Rapid estimation of Salmonella enterica contamination level in ground beef - Application of the time-to-positivity method using a combination of molecular detection and direct plating.

Little progress has been made in decreasing the incidence rate of salmonellosis in the US over the past decade. Mitigating the contribution of contaminated raw meat to the salmonellosis incidence rate requires rapid methods for quantifying Salmonella, so that highly contaminated products can be removed before entering the food chain. Here we evaluated the use of Time-to-Positivity (TTP) as a rapid, semi-quantitative approach for estimating Salmonella contamination levels in ground beef. Growth rates of 14 Salmonella strains (inoculated at log 1 to -2 CFU/g) were characterized in lean ground beef mTSB enrichments and time-to-detection was determined using culture and molecular detection methods. Enrichments were sampled at five timepoints and results were used to construct a prediction model of estimated contamination level by TTP (superscript indicates time in hours) defined as TTP4: ≥5 CFU/g; TTP6: ≤5, ≥1 CFU/g; TTP8: ≤1, ≥0.01 CFU/g; with samples negative at 8 h estimated ≤0.01 CFU/g. Model performance measures showed high sensitivity (100%) and specificity (83% and 93% for two detection methods) for samples with a TTP4, with false negative rates of 0%.

Genomic signatures of Mannheimia haemolytica that associate with the lungs of cattle with respiratory disease, an integrative conjugative element, and antibiotic resistance genes.

BACKGROUND: Mannheimia haemolytica typically resides in cattle as a commensal member of the upper respiratory tract microbiome. However, some strains can invade their lungs and cause respiratory disease and death, including those with multi-drug resistance. A nucleotide polymorphism typing system was developed for M. haemolytica from the genome sequences of 1133 North American isolates, and used to identify genetic differences between isolates from the lungs and upper respiratory tract of cattle with and without clinical signs of respiratory disease. RESULTS: A total of 26,081 nucleotide polymorphisms were characterized after quality control filtering of 48,403 putative polymorphisms. Phylogenetic analyses of nucleotide polymorphism genotypes split M. haemolytica into two major genotypes (1 and 2) that each were further divided into multiple subtypes. Multiple polymorphisms were identified with alleles that tagged genotypes 1 or 2, and their respective subtypes. Only genotype 2 M. haemolytica associated with the lungs of diseased cattle and the sequence of a particular integrative and conjugative element (ICE). Additionally, isolates belonging to one subtype of genotype 2 (2b), had the majority of antibiotic resistance genes detected in this study, which were assorted into seven combinations that ranged from 1 to 12 resistance genes. CONCLUSIONS: Typing of diverse M. haemolytica by nucleotide polymorphism genotypes successfully identified associations with diseased cattle lungs, ICE sequence, and antibiotic resistance genes. Management of cattle by their carriage of M. haemolytica could be an effective intervention strategy to reduce the prevalence of respiratory disease and supplemental needs for antibiotic treatments in North American herds.

Reduced lentivirus susceptibility in sheep with TMEM154 mutations.

Visna/Maedi, or ovine progressive pneumonia (OPP) as it is known in the United States, is an incurable slow-acting disease of sheep caused by persistent lentivirus infection. This disease affects multiple tissues, including those of the respiratory and central nervous systems. Our aim was to identify ovine genetic risk factors for lentivirus infection. Sixty-nine matched pairs of infected cases and uninfected controls were identified among 736 naturally exposed sheep older than five years of age. These pairs were used in a genome-wide association study with 50,614 markers. A single SNP was identified in the ovine transmembrane protein (TMEM154) that exceeded genome-wide significance (unadjusted p-value 3×10(-9)). Sanger sequencing of the ovine TMEM154 coding region identified six missense and two frameshift deletion mutations in the predicted signal peptide and extracellular domain. Two TMEM154 haplotypes encoding glutamate (E) at position 35 were associated with infection while a third haplotype with lysine (K) at position 35 was not. Haplotypes encoding full-length E35 isoforms were analyzed together as genetic risk factors in a multi-breed, matched case-control design, with 61 pairs of 4-year-old ewes. The odds of infection for ewes with one copy of a full-length TMEM154 E35 allele were 28 times greater than the odds for those without (p-value<0.0001, 95% CI 5-1,100). In a combined analysis of nine cohorts with 2,705 sheep from Nebraska, Idaho, and Iowa, the relative risk of infection was 2.85 times greater for sheep with a full-length TMEM154 E35 allele (p-value<0.0001, 95% CI 2.36-3.43). Although rare, some sheep were homozygous for TMEM154 deletion mutations and remained uninfected despite a lifetime of significant exposure. Together, these findings indicate that TMEM154 may play a central role in ovine lentivirus infection and removing sheep with the most susceptible genotypes may help eradicate OPP and protect flocks from reinfection.

Two bovine hepacivirus genome sequences from U.S. cattle.

Bovine hepacivirus (BoHV) is closely related to the hepatitis C virus (HCV) in humans and can cause both acute and chronic liver infections in cattle. BoHV was first identified in Ghana and Germany in 2015 and since then it has been detected and characterized in other countries around the world, but no strains have been sequenced from U.S. cattle. To date, BoHV has been classified into 2 genotypes (1 and 2), with genotype 1 being further divided into 11 subtypes (A-K). However, the true genetic diversity of BoHV is likely underestimated given limited surveillance and a lack of published genome sequences. Here, we sequenced 2 nearly complete BoHV genomes from serum samples collected in 2019 from beef cattle in Missouri. Sequence comparisons and phylogenetic analysis showed that isolate MARC/2019/60 had high sequence homology with genotype 1, subtype E isolates from China. In contrast, isolate MARC/2019/50 represented a novel BoHV subtype within genotype 2. Thus, we report the first genomic characterization of BoHV isolates from U.S. cattle, and the second complete BoHV2 genome worldwide. This work increases our knowledge of the global genetic diversity of BoHV and demonstrates the co-circulation of divergent BoHV strains in U.S. cattle.

Phylogeny of Shiga toxin-producing Escherichia coli O157 isolated from cattle and clinically ill humans.

Cattle are a major reservoir for Shiga toxin-producing Escherichia coli O157 (STEC O157) and harbor multiple genetic subtypes that do not all associate with human disease. STEC O157 evolved from an E. coli O55:H7 progenitor; however, a lack of genome sequence has hindered investigations on the divergence of human- and/or cattle-associated subtypes. Our goals were to 1) identify nucleotide polymorphisms for STEC O157 genetic subtype detection, 2) determine the phylogeny of STEC O157 genetic subtypes using polymorphism-derived genotypes and a phage insertion typing system, and 3) compare polymorphism-derived genotypes identified in this study with pulsed field gel electrophoresis (PFGE), the current gold standard for evaluating STEC O157 diversity. Using 762 nucleotide polymorphisms that were originally identified through whole-genome sequencing of 189 STEC O157 human- and cattle-isolated strains, we genotyped a collection of 426 STEC O157 strains. Concatenated polymorphism alleles defined 175 genotypes that were tagged by a minimal set of 138 polymorphisms. Eight major lineages of STEC O157 were identified, of which cattle are a reservoir for seven. Two lineages regularly harbored by cattle accounted for the majority of human disease in this study, whereas another was rarely represented in humans and may have evolved toward reduced human virulence. Notably, cattle are not a known reservoir for E. coli O55:H7 or STEC O157:H(-) (the first lineage to diverge within the STEC O157 serogroup), which both cause human disease. This result calls into question how cattle may have originally acquired STEC O157. The polymorphism-derived genotypes identified in this study did not surpass PFGE diversity assessed by BlnI and XbaI digestions in a subset of 93 strains. However, our results show that they are highly effective in assessing the evolutionary relatedness of epidemiologically unrelated STEC O157 genetic subtypes, including those associated with the cattle reservoir and human disease.

Escherichia coli O157:H7 tir 255 T > A allele strains differ in chromosomal and plasmid composition.

Shiga toxin-producing Escherichia coli (STEC) O157:H7 strains with the T allele in the translocated intimin receptor polymorphism (tir) 255 A > T gene associate with human disease more than strains with an A allele; however, the allele is not thought to be the direct cause of this difference. We sequenced a diverse set of STEC O157:H7 strains (26% A allele, 74% T allele) to identify linked differences that might underlie disease association. The average chromosome and pO157 plasmid size and gene content were significantly greater within the tir 255 A allele strains. Eighteen coding sequences were unique to tir 255 A allele chromosomes, and three were unique to tir 255 T allele chromosomes. There also were non-pO157 plasmids that were unique to each tir 255 allele variant. The overall average number of prophages did not differ between tir 255 allele strains; however, there were different types between the strains. Genomic and mobile element variation linked to the tir 255 polymorphism may account for the increased frequency of the T allele isolates in human disease.

First gene-edited calf with reduced susceptibility to a major viral pathogen.

Bovine viral diarrhea virus (BVDV) is one of the most important viruses affecting the health and well-being of bovine species throughout the world. Here, we used CRISPR-mediated homology-directed repair and somatic cell nuclear transfer to produce a live calf with a six amino acid substitution in the BVDV binding domain of bovine CD46. The result was a gene-edited calf with dramatically reduced susceptibility to infection as measured by reduced clinical signs and the lack of viral infection in white blood cells. The edited calf has no off-target edits and appears normal and healthy at 20 months of age without obvious adverse effects from the on-target edit. This precision bred, proof-of-concept animal provides the first evidence that intentional genome alterations in the CD46 gene may reduce the burden of BVDV-associated diseases in cattle and is consistent with our stepwise, in vitro and ex vivo experiments with cell lines and matched fetal clones.

Recent Emergence of Bovine Coronavirus Variants with Mutations in the Hemagglutinin-Esterase Receptor Binding Domain in U.S. Cattle.

Bovine coronavirus (BCoV) has spilled over to many species, including humans, where the host range variant coronavirus OC43 is endemic. The balance of the opposing activities of the surface spike (S) and hemagglutinin-esterase (HE) glycoproteins controls BCoV avidity, which is critical for interspecies transmission and host adaptation. Here, 78 genomes were sequenced directly from clinical samples collected between 2013 and 2022 from cattle in 12 states, primarily in the Midwestern U.S. Relatively little genetic diversity was observed, with genomes having >98% nucleotide identity. Eleven isolates collected between 2020 and 2022 from four states (Nebraska, Colorado, California, and Wisconsin) contained a 12 nucleotide insertion in the receptor-binding domain (RBD) of the HE gene similar to one recently reported in China, and a single genome from Nebraska collected in 2020 contained a novel 12 nucleotide deletion in the HE gene RBD. Isogenic HE proteins containing either the insertion or deletion in the HE RBD maintained esterase activity and could bind bovine submaxillary mucin, a substrate enriched in the receptor 9-O-acetylated-sialic acid, despite modeling that predicted structural changes in the HE R3 loop critical for receptor binding. The emergence of BCoV with structural variants in the RBD raises the possibility of further interspecies transmission.

Association of ARRDC3 and NFIA variants with bovine congestive heart failure in feedlot cattle.

Background: Bovine congestive heart failure (BCHF) has become increasingly prevalent among feedlot cattle in the Western Great Plains of North America with up to 7% mortality in affected herds. BCHF is an untreatable complex condition involving pulmonary hypertension that culminates in right ventricular failure and death. Genes associated with BCHF in feedlot cattle have not been previously identified. Our aim was to search for genomic regions associated with this disease. Methods: A retrospective, matched case-control design with 102 clinical BCHF cases and their unaffected pen mates was used in a genome-wide association study. Paired nominal data from approximately 560,000 filtered single nucleotide polymorphisms (SNPs) were analyzed with McNemar's test. Results: Two independent genomic regions were identified as having the most significant association with BCHF: the arrestin domain-containing protein 3 gene ( ARRDC3), and the nuclear factor IA gene ( NFIA, mid- p-values, 1x10 -8 and 2x10 -7, respectively). Animals with two copies of risk alleles at either gene were approximately eight-fold more likely to have BCHF than their matched pen mates with either one or zero risk alleles at both genes (CI 95 = 3-17). Further, animals with two copies of risk alleles at both genes were 28-fold more likely to have BCHF than all others ( p-value = 1×10 -7, CI 95 = 4-206). A missense variant in ARRDC3 (C182Y) represents a potential functional variant since the C182 codon is conserved among all other jawed vertebrate species observed. A two-SNP test with markers in both genes showed 29% of 273 BCHF cases had homozygous risk genotypes in both genes, compared to 2.5% in 198 similar unaffected feedlot cattle. This and other DNA tests may be useful for identifying feedlot animals with the highest risk for BCHF in the environments described here. Conclusions: Although pathogenic roles for variants in the ARRDC3 and NFIA genes are unknown, their discovery facilitates classifying animals by genetic risk and allows cattle producers to make informed decisions for selective breeding and animal health management.

Bacterial community analysis of beef cattle feedlots reveals that pen surface is distinct from feces.

The surface of beef cattle feedlot pens is commonly conceptualized as being packed uncomposted manure. Despite the important role that the feedlot pen may play in the transmission of veterinary and zoonotic pathogens, the bacterial ecology of feedlot surface material is not well understood. Our present study characterized the bacterial communities of the beef cattle feedlot pen surface material using 3647 full-length 16S rDNA sequences, and we compared the community composition of feedlot pens to the fecal source material. The feedlot surface composite was represented by members of the phylum Actinobacteria (42%), followed by Firmicutes (24%), Bacteroidetes (24%), and Proteobacteria (9%). The feedlot pen surface material bacterial communities were clearly distinct from those of the feces from animals in the same pen. Comparisons with previously published results of feces from the animals in the same pen reveal that, of 139 genera identified, only 25 were present in both habitats. These results indicate that, microbiologically, the feedlot pen surface material is separate and distinct from the fecal source material, suggesting that bacteria that originate in cattle feces face different selection pressures and survival challenges during their tenure in the feedlot pen, as compared to their residence in the gastrointestinal tract.

A Conserved Histophilus somni 23S Intervening Sequence Yields Functional, Fragmented 23S rRNA.

Histophilus somni is a Gram-negative bacterial organism that acts as an opportunistic pathogen and is a fastidious member of the Pasteurellaceae family associated with diseases of respiratory, reproductive, cardiac, and other tissues of ruminants. We identified an intervening sequence (IVS) embedded in all five copies of the 23S rRNA gene in the closed genome sequence of the H. somni isolate USDA-ARS-USMARC-63250 that may play an important role in affecting the biology of the organism. Sequencing the RNA from this isolate shows that most of the IVS is cleaved from the transcript, resulting in independent fragments of this structural rRNA that remain functional within the bacterial ribosome. The IVS lies between positions 1170 and 1278 bp of the 3,017-bp gene and exhibits self-complementarity between its 5' and 3' ends that predicts a stem-loop structure interrupting helix-45 in the transcribed 23S rRNA. Excision removes a 94-nucleotide (nt) stem-loop structure that displays an unusual 1-nt 3' end overhang instead of the more typical 2-nt overhang commonly observed at the ends of other excised IVS stem-loops. A comparison with genomes of other H. somni isolates indicates that this IVS is highly conserved, with 31 of 32 complete genomes having similar interruptions of canonical 23S rRNA genes. The potential biological effects of either the released IVS or the fragmentation of the functional 23S rRNA are unknown, but fragmentation may enhance rRNA degradation in ways that contribute to the regulation of gene expression. IMPORTANCE The genome biology underlying H. somni virulence, pathogenicity, environmental adaptability, and broad tissue tropism is understood poorly. We identified a novel H. somni 109-nt IVS stem-loop structure, of which the central portion is excised from the 23S rRNA transcript, resulting in the fragmentation of this rRNA in the H. somni isolate USDA-ARS-USMARC-63250 and the release of a 94-nt structured RNA of unknown function. We determined that this peculiar rRNA biology is widespread among sequenced H. somni isolates, suggesting it has importance to organism biology. The fragmented 23S rRNA molecules remain functional in the ribosome, given that the isolate grows in culture. The structured excised portion of the IVS, presumably due to the action of the endoribonuclease III, has an unusual 1-nt 3' end overhang. This newly discovered H. somni 23S rRNA fragmentation may enhance rRNA degradation providing a previously unrecognized avenue for regulating H. somni biological processes.

Evaluating Large Spontaneous Deletions in a Bovine Cell Line Selected for Bovine Viral Diarrhea Virus Resistance.

Bovine viral diarrhea virus's (BVDV) entry into bovine cells involves attachment of virions to cellular receptors, internalization, and pH-dependent fusion with endosomal membranes. The primary host receptor for BVDV is CD46; however, the complete set of host factors required for virus entry is unknown. The Madin-Darby bovine kidney (MDBK) cell line is susceptible to BVDV infection, while a derivative cell line (CRIB) is resistant at the level of virus entry. We performed complete genome sequencing of each to identify genomic variation underlying the resistant phenotype with the aim of identifying host factors essential for BVDV entry. Three large compound deletions in the BVDV-resistant CRIB cell line were identified and predicted to disrupt the function or expression of the genes PTPN12, GRID2, and RABGAP1L. However, CRISPR/Cas9 mediated knockout of these genes, individually or in combination, in the parental MDBK cell line did not impact virus entry or replication. Therefore, resistance to BVDV in the CRIB cell line is not due to the apparent spontaneous loss of PTPN12, GRID2, or RABGAP1L gene function. Identifying the functional cause of BVDV resistance in the CRIB cell line may require more detailed comparisons of the genomes and epigenomes.

Detection of bovine inflammatory cytokines IL-1ß, IL-6, and TNF-α with a multiplex electrochemiluminescent assay platform.

Commercially available bovine-specific assays are limited in number, and multiplex assays for this species are rare. Our objective was to develop a multiplex assay for the bovine inflammatory cytokines IL-1ß, IL-6, and TNF-α using the Meso Scale Discovery U-PLEX platform. "Do-It-Yourself" ELISA kits that contained polyclonal antibodies, both unlabeled and biotinylated, and the specific recombinant bovine cytokine standard, were purchased for each of these three cytokines. The biotinylated antibodies were coupled to linkers that bind to specific locations within each well of the U-PLEX plate. Unique linkers were used for each of the cytokines. The unlabeled antibodies were conjugated with electrochemiluminescent labels to serve as detection antibodies. Each cytokine assay was optimized individually prior to performing an optimization on the multiplex assay containing reagents for all three cytokines. To calculate cytokine concentrations, standard curves were developed using the recombinant cytokines and were run concurrently on each plate. Standard curves for IL-1ß and TNF-α were run at concentrations ranging from 0 to 50,000 pg/mL, and for IL-6 from 0 to 10,000 pg/mL. The average lowest level of detection concentration measured by the standard curves were 5.3 pg/mL, 0.92 pg/mL, and 22.34 pg/mL for IL-1ß, IL-6, and TNF-α respectively, as determined by data from seven plates containing bovine plasma samples from a combination of healthy and diseased cattle. The U-PLEX platform was a viable means to develop custom analyte- and species-specific multiplex assays using privately developed or purchased sets of commercially available reagents.

Cytokine and Haptoglobin Profiles From Shipping Through Sickness and Recovery in Metaphylaxis- or Un-Treated Cattle.

Fifty-six head of cattle, 28 animals with bovine respiratory disease complex (BRDC), and 28 healthy animals that were matched by treatment, sale barn of origin, day, and interactions among these variables, were identified from a population of 180 animals (60 each purchased at three sale barns located in Missouri, Tennessee, and Kentucky) enrolled in a study comparing animals receiving metaphylaxis to saline-treated controls. Cattle were transported to a feedlot in KS and assigned to treatment group. Blood samples were collected at Day 0 (at sale barn), Day 1, Day 9, and Day 28 (at KS feedlot), and transported to the US Meat Animal Research Center in Clay Center, NE where plasma was harvested and stored at -80°C until assayed for the cytokines IFN-γ, IL-1ß, IL-6, and TNF-α, and the acute stress protein haptoglobin (HPT). Our objectives were to determine if cytokine and haptoglobin profiles differed between control and metaphylaxis treatment groups over time, and if profiles differed between animals presenting with BRDC and those that remained healthy. There was no difference between the treated animals and their non-treated counterparts for any of the analytes measured. Sale barn of origin tended to affect TNF-α concentration. Differences for all analytes changed over days, and on specific days was associated with state of origin and treatment. The Treatment by Day by Case interaction was significant for HPT. The analyte most associated with BRDC was HPT on D9, possibly indicating that many of the cattle were not exposed to respiratory pathogens prior to entering the feedlot.

Animal-to-animal variation in fecal microbial diversity among beef cattle.

The intestinal microbiota of beef cattle are important for animal health, food safety, and methane emissions. This full-length sequencing survey of 11,171 16S rRNA genes reveals animal-to-animal variation in communities that cannot be attributed to breed, gender, diet, age, or weather. Beef communities differ from those of dairy. Core bovine taxa are identified.

Publisher Correction: A Computational Method to Quantify the Effects of Slipped Strand Mispairing on Bacterial Tetranucleotide Repeats.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Classification of 16S rRNA reads is improved using a niche-specific database constructed by near-full length sequencing.

Surveys of microbial populations in environmental niches of interest often utilize sequence variation in the gene encoding the ribosomal small subunit (the 16S rRNA gene). Generally, these surveys target the 16S genes using semi-degenerate primers to amplify portions of a subset of bacterial species, sequence the amplicons in bulk, and assign to putative taxonomic categories by comparison to databases purporting to connect specific sequences in the main variable regions of the gene to specific organisms. Due to sequence length constraints of the most popular bulk sequencing platforms, the primers selected amplify one to three of the nine variable regions, and taxonomic assignment is based on relatively short stretches of sequence (150-500 bases). We demonstrate that taxonomic assignment is improved through reduced unassigned reads by including a survey of near-full-length sequences specific to the target environment, using a niche of interest represented by the upper respiratory tract (URT) of cattle. We created a custom Bovine URT database from these longer sequences for assignment of shorter, less expensive reads in comparisons of the upper respiratory tract among individual animals. This process improves the ability to detect changes in the microbial populations of a given environment, and the accuracy of defining the content of that environment at increasingly higher taxonomic resolution.

Complete Genome Sequence of a Salmonella enterica subsp. enterica Serovar Fresno Isolate Recovered from a Bovine Lymph Node.

Salmonella enterica serovar Fresno is an infrequently isolated serovar whose ecology and genomic characteristics have not yet been described. To further understand the genomic characteristics of this serovar, we sequenced the complete genome of a single isolate recovered from a bovine lymph node at harvest.

A Computational Method to Quantify the Effects of Slipped Strand Mispairing on Bacterial Tetranucleotide Repeats.

The virulence and pathogenicity of bacterial pathogens are related to their adaptability to changing environments. One process enabling adaptation is based on minor changes in genome sequence, as small as a few base pairs, within segments of genome called simple sequence repeats (SSRs) that consist of multiple copies of a short sequence (from one to several nucleotides), repeated in series. SSRs are found in eukaryotes as well as prokaryotes, and length variation in them occurs at frequencies up to a million-fold higher than bacterial point mutations through the process of slipped strand mispairing (SSM) by DNA polymerase during replication. The characterization of SSR length by standard sequencing methods is complicated by the appearance of length variation introduced during the sequencing process that obscures the lower abundance repeat number variants in a population. Here we report a computational approach to correct for sequencing process-induced artifacts, validated for tetranucleotide repeats by use of synthetic constructs of fixed, known length. We apply this method to a laboratory culture of Histophilus somni, prepared from a single colony, and demonstrate that the culture consists of populations of distinct sequence phase and length variants at individual tetranucleotide SSR loci.

Prevalence of the prion protein gene E211K variant in U.S. cattle.

BACKGROUND: In 2006, an atypical U.S. case of bovine spongiform encephalopathy (BSE) was discovered in Alabama and later reported to be polymorphic for glutamate (E) and lysine (K) codons at position 211 in the bovine prion protein gene (Prnp) coding sequence. A bovine E211K mutation is important because it is analogous to the most common pathogenic mutation in humans (E200K) which causes hereditary Creutzfeldt - Jakob disease, an autosomal dominant form of prion disease. The present report describes a high-throughput matrix-associated laser desorption/ionization-time-of-flight mass spectrometry assay for scoring the Prnp E211K variant and its use to determine an upper limit for the K211 allele frequency in U.S. cattle. RESULTS: The K211 allele was not detected in 6062 cattle, including those from five commercial beef processing plants (3892 carcasses) and 2170 registered cattle from 42 breeds. Multiple nearby polymorphisms in Prnp coding sequence of 1456 diverse purebred cattle (42 breeds) did not interfere with scoring E211 or K211 alleles. Based on these results, the upper bounds for prevalence of the E211K variant was estimated to be extremely low, less than 1 in 2000 cattle (Bayesian analysis based on 95% quantile of the posterior distribution with a uniform prior). CONCLUSION: No groups or breeds of U.S. cattle are presently known to harbor the Prnp K211 allele. Because a carrier was not detected, the number of additional atypical BSE cases with K211 will also be vanishingly low.