Digital Commons Network^™

Leptospirosis In The Azores: The Rodent Connection, Gary W. Witmer, Hernani Martins, Lidia Flor

United States Department of Agriculture Wildlife Services: Staff Publications

The Azores are Portuguese islands in the North Atlantic Ocean. The culture is very agrarian with a large cattle industry. Unfortunately, there is a chronic leptospirosis problem within the people, livestock, companion animals, and wildlife of the Azores. Introduced rodents play a significant role as maintenance hosts of this disease. We review the situation and make recommendations for reducing the occurrence and hazard of leptospirosis in the Azores. Areas addressed include the need for a better understanding of the epidemiology of the disease and the role of rodents, development of an effective rodent control program, improvements in farm practices and …

Gene Expression Profiling Of Bovine Macrophages In Response To Escherichia Coli O157:H7 Lipopolysaccharide, Carol G. Chitko-Mckown, James M. Fox, Laura C. Miller, Michael P. Heaton, James L. Bono, James E. Keen, William M. Grosse, William W. Laegried

United States Department of Agriculture-Agricultural Research Service / University of Nebraska-Lincoln: Faculty Publications

The aim of this study was to identify changes in bovine macrophage gene expression in response to treatment with Escherichia coli 0157:H7 lipopolysaccharide (LPS), utilizing a human gene microarray. Bovine cDNA from control and LPStreated primary macrophages hybridized to greater than 5644 (79.8%) of the non-control gene targets on a commercially available microarray containing greater than 7075 targets (Incyte Genomics, St. Louis, MO). Of these target sequences, 44 were differentially expressed upon exposure to LPS, including 18 genes not previously reported to exist in cattle. These included a pentaxin-related gene, CASP8, TNF-induced genes, interferon-induced genes, and inhibitors of apoptosis. Using …

Phylogenetic Analyses Of Texas Isolates Indicate An Evolving Subtype Of The Clade B Feline Immunodeficiency Viruses, Eric A. Weaver, Ellen W. Collisson, Margaret Slater, Guan Zhu

Nebraska Center for Virology: Faculty Publications

Rigorous phylogenetic analyses were used to compare the nucleotide sequences of feline immunodeficiency virus strains isolated from Texas and throughout the world. The envelope V3-V4 sequences and capsid gene of the Texas isolates formed a cluster between subtypes B and E. Statistical comparisons with other published sequences confirmed that the Texas group is a unique cluster, possibly a new subtype, arising from subtype B.

Feline immunodeficiency virus (FIV) was initially isolated in 1987 from a cat in California with severe immunodeficiency and has been recognized as a common worldwide feline pathogen (11, 14, 19, 20, 33). FIV-infected cats exhibit a …

Mapping The Presence Of Wolbachia Pipientis On The Phylogeny Of Filarial Nematodes: Evidence For Symbiont Loss During Evolution, Maurizio Casiraghi, Odile Bain, Ricardo Guerrero, Coralie Martin, Vanessa Pocacqua, Scott Lyell Gardner, Alberto Francheschi, Claudio Bandi

Harold W. Manter Laboratory of Parasitology: Faculty Publications

Wolbachia pipientis is a bacterial endosymbiont associated with arthropods and filarial nematodes. In filarial nematodes, W. pipientis has been shown to play an important role in the biology of the host and in the immuno-pathology of filariasis. Several species of filariae, including the most important parasites of humans and animals (e.g. Onchocerca volvulus, Wuchereria bancrofti and Dirofilaria immitis) have been shown to harbor these bacteria. Other filarial species, including an important rodent species (Acanthocheilonema viteae), which has been used as a model for the study of filariasis, do not appear to harbor these symbionts. There are …

Parapharyngodon Duniae N. Sp. (Nematoda: Pharyngodonidae) In Phrynohyas Venulosa (Anura: Hylidae) From The Área De Conservación Guanacaste, Guanacaste, Costa Rica, Charles R. Bursey, Daniel R. Brooks

Harold W. Manter Laboratory of Parasitology: Faculty Publications

Parapharyngodon duniae n. sp. (Nematoda: Pharyngodonidae) in the large intestine of the veined tree frog, Phrynohyas venulosa, from the Área de Conservación Guanacaste in northwestern Costa Rica is described and illustrated. Parapharyngodon duniae n. sp. represents the 34th species assigned to the genus, the 10th species from the Neotropical Realm, and only the third species to parasitize anurans. It is distinguished from the other Neotropical species by having postbulbar ovaries and a prebulbar excretory pore.

Erroneous Reports Of The Neotropical Echinococcus Oligarthrus As A Cause Of Echinococcosis In India [Critical Comment], Antonio D'Alessandro, Robert L. Rausch

Harold W. Manter Laboratory of Parasitology: Faculty Publications

Two cases of submandibular echinococcosis have been reported in adult residents of India (Kini et al., 1997; Sahni et al., 2000). It was stated in both publications that the cestode was E. oligarthrus, "a rare variety of E. granulosus", based on morphogical characteristics. It was also mentioned that three cases of E. granulosus with similar submandibular metacestode had been previously diagnosed in India. The metacestode is readily distinguishable from that of E. oligarthrus. The final hosts of E. oligarthrus are several neotropical species of wild cats and metacestode occurs in hystricognath, all of them absent in India. …

Role Of Interleukin 6 In Myocardial Dysfunction Of Meningococcal Septic Shock, Nazima Pathan, Cheryl A. Hemingway, Ash A. Alizadeh, Alick C. Stephens, Jennifer C. Boldrick, Emmanuelle E. Oragui, Colm Mccabe, Steven B. Welch, Adeline Whitney, Peter O’Gara, Simon Nadel, David A. Relman, Sian E. Harding, Michael Levin

United States Department of Veterans Affairs: Publications

Background Myocardial failure has a central role in the complex pathophysiology of septic shock and contributes to organ failure and death. During the sepsis-induced inflammatory process, specific factors are released that depress myocardial contractile function. We aimed to identify these mediators of myocardial depression in meningococcal septic shock.

Methods We combined gene-expression profiling with protein and cellular methods to identify a serum factor causing cardiac dysfunction in meningococcal septic shock. We identified genes that were significantly upregulated in blood after exposure to meningococci. We then selected for further analysis those genes whose protein products had properties of a myocardial depressant …

Chronic Wasting Disease Management In Nebraska

Zoonotics and Wildlife Disease: Publications

The presence of Chronic Wasting Disease (CWD) in free-roaming and domesticated cervine animal populations of deer and elk in the state of Nebraska presents a threat to the natural resources of the state. The presence of this disease could drastically restrict the successful achievement of the goals and objectives of the Nebraska Game and Parks Commission (NGPC) as set forth in their long range plan “Focusing on the Future…A Plan for Nebraska’s Fish, Wildlife and Parkland Resources”. Objective 3 in the Big Game section of this plan is to “Monitor the status and health of deer, so reasonable assumptions may …

Inhibition Of Bovine T Lymphocyte Responses By Extracts Of The Stomach Worm Ostertagia Ostertagi, M.T. Gómez-Muñoz, A. Canals-Caballero, S. Almeria, P. Pasquali, D.S. Zarlenga, L.C. Gasbarre

United States Department of Agriculture-Agricultural Research Service / University of Nebraska-Lincoln: Faculty Publications

Lowered immune responses during bovine ostertagiosis have been reported in both in vivo and in vitro assay systems. In the present study we have employed three different life cycle stages of the ne- matode Ostertagia ostertagi to determine if products of this economically important parasite inhibit in vitro proliferation of Con A-stimulated cells from uninfected animals. We have demonstrated an inhibitory effect upon the growth of Con A-stimulated lymphocytes after addition of fourth stage larval (L4) soluble extract (L4SE) to the cultures. In contrast, extracts from the third stage larvae (L3) had little or no inhibitory activity. The suppressive products …

Decreased Shedding Of Escherichia Coli O157:H7 By Cattle Following Vaccination With Type Iii Secreted Proteins, Andrew A. Potter, Sandra Klashinsky, Yuling Li, Elizabeth Frey, Hugh Townsend, Dragan Rogan, Galen E. Erickson, Susanne Hinkley, Terry J. Klopfenstein, Rodney A. Moxley, David R. Smith, B. Brett Finlay

School of Veterinary and Biomedical Sciences: Faculty Publications

Cattle are an important reservoir of Escherichia coli O157:H7 leading to contamination of food and water, and subsequent human disease. This pathogen colonizes its hosts by producing several proteins such as Tir and EspA that are secreted by a type III secretion system. These proteins play a role in colonization of the intestine, suggesting that they might be useful targets for the development of a vaccine to reduce levels of this organism in cattle. Vaccination of cattle with proteins secreted by E. coli O157:H7 significantly reduced the numbers of bacteria shed in feces, the numbers of animals that shed, and …

Estimating The True Prevalence Of Mycobacterium Bovis In Hunter-Harvested White-Tailed Deer In Michigan, Daniel J. O’Brien, Stephen M. Schmitt, Dale E. Berry, Scott D. Fitzgerald, Jolene R. Vanneste, Timothy J. Lyon, Diane Magsig, Jean S. Fierke, Thomas M. Cooley, Laura S. Zwick, Bruce V. Thomsen

Michigan Bovine Tuberculosis Bibliography and Database

Apparent prevalence, although useful as a consistent index, may underestimate the true prevalence of disease. In Michigan, the ability to estimate the true prevalence of bovine tuberculosis (TB; caused by Mycobacterium bovis) in free-ranging white-tailed deer (Odocoileus virginianus) will become increasingly important to accurately assess progress towards eradication. Our objectives were threefold: to estimate the true prevalence of M. bovis in free-ranging deer in Michigan, to evaluate the effectiveness of existing TB surveillance methods, and to indirectly assess whether TB epidemiologic data from captive cervid herds can be meaningfully extrapolated to free-ranging populations. The study population consisted …

Fourth Annual Symposium In Virology, John Owens

John Owens: Speeches and Appearances

What a pleasure it is to take part in welcoming you to this Fourth Annual Symposium in Virology. Such a tremendous program lies ahead! And how pleased and proud we are that this year's symposium is a special tribute to our colleague Dr. James Van Etten, Professor of Plant Pathology in our Institute of Agriculture and Natural Resources here at the University of Nebraska-Lincoln, who last-year was elected to membership in the National Academy of Sciences.

Dihydrofolate Reductase Mutations In Plasmodium Vivax From Indonesia And Therapeutic Response To Sulfadoxine Plus Pyrimethamine, Michele D. Hastings, Kathryn M. Porter, Jason D. Maguire, Ika Susanti, Winni Kania, Michael J. Bangs, Carol Hopkins Sibley, J. Kevin Baird

United States Navy: Publications

Background. The target enzyme of pyrimethamine is dihydrofolate reductase (DHFR), but little is known about allelic variants of dhfr in Plasmodium vivax populations. Still less is known about associations between specific alleles and the failure of sulfadoxine/pyrimethamine (S/P) to clear the erythrocytic stages of P. vivax in vivo.

Methods. We studied P. vivax dhfr mutations in 24 patients who received S/P therapy in Papua or Central Java, Indonesia, and we measured the resistance of the alleles in vitro in a dhfr yeast expression assay.

Results. Fourteen (58%) of 24 patients had an inadequate therapeutic response. Two of 6 alleles that …

Hepatocellular Carcinoma In The Woodchuck Model Of Hepatitis B Virus Infection, Bud C. Tennant, Ilia A. Toshkov, Simon F. Peek, James R. Jacob, Stephan Menne, William E. Hornbuckle, Raymond D. Schinazi, Brent E. Korba, Paul J. Cote, John L. Gerin

United States Department of Veterans Affairs: Publications

The Eastern woodchuck (Marmota monax) harbors a DNA virus (Woodchuck hepatitis virus [WHV]) that is similar in structure and replicative life cycle to the human hepatitis B virus (HBV). Like HBV, WHV infects the liver and can cause acute and chronic hepatitis. Furthermore, chronic WHV infection in woodchucks usually leads to development of hepatocellular carcinoma (HCC) within the first 2–4 years of life. The woodchuck model has been important in the preclinical evaluation of safety and efficacy of the antiviral drugs now in use for treatment of HBV infection and continues to serve as an important, predictive model …

Apoptosis And Porcine Reproductive And Respiratory Syndrome Virus, Laura C. Miller, James M. Fox

Roman L. Hruska U.S. Meat Animal Research Center: Reports

Despite numerous studies examining the possible induction of apoptosis in porcine reproductive and respiratory syndrome virus (PRRSV)-infected cells, it remains unclear if PRRSV infection results in direct apoptotic induction. There is clear evidence that apoptotic cells are present in tissues from PRRSV-infected pigs. However, many of these studies have failed to show that the apoptotic cells are infected with PRRSV. This has led some investigators to propose that ‘‘bystander’’ cells, not infected cells, become apoptotic during PRRSV infection by a yet undetermined mechanism. Studies examining the induction of the apoptotic gene expression response to PRRSV infection are needed to determine …

Beta-2-Microglobulin Haplotypes In U.S. Beef Cattle And Association With Failure Of Passive Transfer In Newborn Calves, Michael L. Clawson, Michael P. Heaton, Carol G. Chitko-Mckown, James M. Fox, Timothy P. L. Smith, Warren M. Snelling, John W. Keele, William W. Laegreid

Roman L. Hruska U.S. Meat Animal Research Center: Reports

Failure of passive transfer (FPT) is a condition in which neonates do not acquire protective serum levels of maternal antibodies. A principal component of antibody transport is the neonatal receptor for the Fc portion of immunoglobulin, a heterodimer of a MHC-1 alpha-chain homolog (FCGRT) and beta- 2-microglobulin (B2M). Previously, two FCGRT haplotypes were associated with differences in immunoglobulin G (IgG) passive transfer in cattle (Laegreid et al. (2002) Mamm Genome 13, 704–710). The present study had two objectives: first, to characterize the B2M haplotype structure in a diverse group of U.S. beef cattle, and second, to evaluate those haplotypes for …

Research At Nebraska, 2003-2004 Report, University Of Nebraska-Lincoln

Office of Research and Economic Development: Publications

This publication is a snapshot of the fiscal year’s activities. To read more stories about research and creativity at Nebraska, visit the online edition at http://research.unl.edu/news/04-ORG-005_AR_Final.pdf. Additional multimedia are available for many stories.

Empirical Measurement Of Parasite Transmission Between Groups In A Colonial Bird, Charles R. Brown, Mary Bomberger Brown

School of Natural Resources: Faculty Publications

While epidemiological models have suggested that the spread of parasites
and infectious diseases often depends critically on the extent of movement by infected
individuals between populations, there is little empirical information for any organism on
the frequency of between-group parasite transmission or how it varies spatially. The transmission
of parasites between discrete social groups or populations may also help determine
a host’s total parasite or pathogen exposure. We measured the introduction of parasitic bugs
(Oeciacus vicarius) into colonies of Cliff Swallows (Petrochelidon pyrrhonota) by transient
birds from outside each group. The transmission of bugs increased strongly …

A Processed Pseudogene Contributes To Apparent Mule Deer Prion Gene Heterogeneity, Kelly A. Brayton, Katherine I. O'Rourke, Amy K. Lyda, Michael W. Miller, Donald P. Knowles Jr.

Zoonotics and Wildlife Disease: Publications

Pathogenesis and transmission of the prion disorders (transmissible spongiform encephalopathies, TSEs) are mediated by a modified isoform of the prion protein (PrP). Prion protein gene (PRNP) alleles associated with relative susceptibility to TSE have been identified in sheep, humans and possibly elk. Comparable data have not been derived for mule deer, a species susceptible to the TSE chronic wasting disease (CWD). Initial analysis of the open reading frame (ORF) in exon 3 of the mule deer PRNP gene revealed polymorphisms in all 145 samples analyzed, with 10 potential polymorphic sites. Because 144/145 (99.3%) of the samples were heterozygous …

Resistance To Scrapie In Prp Arr/Arq Heterozygous Sheep Is Not Caused By Preferential Allelic Use, P. A. Caplazi, Katherine I. O'Rourke, T. V. Baszler

Zoonotics and Wildlife Disease: Publications

Scrapie is a prion disease in sheep that, similar to Kuru or variant Creutzfeldt-Jakob disease (vCJD), requires both prion exposure and a genetic susceptibility. Susceptibility is defined by amino acid polymorphisms in the single copy prion protein (PrP) gene, Prnp. Specifically, in sheep homozygous for A at position 136 and R at position 154, susceptibility is defined by R or Q at position 171. The genotype ARQ/ARQ confers susceptibility, whereas (apart from a few reported exceptions) genotypes ARR/ARQ and ARR/ARR confer resistance. Similarly, a M/V polymorphism at Prnp codon 129 determines susceptibility to vCJD and Kuru,4 with partial resistance conferred …

Polymorphisms In The Prion Precursor Functional Gene But Not The Pseudogene Are Associated With Susceptibility To Chronic Wasting Disease In White-Tailed Deer, Katherine I. O'Rourke, Terry R. Spraker, Linda K. Hamburg, Thomas E. Besser, Kelly A. Brayton, Donald P. Knowles

Zoonotics and Wildlife Disease: Publications

Chronic wasting disease (CWD) status and PrP genotypes were determined for a group of 133 wild white-tailed deer in a 780 acre enclosure in western Nebraska, USA. Approximately half of the deer tested showed evidence of PrP^d in the brainstem or lymphoid tissues. Four PRNP alleles encoding amino acid substitutions were identified, with substitutions at residues 95 (Q→H), 96 (G→S) or 116 (A→G), each with serine (S) at residue 138. In addition, a processed pseudogene with two alleles encoding five or six copies of the octapeptide repeat was identified in 26% of the deer. Both alleles encoded asparagine (N) …

Transmission Of Transmissible Mink Encephalopathy To Raccoons (Procyon Lotor) By Intracerebral Inoculation, Amir N. Hamir, Janice M. Miller, Katherine I. O'Rourke, Jason C. Bartz, Mick J. Stack, Melanie J. Chaplin

Zoonotics and Wildlife Disease: Publications

To determine the transmissibility of transmissible mink encephalopathy (TME) agent to raccoons and to provide information about clinical course, lesions, and suitability of currently used diagnostic procedures for detection of transmissible spongiform encephalopathies (TSEs) in raccoons, 4 raccoon kits were inoculated intracerebrally with a brain suspension from mink experimentally infected with TME. One uninoculated raccoon kit served as a control. All 4 animals in the TME-inoculated group showed clinical signs of neurologic disorder and were euthanized between 21 and 23 weeks postinoculation (PI). Necropsy examinations revealed no gross lesions. Spongiform encephalopathy was observed by light microscopy, and the presence of …

Variable Patterns Of Distribution Of Prpcwd In The Obex And Cranial Lymphoid Tissues Of Rocky Mountain Elk (Cervus Elaphus Nelsoni) With Subclinical Chronic Wasting Disease, T. R. Spraker, A. Balachandran, D. Zhuang, Katherine I. O'Rourke

Zoonotics and Wildlife Disease: Publications

Sections of medulla oblongata. taken at the level of the obex, palatine tonsil and medial retropharyngeal lymph node from 10,269 captive Rocky Mountain elk (Cervus elaphus nelsoni), were examined by immunohistochemical staining with monoclonal antibody for the prion protein associated with the transmissible spongiform encephalopathy of cervids, chronic wasting disease (PrP^CWD). The protein was detected in 226 of them. On the basis of the anatomical location of the deposits in the brainstem of 183 elk, four distinct patterns of distribution of PrP^CWD within the parasympathetic region of the dorsal motor nucleus of the vagus nerve …

Detection Of Antibodies To Squalene Iii. Naturally Occurring Antibodies To Squalene In Humans And Mice, Gary R. Matyas, Mangala Rao, Phillip R. Pittman, Robert Burge, Iris E. Robbins, Nabila M. Wassef, Brandie Thivierge, Carl R. Alving

United States Army: Publications

An ELISA-based assay is described for the measurement of antibodies to squalene (SQE) in human serum and plasma. The assay was adapted from the previously described assay for murine antibodies to SQE (J. Immunol. Methods 267 (2002) 119). Like the murine SQE antibody assay, the human antibody assay used sterile cell culture 96-well plates coated with SQE (20 nmol/well). Phosphate-buffered saline (PBS)–0.5% casein was used as both a blocking agent and dilution buffer. The assay has a high through-put capacity and is reproducible and quantitative. This assay was used to evaluate samples from three different human cohorts. The first cohort …

Biology Of PrpSc Accumulation In Two Natural Scrapie-Infected Sheep Flocks, Patrick Caplazi, Katherine I. O'Rourke, Cynthia Wolf, Daniel Shaw, Timothy V. Baszler

Zoonotics and Wildlife Disease: Publications

Sheep scrapie is a prion disease that requires interaction of exogenous prions with host prion protein (PrP) supporting prion formation. Disease is associated with deposition of a host-generated conformational variant of PrP, PrP^sc, in a variety of tissues, including brain, resulting in fatal spongiform encephalopathy. Efficiency of PrP^sc formation is determined by polymorphisms in the PrP-coding sequence. This article adds to previous data of natural sheep scrapie, concentrating on the effect of host genotype and age on PrP^sc accumulation patterns during preclinical and clinical disease. Two entire scrapie-infected, predominantly Suffolk-cross, sheep flocks euthanized for regulatory purposes …

Heterotrophic Plate Count Bacteria—What Is Their Significance In Drinking Water?, Martin J. Allen, Stephen C. Edberg, Donald J. Reasoner

United States Environmental Protection Agency: Publications

While the literature documents the universal occurrence of heterotrophic plate count (HPC) bacteria in soils, foods, air, and all sources of water, there is a lingering question as to whether this group of organisms may signal an increased health risk when elevated populations are present in drinking water. This paper reviews the relevant literature on HPC bacteria in drinking water, the lack of clinical evidence that elevated populations or specific genera within the HPC flora pose an increased health risk to any segment of the population, and the appropriate uses of HPC data as a tool to monitor drinking water …

Distribution Of Six Virulence Factors In Aeromonas Species Isolated From Us Drinking Water Utilities: A Pcr Identification, K. Sen, M. Rodgers

United States Environmental Protection Agency: Publications

Aims: To examine whether Aeromonas bacteria isolated from municipally treated water had virulence factor genes.

Methods and Results: A polymerase chain reaction-based genetic characterization determined the presence of six virulence factors genes, elastase (ahyB), lipase (pla/lip/lipH3/alp-1) flagella A and B (flaA and flaB), the enterotoxins, act, alt and ast, in these isolates. New primer sets were designed for all the target genes, except for act. The genes were present in 88% (ahyB), 88% (lip), 59% (fla), 43% (alt), 70% (act) and 30% …

Plant Cell-Based Intimin Vaccine Given Orally To Mice Primed With Intimin Reduces Time Of Escherichia Coli O157:H7 Shedding In Feces, Nicole A. Judge, Hugh S. Mason, Alison D. O'Brien

Uniformed Services University of the Health Sciences

Intimin is the primary adhesin of Escherichia coli O157:H7, the most common infectious cause of bloody diarrhea in the United States and the leading cause of acute kidney failure in children who develop hemolytic uremic syndrome. Cattle are the primary reservoir of E. coli O157:H7. Indeed, most cases of E. coli O157:H7 infection in the United States occur after ingestion of contaminated undercooked hamburger or produce that had contact with bovine manure. Because intimin is required for persistent colonization of neonatal calves and adult cattle, we hypothesized that an intimin-based vaccination strategy in calves would reduce colonization of cattle with …

Bovine Tuberculosis In Swedish Farmed Deer: Detection And Control Of The Disease, Helene Wahlström

Michigan Bovine Tuberculosis Bibliography and Database

Bovine tuberculosis (BTB) was introduced into Swedish farmed deer herds in 1987. Epidemiological investigations showed that 10 deer herds had become infected (July 1994) and a common source of infection, a consignment of 168 imported farmed fallow deer, was identified (I).

As trace-back of all imported and in-contact deer was not possible, a control program, based on tuberculin testing, was implemented in July 1994. As Sweden has been free from BTB since 1958, few practicing veterinarians had experience in tuberculin testing. In this test, result relies on the skill, experience and conscientiousness of the testing veterinarian. Deficiencies in performing the …

Different Biological Responses Of Pigs Of Two Genetic Populations To Prrsv Challenge Suggests Underlying Genetic Variation In Susceptibility/Resistance To Prrsv, Derek B. Petry, Justin W. Holl, John Weber, Rodger K. Johnson, Alan R. Doster, Fernando Osario

Nebraska Swine Reports

The objective was to determine whether genetic variation in susceptibility to Porcine Respiratory and Reproductive Syndrome virus (PRRSV) exists. One hundred pigs from each of two distinctly different populations (NE Index Line, I, and Duroc-Hampshire cross pigs, DH) were challenged with PRRSV at 26 days old. A littermate to each challenged pig was included in the experiment without PRRSV challenge to serve as a control. Body weight and temperature were recorded and blood samples were drawn from all pigs on the day of challenge and 4, 7 and 14 days post-challenge. All pigs were sacrificed and a necropsy was performed …