Analysis of the Population Structure of Anaplasma Phagocytophilum Using Multilocus Sequence Typing

Anaplasma phagocytophilum is a Gram-negative obligate intracellular bacterium that replicates in neutrophils. It is transmitted via tick-bite and causes febrile disease in humans and animals. Human granulocytic anaplasmosis is regarded as an emerging infectious disease in North America, Europe and Asia. However, although increasingly detected, it is still rare in Europe. Clinically apparent A. phagocytophilum infections in animals are mainly found in horses, dogs, cats, sheep and cattle. Evidence from cross-infection experiments that A. phagocytophilum isolates of distinct host origin are not uniformly infectious for heterologous hosts has led to several approaches of molecular strain characterization. Unfortunately, the results of these studies are not always easily comparable, because different gene regions and fragment lengths were investigated. Multilocus sequence typing is a widely accepted method for molecular characterization of bacteria. We here provide for the first time a universal typing method that is easily transferable between different laboratories. We validated our approach on an unprecedented large data set of almost 400 A. phagocytophilum strains from humans and animals mostly from Europe. The typability was 74% (284/383). One major clonal complex containing 177 strains was detected. However, 54% (49/90) of the sequence types were not part of a clonal complex indicating that the population structure of A. phagocytophilum is probably semiclonal. All strains from humans, dogs and horses from Europe belonged to the same clonal complex. As canine and equine granulocytic anaplasmosis occurs frequently in Europe, human granulocytic anaplasmosis is likely to be underdiagnosed in Europe. Further, wild boars and hedgehogs may serve as reservoir hosts of the disease in humans and domestic animals in Europe, because their strains belonged to the same clonal complex. In contrast, as they were only distantly related, roe deer, voles and shrews are unlikely to harbor A. phagocytophilum strains infectious for humans, domestic or farm animals

Analysis of the population structure of Anaplasma phagocytophilum using multilocus sequence typing.

Anaplasma phagocytophilum is a Gram-negative obligate intracellular bacterium that replicates in neutrophils. It is transmitted via tick-bite and causes febrile disease in humans and animals. Human granulocytic anaplasmosis is regarded as an emerging infectious disease in North America, Europe and Asia. However, although increasingly detected, it is still rare in Europe. Clinically apparent A. phagocytophilum infections in animals are mainly found in horses, dogs, cats, sheep and cattle. Evidence from cross-infection experiments that A. phagocytophilum isolates of distinct host origin are not uniformly infectious for heterologous hosts has led to several approaches of molecular strain characterization. Unfortunately, the results of these studies are not always easily comparable, because different gene regions and fragment lengths were investigated. Multilocus sequence typing is a widely accepted method for molecular characterization of bacteria. We here provide for the first time a universal typing method that is easily transferable between different laboratories. We validated our approach on an unprecedented large data set of almost 400 A. phagocytophilum strains from humans and animals mostly from Europe. The typability was 74% (284/383). One major clonal complex containing 177 strains was detected. However, 54% (49/90) of the sequence types were not part of a clonal complex indicating that the population structure of A. phagocytophilum is probably semiclonal. All strains from humans, dogs and horses from Europe belonged to the same clonal complex. As canine and equine granulocytic anaplasmosis occurs frequently in Europe, human granulocytic anaplasmosis is likely to be underdiagnosed in Europe. Further, wild boars and hedgehogs may serve as reservoir hosts of the disease in humans and domestic animals in Europe, because their strains belonged to the same clonal complex. In contrast, as they were only distantly related, roe deer, voles and shrews are unlikely to harbor A. phagocytophilum strains infectious for humans, domestic or farm animals

Allele frequency and number of polymorphic sites of the seven loci of the <i>A. phagocytophilum</i> MLST scheme¹ and of the partial 16S rRNA² and <i>ankA</i>³ gene sequences.

1<p>284 strains typeable by MLST were included.</p>2<p>369 strains without ambiguous nucleotides were included.</p>3<p>369 sequences without ambiguous nucleotides from 363 samples were included.</p>4<p>not applicable.</p

<i>ankA</i> gene cluster and host species of the <i>A. phagocytophilum</i> strains, in which <i>ankA</i> could be amplified (n = 386).

1<p>Prevalence of strains in the data set.</p>2<p>NT = nontypeable. In these samples sequences belonging to two different <i>ankA</i> gene clusters were found.</p>3<p>n (n) = nontypeable strains (all strains).</p>4<p>n (n) = strains belonging to the respective <i>ankA</i> gene cluster (typeable strains).</p

ST and host species of the <i>A. phagocytophilum</i> positive samples (n = 383).

1<p>Prevalence of the respective STs in the dataset.</p>2<p>NT = nontypeable.</p>3<p>n (n) = nontypeable strains (all strains).</p>4<p>n (n) = strains with respective ST (typeable strains).</p

Tyability and discriminatory indices of MLST, 16S rRNA and <i>ankA</i> gene-based typing of <i>A. phagocytophilum</i> strains.

1<p>NT = nontypeable.</p>2<p>CI = confidence interval.</p>3<p>CC = clonal complex.</p>4<p>Concatenated allele gene cluster.</p

Wallace coefficients for the different typing methods (n = 357)¹.

1<p><i>A. phagocytophilum</i> strains from ticks were excluded.</p>2<p>CC = clonal complex.</p>3<p>Concatenated allele gene cluster.</p>4<p>Wallace coefficients >0.750 are shown in bold.</p

Results of the four tests for recombination.

1<p>Number of unique sequences.</p>2<p>IS = informative sites.</p>3<p>PHI = pairwise homoplasy index, perm. = permutated.</p>4<p>obs. = observed. *Maximum χ².</p>5<p>NSS = neighborhood similarity score.</p>6<p>Significant <i>p</i> values (α <0.05) are shown in bold.</p

Clonal complexes and host species of the <i>A. phagocytophilum</i> strains typeable by MLST (n = 284).

1<p>Clonal complexes were defined by sharing identical alleles at five of the seven loci with at least one other member of the group.</p>2<p>Prevalence of strains in the data set.</p>3<p>n (n) = strains belonging to the respective clonal complex (typeable strains).</p>4<p>60 (60) dogs from Europe.</p>5<p>32 (32) humans from Europe.</p>6<p>10 (10) humans from the USA.</p>7<p>1 (1) dog from the USA.</p

Adjusted Wallace coefficients for the different typing methods (n = 247)¹.

1<p><i>A. phagocytophilum</i> strains from ticks were excluded. Only typeable strains with data for all partitions were included. The clonal complex as partition was excluded, because only 223 of 383 strains were part of a clonal complex.</p>2<p>Concatenated allele gene cluster.</p>3<p>Wallace coefficients >0.750 are shown in bold.</p