Limitations of variable number of tandem repeat typing identified through whole genome sequencing of Mycobacterium avium subsp. paratuberculosis on a national and herd level

Background: Mycobacterium avium subsp. paratuberculosis (MAP), the causative bacterium of Johne’s disease in dairy cattle, is widespread in the Canadian dairy industry and has significant economic and animal welfare implications. An understanding of the population dynamics of MAP can be used to identify introduction events, improve control efforts and target transmission pathways, although this requires an adequate understanding of MAP diversity and distribution between herds and across the country. Whole genome sequencing (WGS) offers a detailed assessment of the SNP-level diversity and genetic relationship of isolates, whereas several molecular typing techniques used to investigate the molecular epidemiology of MAP, such as variable number of tandem repeat (VNTR) typing, target relatively unstable repetitive elements in the genome that may be too unpredictable to draw accurate conclusions. The objective of this study was to evaluate the diversity of bovine MAP isolates in Canadian dairy herds using WGS and then determine if VNTR typing can distinguish truly related and unrelated isolates.<p></p> Results: Phylogenetic analysis based on 3,039 SNPs identified through WGS of 124 MAP isolates identified eight genetically distinct subtypes in dairy herds from seven Canadian provinces, with the dominant type including over 80% of MAP isolates. VNTR typing of 527 MAP isolates identified 12 types, including “bison type” isolates, from seven different herds. At a national level, MAP isolates differed from each other by 1–2 to 239–240 SNPs, regardless of whether they belonged to the same or different VNTR types. A herd-level analysis of MAP isolates demonstrated that VNTR typing may both over-estimate and under-estimate the relatedness of MAP isolates found within a single herd.<p></p> Conclusions: The presence of multiple MAP subtypes in Canada suggests multiple introductions into the country including what has now become one dominant type, an important finding for Johne’s disease control. VNTR typing often failed to identify closely and distantly related isolates, limiting the applicability of using this typing scheme to study the molecular epidemiology of MAP at a national and herd-level.<p></p&gt

Management of cull dairy cows—Consensus of an expert consultation in Canada

Many cull dairy cows enter the marketing system and travel to widely dispersed and specialized slaughter plants, and they may experience multiple handling events (e.g., loading, unloading, mixing), change of ownership among dealers, and feed and water deprivation during transport and at livestock markets. The objectives of this study were to describe the diverse management of cull dairy cows in Canada and establish consensus on ways to achieve improvements. A 2-day expert consultation meeting was convened, involving farmers, veterinarians, regulators, and experts in animal transport, livestock auction, and slaughter. The 15 participants, recruited from across Canada, discussed regional management practices for cull cattle, related risk factors, animal welfare problems, and recommendations. An audio recording of the meeting was used to extract descriptive data on cull cattle management and identify points of agreement. Eight consensus points were reached: (1) to assemble information on travel times and delays from farm to slaughter; (2) to increase awareness among producers and herd veterinarians of potential travel distances and delays; (3) to promote pro-active culling; (4) to improve the ability of personnel to assess animal condition before loading; (5) to identify local options for slaughter of cull dairy cows; (6) to investigate different management options such as emergency slaughter and mobile slaughter; (7) to ensure that all farms and auctions have, or can access, personnel trained and equipped for euthanasia; and (8) to promote cooperation among enforcement agencies and wider adoption of beneficial regulatory options

Genome-wide diversity and phylogeography of Mycobacterium avium subsp. paratuberculosis in Canadian dairy cattle

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative bacterium of Johne’s disease (JD) in ruminants. The control of JD in the dairy industry is challenging, but can be improved with a better understanding of the diversity and distribution of MAP subtypes. Previously established molecular typing techniques used to differentiate MAP have not been sufficiently discriminatory and/or reliable to accurately assess the population structure. In this study, the genetic diversity of 182 MAP isolates representing all Canadian provinces was compared to the known global diversity, using single nucleotide polymorphisms identified through whole genome sequencing. MAP isolates from Canada represented a subset of the known global diversity, as there were global isolates intermingled with Canadian isolates, as well as multiple global subtypes that were not found in Canada. One Type III and six “Bison type” isolates were found in Canada as well as one Type II subtype that represented 86% of all Canadian isolates. Rarefaction estimated larger subtype richness in Québec than in other Canadian provinces using a strict definition of MAP subtypes and lower subtype richness in the Atlantic region using a relaxed definition. Significant phylogeographic clustering was observed at the inter-provincial but not at the intra-provincial level, although most major clades were found in all provinces. The large number of shared subtypes among provinces suggests that cattle movement is a major driver of MAP transmission at the herd level, which is further supported by the lack of spatial clustering on an intra-provincial scale

Management of cull dairy cows—Consensus of an expert consultation in Canada

Many cull dairy cows enter the marketing system and travel to widely dispersed and specialized slaughter plants, and they may experience multiple handling events (e.g., loading, unloading, mixing), change of ownership among dealers, and feed and water deprivation during transport and at livestock markets. The objectives of this study were to describe the diverse management of cull dairy cows in Canada and establish consensus on ways to achieve improvements. A 2-day expert consultation meeting was convened, involving farmers, veterinarians, regulators, and experts in animal transport, livestock auction, and slaughter. The 15 participants, recruited from across Canada, discussed regional management practices for cull cattle, related risk factors, animal welfare problems, and recommendations. An audio recording of the meeting was used to extract descriptive data on cull cattle management and identify points of agreement. Eight consensus points were reached: (1) to assemble information on travel times and delays from farm to slaughter; (2) to increase awareness among producers and herd veterinarians of potential travel distances and delays; (3) to promote pro-active culling; (4) to improve the ability of personnel to assess animal condition before loading; (5) to identify local options for slaughter of cull dairy cows; (6) to investigate different management options such as emergency slaughter and mobile slaughter; (7) to ensure that all farms and auctions have, or can access, personnel trained and equipped for euthanasia; and (8) to promote cooperation among enforcement agencies and wider adoption of beneficial regulatory options

Phylogenetic clustering of MAP isolates among Canadian regions.

<p>A) Circularized maximum likelihood phylogenetic tree of 182 Canadian MAP isolates, rooted to the Type III isolate. Tips are colored according to province of origin (Alberta = blue, Ontario = light blue, British Colombia = red, Québec = orange, Saskatchewan = yellow, the Atlantic provinces = pink, Manitoba = purple). The branches leading to the nine subtypes are labeled with a square. Dotted lines indicate the threshold for the different subtype definitions used in the rarefaction analysis. B) Map of the 7 Canadian regions in which MAP isolates were obtained. C) The statistics (AI = association index, PS = parsimony score, MC = monophyletic clade size statistic), number of samples from each of the 7 regions, observed mean, null mean, and significance (p-value) are presented. Low AI and PS scores indicate a strong association, whereas high MC scores indicate a strong association.</p

Maximum likelihood phylogenetic tree of sequenced MAP isolates.

<p>Phylogenetic tree of global (n = 26, labeled with a black dot (•)) and Canadian (n = 182) isolates based on 9,670 variant sites using the TPM1uf nucleotide substitution model. The tree is rooted to the Type I sequence. A magnification of the phylogeny excluding the Type I, III, and B isolates is outlined in dotted lines. The dominant subtype is shaded in grey. Bootstrap values with branch support ≥ 70% are displayed.</p

Maximum likelihood phylogenetic trees of Canadian and global MAP isolates.

<p>A) Phylogenetic tree of one Canadian and ten global Type III isolates based on 5,416 concatenated variant sites. B) Phylogenetic tree of six Canadian and four global “Bison type” isolates based on 403 concatenated variant sites. The global sequences are shaded in grey and branches are labeled according to the isolate ID.</p

Phylogenetic clustering of MAP isolates in three regions in Alberta.

<p>A) Circularized maximum likelihood phylogenetic tree of 58 Alberta MAP isolates. Tips are colored according to the geographical location of originating farms (Region 1 = light blue, Region 2 = dark blue, Region 3 = blue). B) Map of Alberta with the three regions indicated by their respective colors. C) BaTS analysis of the phylogeny-region association. The statistics (AI = association index, PS = parsimony score, MC = monophyletic clade size statistic), number of samples from each region, observed mean, null mean, and significance (p-value) are presented. Low AI and PS scores indicate a strong association, whereas high MC scores indicate a strong association.</p

Rarefaction curves indicating the mean subtype richness of each region at different sampling efforts.

<p>Rarefaction curves were generated using the function rarefy in the R package Vegan using A) a strict subtype definition (9 total subtypes) and B) a relaxed subtype definition (32 total subtypes). 95% confidence intervals are indicated by a dotted line. The 7 regions are labeled with shapes indicated in the key at the bottom of the figure. Regions are abbreviated as follows: Québec = QC, Alberta = AB, Ontario = ON, the Atlantic region = AT, Manitoba = MB, British Columbia = BC, and Saskatchewan = SK.</p