia, with 11 validly published species and seven more taxa. It includes the human pathogens Chlamydia (C.) trachomatis, C. pneumoniae and C. psittaci, a zoonotic agent causing avian chlamydiosis and human psittacosis, as well as other proven or potential pathogens in ruminants, birds, snakes, reptiles and turtles. During routine testing of 15 apparently healthy captive flamingos in a zoo in 2011, an atypical strain of Chlamydiaceae was detected by real-time PCR of cloacal swab samples. Sequence analysis of the 16S rRNA gene revealed high similarity to the uncultured Chlamydiales bacterium clone 122, which previously had been found in gulls. As more samples were collected during annual campaigns of the flamingo ringing program in southern France from 2012 to 2015, Chlamydiaceae-specific DNA was detected by PCR in 30.9% of wild birds. From these samples, three strains were successfully grown in cell culture. Ultrastructural analysis, comparison of 16S and 23S rRNA gene sequences, whole-genome analysis based on de novo hybrid-assembled sequences of the new strains as well as subsequent calculation of taxonomic parameters revealed that the relatedness of the flamingo isolates to established members of the family Chlamydiaceae was sufficiently distant to indicate that the three strains belong to two distinct species within a new genus. Based on these data, we propose the introduction of Chlamydiifrater gen. nov., as a new genus, and Chlamydiifrater phoenicopteri sp. nov. and Chlamydiifrater volucris sp. nov., as two new species of the genus.

Experimental Infection of Goats with Mycobacterium avium subsp. hominissuis: a Model for Comparative Tuberculosis Research.

Mycobacterium avium subsp. hominissuis (MAH) is an opportunistic pathogen that causes infections in man and animals. In this study, 18 goat kids were inoculated orally with a high dose of MAH. One group of goats (n = 9) developed severe clinical disease for up to 2-3 months post inoculation (mpi). At necropsy examination, there were ulcerative and granulomatous lesions in gut-associated lymphoid tissue and granulomas with extensive necrosis in the lymph nodes (LNs) of the cranial mesenteric lymphocentre (CMLNs). Culture revealed growth of MAH in all lesions with systemic spread. A second group of goats were healthy at the end of the trial (13 mpi); however, all had extensive granulomas in the CMLNs, but no extra-intestinal spread of bacteria. Moderate faecal shedding occurred in all goats up to 2 mpi. Microscopical characterization of the granulomas revealed solid non-necrotic, necrotic, calcified and fibrocalcified granulomas with resemblance to those seen in human and bovine tuberculosis. The two different courses of disease, with highly heterogenic lesions, systemic spread in goats with severe clinical disease and the development of granulomas of all stages in the surviving goats, makes the experimental infection of goats with MAH a valuable model for tuberculosis research. This model might allow new insights into host-pathogen interaction and anti-mycobacterial compound testing.

Cellular composition of granulomatous lesions in gut-associated lymphoid tissues of goats during the first year after experimental infection with Mycobacterium avium subsp. paratuberculosis.

Mycobacterium avium subsp. paratuberculosis (MAP) causes lesions in naturally and experimentally infected ruminants which greatly differ in severity, cellular composition and number of mycobacteria. Morphologically distinct lesions are already found during the clinically inapparent phase of infection. The complex local host response and number of MAP were characterized at the initial sites of lesions, organized gut-associated lymphoid tissue, in experimentally infected goats. Tissues were collected at 3, 6, 9 and 12 month post-inoculation (mpi) from goat kids that had orally received 10 times 10mg of bacterial wet mass of MAP (JII-1961). The cellular composition of lesions in Peyer's patches in the jejunum and next to the ileocecal valve was evaluated in 21 MAP-inoculated goats, where lesions were compared with unaltered tissue of six control goats. CD68+, CD4+, CD8+, γδ T lymphocytes, B lymphocytes and plasma cells, MHC class II+ and CD25+ cells were demonstrated by immunohistochemistry in serial cryostat sections. At 3 mpi, extensive granulomatous infiltrates predominated, consisting of numerous epitheloid cells admixed with many CD4 and γδ T lymphocytes. Only single MAP were detected. This indicates a strong cellular immune reaction able to control MAP infection. γδ T lymphocytes were markedly increased in this type of lesion which may reflect their important role early in the pathogenesis of paratuberculosis. At 9 and 12 mpi, divergent lesions were observed which may reflect different outcomes of host-pathogen interactions. In five goats, minimal granulomatous lesions were surrounded by extensive lymphoplasmacytic infiltrates and no MAP were detected by immunohistochemistry. This was interpreted as effective host response that was able to eliminate MAP locally. In three goats, decreased numbers of lymphocytes, but extensive granulomatous infiltrates with numerous epitheloid cells containing increased numbers of mycobacteria were seen. This shift of the immune response resulted in uncontrolled mycobacterial multiplication. Focal and multifocal circumscribed granulomatous infiltrates of mainly epitheloid cells may represent sites of new infection, since they were observed in goats at all times after inoculation. Their presence in goats with minimal granulomatous lesions surrounded by extensive lymphoplasmacytic infiltrates may indicate that despite the local clearance, the infection may be perpetuated. The complex cellular immune reactions postulated for the pathogenesis of paratuberculosis were demonstrated at the local sites of infection. These early host-pathogen interactions are most likely essential for the eventual outcome of the MAP infection.

Sequential development of lesions 3, 6, 9, and 12 months after experimental infection of goat kids with Mycobacterium avium subsp paratuberculosis.

The development of lesions after infection with Mycobacterium avium subsp paratuberculosis (MAP) was examined in an experimental infection model. Goat kids were orally inoculated 10 times with 10 mg bacterial wet mass of MAP (total dose 2.6 × 10(8) colony-forming units). Six to 7 inoculated goats and 3 controls were autopsied 3, 6, 9, and 12 months postinoculation (mpi), lesions were documented, and samples were collected for histology, immunohistochemistry (IHC), and bacterial culture. Twenty-five of the 26 inoculated goats did not develop clinical signs. Macroscopic lesions were detected in 3 of the 7 inoculated goats as soon as 3 mpi. Jejunal Peyer's patches (JPPs) were thickened and had ulcerated surfaces and circumscribed serositis. Characteristic granulomatous infiltrates were seen in all goats in gut-associated lymphoid tissues (GALTs), especially JPPs and lymphoid tissue at the ileocecal valve and in intestinal lymph nodes. Granulomatous intestinal infiltrates not associated with GALT were seen beginning at 6 mpi and with increasing frequency thereafter. Interindividual differences in lesions were most pronounced at 12 mpi, varying from mild focal paucibacillary to severe diffuse multibacillary patterns. Bacterial culture of MAP confirmed the IHC findings but was more sensitive and revealed widespread dissemination at 3 and 12 mpi. Granulomatous arteritis was found in intestinal submucosa of several goats. This may contribute to the spreading of MAP to the intestinal wall and possibly systemically. The different lesions observed during the clinically inapparent period of paratuberculosis are most likely indicators for the later progression of infection and development of clinical disease.

Bovine tuberculosis: making a case for effective surveillance.

In 2008, a cow with marked gross lesions suspicious for bovine tuberculosis (bTB) was identified by meat inspection at home slaughtering in north-western Germany. Epidemiological investigations led to the identification of another 11 affected farms with a total of 135 animals which reacted positive to the skin test. Eight affected farms had been in trade contact with the putative index farm. While the source for the initial introduction remained unknown, it was shown that all isolates tested shared the same molecular characteristics suggesting a common source of infection. The findings demonstrate that bTB can easily be transmitted via animal trade and may remain undetected for years in herds in the absence of tuberculin testing. Hence, we believe that bTB surveillance should not rely only on meat inspection, but on a combination of both meat inspection and intradermal tuberculin testing.

Genomic and phenotypic changes of Campylobacter jejuni strains after passage of the chicken gut.

The ability to colonize the chicken gut was determined for 17 Campylobacter jejuni strains of human and bovine origin. The level of colonization varied according to the strain used for experimental infection. Two Campylobacter isolates from patients suffering from gastroenteritis were found in the group of non-colonizing strains, suggesting that other reservoirs as poultry are also important sources of human Campylobacter infections. Bovine Campylobacter isolates can also effective colonize the chicken intestine and may be a source for poultry infection. The invasion ability of the strains as determined in the cell culture model using Caco-2 cells correlates with their colonization capacity in the chicken gut. The genomic and phenotypic stability of the selected strains were evaluated by analysis of their pulsed-field gel electrophoresis (PFGE) patterns, flaA-typing and in vitro determination of motility, adhesion and invasion abilities after colonizing chickens for up to 21 days. Changes were identified in flaA-types of six isolates and three isolates from chicken showed different patterns by PFGE using SmaI or KpnI as restriction enzymes. One isolate showed phenotypic differences after in vivo passage which were seen in enhancement of adherence to eukaryotic cells, decrease of motility and changes in morphology. These phenotypic changes were not associated with the observed genomic instabilities.

Incidence of BVDV1 and BVDV2 infections in cattle submitted for necropsy in Northern Germany.

The incidence of bovine viral diarrhoea virus (BVDV) 1 and 2 infections was determined in calves, young cattle and older cattle with signs of mucosal disease (MD) submitted for necropsy to three laboratories in Northern Germany between June 2000 and May 2001. At necropsy, tonsils, retropharyngeal lymph nodes, mesenteric lymph nodes, ileal Peyer's patch and spleen were collected and examined by immunohistochemistry and virus isolation. From 311 animals examined, 30 (9.6%) were positive for BVDV. All viral isolates were typed by polymerase chain reaction after reverse transcription using species-specific primers and determined to be BVDV1. Based on the distribution of lesions and viral antigen, animals with MD, persistent infection (PI) and acute, transient infection could be distinguished. Twelve of the positive animals had characteristic signs of MD: severe diarrhoea, erosive to ulcerative lesions throughout the digestive tract and severe depletion of all lymphoid tissues. Viral antigen was present in all tissues and cell types, but particularly in depleted lymphoid follicles and altered epithelium. In seven calves, viral antigen was detectable in all tissues and cell types, but lesions were mild or missing. This is typical for PI. The remaining 11 calves most likely represent animals with acute, transient infection. Distribution of antigen was more variable, predominantly restricted to lymphoid follicles and often not seen in all tissues examined. Clinical findings were combined bronchopneumonia and enteritis. The detection of BVDV1 in young calves with pneumonia and enteritis emphasizes the importance of BVDV1 and not only BVDV2 for severe respiratory and enteric diseases of calves.

Lesions and tissue distribution of viral antigen in severe acute versus subclinical acute infection with BVDV2.

Differences in the distribution and spread of viral antigen, development of lesions and correlation between presence of viral antigen and lesions were compared between a low and highly virulent strain of BVDV2. Two groups of two-week- to two-month-old colostrum-deprived calves were inoculated intranasally with the naturally occurring low virulent BVDV2 strain 28508-5 or the highly virulent strain 1373. To study the sequence of virus spread and lesion development, calves were necropsied at days three, six, eight-nine and 12 to 14 post inoculation (pi). Viral antigen was detected by the indirect immunoperoxidase method in cryostat sections and lesions were evaluated in H&E-stained paraffin sections. Clinical signs and changes in lymphocyte and thrombocyte numbers confirmed the difference in virulence between the two strains. Both strains showed comparable initial infection and spread at day three pi. At day six pi, they were found widespread in lymphoid tissues and multifocally in intestinal mucosa. Lesions were very mild despite the large amount of antigen in the lymphoid tissues. After day six pi, differences between the low and highly virulent strains became more prominent. The strain of low virulence was cleared from the tissues, but there was a transient phase of depletion. The highly virulent strain continued to spread to different organs and there was severe depletion of lymphoid tissues without recovery.

Changes in distribution and numbers of CD4+ and CD8+ T-lymphocytes in lymphoid tissues and intestinal mucosa in the early phase of experimentally induced early onset mucosal disease in cattle.

Mucosal disease (MD), one sequelae of bovine virus diarrhoea virus (BVDV) infection, causes severe lesions in lymphoid tissues and mucosal surfaces. Lesions are associated with the presence of cytopathogenic (cp) BVDV and initially characterized by apoptotic cell death. The objective of this investigation was to determine if this cell death is mediated only by the cp BVDV, which is known to induce apoptosis in cell culture or if immune-mediated host reactions might also contribute. Early onset MD was experimentally induced in calves by inoculation of persistently viremic calves with a closely related cp BVDV. Calves were euthanized in the early phase of infection between days 5 and 13 post-inoculation and tissues from tonsils, lymph nodes, Peyer's patches, jejunum and colon were collected. Presence of cp BVDV antigen was correlated with distribution of lymphocyte subpopulations in consecutive cryostat sections. In the lymphoid tissues, cp BVDV antigen was predominantly found in the lymphoid follicles. The increase of infected cells with time post-inoculation was paralleled by a decrease of B-lymphocytes and an increase of CD4+ T-lymphocytes. An increased number of CD8+ T-lymphocytes was seen in progressed lesions only. In the intestinal mucosa, initially multifocal, later diffuse infection with cp BVDV was accompanied by a multifocal or diffuse increase of CD4+ T-lymphocytes, respectively. Numbers of IgA+ plasma cells and CD8+ T-lymphocytes were decreased. The common change observed in lymphoid tissues and mucosa was the increase of CD4+ T-lymphocytes in sites with lesions. This might indicate a cell-mediated immune response to the cp BVDV. Besides their helper function to other cells of the immune system, activated CD4+ T-lymphocytes might also exert cytotoxic activity, induce apoptosis in target cells via Fas/Fas ligand binding and thus contribute to the severity of tissue lesions in MD.

Leukopenia and thrombocytopenia in pigs after infection with bovine viral diarrhoea virus-2 (BVDV-2).

The aim of this study was to investigate whether bovine viral diarrhoea virus-2 (BVDV-2) is pathogenic for pigs, which organs become infected and whether or to which extent the virus is excreted into the environment. Ten pigs were observed for clinical reactions after infection with a BVDV-2 strain, that has been shown to be pathogenic in calves under experimental conditions. Samples were taken to monitor thrombocyte and leukocyte counts as well as antibody development. Post mortem examinations were performed at 7, 11 and 27 days after infection. Tissue samples were collected for virus isolation, histological and immunohistological examination. All ten pigs became infected and BVDV could be re-isolated from the lymphocytes, the plasma and different lymphatic organs. The infection passed clinically inapparent, apart from a slight increase in body temperature in some animals. Some animals developed a slight leukopenia and/or thrombocytopenia. There were no macroscopic or histological lesions observed that could specifically be related to the inoculation of BVDV-2. With respect to all parameters studied, the infection and the consequences thereof were clearly less pronounced in pigs as compared to cattle, the natural host. Our results indicate, that pigs infected with BVDV-2 might develop antibodies that cross-react in tests for antibodies against classical swine fever virus.

Saccharomyces boulardii and bacillus cereus var. Toyoi influence the morphology and the mucins of the intestine of pigs.

The mode of action of probiotics is still incompletely understood. To study the interactions between probiotic micro-organisms and the host their effects on morphology and mucins of the intestinal mucosa were investigated. Fifteen clinically healthy weaned pigs were divided into three groups and received either Saccharomyces boulardii or Bacillus cereus var. toyoi or were left untreated. Sections of duodenum, proximal and mid jejunum, ileum, caecum, and colon were examined. An increase of villus length in the small intestine and a decrease in the number of goblet cells with 2.6-sialylated mucins in the large intestine were observed in both treatment groups. There were no differences in crypt morphology, number of Ki67-positive cells, total number of goblet cells and number of goblet cells with acidic, neutral, sulphated, or 2.3-sialylated mucins between groups. The results indicate an effect of Saccharomyces boulardii and Bacillus cereus var. toyoi on the intestinal architecture of pigs.

Uptake of colostral leukocytes in the intestinal tract of newborn calves.

The role of colostral immunoglobulins for the protection of newborn calves has been studied extensively, but little is known about the importance of colostral leukocytes. To study the uptake of colostral leukocytes in the intestine of calves and to determine preferential sites for this uptake, FITC-labelled colostral cells derived from the respective dams were injected into intestinal loops with/without Peyer's patches of three male Holstein Frisian calves about 5h post natum. In adjacent loops, PBS was injected as control. Loops were excised after an exposure of 1.5-2h. FITC-labelled material and cells were detected by the direct immunoperoxidase method in paraplast sections. Twenty-five consecutive sections were evaluated from each localization. Uptake of labelled material and cells was observed in all three calves in the jejunal Peyer's patch and in two calves in the ileal Peyer's patch as well. In the jejunal Peyer's patch, labelled material and cells were present in epithelium, domes and sinuses around lymphoid follicles, whereas in the ileal Peyer's patch, they were found in the sinuses only. These findings confirm that uptake of colostral leukocytes through the intestinal barrier is possible and that the preferential route of uptake is through follicle-associated epithelium of Peyer's patches.

[Nerve sheath tumors in cattle: literature review and case report]. / Nervenscheidentumoren beim Rind: Schrifttumsübersicht und Fallbericht.

Incidence, clinical signs, pathomorphological findings, differential diagnosis and prognosis of bovine nerve sheath tumours are reviewed. Own clinical and pathomorphological findings are described in an eight years old cow with a benign SCHWANNoma of the eight spinal nerve.

Comparative investigation of tissue alterations and distribution of BVD-viral antigen in cattle with early onset versus late onset mucosal disease.

Tissue alterations and distribution of BVDV antigen were examined in nine cattle with early onset and five cattle with late onset mucosal disease (MD) to evaluate the possibility to differentiate between the two disease entities. MD was induced by inoculation of persistently viremic cattle with different strains of cytopathogenic BVDV. Animals which developed early onset MD became moribund approximately 2 weeks post-inoculation (pi); animals with late onset MD 42-115 days pi. All animals were euthanized and necropsied when moribund. Macroscopic lesions were found in the upper and lower digestive tract of cattle with early and late onset MD. In cattle with late onset MD, lesions in the oral cavity were generally milder and in the intestinal tract they were not only associated with GALT, but frequently affected the mucosa outside. Histologically, the abrupt changes between hyperplastic and atrophic areas of mucosa were striking in the cattle with late onset MD. This corresponded with the multifocal distribution of areas of mucosa in which intense staining for BVD-virus antigen could be demonstrated. In both courses of MD, a severe depletion of Peyer's patches was noted, but only in late onset MD, there was a complete loss of architecture. The most distinctive difference was the presence of vascular lesions which were observed in all five cattle with late onset MD, but in none of the animals with early onset MD. The vasculopathy was characterized by segmental necrosis of vascular walls and lymphohistiocytic perivasculitis in arterioles and small arteries in the submucosa of the intestine.

Ultrastructural changes in follicles of small-intestinal aggregated lymphoid nodules in early and advanced phases of experimentally induced mucosal diseases in calves.

OBJECTIVE: To investigate ultrastructural changes in follicles of small-intestinal aggregated lymphoid nodules (Peyer's patches) of calves with early and advanced phases of experimentally induced mucosal disease (MD). ANIMALS: Twenty 2.5- to 7-month-old Holstein-Friesian calves (11 females, 9 males). PROCEDURE: MD was induced in 13 of 18 calves that were persistently viremic with bovine viral diarrhea virus (BVDV). Eight of the 13 calves were euthanatized before the onset of clinical signs of MD, and 5 were euthanatized after becoming moribund with MD. Five persistently viremic calves and 2 calves without BVDV served as controls. Specimens of small-intestinal aggregated lymphoid nodules were prepared for transmission electron microscopy. RESULTS: The ultrastructure of follicles of small-intestinal aggregated lymphoid nodules from healthy calves was consistent with that in sheep. In the early phase of MD, changes were characterized by numerous apoptotic lymphocytes and macrophages with apoptotic bodies. In more advanced lesions, affected lymphoid follicles consisted of macrophages and variable numbers of follicular dendritic cells (FDC), whereas others did not contain FDC. In moribund calves, small follicles consisting predominantly of FDC and follicles with central cavities surrounded by macrophages, and few neutrophils were observed. CONCLUSIONS AND CLINICAL RELEVANCE: The ultrastructural changes in lymphoid follicles of small-intestinal aggregated lymphoid nodules indicate apoptosis of lymphocytes as an initial event. The development of small follicles consisting predominantly of FDC or the complete loss of follicular architecture in advanced phases of MD is determined by the intensity of apoptosis of lymphocytes, the capacity of the macrophages for uptake, and the reorganization of a stromal network.

Organ and tissue distribution of the antigen of the cytopathogenic bovine virus diarrhea virus in the early and advanced phase of experimental mucosal disease.

To study the development of lesions in mucosal disease, the spread of cytopathogenic (cp) bovine virus diarrhea virus (BVDV) to different organs was examined in relation to the time post inoculation (pi). Mucosal disease was induced in 15 persistently viremic cattle from two herds by intranasal inoculation with antigenically similar cp BVDV strain. This strain reacted with one additional monoclonal antibody when compared to the corresponding herd-specific non cytopathogenic (ncp) isolate. Twelve cattle were euthanized at days 3, 5, 7, 9 and 13 pi in the early phase before they developed clinical signs of mucosal disease, three in the advanced phase when they were moribund and three served as controls. Antigen of the cp BVDV strains was selectively detected in tissue sections by immunohistochemistry. In the early phase, varying amounts cp BVDV were present most consistently in tonsils, lymph nodes, Peyer's patches and lymphoid nodules in the large intestine. In the lymphoid tissues, first a few cells in single lymphoid follicles, then groups of lymphoid follicles contained antigen. In intestinal epithelium, cp BVDV antigen was found focally in the early phase of mucosal disease. Its diffuse distribution in the late phase corresponded with clinical signs of diarrhea.

Experimental mucosal disease of cattle: changes in cell proliferation in lymphoid tissues and intestinal epithelium.

In mucosal disease of cattle, the initial and most severe lesions are found in the lymphoid follicles and intestinal crypts, both sites showing a high cell proliferation rate. In the present study, the changes in the number and distribution of proliferating cells were investigated immunohistologically, by demonstrating the proliferation-associated nuclear antigen, Ki-67. Tissues were obtained from 30 cattle, all of which had a persistent natural infection with non-cytopathogenic (ncp) bovine virus diarrhoea virus (BVDV), and 22 of which were subsequently inoculated with antigenically closely related strains of cytopathogenic (cp) BVDV to produce mucosal disease (MD); the remaining eight cattle served as uninoculated controls. Twelve of the inoculated cattle were killed before the onset of clinical signs of MD ("early phase"), and 10 when they were moribund ("late phase"). In the controls, the lymphoid follicles in lymph nodes and Peyer's patches consisted predominantly of Ki-67-positive cells; high numbers of such cells were observed in the crypts of the small intestine and moderate numbers in the crypts of the large intestine. In the early phase of MD, the number of Ki-67-positive cells in the lymphoid follicles and in the domes of the Peyer's patches gradually decreased; but in the mucosa, foci of crypts with increased numbers of Ki-67-positive cells were observed. In the late phase of MD, only a few Ki-67-positive cells were present in the lymphoid follicles. There was an increase in the number of Ki-67-positive cells in most crypts in the small and large intestine, but foci of crypts without Ki-67-positive cells occurred.