Effect of Histophilus somni on Heart and Brain Microvascular Endothelial Cells.

Histophilus somni is a pathogenic gram-negative bacterium responsible for pneumonia and septicemia in cattle. Sequelae include infectious thrombotic meningoencephalitis (ITME), myocarditis, arthritis, and abortion. These syndromes are associated with widespread vasculitis and thrombosis, implicating a role for endothelium in pathogenesis. Histopathologic and immunohistochemical investigation of 10 natural cases of bovine H. somni myocarditis and 1 case of ITME revealed intravascular H. somni in large biofilm-like aggregates adherent to the luminal surface of microvascular endothelium. Ultrastructurally, bacterial communities were extracellular and closely associated with degenerating or contracted endothelial cells. Histophilus somni was identified by bacterial culture and/or immunohistochemistry. Western blots of the bacterial isolates revealed that they expressed the immunodominant protective 40 kDa OMP and immunoglobulin-binding protein A (IbpA) antigens. The latter is a large surface antigen and shed fibrillar antigen with multiple domains. The cytotoxic DR2Fic domain of IbpA was conserved as demonstrated by polymerase chain reaction. Treatment of endothelial cells in vitro with IbpA in crude culture supernatants or purified recombinant GST-IbpA DR2Fic (rDR2) cytotoxin induced retraction of cultured bovine brain microvascular endothelial cells. By contrast, no retraction of bovine endothelium was induced by mutant rDR2H/A with an inactive Fic motif or by a GST control, indicating that the cytotoxic DR2Fic motif plays an important role in endothelial cell retraction in vasculitis. The formation of biofilm-like aggregates by H. somni on bovine microvascular endothelium may be fundamental to its pathogenesis in heart and brain.

Bovine respiratory syncytial virus and Histophilus somni interaction at the alveolar barrier.

Our previous studies showed that Histophilus somni and bovine respiratory syncytial virus (BRSV) act synergistically in vivo to cause more severe bovine respiratory disease than either agent alone causes. Since H. somni surface and secreted immunoglobulin binding protein A (IbpA) causes retraction of bovine alveolar type 2 (BAT2) cells and invasion between BAT2 cells in vitro, we investigated mechanisms of BRSV-plus-H. somni infection at the alveolar barrier. BRSV treatment of BAT2 cells prior to treatment with IbpA-rich H. somni concentrated culture supernatant (CCS) resulted in increased BAT2 cell rounding and retraction compared to those with either treatment alone. This mimicked the increased alveolar cell thickening in calves experimentally infected with BRSV followed by H. somni compared to that in calves infected with BRSV or H. somni alone. BRSV-plus-H. somni CCS treatment of BAT2 cells also enhanced paracellular migration. The effect of matrix metalloproteinases (MMPs) was investigated as well because microarray analysis revealed that treatment with BRSV plus H. somni synergistically upregulated BAT2 cell expression of mmp1 and mmp3 compared to that in cells treated with either agent alone. Enzyme-linked immunosorbent assay (ELISA) confirmed that MMP1 and MMP3 protein levels were similarly upregulated. In collagen I and collagen IV (targets for MMP1 and MMP3, respectively) substrate zymography, digestion was increased with supernatants from dually treated BAT2 cells compared with those from singly treated cells. Enhanced breakdown of collagen IV in the basal lamina and of fibrillar collagen I in the adjacent interstitium in the dual infection may facilitate dissemination of H. somni infection.

IbpA DR2 subunit immunization protects calves against Histophilus somni pneumonia.

Histophilus somni is a prevalent cause of pneumonia and septicemia in cattle. Yet evidence for protection against pneumonia by current vaccines is controversial. We have identified a new H. somni virulence factor, IbpA. Previous studies implicated three likely protective subunits or domains in IbpA (A3, A5, and DR2), which were expressed as recombinant GST fusion proteins and purified for systemic vaccination of calves. After two subcutaneous immunizations, calves were challenged intrabronchially with virulent H. somni strain 2336 and clinical signs were monitored for four days before necropsy. Serum samples were collected throughout. At necropsy, the area of gross pneumonia was estimated, bronchial lavage fluid was collected, lesions were cultured and tissue samples were fixed for histopathology. Results showed that calves immunized with IbpA DR2 had a statistically lower percentage of lung with gross lesions than controls, fewer histologic abnormalities in affected areas and no H. somni isolated from residual pneumonic lesions. Calves immunized with the control GST vaccine, IbpA3 or IbpA5 had larger H. somni positive pneumonic lesions. ELISA results for serum antibodies showed that calves immunized with the IbpA DR2 antigen had high IgG1 and IgG2 and lowest IgE responses to the immunizing antigen. Specific IgG responses were also high in the bronchial lavage fluid. High specific serum IgE responses were previously shown to be associated with more severe pneumonia, but high IgG specific anti-IbpA DR2 responses seem to be critically related to protection. Since the IbpA DR2 Fic motif has been shown to cause bovine alveolar cells to retract, we tested the neutralizing ability of pooled serum from the IbpA DR2 immunized group. This pooled serum reduced cytotoxicity by 75-80%, suggesting that the protection was due to antibody neutralization of IbpA cytotoxicity, at least in part. Therefore, IbpA DR2 appears to be an important protective antigen of H. somni. The study shows, for the first time, that immunization with a purified Fic protein protects against disease in a natural host.

Histophilus somni IbpA Fic cytotoxin is conserved in disease strains and most carrier strains from cattle, sheep and bison.

Histophilus somni causes bovine pneumonia, septicemia, myocarditis, thrombotic meningoencephalitis and arthritis, as well as a genital or upper respiratory carrier state in normal animals. However, differences in virulence factors among strains are not well studied. The surface and secreted immunoglobulin binding protein A (IbpA) Fic motif of H. somni causes bovine alveolar type 2 (BAT2) cells to retract, allowing virulent bacteria to cross the alveolar monolayer. Because H. somni IbpA is an important virulence factor, its presence was evaluated in different strains from cattle, sheep and bison to define whether there are syndrome specific markers and whether antigenic/molecular/functional conservation occurs. A few preputial carrier strains lacked IbpA by Western blotting but all other tested disease or carrier strains were IbpA positive. These positive strains had either both IbpA DR1/Fic and IbpA DR2/Fic or only IbpA DR2/Fic by PCR. IbpA Fic mediated cytotoxicity for BAT2 cells and sequence analysis of IbpA DR2/Fic from selected strains revealed conservation of sequence and function in disease and IbpA positive carrier strains. Passive protection of mice against H. somni septicemia with antibody to IbpA DR2/Fic, along with previous data, indicates that the IbpA DR1/Fic and/or DR2/Fic domains are candidate vaccine antigens for protection against many strains of H. somni. Since IbpA DR2/Fic is conserved in most carrier strains, they may be virulent if introduced to susceptible animals at susceptible sites. Conservation of the protective IbpA antigen in all disease isolates tested is encouraging for development of protective vaccines and diagnostic assays.

Vitamin and trace mineral content in feed of breeders and their progeny: effects of growth, feed conversion and severity of malabsorption syndrome of broilers.

1. A study was conducted to investigate the effects of several vitamins and trace elements chickens and in chickens experimentally infected with malabsorption syndrome (MAS). 2. Vitamins and trace minerals in feed were varied. Breeders received either a basal amount of vitamins and trace minerals (low mix) or an increased amount (high mix). Their progeny also received either a low mix or a high mix. Effects of different breeder and broiler mix combinations on broiler performance, heamatology, spleen weight and humoral response were examined in control chickens. The effects of the different feeds and breeder, broiler combinations at the severity and recovery of MAS infection were also studied. 3. In general, the immune system can be stimulated by addition of vitamins and trace minerals, without affecting the growth potential of the controls. The number of leukocytes increased on d 1 in the broilers descended from breeders receiving high mix. The response to Newcastle disease virus boost was affected by the different amount of vitamins. 4. When breeders received a high mix the number of infiltrating polymorphonuclear leukocytes in the intestine was higher compared with breeders receiving basal amounts of minerals and vitamins. Also the recovery rate of intestinal lesions, cystic crypts of Lieberkühn and villus atrophy, as observed by histopathology, was faster in the groups where the breeders received high mix.

Development of organs and intestinal mucosa leukocytes in four broiler lines that differ in susceptibility to malabsorption syndrome.

Growth retardation in young broiler chicks due to poor nutrient metabolism, commonly known as malabsorption syndrome (MAS), is a widespread problem caused by enteric infections with a combination of pathogens mainly viruses. Genetic lines of broiler chickens differ in susceptibility to the syndrome. A difference in growth retardation was observed among four broiler lines (BL) after oral inoculation at 1 d of age with intestinal homogenates obtained from MAS-affected birds. Two of the lines that are more susceptible to MAS had severe weight gain depression. To uncover the factors that play a role in the susceptibility to MAS, we analyzed the growth rate of the body and vital organs and the quantity of leukocytes in the peripheral blood and intestinal mucosa. The development of the intestine, liver, bursa of Fabricius, and spleen was similar among the BL. The resistant BL had higher numbers of peripheral blood leukocytes, especially lymphocytes, at 1 d of age. A significant difference was noted in the numbers of CD4+ T cells and CD8+ T cells in the intestinal villi. At the ages of 3 and 8 d, the susceptible BL had more CD8+ T cells in the villi, whereas the ratios of CD4+:CD8+ T cells were higher in the resistant BL. This difference in the number of T-cell subpopulations in the intestinal mucosa might be an important factor in the difference in susceptibility to the enteric infections associated with MAS.

Experimental reproduction of malabsorption syndrome with different combinations of reovirus, Escherichia coli, and treated homogenates obtained from broilers.

Attempts to reproduce malabsorption syndrome (MAS) by oral inoculation with several different combinations including intestinal homogenate, reovirus, and hemolytic Escherichia coli obtained from MAS-affected chickens and intestinal homogenate from healthy chickens (healthy homogenate) were performed in 1-day-old specific-pathogen-free (SPF) broilers. The MAS homogenate, serving as a positive control, induced weight gain depression and intestinal lesions such as cystic crypts of Lieberkuhn, villus atrophy, and lymphoid and/or granulocytic infiltration. The healthy homogenate, the formalin-treated MAS homogenate, the formalin-treated healthy homogenate, and phosphate-buffered saline caused neither weight gain depression nor intestinal lesions. We were able to reproduce both weight gain depression and intestinal lesions by inoculation of reovirus either combined with the formalin-treated MAS homogenate or combined with healthy homogenate. Surprisingly, when hemolytic E. coli was added to the combination of reovirus with formalin-treated MAS homogenate, this did not cause weight gain depression although this combination caused the described intestinal lesions. Identical results were obtained with the combination of formalin-treated MAS homogenate with hemolytic E coli or the combination of reovirus with hemolytic E. coli. The intestinal lesions were more severe and developed faster by combinations including reovirus and formalin-treated MAS homogenate. This study indicates that a combination of enteropathogenic reovirus with other agents or substances that are present in an intestinal homogenate from MAS-affected and healthy chickens can induce MAS in SPF broilers. Escherichia coli is not essential for induction of weight gain depression but can play a role in development of intestinal lesions. Furthermore, intestinal lesions alone will not always result in weight gain depression.

Evaluation of 99mTc-MAMA-chrysamine G as an in vivo probe for amyloidosis.

To date, systemic amyloidosis is diagnosed histologically using Congo red staining or in vivo using iodine-123 labelled serum amyloid P component (123I-SAP) scintigraphy. We developed 99mTc-MAMA-CG, a 99mTc-labelled derivative of the lipophilic Congo red analogue chrysamine G (CG), as a possible alternative to 123I-SAP. In vivo 99mTc-MAMA-CG scintigraphy, performed in chickens with spontaneous joint amyloidosis, resulted as soon as 10 min after injection in scintigraphic images showing uptake of activity in amyloid-loaded organs (liver, joints). One of these chickens was studied also with 123I-SAP resulting in scintigraphic images revealing 123I-SAP binding to amyloid deposits in the liver. However, up to 11 h after injection no radioactivity was visible in the amyloid positive joints. In vitro autoradiography, performed on sections of chicken joints with Enterococcus faecalis induced amyloid arthropathy (chjAA), demonstrated the failure of 99mTc-MAMA-CG to bind significantly to amyloid deposits in the presence of 10 microM Congo red The specificity of 99mTc-MAMA-CG localisation was also established by the absence of 99mTc-MAMA-CG binding in non-amyloidotic organs in vitro and in vivo. 99mTc-MAMA-CG did not show any sign of acute toxicity. These findings establish the usefulness of 99mTc-MAMA-CG as a non-invasive in vivo diagnostic probe in chickens with amyloid arthropathy and suggest that it may also be applicable to human amyloidosis.

Leukocyte responses in two breeds of layer chicken that differ in susceptibility to induced amyloid arthropathy.

Amyloid arthropathy in chicken can be induced by intravenous inoculation of an arthropathic and amyloidogenic Enterococcus faecalis in susceptible breeds. The commercial brown layer hybrids (BL) are more susceptible to the disease compared to their white counterparts (WL). The precursor of amyloid-A protein, which is serum amyloid-A (SAA), is identical in WL and BL. To investigate the factors involved in the breed-restricted susceptibility to amyloid arthropathy, we studied the type of leukocyte response and inflammatory reactions in E. faecalis-induced disease. In the BL, a significant dose dependent peripheral leukocytosis mainly by heterophils, and plasma cell infiltration in arthritic joints was found. In contrast, secondary lymphoid nodular aggregates in the synovial membrane were prominent in the WL. The aggregates consisted mainly of CD8+ T cells. The high number of circulating leukocyte and prolific plasma cell responses in the BL predict extensive humoral and acute phase reactions. This is in agreement with literature data on suppressed T-cell function in casein-induced amyloid-susceptible mice strains. The difference in leukocyte response and type of inflammation between WL and BL, when arthropathic and amyloidogenic bacteria induce infection, in conjunction with susceptibility to amyloid arthropathy, is discussed in view of the murine T-helper responses.