Lucy A. Ward

Cryptosporidium hominis n. sp. (Apicomplexa: Cryptosporidiidae) from Homo sapiens

Abstract The structure and infectivity of the oocysts of a new species of Cryptosporidium from the feces of humans are described. Oocysts are structurally indistinguishable from those of Cryptosporidium parvum. Oocysts of the new species are passed fully sporulated, lack sporocysts, and measure 4.4–5.4 μm (mean = 4.86) × 4.4–5.9 μm (mean = 5.2 μm) with a length to width ratio 1.0–1.09 (mean 1.07) (n = 100). Oocysts were not infectious for ARC Swiss mice, nude mice, Wistar rat pups, puppies, kittens or calves, but were infectious to neonatal gnotobiotic pigs. Pathogenicity studies in the gnotobiotic pig model revealed significant differences in parasite-associated lesion distribution (P = 0.005 to P = 0.02) and intensity of infection (P = 0.04) between C. parvum and this newly described species from humans. In vitro cultivation studies have also revealed growth differences between the two species. Multi-locus analysis of numerous unlinked loci, including a preliminary sequence scan of the entire genome demonstrated this species to be distinct from C. parvum and also demonstrated a lack of recombination, providing further support for its species status. Based on biological and molecular data, this Cryptosporidium infecting the intestine of humans is proposed to be a new species Cryptosporidium hominis n. sp.

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

ABSTRACT 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 mutantEscherichia 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.

The gnotobiotic piglet as a model for studies of disease pathogenesis and immunity to human rotaviruses

Gnotobiotic piglets serve as a useful animal model for studies of human rotavirus infections, including disease pathogenesis and immunity. An advantage of piglets over laboratory animal models is their prolonged susceptibility to human rotavirus-induced disease, permitting cross-protection studies and an analysis of active immunity. Major advances in rotavirus research resulting from gnotobiotic piglet studies include: 1) the adaptation of the first human rotavirus to cell culture after passage and amplification in piglets; 2) delineation of the independent roles of the two rotavirus outer capsid proteins (VP4 and VP7) in induction of neutralizing antibodies and cross-protection; and 3) recognition of a potential role for a nonstructural protein (NSP4) in addition to VP4 and VP7, in rotavirus virulence. Current studies of the pathogenesis of group A human rotavirus infections in gnotobiotic piglets in our laboratory have confirmed that villous atrophy is induced in piglets given virulent but not cell culture attenuated human rotavirus (G1, P1A, Wa strain) and have revealed that factors other than villous atrophy may contribute to the early diarrhea induced. A comprehensive examination of these factors, including a proposed role for NSP4 in viral-induced cytopathology, may reveal new mechanisms for induction of viral diarrhea. Finally, to facilitate and improve rotavirus vaccination strategies, our current emphasis is on the identification of correlates of protective active immunity in the piglet model of human rotavirus-induced diarrhea. Comparison of cell-mediated and antibody immune responses induced by infection with a virulent human rotavirus (to mimic host response to natural infection) with those induced by a live attenuated human rotavirus (to mimic attenuated oral vaccines) in the context of homotypic protection has permitted an analysis of correlates of protective immunity. Results of these studies have indicated that the magnitude of the immune response is greatest in lymphoid tissues adjacent to the local site of viral replication (small intestine). Secondly, there was a direct correlation between the degree of protection induced and the level of the intestinal immune response, with significantly higher local immune responses and complete protection induced only after primary exposure to virulent human rotavirus. These studies thus have established basic parameters related to immune protection in the piglet model of human rotavirus-induced disease, verifying the usefulness of this model to examine new strategies for the design and improvement of human rotavirus vaccines.

Pathogenesis of Human and Bovine Cryptosporidium parvum in Gnotobiotic Pigs

To compare the pathogenesis of human genotype 1 (HuG1) and bovine genotype 2 (BoG2) Cryptosporidium parvum, neonatal gnotobiotic pigs were given 1-10 HuG1 or BoG2 oocysts. The prepatent and patent periods were significantly longer for HuG1 than for BoG2 C. parvum (prepatent, 8.6 vs. 5.6 days; patent, 16.6 vs. 10.3 days). BoG2-infected pigs developed significantly more severe disease than did HuG1-infected pigs. BoG2 parasites were seen microscopically throughout the intestines during the prepatent and patent periods. HuG1 parasites were only detected during the patent period in the ileum and colon but colonized the mucosal surface in significantly larger numbers than did BoG2. Moderate-to-severe villus/mucosal attenuation with lymphoid hyperplasia was seen throughout the intestines of BoG2-infected pigs, whereas lesions in HuG1-infected pigs were mild to moderate and restricted to the ileum and colon. These findings provide additional support for the hypothesis that human and bovine C. parvum genotypes may be separate species.

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.

Comparative Studies of the Pathogenesis, Antibody Immune Responses, and Homologous Protection to Porcine and Human Rotaviruses in Gnotobiotic Piglets

Gnotobiotic piglets serve as a useful animal model for studies of rotavirus pathogenesis and immunity. An advantage over laboratory animal models is the prolonged susceptibility of piglets to rotavirus-induced disease, permitting an analysis of cross-protection and active immunity. Studies from our laboratory of the pathogenesis of human rotavirus infections in gnotobiotic piglets have confirmed that villous atrophy is induced in piglets given virulent but not attenuated human rotavirus (Wa strain) and have revealed that factors other than villous atrophy may contribute to the early diarrhea induced. To facilitate and improve rotavirus vaccination strategies, it is important to identify correlates of protective immunity. Comparison of antibody immune responses induced by infection with virulent porcine and human rotaviruses (mimic host response to natural infection) with those induced by live attenuated human rotavirus (mimic attenuated oral vaccines) in the context of homotypic protection has permitted an analysis of correlates of protective immunity. Our results indicate that the magnitude of the immune response is greatest in lymphoid tissues adjacent to the site of viral replication (small intestine). Secondly there was a direct association between the degree of protection induced and the level of the intestinal immune response, with primary exposure to virulent rotaviruses inducing significantly higher numbers of IgA ASC and complete protection against challenge. These studies thus have established basic parameters related to immune protection in the piglet model of rotavirus-induced disease, verifying the usefulness of this model to apply new strategies for the design and improvement of rotavirus vaccines.

Detection of Cytopathic Bovine Viral Diarrhea Virus in the Ovaries of Cattle following Immunization with a Modified Live Bovine Viral Diarrhea Virus Vaccine

Economic loss from infection with bovine viral diarrhea virus (BVDV) is of worldwide concern. The unique pathogenesis and antigenic variability of BVDV have made this virus challenging to control. Vaccination programs are a major component of control and prevention strategies. Both killed and modified live vaccines are commercially available. Choice between killed and modified live vaccines is controversial. Of major concern is the safety of modified live vaccines. Little information is available on their tissue tropism and potential for causing pathology, especially with respect to the reproductive system. The objective of this study was to determine if BVDV could be detected in the ovary of cattle following immunization with a modified live BVDV vaccine. In 2 separate trials, 6 heifers and 4 mature cows were immunized with a modified live BVDV vaccine and ovaries were removed between 7 and 30 days postvaccination. Cytopathic BVDV was isolated from ovaries removed on days 8, 10, and 12. BVDV antigen was detected using immunohistochemistry on days 10–30. These findings are significant because replication of virus in the ovary could cause ovarian dysfunction, resulting in reduced fertility.

Enhanced Detection of Cryptosporidium Parvum in the Acid-Fast Stain

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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).