Unraveling clonal CD8 T cell expansion and identification of essential factors in γ-herpesvirus-induced lymphomagenesis.

Alcelaphine gammaherpesvirus 1 (AlHV-1) asymptomatically persists in its natural host, the wildebeest. However, cross-species transmission to cattle results in the induction of an acute and lethal peripheral T cell lymphoma-like disease (PTCL), named malignant catarrhal fever (MCF). Our previous findings demonstrated an essential role for viral genome maintenance in infected CD8+ T lymphocytes but the exact mechanism(s) leading to lymphoproliferation and MCF remained unknown. To decipher how AlHV-1 dysregulates T lymphocytes, we first examined the global phenotypic changes in circulating CD8+ T cells after experimental infection of calves. T cell receptor repertoire together with transcriptomics and epigenomics analyses demonstrated an oligoclonal expansion of infected CD8+ T cells displaying effector and exhaustion gene signatures, including GZMA, GNLY, PD-1, and TOX2 expression. Then, among viral genes expressed in infected CD8+ T cells, we uncovered A10 that encodes a transmembrane signaling protein displaying multiple tyrosine residues, with predicted ITAM and SH3 motifs. Impaired A10 expression did not affect AlHV-1 replication in vitro but rendered AlHV-1 unable to induce MCF. Furthermore, A10 was phosphorylated in T lymphocytes in vitro and affected T cell signaling. Finally, while AlHV-1 mutants expressing mutated forms of A10 devoid of ITAM or SH3 motifs (or both) were able to induce MCF, a recombinant virus expressing a mutated form of A10 unable to phosphorylate its tyrosine residues resulted in the lack of MCF and protected against a wild-type virus challenge. Thus, we could characterize the nature of this γ-herpesvirus-induced PTCL-like disease and identify an essential mechanism explaining its development.

Hemorrhagic bowel syndrome in dairy cattle: Gross, histological, and microbiological characterization.

Hemorrhagic bowel syndrome (HBS) is a sporadic and fatal disease of predominantly lactating dairy cattle, characterized by segmental hemorrhage and luminal clot formation in the small intestine. Although, Clostridium perfringens and Aspergillus fumigatus have been associated with HBS, the pathogenesis and cause are currently unknown. In this study, 18 naturally occurring cases of HBS (7 necropsied immediately following euthanasia, 11 with 12-48 hour postmortem intervals) were investigated to characterize the pathology and the intestinal microbiome. Hemorrhagic bowel syndrome was characterized by a single small-intestinal, intramucosal hematoma with dissection of the lamina muscularis mucosae. In most cases necropsied immediately after euthanasia (4/7), the intestinal mucosa proximal to the hematoma contained 9 to 14, dispersed, solitary or clustered, erosions or lacerations measuring 4 to 45 mm. In 77% (37/48) of these mucosal lesions, microscopic splitting of the lamina muscularis mucosae comparable to the hematoma was present. These findings suggest the intramucosal hematoma to originate from small mucosal erosions through dissecting hemorrhage within the lamina muscularis mucosae. No invasive fungal growth was observed in any tissue. Bacteriological cultivation and nanopore sequencing showed a polymicrobial population at the hematoma and unaffected intestine, with mostly mild presence of C perfringens at selective culture. Gross and microscopic lesions, as well as the culture and sequencing results, were not in support of involvement of C perfringens or A fumigatus in the pathogenesis of HBS.

Species-Specific Humoral Immune Responses in Sheep and Goats upon Small Ruminant Lentivirus Infections Inversely Correlate with Protection against Virus Replication and Pathological Lesions.

Maedi-Visna-like genotype A strains and Caprine arthritis encephaltis-like genotype B strains are small ruminant lentiviruses (SRLV) which, for incompletely understood reasons, appear to be more virulent in sheep and goats, respectively. A 9-month in vivo infection experiment using Belgian genotype A and B SRLV strains showed that almost all homologous (genotype A in sheep; genotype B in goats) and heterologous (genotype A in goats; genotype B in sheep) intratracheal inoculations resulted in productive infection. No differences in viremia and time to seroconversion were observed between homologous and heterologous infections. Higher viral loads and more severe lesions in the mammary gland and lung were however detected at 9 months post homologous compared to heterologous infection which coincided with strongly increased IFN-γ mRNA expression levels upon homologous infection. Pepscan analysis revealed a strong antibody response against immune-dominant regions of the capsid and surface proteins upon homologous infection, which was absent after heterologous infection. These results inversely correlated with protection against virus replication in target organs and observed histopathological lesions, and thus require an in-depth evaluation of a potential role of antibody dependent enhancement in SRLV infection. Finally, no horizontal intra- and cross-species SRLV transmission to contact animals was detected.

An essential role for γ-herpesvirus latency-associated nuclear antigen homolog in an acute lymphoproliferative disease of cattle.

Wildebeests carry asymptomatically alcelaphine herpesvirus 1 (AlHV-1), a γ-herpesvirus inducing malignant catarrhal fever (MCF) to several ruminant species (including cattle). This acute and lethal lymphoproliferative disease occurs after a prolonged asymptomatic incubation period after transmission. Our recent findings with the rabbit model indicated that AlHV-1 infection is not productive during MCF. Here, we investigated whether latency establishment could explain this apparent absence of productive infection and sought to determine its role in MCF pathogenesis. First, whole-genome cellular and viral gene expression analyses were performed in lymph nodes of MCF-developing calves. Whereas a severe disruption in cellular genes was observed, only 10% of the entire AlHV-1 genome was expressed, contrasting with the 45% observed during productive infection in vitro. In vivo, the expressed viral genes included the latency-associated nuclear antigen homolog ORF73 but none of the regions known to be essential for productive infection. Next, genomic conformation analyses revealed that AlHV-1 was essentially episomal, further suggesting that MCF might be the consequence of a latent infection rather than abortive lytic infection. This hypothesis was further supported by the high frequencies of infected CD8(+) T cells during MCF using immunodetection of ORF73 protein and single-cell RT-PCR approaches. Finally, the role of latency-associated ORF73 was addressed. A lack of ORF73 did not impair initial virus replication in vivo, but it rendered AlHV-1 unable to induce MCF and persist in vivo and conferred protection against a lethal challenge with a WT virus. Together, these findings suggest that a latent infection is essential for MCF induction.

Two cases of congenital lipomatosis in calves.

Fetal tumours in domestic animals are considered rare. Congenital lipomatous changes have been sporadically reported in fetal, neonatal and young bovids. Their prognosis is often guarded compared with benign lipocytic tumours and depends on their location and degree of infiltration. They can infiltrate the subcutis, deeper musculature and even the skeleton. Four cases of congenital infiltrative lipomatosis have been described in bovine fetuses and young calves. In this report we describe an additional two cases in a neonatal Belgian Blue White calf and a fetal dairy calf. A potential role in bovine abortion needs further investigation. Furthermore, a suggestion is made to adapt the nomenclature from infiltrative lipoma to lipomatosis in order to emphasize the poor prognosis due to the local infiltrative behaviour of these lesions.

Effect of a DIVA vaccine with and without in-feed use of coated calcium-butyrate on transmission of Salmonella Typhimurium in pigs.

BACKGROUND: For satisfactory Salmonella control, good biosecurity along the pork production chain is crucial, although additional control measures on-farm need to be considered. This study evaluated the effect of two potential control measures against the spread of Salmonella Typhimurium via a transmission experiment with 56 piglets (3-15 weeks of age): two groups were orally vaccinated with 107 - 108 Colony Forming Units (CFU)/2 mL of a new attenuated Salmonella Typhimurium vaccine 'Salmoporc-∆rfaJ' with DIVA capacities (Differentiation between Infected and Vaccinated Animals) (n = 2x16); the feed of one group was additionally supplemented with coated calcium-butyrate salt. Two weeks post vaccination, four pigs per group were orally challenged with 107 CFU/2 mL of a Salmonella Typhimurium strain 112910a. Both groups were compared with a positive (challenged/untreated; n = 16) and negative (unchallenged/untreated; n = 8) control group. Until six weeks post challenge, blood, individual faecal and finally tissue samples were examined. Adjusted transmission ratios 'Ra' were estimated, based on the challenge strain isolation from faecal and/or tissue samples. RESULTS: In both intervention groups, Ra values were lower compared to the positive control group, although these differences were not significant. In the combination group DIVA vaccine + coated butyrate, less non-challenged contact animals excreted Salmonella and less tissue samples were found Salmonella-positive in all pigs, when compared to the positive control group (P < 0.01). Seroconversion was detected in none of the vaccinated animals before challenge, when using a commercial lipopolysaccharide (LPS) ELISA targeting only Salmonella O-antigens, deleted in this vaccine. This was in contrast with an in-house whole-cell ELISA testing for various Salmonella antigens, in which Salmonella-specific antibodies were found pre-challenge in the serum of the vaccinated pigs. CONCLUSIONS: Both interventions showed a limited, non-significant reduction of Salmonella transmission between piglets. They may have applications towards Salmonella control and surveillance. Firstly, the number of Salmonella excreting contact pigs was significantly lower in the group where vaccination was combined with coated calcium-butyrate salt in the feed; secondly, the new vaccine confirmed its DIVA capacity. Therefore, these interventions merit further research with larger sample sizes, to optimize their use for Salmonella programmes.

Cholesterol granuloma associated with otitis media and leptomeningitis in a cat due to a Streptococcus canis infection.

Cholesterol granuloma in the middle ear is a pathological condition often associated with otitis media in humans. Cholesterol granulomas in cats are rarely described. To our knowledge, this is the first report of middle ear cholesterol granuloma in a cat, associated with otitis media and leptomeningitis due to a Streptococcus canis septicemia.

Granulome à cholestérol associé à une otite moyenne et à une leptoméningite chez un chat causé par une infection parStreptococcus canis. Un granulome à cholestérol dans l'oreille moyenne est une affection pathologique souvent associée à l'otite moyenne chez les humains. Les granulomes à cholestérol chez les chats sont rarement décrits. À notre connaissance, il s'agit du premier rapport d'un granulome à cholestérol de l'oreille moyenne chez un chat, associé à l'otite moyenne et à la letpoméningite, causé par une septicémie à Streptococcus canis.(Traduit par Isabelle Vallières).

Wildebeest-Derived Malignant Catarrhal Fever: A Bovine Peripheral T Cell Lymphoma Caused by Cross-Species Transmission of Alcelaphine Gammaherpesvirus 1.

Gammaherpesviruses (γHVs) include viruses that can induce lymphoproliferative diseases and tumors. These viruses can persist in the long term in the absence of any pathological manifestation in their natural host. Alcelaphine gammaherpesvirus 1 (AlHV-1) belongs to the genus Macavirus and asymptomatically infects its natural host, the wildebeest (Connochaetes spp.). However, when transmitted to several susceptible species belonging to the order Artiodactyla, AlHV-1 is responsible for the induction of a lethal lymphoproliferative disease, named wildebeest-derived malignant catarrhal fever (WD-MCF). Understanding the pathogenic mechanisms responsible for the induction of WD-MCF is important to better control the risks of transmission and disease development in susceptible species. The aim of this review is to synthesize the current knowledge on WD-MCF with a particular focus on the mechanisms by which AlHV-1 induces the disease. We discuss the potential mechanisms of pathogenesis from viral entry into the host to the maintenance of viral genomes in infected CD8+ T lymphocytes, and we present current hypotheses to explain how AlHV-1 infection induces a peripheral T cell lymphoma-like disease.

Epidemiology of Bovine Hemorrhagic Bowel Syndrome in Belgium and The Netherlands.

Hemorrhagic bowel syndrome (HBS) is a poorly understood, sporadic and often fatal disease in cattle. Although, HBS is considered an important disease in dairy cattle, epidemiological data is largely lacking. This study describes the epidemiology of HBS in Belgium and the Netherlands, based on necropsy records from 2009 to 2022, and reports characteristics from 27 cows and 35 dairy operations with HBS, gathered through a survey. The annual incidence of HBS has a significantly increasing trend both at cow and herd level, with incidence above 3.2% in necropsied mature dairy cattle in the most recent years. Estimated herd-level incidence in the Netherlands was double the estimated incidence in Belgium, which might be explained by higher herd size in the Netherlands. Occurrence of HBS was most prevalent in fall, being 40.1% higher than the average of the other seasons. In 35 Flemish (Belgian) surveyed dairy herds with HBS, manifestation of HBS was mostly as solitary cases, and if multiple cases occurred, the time interval was highly variable. In addition, the majority of cows with HBS (61.1%; 16/26) were in more than 100 days lactation. In conclusion, HBS is an important and possibly emerging disease in dairy cattle in Belgium and the Netherlands.

Antiserum against the conserved nine amino acid N-terminal peptide of influenza A virus matrix protein 2 is not immunoprotective.

The recent emergence and rapid spread of the pandemic H1N1 swine influenza virus reminded us once again of the need for a universal influenza vaccine that can elicit heterosubtypic protection. Here, we show the superior immunogenicity and immunoprotective capacity of the full-length matrix protein 2 ectodomain (M2e) peptide coupled to keyhole limpet haemocyanin (KLH) compared with the N-terminal 9 aa residues of M2e (SP1). Immunization with M2e-KLH protected mice against a lethal challenge with influenza A virus and significantly reduced weight loss and lung virus titres. In addition, passive transfer of serum raised in rabbits against M2e-KLH protected mice against a lethal influenza virus challenge, whereas serum from rabbits immunized with SP1-KLH did not. Nevertheless, immunofluorescence staining revealed that rabbit serum raised against SP1-KLH bound specifically to infected Madin-Darby canine kidney cells. We conclude that the peptide SP1 contains an immunogenic epitope that is not sufficient for immunoprotection.

No H- and L-type cases in Belgium in cattle diagnosed with bovine spongiform encephalopathy (1999-2008) aging seven years and older.

BACKGROUND: The bovine spongiform encephalopathy (BSE) epidemic presented homogeneity of the phenotype. This classical BSE (called C-type) was probably due to the contamination of the food chain by a single prion strain. However, due to the active surveillance and better techniques, two rare variants of BSE have been recently reported in different continents without a clear correlation to the BSE epidemic. These emerging types behave as different strains of BSE and were named H-type and L-type according to the high and low molecular mass of the unglycosylated fragment of their proteinase K resistant prion protein (PrPres). In these types, the proportion of the un-, mono- and di-glycosylated fragments of PrP (glycoprofile) is also atypical and represents an effective diagnostic parameter. This study evaluated the presence of such types in bovine of 7 years and older in Belgium. RESULTS: The Belgian BSE archive contained 41 bovines of at least 7 years of age. The biochemical features of their PrPres were analyzed by Western blot with five antibodies recognising different regions of PrPres, from N- to C-terminus: 12B2, 9A2, Sha31, SAF84 and 94B4. All antibodies clearly detected PrPres except 12B2 antibody, which is specific for N-terminal region 101-105, a PrP region that is only retained in H-types. The glycoprofiles did correspond to that of C-type (with more than 55% of diglycosylated PrPres using antibody 94B4). Therefore, all cases have the features of C-type BSE. CONCLUSIONS: This study supports that, among the BSE cases of 7 years and older identified in Belgium, none was apparently of the H- or L- type. This is consistent with the very rare occurrence of atypical BSE and the restricted dimension of Belgium. These results shed some light on the worldwide prevalence of atypical BSE.

Lethal ricin intoxication in two adult dogs: toxicologic and histopathologic findings.

Two adult dogs with the same owner were intoxicated by ingestion of fertilizer composed of residual plant material of the castor bean plant (Ricinus communis L.). Both dogs died within 2 and 3 days, respectively, after the first signs of vomiting and abundant hemorrhagic diarrhea. Toxicologic and histopathologic examinations were performed on different organs. Histopathologic examination of the kidneys revealed tubular degeneration and necrosis and membranous glomerulonephritis. Additionally, myocardial degeneration with localized inflammation, lymphoid necrosis, and depletion in the spleen and mesenteric lymph nodes and hemorrhagic ulcerative gastroenteritis were found. The 2 cases could be used to elucidate the lethal dose of ricin and the histopathologic lesions in dogs.

Nationwide Screening for Bee Viruses and Parasites in Belgian Honey Bees.

The health of honey bees is threatened by multiple factors, including viruses and parasites. We screened 557 honey bee (Apis mellifera) colonies from 155 beekeepers distributed all over Belgium to determine the prevalence of seven widespread viruses and two parasites (Varroa sp. and Nosema sp.). Deformed wing virus B (DWV-B), black queen cell virus (BQCV), and sacbrood virus (SBV) were highly prevalent and detected by real-time RT-PCR in more than 95% of the colonies. Acute bee paralysis virus (ABPV), chronic bee paralysis virus (CBPV) and deformed wing virus A (DWV-A) were prevalent to a lower extent (between 18 and 29%). Most viruses were only present at low or moderate viral loads. Nevertheless, about 50% of the colonies harbored at least one virus at high viral load (>107 genome copies/bee). Varroa mites and Nosema sp. were found in 81.5% and 59.7% of the honey bee colonies, respectively, and all Nosema were identified as Nosema ceranae by real time PCR. Interestingly, we found a significant correlation between the number of Varroa mites and DWV-B viral load. To determine the combined effect of these and other factors on honey bee health in Belgium, a follow up of colonies over multiple years is necessary.

Short-term biodistribution and clearance of intravenously administered silica nanoparticles.

Recently, concerns have been raised about potential adverse effects of synthetic amorphous silica, commonly used as food additive (E551), since silica nanoparticles have been detected in food containing E551. We examined the biodistribution and excretion in female Sprague-Dawley rats of NM-200, a well characterized nanostructured silica representative for food applications. A single intravenous injection of NM-200 was applied at a dose of 20 mg/kgbw, followed by autopsy after 6 and 24 h. The main organs where silicon accumulated were liver and spleen. The silicon concentration significantly decreased in spleen between 6 and 24 h. In liver the tendency was the same but the effect was not significant. This could be due to clearance of the spleen to the liver via the splenic vein, while liver clearance takes more time due to hepatic processing and biliary excretion. In treated animals the liver showed in addition a prominent increase of macrophages between both evaluation moments. Within the first 24 h, silicon was mainly excreted through urine. Further studies are necessary to evaluate the toxicokinetics of different types of silica nanomaterials at lower exposure doses in order to be able to predict kinetics and toxicity of silica nanoparticles depending on their physicochemical characteristics.

Evaluation of mycobacteria-specific gamma interferon and antibody responses before and after a single intradermal skin test in cattle naturally exposed to M. avium subsp. paratuberculosis and experimentally infected with M. bovis.

This study reports on the diagnostic potential of IFN-γ release assays and serology for Mycobacterium bovis in six naturally M. avium subsp. paratuberculosis (Map) exposed bulls of which four were intratracheally infected with a Belgian field strain of M. bovis. Heparinized blood, serum and fecal samples were collected at regular time intervals for mycobacteria-specific IFN-γ release assays, antibody analysis and for Map culture respectively. Single intradermal skin test (SIT) with bovine tuberculin (PPD-B) was performed on day 115 and animals were sacrificed on day 133 after M. bovis infection. Organs were collected and stored for histopathological examination, modified Ziehl-Neelsen staining and bacteriological analysis of M. bovis and Map by culture and RT-PCR. Prior to infection five animals showed positive IFN-γ responses to avian PPD (PPD-A) and four were positive in Map PCR (IS900) on faeces. Three M. bovis infected animals reacted as early as day 14 with sustained higher PPD-B than PPD-A specific IFN-γ responses, whereas the fourth animal (with the strongest PPD-A response prior to infection) showed sustained higher PPD-B specific IFN-γ levels only a day 56 after infection. Two of the infected animals had a sustained positive IFN-γ response to the ESAT-6/CFP-10/TB7.7 (QuantiFERON®-TB Gold) peptide cocktail as early as day 14, among which the animal with the initial high PPD-A response. Later during infection, positive responses were found to ESAT-6 peptides in three infected bulls and to CFP-10 peptides in all four infected bulls. One of the control animals reacted intermittently to the ESAT-6/CFP10/TB7.7 cocktail. Prior to SIT, weak but positive MPB83/MBP70 specific antibody responses were detected in two of the infected bulls. All four M. bovis infected bulls reacted with a positive skin test and showed, as reported by others, increased mycobacteria specific IFN-γ production and increased positive responses in MPB83/MBP70 specific serology after SIT. At autopsy, M. bovis lesions were detected in all four experimentally infected bulls. Our results indicate that in Map exposed cattle, M. bovis diagnosis using IFN-γ assays needs a combination of PPD-B/A and ESAT-6/CFP10 for early and optimal sensitivity and that sensitivity of MPB83/MBP70 serodiagnosis is dramatically increased by prior skin testing. Map exposure did not interfere with the development of SIT in M. bovis infected animals, but resulted in a false positive M. bovis specific IFN-γ and antibody response after SIT in one of the two control animals (which remained negative in skin-test).

TSE detected in a Belgian ARR-homozygous sheep via active surveillance.

It is commonly accepted that scrapie-resistance and -susceptibility in sheep are genetically controlled. Consequently, the selection of sheep with scrapie-resistant genotypes is currently one of the most important objectives of the sheep breeding associations. However, during the last two years, new data have become available on transmissible spongiform encephalopathy (TSE) cases in TSE-resistant sheep in several European Union member states. The present paper describes the first Belgian natural "atypical" TSE case in a sheep with a scrapie-resistant genotype (ARR/ARR) detected via active surveillance. No other infections or diseases were detected in the source flock. The continued finding of new "atypical" TSE cases in sheep with scrapie-resistant genotypes undermines the purpose and efficacy of the breeding programs.

First TBEV serological screening in Flemish wild boar.

In the frame of a Flemish wildlife surveillance in 2013, a serological screening was performed on sera from wild boar (Sus scrofa; n=238) in order to detect tick-borne encephalitis virus (TBEV)-specific antibodies. Neutralising antibodies were titrated with a seroneutralisation test (SNT), using two cut-off titres (1/10-1/15). Seven wild boars were found TBEV-seropositive and showed moderate (>1/15) to high (>1/125) SNT-titres; three individuals had borderline results (1/10-1/15). This study demonstrated the presence of TBEV-specific antibodies in wild boar and highlighted potential TBEV-foci in Flanders. Additional surveillance including direct virus testing is now recommended.

Serologic screening for 13 infectious agents in roe deer (Capreolus capreolus) in Flanders.

INTRODUCTION: In order to investigate the role of roe deer in the maintenance and transmission of infectious animal and human diseases in Flanders, we conducted a serologic screening in 12 hunting areas. MATERIALS AND METHODS: Roe deer sera collected between 2008 and 2013 (n=190) were examined for antibodies against 13 infectious agents, using indirect enzyme-linked immunosorbent assay, virus neutralisation, immunofluorescence, or microagglutination test, depending on the agent. RESULTS AND DISCUSSION: High numbers of seropositives were found for Anaplasma phagocytophilum (45.8%), Toxoplasma gondii (43.2%) and Schmallenberg virus (27.9%), the latter with a distinct temporal distribution pattern following the outbreak in domestic ruminants. Lower antibody prevalence was found for Chlamydia abortus (6.7%), tick-borne encephalitis virus (5.1%), Neospora caninum (4.8%), and Mycobacterium avium subsp paratuberculosis (4.1%). The lowest prevalences were found for Leptospira (1.7%), bovine viral diarrhoea virus 1 (1.3%), and Coxiella burnetii (1.2%). No antibodies were found against Brucella sp., bovine herpesvirus 1, and bluetongue virus. A significant difference in seroprevalence between ages (higher in adults >1 year) was found for N. caninum. Four doubtful reacting sera accounted for a significant difference in seroprevalence between sexes for C. abortus (higher in females). CONCLUSIONS: Despite the more intensive landscape use in Flanders, the results are consistent with other European studies. Apart from maintaining C. abortus and MAP, roe deer do not seem to play an important role in the epidemiology of the examined zoonotic and domestic animal pathogens. Nevertheless, their meaning as sentinels should not be neglected in the absence of other wild cervid species.

Putative roles of the CNF2 and CDTIII toxins in experimental infections with necrotoxigenic Escherichia coli type 2 (NTEC2) strains in calves.

Newborn colostrum-restricted calves were orally inoculated with an Escherichia coli strain, identified originally as non-pathogenic, and into which the plasmid pVir was conjugally transferred. This resulted in diarrhea, intestinal lesions and extra-intestinal invasion, suggesting that factors affecting these pathogenic properties are located on pVir. In order to analyze the respective roles of the toxins CNF2 and CDTIII in the pathogenesis, colostrum-restricted calves were inoculated with isogenic mutants in the cnf2 and the cdt-III genes. The loss of cnf2 is associated with a reduction in the pathogenicity, since diarrhea does not occur in calves challenged, in spite of successful colonization of the intestine. Nevertheless, the mutant strain remains able to invade the bloodstream and to localize in the internal organs. Conversely, the calves inoculated with mutant in the cdt-III gene evolved in the same way as wild-type strain-inoculated calves with regard to clinical signs and macroscopic or microscopic lesions.