Reconstitution of Human Necrosome Interactions in Saccharomyces cerevisiae.

The necrosome is a large-molecular-weight complex in which the terminal effector of the necroptotic pathway, Mixed Lineage Kinase Domain-Like protein (MLKL), is activated to induce necroptotic cell death. The precise mechanism of MLKL activation by the upstream kinase, Receptor Interacting Serine/Threonine Protein Kinase 3 (RIPK3) and the role of Receptor Interacting Serine/Threonine Protein Kinase 1 (RIPK1) in mediating MLKL activation remain incompletely understood. Here, we reconstituted human necrosome interactions in yeast by inducible expression of these necrosome effectors. Functional interactions were reflected by the detection of phosphorylated MLKL, plasma membrane permeabilization, and reduced proliferative potential. Following overexpression of human necrosome effectors in yeast, MLKL aggregated in the periphery of the cell, permeabilized the plasma membrane and compromised clonogenic potential. RIPK1 had little impact on RIPK3/MLKL-mediated yeast lethality; however, it exacerbated the toxicity provoked by co-expression of MLKL with a RIPK3 variant bearing a mutated RHIM-domain. Small molecule necroptotic inhibitors necrostatin-1 and TC13172, and viral inhibitors M45 (residues 1-90) and BAV_Rmil, abated the yeast toxicity triggered by the reconstituted necrosome. This yeast model provides a convenient tool to study necrosome protein interactions and to screen for and characterize potential necroptotic inhibitors.

Morphometrics of the Southern Green Stink Bug [Nezara viridula (L.) (Hemiptera: Pentatomidae)] Stylet Bundle.

The southern green stink bug, Nezara viridula (L.) (Hemiptera: Pentatomidae), is a cosmopolitan pest of high-value cash crops, including cotton (Gossypium hirsutum L.; Malvales: Malvaceae). The pest can ingest and transmit disease-causing bacterial and fungal pathogens of cotton. We hypothesized that the size of the food canal may contribute to selective transmission, as observed in previous reports. The objective of this study was to examine food canal size and other morphometric parameters of the southern green stink bug stylet bundle at two locations (labium and head) to improve our understanding of factors that may contribute to pathogen transmission. For the food canal, females possessed significantly larger canals than males, major axes were significantly longer than minor axes, and canal sizes were numerically higher at the labium compared with the head. For salivary canal, mean axes lengths were similar between sexes and the head and labium. For both food and salivary canals, axes lengths were longer and area was larger at the labium compared with the head. These findings indicate the presence of a "funnel effect" with canals becoming narrower proximally. Sex and location significantly affected the size of the intact stylet bundle. Results indicate the food canal size was not a factor affecting previously observed selective passive transmission. Major and minor axes measurements, coupled with morphological observations of canal shapes and observed "funnel effect" in the food and salivary canals, improve our understanding of the hemipteran stylet bundle and its relationship with the insect's internal morphology.

Intestinal Batf3-dependent dendritic cells are required for optimal antiviral T-cell responses in adult and neonatal mice.

Although we know a great deal about which types of dendritic cells (DCs) promote T-cell priming in the periphery, less is known about which DC subset(s) provoke antiviral responses within the gut. Here we report that conventional Zbtb46-dependent DCs were critically required for antiviral CD8+ T-cell responses against rotavirus (RV), the major cause of childhood gastroenteritis worldwide. Furthermore, we found that in adult mice, Batf3-dependent DCs were required for generating optimal RV-specific CD8+ T-cell responses. However, in contrast to mice that lack Zbtb46-dependent DCs, a significant amount of interferon gamma-producing RV-specific CD8+ T cells were still detected in the small intestine of RV-infected adult Batf3-/- mice, suggesting the existence of compensatory cross-presentation mechanisms in the absence of Batf3-dependent DCs. In contrast to adult mice, we found that Batf3-dependent DCs were absolutely required for generating RV-specific CD8+ T-cell responses in neonates. Loss of Batf3-dependent DCs also resulted in a skewed polyclonal CD4+ T-cell response in both adult and neonatal mice upon RV infection, although local and systemic RV-specific immunoglobulin A production kinetics and titers were unimpaired. Our results provide insights that inform early-life vaccination strategies against RV infection.

FlaC, a protein of Campylobacter jejuni TGH9011 (ATCC43431) secreted through the flagellar apparatus, binds epithelial cells and influences cell invasion.

Type III secretion systems identified in bacterial pathogens of animals and plants transpose effectors and toxins directly into the cytosol of host cells or into the extracellular milieu. Proteins of the type III secretion apparatus are conserved among diverse and distantly related bacteria. Many type III apparatus proteins have homologues in the flagellar export apparatus, supporting the notion that type III secretion systems evolved from the flagellar export apparatus. No type III secretion apparatus genes have been found in the complete genomic sequence of Campylobacter jejuni NCTC11168. In this study, we report the characterization of a protein designated FlaC of C. jejuni TGH9011. FlaC is homologous to the N- and C-terminus of the C. jejuni flagellin proteins, FlaA and FlaB, but lacks the central portion of these proteins. flaC null mutants form a morphologically normal flagellum and are highly motile. In wild-type C. jejuni cultures, FlaC is found predominantly in the extracellular milieu as a secreted protein. Null mutants of the flagellar basal rod gene (flgF) and hook gene (flgE) do not secrete FlaC, suggesting that a functional flagellar export apparatus is required for FlaC secretion. During C. jejuni infection in vitro, secreted FlaC and purified recombinant FlaC bind to HEp-2 cells. Invasion of HEp-2 cells by flaC null mutants was reduced to a level of 14% compared with wild type, suggesting that FlaC plays an important role in cell invasion.

Effect of diffferent levels of maternally derived antibodies on protection against infectious bursal disease virus.

Fertile eggs were obtained from three different broiler breeder flocks with different levels of virus neutralizing antibodies to infectious bursal disease virus. Egg yolk from these flocks was tested for antibody titers by the virus neutralization test. Flock I eggs had no antibodies, flock II had medium level antibodies (1:200-1600; geometric mean = 1:975), and flock III had a high level of antibodies (1:1600-6400; geometric mean = 1:3365). Chicks from the above flocks were challenged each with 10(2) 50% embryo infective dose of the IN serotype 1 variant virus at 1, 2, and 4 wk of age and examined at 5 and 11 days postchallenge. The average organ/body weight ratios were calculated and statistically analyzed. Chicks with no maternal antibodies were not protected at any age. Chicks with medium levels of maternal antibodies were protected when challenged at 1 and 2 wk of age. Chicks with high levels of maternally derived antibodies were protected when challenged at all the ages tested. The above results were statistically significant (P < 0.05).

Pathogenicity, transmissibility, and tissue distribution of avian pneumovirus in turkey poults.

The pathogenicity, transmissibility, tissue distribution, and persistence of avian pneumovirus (APV) in turkey poults were investigated in three experiments. In the first experiment, we inoculated 2-wk-old commercial turkey poults oculonasally with APV alone or in combination with Bordetella avium. In the dually infected group, clinical signs were more severe, the virus persisted longer, the bacteria invaded more respiratory tissues, and the birds had higher antibody titer than the group exposed to APV or B. avium alone. In the second experiment, we studied the distribution of APV in different tissues in experimentally inoculated 2-wk-old commercial turkey poults. Only samples from sinuses, tracheas, and lungs were positive for APV by both reverse transcriptase-polymerase chain reaction and virus isolation. In the third experiment, we studied the ability of APV to spread among birds in 1-wk-old commercial turkey poults inoculated oculonasally. The virus was isolated and the viral RNA was detected in the inoculated and direct contact birds. The virus was not isolated, viral RNA was not detected, and no antibodies were detected in the indirect contact birds. These birds were placed in different cages in the same room where the airflow was directed from the infected toward the uninfected indirect contact group.

Variability among Cryptosporidium parvum genotype 1 and 2 immunodominant surface glycoproteins.

Published genomic differences between Cryptosporidium parvum genotype 1 (human-derived) and genotype 2 (animal and human-derived) isolates suggest that these may belong to two distinct species. This is of significant interest since genotype 1 isolates are associated with sporadic cases of human cryptosporidiosis in 30-40 % of cases in contrast to 60-70 % of cases caused by genotype 2. The lower genetic sequence similarity between genotype 1 and 2 surface glycoproteins (gp40/15) suggests that antigenic differences should also occur, a feature that was investigated in this study. Using immune and convalescent serum samples from gnotobiotic piglets previously inoculated with genotype 1 and 2 isolates, we demonstrated that C. parvum gp15 was immunodominant for both genotype 1 and 2 isolates. Lower genetic sequence similarity between genotype 1 and 2 Cpgp40/15 did correspond to gp15 protein differences as detected by Western blot. Moreover, we confirmed that gp15 contains epitopes that are also immunodominant. Deglycosylation of C. parvum proteins resulted in decreased ability of gp15, gp23 and gp900 to react with homologous polyclonal antibodies, suggesting that these proteins also express carbohydrate epitopes. Taken together, our data suggest that there is a high phenotypic variability between C. parvum genotype 1 and 2 isolates at the level of gp15. We contemplate that gp15 surface glycoprotein plays an important role in the biology of C. parvum as a potent inducer of immune response and a possible virulence factor.

Rapid methods to isolate Cryptosporidium DNA from frozen feces for PCR.

The polymerase chain reaction (PCR) offers one of the most sensitive methods for detecting Cryptosporidium parvum but its sensitivity in fecal material can be greatly reduced by a variety of poorly defined 'inhibitors'. Methods that separate the parasitic oocyst from fecal material prior to DNA extraction circumvent inhibitor interference but are problematic for frozen specimens since 'intact' oocysts are required for separation. We report here a relatively low-cost, rapid method for extracting C. parvum DNA from frozen fecal materials that can be used in a PCR assay for detection of single parasitic oocysts.

Experimental bovine coronavirus in turkey poults and young chickens.

The DB2 calf strain of bovine coronavirus (BCV) was used to inoculate 1-day-old specific-pathogen-free (SPF) turkey poults in three trials. In all trials, the birds developed clinical signs of enteritis at 48-72 hr postinoculation. Birds euthanatized at 3, 5, and 7 days postinoculation (DPI) had flaccid, pale intestines with watery contents, and the ceca were markedly enlarged with frothy contents. Coronavirus particles were detected by immune electron microscopy with BCV antibodies from the intestinal contents of birds killed at 3, 5, 7, and 12 DPI. Body weights of inoculated poults killed at 3, 5, and 7 DPI were significantly reduced as compared with controls. Hemagglutinating antibodies were detected in sera of convalescent birds at 12 DPI. However, experimental inoculation of 1-day-old SPF chicks in two trials with the same virus resulted in no clinical signs or macroscopic or microscopic lesions. No coronaviruses were detected from intestinal contents, and there were no significant differences in body weights of inoculated and noninoculated control chicks.

Spasticity in kids. An intrathecal option.

The painful muscle rigidity and severe spasms suffered by patients with brain and spinal injury can be managed even more successfully than in the past with intrathecal baclofen. For many, it's provided effective relief and improved levels of function.

Intranasal administration of 2/6-rotavirus-like particles with mutant Escherichia coli heat-labile toxin (LT-R192G) induces antibody-secreting cell responses but not protective immunity in gnotobiotic pigs.

We investigated the immunogenicity of recombinant double-layered rotavirus-like particle (2/6-VLPs) vaccines derived from simian SA11 or human (VP6) Wa and bovine RF (VP2) rotavirus strains. The 2/6-VLPs were administered to gnotobiotic pigs intranasally (i.n.) with a mutant Escherichia coli heat-labile toxin, LT-R192G (mLT), as mucosal adjuvant. Pigs were challenged with virulent Wa (P1A[8],G1) human rotavirus at postinoculation day (PID) 21 (two-dose VLP regimen) or 28 (three-dose VLP regimen). In vivo antigen-activated antibody-secreting cells (ASC) (effector B cells) and in vitro antigen-reactivated ASC (derived from memory B cells) from intestinal and systemic lymphoid tissues (duodenum, ileum, mesenteric lymph nodes [MLN], spleen, peripheral blood lymphocytes [PBL], and bone marrow lymphocytes) collected at selected times were quantitated by enzyme-linked immunospot assays. Rotavirus-specific immunoglobulin M (IgM), IgA, and IgG ASC and memory B-cell responses were detected by PID 21 or 28 in intestinal and systemic lymphoid tissues after i.n. inoculation with two or three doses of 2/6-VLPs with or without mLT. Greater mean numbers of virus-specific ASC and memory B cells in all tissues prechallenge were induced in pigs inoculated with two doses of SA11 2/6-VLPs plus mLT compared to SA11 2/6-VLPs without mLT. After challenge, anamnestic IgA and IgG ASC and memory B-cell responses were detected in intestinal lymphoid tissues of all VLP-inoculated groups, but serum virus-neutralizing antibody titers were not significantly enhanced compared to the challenged controls. Pigs inoculated with Wa-RF 2/6-VLPs (with or without mLT) developed higher anamnestic IgA and IgG ASC responses in ileum after challenge compared to pigs inoculated with SA11 2/6-VLPs (with or without mLT). Three doses of SA 11 2/6-VLP plus mLT induced the highest mean numbers of IgG memory B cells in MLN, spleen, and PBL among all groups postchallenge. However, no significant protection against diarrhea or virus shedding was evident in any of the 2/6-VLP (with or without mLT)-inoculated pigs after challenge with virulent Wa human rotavirus. These results indicate that 2/6-VLP vaccines are immunogenic in gnotobiotic pigs when inoculated i.n. and that the adjuvant mLT enhanced their immunogenicity. However, i.n. inoculation of gnotobiotic pigs with 2/6-VLPs did not confer protection against human rotavirus challenge.

Pathogenesis and animal models.

Because of the limitations in studying human rotavirus (HRV) pathogenesis and mucosal immunity in the natural host (infants and children), various animal models have been utilized to investigate rotavirus (RV) disease pathogenesis and immunity. Mice and rabbits serve as useful models to evaluate and dissect immune responses to RV (see Chapter 9 ). However, because older mice (>14 d) and rabbits are not susceptible to diarrhea after inoculation with either homologous (murine or lapine, respectively) or heterologous (human) RVs, assessment of protective immunity is restricted to prevention of virus shedding only (1-3).

Dual infection of gnotobiotic calves with bovine strains of group A and porcine-like group C rotaviruses influences pathogenesis of the group C rotavirus.

There is serological evidence that bovine group C rotaviruses exist in the United States, but there are no reports of their isolation. Ninety fecal samples from calves with diarrhea, 81 samples from adult cows with diarrhea (winter dysentery), and 20 fecal samples from healthy adult cows were tested for group C rotaviruses by polyacrylamide gel electrophoresis, immune electron microscopy, and reverse transcription-PCR (RT-PCR). Three samples from adult cow diarrhea cases were positive only by RT-PCR, and a group C rotavirus was isolated from a positive sample in monkey kidney (MA104) cells (WD534tc/C). Genetically and serologically, the WD534tc/C strain was more closely related to the Cowden porcine group C strain than to the Shintoku bovine strain. Because the original cow feces also contained a group A rotavirus (detected after passage in cell culture), we hypothesized that such dual-rotavirus infections might play a role in the pathogenesis and host adaptation of rotaviruses. Thus, we examined the pathogenesis of WD534tc/C alone or combined with virulent (IND/A) or attenuated (NCDV/A) bovine group A rotaviruses in gnotobiotic calves. WD534tc/C alone induced diarrhea without (or with limited) virus shedding in inoculated calves (n = 3). In contrast, all calves coinfected with WD534tc/C and IND/A (n = 2) developed diarrhea and shed both viruses, whereas calves coinfected with WD534tc/C and NCDV/A (n = 3) developed diarrhea but did not shed either virus. Infection with WD534tc/C or NCDV/A alone caused only mild villous atrophy (jejunum and/or ileum), whereas dual infection with both viruses induced lesions throughout the small intestine. Although IND/A alone caused villous atrophy, more-widespread small intestinal lesions occurred in calves coinfected with WD534tc/C and IND/A. In conclusion, coinfection of calves with group A rotaviruses enhanced fecal shedding of a bovine group C rotavirus and the extent of histopathological lesions in the small intestines. Thus, our findings suggest a potential novel hypothesis involving dual infections for the adaptation of heterologous rotaviruses to new host species.

Serum and intestinal isotype antibody responses to Wa human rotavirus in gnotobiotic pigs are modulated by maternal antibodies.

The effects of passive antibodies on protection and active immune responses to human rotavirus were studied in gnotobiotic pigs. Pigs were injected at birth with saline or sow serum of high (immunized) or low (control) antibody titre and subsets of pigs were fed colostrum and milk from immunized or control sows. Pigs were inoculated at 3-5 days of age and challenged at 21 days post-inoculation (p.i.) with virulent Wa human rotavirus. Pigs receiving immune serum with or without immune colostrum/milk were partially protected against diarrhoea and virus shedding after inoculation, but had significantly lower IgA antibody titres in serum and small intestinal contents at 21 days p.i. and lower protection rates after challenge compared with pigs given control or no maternal antibodies. IgG antibody titres were consistently higher in small than in large intestinal contents. Pigs given control serum with control colostrum/milk had lower rates of virus shedding after inoculation than those given control serum alone. In summary, high titres of circulating maternal antibodies with or without local (milk) antibodies provided passive protection after inoculation but suppressed active mucosal antibody responses. These findings may have implications for the use of live, oral rotavirus vaccines in breast-fed infants.

International travel and vaccinations.

With the increase in global travel, no disease is beyond the reach of any population. Traveling patients should be advised to follow food and water precautions and encouraged to receive the recommended immunizations. Travel medicine plays a vital role not only in limiting the morbidity of travel-related illnesses but also in limiting the spread of diseases. This article addresses the common issues related to travel, reviews the care of the immunocompromised traveler, and updates the available vaccinations and prophylactic regimens available to limit sickness abroad.

Effects of maternal antibodies on protection and development of antibody responses to human rotavirus in gnotobiotic pigs.

Although maternal antibodies can protect against infectious disease in infancy, they can also suppress active immune responses. The effects of circulating maternal antibodies, with and without colostrum and milk antibodies, on passive protection and active immunity to human rotavirus (HRV) were examined in gnotobiotic pigs. Pigs received intraperitoneal injections of high-titer serum (immune pigs [groups 1 and 2]) from immunized sows, low-titer serum from naturally infected sows (control pigs [groups 3 and 4]), or no serum (group 5). Immune or control colostrum and milk were added to the diet of groups 2 and 4, respectively. After inoculation (3 to 5 days of age) and challenge (postinoculation day [PID] 21) with virulent HRV, the effects of maternal antibodies on protection (from diarrhea and virus shedding), and on active antibody responses (measured by quantitation of antibody-secreting cells [ASC] in intestinal and systemic lymphoid tissues by ELISPOT) were evaluated. Groups 1 and 2 had significantly less diarrhea and virus shedding after inoculation but higher rates of diarrhea and virus shedding after challenge than did groups 3 and 5. Group 1 and 2 pigs had significantly fewer immunoglobulin A (IgA) ASC in intestinal tissues at PID 21 and at postchallenge day (PCD) 7 compared to group 5. Significantly fewer IgG ASC were present in the intestines of group 2 pigs at PID 21 and PCD 7 compared to group 5. There was a trend towards fewer ASC in intestinal tissues of group 2 than group 1, from PID 21 on, with significantly fewer IgA ASC at PCD 7. IgG ASC in the duodenum and mesenteric lymph nodes of group 3 and 4 pigs were significantly fewer than in group 5 at PCD 7. These decreases in ASC emphasize the role of passive antibodies in impairing induction of ASC rather than in merely suppressing the function of differentiated B cells. To be successful, vaccines intended for populations with high titers of maternal antibodies (infants in developing countries) may require higher titers of virus, multiple doses, or improved delivery systems, such as the use of microencapsulation or immune stimulating complexes, to overcome the suppressive effects of maternal antibodies.

Serum and intestinal isotype antibody responses and correlates of protective immunity to human rotavirus in a gnotobiotic pig model of disease.

We examined the antibody responses and protection to a human rotavirus (HRV) in gnotobiotic (Gn) pigs. Pigs were perorally (p.o.) inoculated with attenuated (group 1), virulent (group 2), or inactivated (group 3) Wa (P1A[8]G1) HRV. A fourth group was inoculated intramuscularly (i.m.) with inactivated Wa HRV in adjuvant. After p.o. challenge with virulent Wa HRV at post-inoculation day 21, most group 1, 3 and 4 pigs shed virus and developed diarrhoea, whereas this occurred in only a few group 2 pigs. Antibodies to HRV (IgM, IgA or IgG) were detected in serum and intestinal contents of pigs of all groups after virus inoculation or challenge, and the antibody titres in intestinal contents, although lower, showed similar kinetics to the serum responses. There was no correlation between protection and neutralizing antibody titres of serum or intestinal contents, but a positive correlation existed between protection and serum IgA, intestinal IgA and intestinal IgG antibody titres. These findings suggest that serum IgA antibodies to HRV could act as an indicator of IgA antibodies in the intestine after rotavirus infection. The virulent HRV elicited protective immunity and higher levels of serum and intestinal IgA antibodies to HRV compared to attenuated and inactivated HRV. These findings suggest that more efficient mucosal delivery systems and/or adjuvants are needed to enhance the intestinal immune responses to attenuated or inactivated HRV if more successful vaccination is to be achieved in neonates.

Immune response of sows vaccinated with attenuated transmissible gastroenteritis virus (TGEV) and recombinant TGEV spike protein vaccines and protection of their suckling pigs against virulent TGEV challenge exposure.

OBJECTIVE: To compare recombinant transmissible gastroenteritis virus (TGEV) spike protein, (SP) R2-2, with attenuated live virus (ALV) vaccine in sows during late pregnancy. ANIMALS: 13 TGEV-seronegative sows and their pigs. PROCEDURE: At prepartum weeks (PPW) 6 and 4, sows of groups 1 and 2 received ALV via the oral/intranasal (O/IN) route. At PPW 2, group-1 sows received ALV IM and group-2 sows received SPR2-2 IM. Group-3 sows received SPR2-2 IM at PPW 4 and ALV O/IN at PPW 2. Sows of group 4 (negative controls) were inoculated O/IN with mock-infected ST cell fluids at PPW 6 and 4 and IM with Sf9 cell lysates at PPW2 (n = 2), or IM with Sf9 cell lysates at PPW4 and O/IN with mock-infected ST cell fluids at PPW2 (2). Serum, colostrum, and milk samples were tested for antibody to TGEV, and a lymphoproliferative (LP) assay was done on blood mononuclear cells. Suckling pigs were challenge exposed with virulent TGEV. RESULTS: Sows of groups 1 and 2 had higher IgG and significantly higher antibody titers in colostrum; their pigs had significantly higher serum antibody titer. At challenge exposure of their pigs, LP responses of group-2 sows were significantly higher than those of sows in the other 3 groups. Mean pig mortality ranged from 43 (group 2) to 92% (group 4). Significant negative correlations were observed among litter mortality and sow LP response, colostral titer, and pig serum titer at time of challenge exposure. CONCLUSIONS: In sows vaccinated twice with attenuated live TGEV, the recombinant SPR2-2 administered IM may be comparable to ALV administered IM as a booster. Vaccination failed to provide complete protection to suckling pigs after challenge exposure.

Detection of bovine viral diarrhea virus in the ovaries of cattle acutely infected with bovine viral diarrhea virus.

Bovine viral diarrhea virus (BVDV) is recognized worldwide as a major cause of economic loss in cattle. Infection with BVDV can result in several clinical outcomes. However, the reproductive consequences may be the most important. Infertility, early embryonic death, abortion, and congenital anomalies have all been reported following acute infection with BVDV. The cause of infertility following acute BVDV infection is not known. BVDV has been isolated from the bovine ovary and has been associated with chronic oophoritis. The purpose of this study was to identify the ovarian cell types infected with BVDV following acute infection. Twelve heifers were acutely infected with noncytopathic BVDV, and ovariectomies were performed between 4 and 60 days postinfection. BVDV was isolated on days 6 and 8 postinfection. Viral antigen was detected in macrophage-like cells and stromal cells in the ovarian cortex and oophoritis was evident from 6 to 60 days postinfection. These findings indicate that acute infection with BVDV may result in changes in ovarian function that could lead to reduced fertility.