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 microm (mean = 4.86) x 4.4-5.9 microm (mean = 5.2 microm) 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.

Neonatal gnotobiotic pigs orally inoculated with virulent (intestinal-suspension) Wa strain human rotavirus (which mimics human natural infection) developed diarrhea, and most pigs which recovered (87% protection rate) were immune to disease upon homologous virulent virus challenge at postinoculation day (PID) 21. Pigs inoculated with cell culture-attenuated Wa rotavirus (which mimics live oral vaccines) developed subclinical infections and seroconverted but were only partially protected against challenge (33% protection rate). Isotypespecific antibody-secreting cells (ASC) were enumerated at selected PID in intestinal (duodenal and ileal lamina propria and mesenteric lymph node [MLN]) and systemic (spleen and blood) lymphoid tissues by using enzyme-linked immunospot assays. At challenge (PID 21), the numbers of virus-specific immunoglobulin A (IgA) ASC, but not IgG ASC, in intestines and blood were significantly greater in virulent-Wa rotavirusinoculated pigs than in attenuated-Wa rotavirus-inoculated pigs and were correlated (correlation coefficients: for duodenum and ileum, 0.9; for MLN, 0.8; for blood, 0.6) with the degree of protection induced. After challenge, the numbers of IgA and IgG virus-specific ASC and serum-neutralizing antibodies increased significantly in the attenuated-Wa rotavirus-inoculated pigs but not in the virulent-Wa rotavirus-inoculated pigs (except in the spleen and except for IgA ASC in the duodenum). The transient appearance of IgA ASC in the blood mirrored the IgA ASC responses in the gut, albeit at a lower level, suggesting that IgA ASC in the blood of humans could serve as an indicator for IgA ASC responses in the intestine after rotavirus infection. To our knowledge, this is the first report to study and identify intestinal IgA ASC as a correlate of protective active immunity in an animal model of human-rotavirus-induced disease. . † Approved as Ohio Agricultural Research and Development Center manuscript 179-95. 3076 YUAN ET AL. J. VIROL. 3080 YUAN ET AL. J. VIROL.

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Summary. Gnotobiotic piglets serve as a useful animal model for studies of human rotavirus infections, including disease pathogenesis and immunity. An advantage of piglets ov~r 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 rota virus 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 (Gl, PIA, Wa strain) and have revealed that factors other than villous atrophy may contribute to the early diarrhea induced. A comprehensive ~xamination 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 rota virus (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 154 L. J. Saif et al. 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 rota virus vaccines.

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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) Rotaviruses are the leading cause of gastroenteritis in infants and young children worldwide (37). Rotavirus particles consist of triple-layered capsids containing a segmented double-stranded RNA genome. The rotavirus core is composed of VP2, which is the most abundant protein of the central core (15% of total virion mass) and a component of the RNA polymerase complex (26). The rotavirus major inner capsid protein is VP6, which is the most abundant structural protein of rotavirus (Ͼ50% of total virion mass) (26). VP6 is highly antigenic and contains antigenic determinants shared by all group A rotaviruses and antigenic determinants unique to the subgroup specificity. In general, most animal group A rotaviruses (including SA11) are subgroup I, whereas most human group A rotaviruses (including Wa) are subgroup II (34). The surface layer of the rotavirus capsid is composed of VP7 with VP4 spikes emanating from the outer surface (26). VP7 and VP4 independently induce virus-neutralizing (VN) antibodies (34). Diversity in VP4 and VP7 neutralizing antigenic determinants determines rotavirus P and G serotype specificity, respectively.,The viral proteins and determinants associated with protective immunity against rotavirus have not been fully delineated. Fecal or intestinal immunoglobulin A (IgA) antibodies or antibody-secreting cells (ASC) directed to rotavirus were suggested as correlates of protection in several studies of different species, including humans (15,20,28,42,64,71). Neutralizing antibodies to VP4 and VP7 were protective against rotavirus infection in mice, using monoclonal antibodies or recombinant

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.