Brief Summary of Minutes of Annual Meeting

Progress of Work and Principal Accomplishments Objective 1. Focus on emerging diseases- Identify, characterize and develop improved detection methods related to newly recognized, novel or emerging causes of zoonotic enteric disease and enteric pathogens of cattle and swine. A. Salmonella enterica We sequenced the genotype JJPX01.0014 found in oysters and are currently annotating the sequence. We have also produced a S. Newport mutant library for use in transposon site hybridization studies. The culturing of cells and genomic extraction were repeated using a non-mutagenized Salmonella Newport JJPX 01.0014 control. PCR to the T7 promoter and linker sites in the mutant library was performed with a positive conformation that the transposon had been randomly inserted. We conducted a survey of oysters purchased from restaurants in Tucson from 2007-2008. Samples were tested for Salmonella using a modified version of the FDA BAM manual. A total of 2281 oysters were tested for the presence of Salmonella. Twenty-eight oysters were found to be positive for Salmonella giving us a 1.2% prevalence rate. In addition, 43% of the oysters positive for Salmonella were of the S. Newport serovar. The genetic diversity of E. coli O157:H7 and Salmonella isolated at the various fecal sampling stages of feedlot steers fed with direct-fed microbial (DFM; Lactobacillus acidophilus; BT1386) and those not fed DFM during finishing was evaluated. A total of 123 of 454 (27.1 %) and 112 of 454 (24.7 %) E. coli O157:H7 were recovered from FG and the FS respectively (P < 0.05). For DFM treated steers, 48 of 66 (72.7 %) and 34 of 66 (51.5 %) tested positive for E. coli O157:H7 in FG and the FS, respectively (P < 0.05). The control steers tested E. coli O157:H7 positive in 57 of 71 (76 %; FG) and 41 of 71 (57.7 %; FS). (P < 0.05). Pulsed field gel electrophoresis (PFGE) analysis was performed on E. coli O157:H7 (n =194) and on fecal Salmonella (n = 58) isolated from the FS and FG. Of the 194 E. coli O157:H7 isolates tested, PFGE revealed 21 distinguishable genotypes. New genotypes were isolated from 1 of 39 (2.6 %) treated and 19 of 55 (34.5 %) control steers (P < 0.05). For Salmonella, 9 distinguishable genotypes were identified by PFGE. New Salmonella genotypes were observed among 1 of 29 (3.5 %) treated and 2 of 29 (6.9 %) control steers (P < 0.05). Based on data from this study, DFM protected cattle from recurrent E. coli O157:H7 infections and not Salmonella. Salmonella Typhimurium serovars isolated from naturally infected feedlot cattle (n = 138) in North Dakota were characterized for antimicrobial resistance (AMR), presence of integrons and genotypic relatedness using PFGE assays. All 58 Salmonella isolates tested were resistant to e1 of the antimicrobials with 56/58 (96.6%) showing multi-drug resistance (resistant to e2 antimicrobials). Twenty-nine were Salmonella serovars Typhimurium var Copenhagen and were positive for class I integron, two of which also tested positive for integron 2. The 58 Salmonella isolates belonged to 9 distinguishable PFGE profiles, with the most prevalent genotype accounting for 46.6% (27/58) of the isolates. These data indicate that Salmonella Typhimurium serovars isolated from naturally infected feedlot cattle in ND showed widespread AMR with presence of class 1 integron and a wide variety of distinguishable PFGE profiles. The rate of new introduction of multidrug resistant Salmonella into Northwestern dairy farms averaged 0.91 new introductions per herd-year but was quite variable among study farms. The main variable that seemed to explain the farm-farm difference was the use of commercial, off-farm heifer raising at calf ranches where animals from many farms are mixed. Contamination of feeds with MDR salmonella was found to be very infrequent and very low concentration, and did not account for any new MDR salmonella introductions. A single strain of MDR Typhimurium was observed to spread to many study herds and a genetic analysis, using multilocus VNTR analysis has been completed that demonstrated multiple acquisitions of Cmy2 bearing plasmids within this strain To test the hypothesis that resistance genes undergo mutations that are selectively neutral, we hybridized 57 E. coli isolates onto our resistance gene microarray. We expected that mutations preventing gene expression would occur more frequently in a low antimicrobial use setting like a cow-calf operation than in an intense antimicrobial use setting like a dairy calf raiser. The results of these hybridizations did not support our hypothesis. Plasmid profiles and transformations onto selective media indicated that the unknown ²-lactam resistance gene or genes are likely to be on the chromosome. One of four isolates with ampicillin resistance and no ²-lactamase genes had no plasmids and the plasmids from the others failed to transform onto ampicillin plates. Plasmid profiles from the other 6 isolates showed plasmids of diverse sizes, including one or two very large (ca. 200 kb) plasmids. The effort to isolate plasmids carrying the targeted unknown resistance genes has met with mixed success. B. Campylobacter jejuni We hypothesized that particular genetic backgrounds enhance susceptibility of IL-10-/- mice to C. jejuni. C. jejuni stably colonized C57BL/6 IL-10-/-, C57BL/6 IL-10+/+, NOD IL-10-/-, NOD IL-10+/+, C3Bir IL-10-/-, and C3H/HeJ IL-10+/+ mice, while only IL-10-/- mice had typhlocolitis that mimicked human campylobacteriosis. C. jejuni was detected in blood and spleens of many infected C3Bir IL-10-/-, NOD IL-10-/-, and NOD IL-10+/+ but not C57BL/6 mice indicating that genetic background influenced extraintestinal spread. In C3H/HeJ IL-10+/+ mice, lack of TLR4 and the Cdcs1 allele were not sufficient for enteritis, but in C3Bir IL-10-/- mice these combined deficiencies enhanced both extraintestinal spread of C. jejuni and sensitivity to colon microbiota. All infected IL-10-/- and IL-10+/+ mice had anti-C. jejuni specific IgG antibody that failed to protect IL-10-/- mice against enteritis. These data demonstrate that lack of IL-10 had a greater effect on C. jejuni induced GI disease than other immune elements such as TLR4 (C3H/HeJ) and MHC H-2g7, diabetogenic genes, and CTLA-4 (NOD) and that host genetic background is in part responsible for disease phenotype. C3Bir IL-10-/- mice where Cdcs1 impairs gut barrier function provide a new murine model of C. jejuni and can serve as surrogates for immunocompromised patients with extraintestinal spread. C. Lawsonia intracelluaris Weve continued to add to our database of L. intracellularis genotypic types (based on VNTR typing) from various proliferative enteropathy outbreaks of pigs, horses, and other animal species. We collected and analyzed samples from each of 42 proliferative enteropathy outbreaks in pig herds, from 14 horse herds, and from outbreaks in 5 other animal species. From the data analyzed so far, no genetic variation was found within clinical types of proliferative enteropathy within a pig outbreak. There are no detectable differences between the PFGE patterns of three pig (North American or European) and a horse isolate using the restriction enzyme Pme I. The Sse8387 I restriction enzyme resulted in an inconsistent PFGE pattern in some isolates because of the presence of variable concentrations of the largest (194 kb) plasmid of L. intracellularis, which is not cut by this enzyme. On the day of sampling, all foals appeared in good health and none were showing clinical signs compatible with EPE. With the exception of one previously diagnosed case of EPE from farm 1, all remaining foals had normal total solid serum concentrations. Exposure rate for the study farms ranged from 29.7% to 45.5% and all fecal samples tested negative by PCR for the presence of L. intracellularis. The present study documents the high exposure rate to L. intracellularis in resident foals from farms with documented cases of EPE. Fresh feces from jack rabbits (100), striped skunks (22), feral cats (14), Brewers blackbirds (10), Virginian opossums (9), raccoons (4), ground squirrels (3) and coyotes (2) were collected either from the ground or after trapping the animals using live traps. Feces from jack rabbits, striped skunks, Virginian opossums and coyotes tested PCR positive for L. intracellularis, while all feces from feral cats, Brewers blackbirds, raccoons and ground squirrels tested PCR negative for L. intracellularis. PCR testing on DNA extracted directly from feces was positive for L. intracellularis in 6 out of 164 feces. The largest number of PCR positive L. intracellularis fecal samples was observed in striped skunks, followed by Virginian opossums, jack rabbits and coyotes. Since the fecal samples were collected at equine farms with confirmed cases of EPE, striped skunks, Virginian opossums, jack rabbits and coyotes may act as potential sources of infection to susceptible weanlings. D. Escherichia coli The objective of this study was to compare the accuracy of the QIAamp DNA Stool Mini Kit (QIAamp Kit) in detecting E. coli O157:H7 in enrichment and non-enrichment cattle feces. Using conventional culture methods (gold standard), 456 fecal samples were analyzed for detectable levels of E. coli O157:H7. QIAamp Kit was used to purify fecal DNA (from enrichment and non-enrichment feces) and E. coli O157:H7 genes (Stx1 and Stx 2) were targeted and amplified by PCR. A total of 199/456 (43.6%) fecal samples were positive for E. coli O157:H7 by culture. Of the 456 enrichment fecal samples, E. coli O157:H7 Shiga toxin-like genes were detected from 159/456 (34.6%) enrichment fecal samples compared to 43/456 (9.4%) targeted from non-enrichment samples. We demonstrated that elaboration of LT promotes a significant increase in E. coli adherence. This phenotype is primarily dependent on the inherent ADP-ribosylation activity of this toxin, with a secondary role observed for the receptor-binding ability to promote subsequent bacterial adherence. Increased adherence was not due to change in the surface expression of the host receptor for the K88ac adhesion. We constructed and tested a mutant LT toxin containing an amino acid substitution at position 192, and prepared a chimeric LT/STb gene by linkage of STb gene to the LTb subunit gene segment. These genes were transformed into an E. coli strain that produced K88ac fimbriae to form a live vaccine. Purified antigens from constructs expressing K88ac or LT were employed in a subunit vaccine delivered by the intranasal route. Both vaccines stimulated IgA antibody production in young pigs. Pigs receiving the living vaccine were protected from virulent challenge. To develop a piglet model for studying diarrhea disease and developing vaccines, we challenged gnotobiotic piglets with constructed isogenic E. coli strains expressing porcine 987P (F6) fimbriae and a heat-labile or a heat-stable enterotoxin to examine clinical outcomes. Piglets developed identical diarrheal disease when inoculated with constructs expressing human or porcine enterotoxins. In this study, we evaluated extracts of the fermented soy product, Dochi, for its ability to block K88 ETEC binding to enterocytes. We found that Dochi extracts were able to significantly inhibit binding of K88 ETEC to brush borders isolated from porcine enterocytes (50% inhibition, P < 0.05) and cultured porcine intestinal epithelial (IPEC-J2) cells (60% inhibition, P < 0.05). The inhibitory compound in the Dochi extract was found to elute during fractionation at an approximate molecular weight of 1300 Da. E. Calicivirus About 50% of foodborne outbreaks of known etiology are caused by HuNoVs. Specific attachment of the tissue culture adapted porcine sapovirus Cowden strain (TC-Po/SaV) to the vegetable surface will be analyzed next by blocking attachment with specific antibodies, synthetic oligosaccharides, etc. For post-harvest processing, the efficacy of different treatments on reduction of infectious enteric caliciviruses in vegetables will be evaluated using the TC-Po/SaV as a HuNoV surrogate. We tested if TC-Po/SaV attaches to lettuce leaves at different pHs. At pH 5.0 and pH 6.0, 82% and 76% of virus bound to the lettuce leaves, respectively. At pH 3.0, 4.0 and 8.0, viral titers were below 10, the detection limit of our infectivity assay. F. Coronaviruses (CoVs We sequenced and analyzed the full-length genomes of four coronaviruses (CoVs) each from a distinct wild ruminant species in Ohio: sambar deer, a waterbuck, a sable antelope and a white-tailed deer. For two of the CoVs (sambar deer and waterbuck), complete genomes from both the cell culture-adapted and gnotobiotic calf-passaged strains were also sequenced and analyzed. Phylogenetically, wild ruminant CoVs belong to group 2a CoVs with the closest relatedness to the most recent BCoV strains. High nucleotide identities (99.4-99.6%) among the wild ruminant strains and the most recent BCoV strains (BCoV-LUN and BCoV-ENT, 1998) further comfirm the close relatedness. Comparative genetic analysis of captive wild ruminant CoVs and BCoV strains from various NC states suggests that no specific genomic markers are present that allow discrimination between the bovine strains and bovine-like CoVs from captive wild ruminants; furthermore, no specific genetic markers were identified that defined cell culture or calf-passaged strains, or host origin of strains. Our findings suggest that cattle may be reservoirs for CoVs that infect captive wild ruminants or vice. G. Cryptosporidiumparvum and Rotavirus We are developing and optimizing a new method for the sensitive detection of environmental microbial pathogens, especially waterborne pathogens, using label-free photonic crystal biosensor technology. The optical biosensor operates by measuring changes in the reflected resonant wavelength. Using this new sensor technology, we have demonstrated in preliminary experiments that porcine rotavirus can be specifically detected in aqueous samples as sensitively as conventional ELISA but without the need for secondary label processing. Objective 2. Focus on effective interventions- Develop and improve interventions and preventative measures to reduce the incidence and prevalence of infections of cattle and swine with enteric and food borne disease agents A. Campylobacter jejuni In our current studies we demonstrated a reduction in colonization and invasion of epithelial cells in vitro by a Cj1534 mutant. Essentially, a 10-fold reduction in adherence was noted in the Cj1534 mutant strain when compared to the adherence of the parent strain. The invasion results were surprising, with average invasion numbers of 1.8 x 103 for the parent M129 strain, but no invasion of the INT 407 cells with the mutant strain. Therefore, the mutation of the Cj1534 gene had a reduction affect on the colonization of the host cells with C. jejuni, but the mutation also dramatically reduced the invasion. We showed that infected C57BL/6 and congenic interleukin (IL)-10-/- mice serve as models of C. jejuni colonization and enteritis, respectively. Thus, C57BL/6 are resistant to C. jejuni-induced disease. We investigated the interaction of C. jejuni with murine bone marrow-derived DCs (BM-DCs) to assess bacterial killing, DC activation, and ability of C. jejuni-infected BM-DCs to stimulate Campylobacter-specific T cell responses in vitro. BM-DCs challenged with C. jejuni efficiently took up and killed C. jejuni 11168 and significantly up-regulated surface MHC-II, CD40, CD80 and CD86 demonstrating a mature phenotype. Infected BM-DCs secreted significant amounts of tumor necrosis factor-alpha, IL-6 and IL-12p70. Formalin-killed C. jejuni also induced maturation of BM-DCs with similar cytokine production but at a significantly lower magnitude than live bacteria. Maximal activation of murine BM-DCs required internalization of C. jejuni; attachment alone was not sufficient to elicit significant responses. Also, various strains of C. jejuni elicited different magnitudes of cytokine production from BM-DCs. Finally, using a coculture system, C. jejuni-infected BM-DCs induced high-level interferon-³ production from CD4+ T cells indicating Th1 polarization. The twin-arginine translocation (TAT) pathway represents an important virulence mechanism in many bacterial pathogens. Our recent study with IA of the C. jejuni TAT system has indicated that the deletion in a TAT system functional gene significantly compromises the bacterial ability to tolerate stress responses including biofilm formation and cause persistent infection in chickens. C. jejuni has a limited number of genes that contribute to stress responses; however, the high prevalence of C. jejuni infections in domestic livestock including poultry suggests that the TAT system may allow this fastidious pathogen to successfully overcome a number of environmental stresses during transmission. B. Salmonella enterica The Cj1534 gene was cloned into a plasmid vector pYA4495-1 and expressed in a lac operon under a PTRC promoter. Hatchlings were purchased and placed in isolation. Birds were immunized orally with ~109 viable cells of Salmonella Typhimurium x9088 (pYA3493-empty vector control; pYA4495-1-vector with Cj1534) on days 3, 10, and 16. The vaccinated birds were challenged on day 26 with ~105 viable cells of C. jejuni strain NCTC11168 and necropsied 10 days later. Birds vaccinated with the Cj1534 protein expressed by S. Typhimurium demonstrated a C. jejuni reduction of 3.5 logs after challenge exposure. C. Escherichia coli A study was completed to compare the importance of heat-labile enterotoxin (LT) and heat-stable enterotoxin-b in piglets <2 weeks old. Based on the PI percent weight change per h and serum bicarbonate concentrations, the virulence of the STb- mutant (?estB) did not significantly differ from that of the parent. However, deletion of the LT genes (?eltAB) in the STb- mutant resulted in complete abrogation of weight loss, dehydration, and metabolic acidosis in inoculated pigs, and LT complementation restored the virulence of this strain. However, in contrast to previous studies in gnotobiotic piglets, there was no evidence that expression of LT enhanced the ability of the F4+ ETEC strain to colonize the small intestine. Recently, we reported the occurrence of E. coli O157:H7 infection of the urinary bladders of gnotobiotic piglets following oral inoculation and causation of attaching-effacing enterocolitis. In the present study, we conducted transmission electron microscopy on formalin-fixed tissues from these pigs and found that the infecting E. coli O157:H7 organisms had caused pedestal formation at the attachment sites to the transitional epithelial cells, similar to the lesions typically caused in the intestinal tract. We began testing the efficacy of a galactooligosaccharide (GOS) for reducing adherence and colonization of the gnotobiotic piglet intestine by enteropathogenic E. coli (EPEC) and E. coli O157:H7. A 2X2 factorial design with four treatment groups was used: GOS vs control X 2 bacterial strains (EPEC strain E2348/69 or E. coli O157:H7 Shigatoxin-negative ATCC strain 43888). All inoculated piglets (both those treated with GOS and the non-GOS controls) developed gross and microscopic intestinal lesions consistent with EPEC and E. coli O157:H7 infection. However, there was a numerical reduction of the respective inoculum strains isolated from the ileum and spiral colon in CFU/g of PBS-rinsed intestine obtained at necropsy. At the concentration of GOS used, piglets developed mild osmotic diarrhea. A trial was conducted to test the efficacy of 2- and 3-dose regimens of an Escherichia coli O157 vaccine product on cattle shedding E. coli O157:H7 in feces, or being colonized at the terminal rectal mucosa (TRM), and having a humoral response, compared to placebo-treated controls. Vaccine efficacy was 33% and 65% for 2- and 3- dose regimens, respectively. Cattle receiving two doses of vaccine had an increase in EspB antibody titers between days 0 and 63 (p=.0.04). Cattle receiving two or three doses of vaccine had an increase in O157 LPS antibody titers between days 0 and 63 (p<0.0001), and between days 0 and 91 (p<0.0001). A dose-response for fecal shedding efficacy, and humoral antibody responses to EspB and O157 LPS following vaccination were detected. A trial was conducted to test vaccinating feedlot steers against type III secreted proteins of E. coli O157:H7 on the proportion of steers shedding E. coli O157:H7 in feces when fed diets with or without corn wet distillers grains plus solubles (WDGS). Steers were vaccinated three times at four-week intervals beginning at trial initiation. Rectal fecal samples were collected 3, 6, 9, and 12 wks post-vaccination. There was no interaction (P=0.97) between diet and vaccination for E. coli O157:H7 shedding. There was no interaction between treatment and sampling time (P > 0.40) and no effect of time (P = 0.17) on E. coli O157:H7 shedding. Vaccinated steers were 43% less likely to shed E. coli O157: than non-vaccinated steers. Steers fed WDGS were 2.1 times more likely to shed E. coli O157:H7 than cattle fed the control diet. Our initial objective was to determine the fitness cost associated with carriage of peH4H. Growth curve experiments and direct competition studies demonstrated that under most conditions the wild-type strain (H4H) is able to out-compete the plasmid cured strain (NC5). This finding suggests that peH4H confers a direct selective advantage on its host. The critical test occurred when we added peH4H plasmid back into strain NC5 by electroporation to make strain NC5p. However, growth curve and competition studies showed that the fitness advantage was not recovered. At this point we assume that NC5 was altered in some manner during the curing/electroporation process. Because plasmid curing is not a viable approach to conduct these studies, we moved peH4H into a lab strain of E. coli, DH10B (designated GH hereafter). We also included a second plasmid, Cp, from strain H4H that harbors resistance to trimethoprim (see below). Cp was included because of the possibility that there was an important interaction between peH4H and Cp. Introduction of these plasmids into GH produced strain H4H-GH, Cp-GH, and H4H-GH-Cp. We then competed these strains against the parental stock (GH) using a serial passage experiment over 8 days. In all cases, addition of either H4H or Cp increased the fitness of GH with a possible additive effect when both plasmids were introduced into GH. We have passaged H4H in LB media over 1,450 generations and strains H4HGH and CpGH over 500 generations and so far we have found no evidence of any plasmid-free strains in these cultures. Thus, both peH4H and Cp appear to convey some kind of fitness advantage to strain GH. This study assessed the effect(s) of ceftiofur on blaCMY-2 transfer and dissemination by: 1) an in vivo experimental study in which calves were inoculated with competent blaCMY-2  bearing plasmid donors and susceptible recipients and subjected to ceftiofur selection, and 2) an observational study to determine if ceftiofur use in dairy herds is associated with the occurrence and frequency of cephalosporin resistance in Salmonella and commensal E. coli. The first study revealed blaCMY-2 plasmid transfer in both ceftiofur-treated and untreated calves, but detected no enhancement of plasmid transfer associated with ceftiofur treatment. The second study detected no association (P=0.22) between ceftiofur use and either the occurrence of ceftiofur-resistant salmonellosis or the frequency of cephalosporin resistance in commensal E. coli. These findings fail to support a major role for ceftiofur use in the maintenance and dissemination of blaCMY-2 bearing plasmid mediated cephalosporin resistance in commensal E. coli and in pathogenic Salmonella in these dairy cattle populations. The proportion of cephalosporin resistant E. coli returned to starting levels by the end of the sampling period (15 days). 86% of the E. coli isolated from calf feces on the first day of sampling (before any antibiotic treatment was administered) were resistant to cephalosporin compared to 23% of E. coli isolated from cow feces on the first day of sampling. Treatment with antibiotics reduced the total number of E. coli in the feces of fresh cows and in the feces of calves. While the trends for cows and calves was similar, the baseline and magnitude of effects were dramatically different. D. Lawsonia intracellularis The disinfectants Synergize®, Roccal®-D plus, Virkon-S®, Tek-Trol®, Nolvasan S®, DC&R®, and Certi-Dine® were tested for activity against two isolates from affected pigs. Both isolates were completely inactivated with Synergize® (1:256), Roccal®-D (1:256) and DC&R® (1:128). Inactivation was e 99% with Virkon-S® (1%) and 90-99% with Tek-Trol® (1:256) and Nolvasan S® (1:128) and <90% with Certi-Dine® (1:128) within 10 minutes. The objectives of this study were to evaluate the humoral immune response and onset and duration of fecal shedding in pregnant broodmares after administration of a modified-live vaccine of L. intracellularis and to determine if specific maternal antibodies against L. intracellularis are passively transferred to foals. All vaccinated mares remained healthy and no adverse vaccine reactions were observed. Fecal shedding was found in 3 mares and lasted from 1 to 3 days. Serologic responses were detected in all vaccinated mares with titers ranging from 60 to 240. Antibodies against L. intracellularis persisted for up to 10 weeks. Colostral antibodies were detected in 2 out of the 8 seropositive mares. Maternally derived antibodies were detected in 6 foals and persisted for up to 8 weeks. E. Porcine Group A Rotavirus We recently synthesized a sialyl-lactosylphosphatidyl-ethanolamine (SLPE) neoglycolipid. In field trials this inhibitor blocked infection, virus shedding and diarrhea using a twice a day dosage administered to newborn pigs at the time of virus inoculation. Results from these studies suggest that specific oligosaccharide/isoflavone mixtures or profiles may be engineered to provide a deliverable nutriceutical for the treatment and prevention of rotavirus disease across susceptible animal species and humans. F. Cornavirus We investigated the effects of a prior and ongoing PRRSV infection (down-regulator of innate immunity), on PRCV (up-regulator of innate immunity) co-infection and clinical disease and the pathologic and immunologic interactions of the two viruses, as a model for respiratory viral co-infections potentially relevant to SARS. To establish a persisting PRRSV infection, weaned PRRSV and PRCV seronegative pigs were initially inoculated intranasally (IN) and intramuscularly with PRRSV SD23983 strain at about 3 wks of age. At 10 days after PRRSV infection, anesthetized pigs were inoculated IN and intratracheally with the PRCV ISU-1 strain. The PRRSV+PRCV pigs had greater reductions in weight gains and more severe gross or histological pneumonic lesions compared to either single infection at PRCV PID 2 to 21. The enhanced disease coincided with higher Th1 (IFN-³, IL-12) but lower Th2 (IL-4) cytokine responses in serum for the dually infected pigs at PRCV PID 6 to 21. Greater amounts of PRCV antigen and RNA were observed in the lungs of the dually-infected pigs at PRCV PID 2 compared to PRCV single infection, possibly due to suppression of innate immune responses (IFN± and NK cell activity) by the ongoing PRRSV infection; however, the opposite trend (decreased PRCV antigen and RNA) was noted at PRCV PID 4 to 21. In contrast, PRRSV RNA levels were higher in the dually-infected pigs compared to PRRSV single infection at PRCV PID 4 to 14, consistent with more severe PRRSV-related alveolar macrophage apoptosis in the lungs detected by in situ Tunnel assay. Antibody (Ab) titers to PRRSV were higher for dually-infected compared to single infected pigs at PRCV PID 4 to 21 (PRRSV PID 14 to 31), reflecting increased PRRSV replication. In contrast, lower virus neutralization Abs to PRCV were observed in the dual-infected pigs compared to single PRCV infection at PRCV PID 21, which coincides with lower PRCV RNA levels in dual-infected pigs (PRCV PID 4 to 21). G. Cryptosporidium parvum Suppressive subtractive hybridization experiments aimed at identifying specific sporozoite genes expressed in response to host cell attachment or exposure to the inhibitory lipid indicate these processes occur without the necessity for attachment- or lipid-induced differential gene expression. This lipid, introduced as part of different dietary regimens, also is being examined for its ability to reduce or eliminate bovine cryptosporidiosis in newborn calves. We determined that cryptosporidium oocysts are effectively retarded from overland transport by vegetative filter strips (VFS) and that the mechanism of this retardation is specific adhesion to the clay particles of the soil that occurs as a consequence of reduced flow over a vegetated surface as compared to bare soil. H. Lactic acid bacteria and probiotics We evaluated the patterns of TLR2- (recognizes peptidoglycan), TLR3- (recognizes dsRNA) and TLR9- (recognizes CpG) expressing antigen presenting cells (APC) (defined by CD14, SWC3, CD11R1 markers) in spleen and blood of gnotobiotic (Gn) pigs after colonization with a mixture of two strains of lactic acid bacteria (LAB), L. acidophilus and L. reuteri or infection with the virulent human rotavirus (HRV) Wa strain. We demonstrated that LAB induced strong TLR2-expressing APC responses in blood and spleen; HRV induced a TLR3 response in spleen; and TLR9 responses were induced by either HRV (in spleen) or LAB (in blood). LAB and HRV had an additive effect on TLR2- and TLR9-expressing APC responses, consistent with the adjuvant effect of LAB. Overall, the frequencies of TLR-expressing CD14+ APCs were higher than CD14- APCs. LAB enhanced the IFN-³ (Th1) and IL-4 (Th2) responses in serum, but they had a suppressive effect on the TLR3- and TLR9-expressing CD14- APC responses in spleen and the serum IFN-± response induced by HRV. We examined rotavirus-specific IFN-³ producing CD4+, CD8+ and CD4+CD8+ T cell responses in gnotobiotic pigs infected with virulent human rotavirus (HRV) or vaccinated with an attenuated HRV vaccine (three or two doses) or an attenuated oral rotavirus vaccine priming and VP 2/6-virus-like particle (VLP) vaccine intranasal boosting regimen. In virulent HRV infected pigs, HRV-specific IFN-³ producing T cells resided primarily in ileum. Attenuated-HRV+2/6VLP induced similar frequencies of intestinal IFN-³ producing T cells as the virulent HRV, whereas 3x or 2x doses of attenuated HRV vaccine were less effective. Protection rates against rotavirus diarrhea upon virulent HRV challenge were significantly correlated with frequencies of intestinal IFN-³ producing T cells, suggesting their role in protective immunity. Objective 3. Focus on disseminating knowledge- Provide training and continuing education opportunities and dissemination of information to students, producers, veterinarians and diagnostic laboratories. NDSU in conjunction with the Department of Veterinary Public Health and Preventive Medicine, Makerere University (Mak), Kampala Uganda, developed a 3-credits short term course (4 weeks) International Animal Production Disease Surveillance and Public Health. The course was designed to facilitate diversity in student training and exposure. In order to produce a broadly inclusive, open minded, and globally engaged science workforce.