Identification, molecular characterization, and occurrence of two bovine hemoplasma species in Swiss cattle and development of real-time TaqMan quantitative PCR assays for diagnosis of bovine hemoplasma infections

Abstract

Concomitantly with an outbreak of fatal anaplasmosis in a cattle herd in Switzerland in 2002, we detected two bovine hemoplasma species in diseased animals: Mycoplasma wenyonii (formerly Eperythrozoon wenyonii) and a second, novel bovine hemoplasma species later designated "Candidatus Mycoplasma haemobos" (synonym, "Candidatus Mycoplasma haemobovis"). The second species was characterized by a shorter 16S rRNA gene. The aims of the present study were to provide a detailed molecular characterization of this species, to develop specific quantitative real-time PCR assays for the two bovine hemoplasma species, and to apply these assays in order to evaluate the prevalence and clinical significance of the hemoplasmas. Sequencing of the near-complete 16S rRNA gene of the second hemoplasma revealed that it was 94% identical to that of Mycoplasma haemofelis, an anemia-inducing feline hemoplasma species, but less than 85% identical to that of the bovine hemoplasma M. wenyonii. Using the newly developed assays, a total of 159 animals from the anaplasmosis outbreak were reexamined. In addition, we tested 57 clinically ill and 61 healthy Swiss cattle, as well as 47 calves. Both hemoplasmas were highly prevalent in adult cattle but occurred rarely in calves. Animals from the herd with the fatal anemia outbreak were more frequently infected with M. wenyonii and exhibited higher M. wenyonii blood loads than animals with unrelated diseases and healthy animals. Coinfections may increase the pathogenicity and clinical significance of bovine hemoplasmosis.

FIG. 1.

Bovine hemoplasma 16S rRNA gene products identified using conventional PCR. Agarose gel electrophoresis (3%) of PCR products that were amplified with primers specific for both feline and bovine hemoplasma 16S rRNAs (MychaeF and MychaeR [25]) is shown. Abbreviations: M, molecular size marker (100-bp ladder); C, “Candidatus Mycoplasma haemominutum”; H, M. haemofelis; K1 to K5, five different samples from cows from the herd with fatal anemia (5).

FIG. 2.

Phylogenetic analysis of near-complete 16S rRNA gene sequences from “Candidatus Mycoplasma haemobos” and related hemoplasmas. A bootstrap phylogenetic tree demonstrating the relationship of the Swiss “Candidatus Mycoplasma haemobos” isolates (CH307 and CH311) to other hemoplasma species using the neighbor-joining method is shown. The numbers at the nodes were generated from 1,000 bootstrap resamplings. The bar represents the mean number of differences per 100 sites. GenBank accession numbers are shown in parentheses.

FIG. 3.

Box plot of the distribution of bacteremia as assessed using real-time qPCR in the following three examined cattle populations: animals from a herd with a fatal anemia outbreak, cows with unrelated diseases, and healthy animals. (A) Singly infected animals; (B) coinfected animals. MW, M. wenyonii; CMhb, “Candidatus Mycoplasma haemobos.” Boxes extend from the 25th to the 75th percentile, a horizontal line represents the median, and the error bars extend down to the smallest and up to the largest value. Only hemoplasma-positive cattle were included in our analysis. Groups were compared using Kruskal-Wallis statistics and Dunn's posttests. A significant difference in the bacterial loads (copy number) was apparent among the three groups for M. wenyonii in both singly infected and coinfected animals. Significant differences in bacterial loads between groups are indicated (*, P < 0.05; **, P < 0.01; ***, P < 0.001).