Prevalence and Diagnosis of Hemotrophic Mycoplasma Infection in Research Sheep and Its Effects on Hematology Variables and Erythrocyte Membrane Fragility

Hemotrophic mycoplasma (hemoplasma) infection in research sheep can confound experimental results and contribute to morbidity and mortality. Prevalence and clinicopathologic studies historically relied on blood-smear diagnosis, but systematic studies using current molecular techniques are warranted. Here we sought to report the prevalence of subclinical infection in our study population, compare diagnostic sensitivity and specificity between blood smears and a PCR assay, and determine the effects of infection on CBC variables and erythrocyte membrane fragility. We collected whole-blood samples from 111 convenience-sampled research sheep. All samples were tested for hemoplasmas by using a PCR assay, blood smears were evaluated for visual presence of hemoplasmas, and CBC and osmotic fragility assays were performed. Subclinical prevalence, according to PCR diagnosis, was 14.1% (14 of 99) in our study population. Relative to the PCR assay, blood-smear diagnosis was 8.3% sensitive and 100% specific for hemoplasma detection. Subclinical infection was associated with changes in MCV, MCHC, RBC distribution width, and absolute monocyte count. Acute infection was associated with changes in RBC mass, Hgb concentration, MCV, MCH, MCHC, and absolute lymphocyte and monocyte counts. Acute infection was associated with increased mean erythrocyte fragility compared with that in uninfected control and treated sheep. We demonstrated that hemoplasma infection is common in our study population, blood-smear evaluation is insensitive at detecting infection, and infection is associated with changes in CBC variables and increased erythrocyte membrane fragility. These findings raise concerns regarding the suitability of hemoplasma-infected sheep for biomedical research.Abbreviations: MEF, mean erythrocyte fragilityHemoplasma infection in sheep is caused by Mycoplasma ovis and ‘Candidatus M. haemovis.’^15,34 Recent work, however, suggests M. ovis and ‘Ca. Mycoplasma haemovis’ represent the same organism with 2 different copies of the 16S rRNA.¹¹ This agent, formerly called Eperythrozoon ovis, is a cell-wall–free bacterial parasite that is intimately associated with the plasma membrane of sheep erythrocytes.²⁵ It typically is considered to be nonpathogenic in chronic infection, but it occasionally is associated with hemolytic anemia during acute infection.²⁴ Hemoplasma infection can confound experimental results and contribute to morbidity and mortality in research animals.²⁰ Infections have been reported worldwide. The clinicopathologic effects in lab animals may be a concern for biomedical research.²The organism cannot be grown in culture, making its diagnosis difficult. Previous prevalence and clinicopathologic studies relied on blood-smear diagnosis or serology.^{5,6,8-10,13,14,16,20,22,25-27,31} Systematic studies on the diagnosis and clinicopathologic effects of ovine hemoplasma infection using current, molecular techniques are unavailable. In addition, the contemporary prevalence of hemoplasma infection in research sheep in the United States is unknown.The purpose of the current study was to evaluate: 1) the prevalence of subclinical hemoplasma infection in our study population of research sheep; 2) the sensitivity and specificity of blood smears to detect hemoplasma infection; 3) the effects of subclinical and acute hemoplasma infection on CBC variables; and 4) the effects of acute hemoplasma infection on erythrocyte membrane fragility. We hypothesized that: 1) subclinical hemoplasma infection is common; 2) the examination of blood smears is not as sensitive or specific as is PCR analysis for detecting hemoplasma infection; 3) subclinical and acute infection alters CBC variables; and 4) acute infection increases erythrocyte membrane fragility.To address these questions, we collected whole-blood samples from 111 convenience-sampled research sheep as part of routine health surveillance. All samples were PCR tested by using hemoplasma-specific primers, blood smears were evaluated, and CBC analyses were performed. Osmotic fragility assays were performed on a subset of animals.