Development and Evaluation of a Polymerase Chain Reaction Assay Using the 16S rRNA Gene for Detection ofEperythrozoon SuisInfection

Abstract

Abstract. The 16S ribosomal RNA (rRNA) gene of Eperythrozoon suis was amplified using gene-specific primers developed from GenBank sequence accession U88565. The gene was subsequently cloned and sequenced. Based on these sequence data, 3 sets of E. suis-specific primers were designed. These primers selectively amplified 1394, 690, and 839 base-pair (bp) fragments of the 16S rRNA gene from DNA of E. suis extracted from the blood of an experimentally infected pig during a parasitemic episode. No polymerase chain reaction (PCR) products were amplified from purified DNA of Haemobartonella felis, Mycoplasma genitalium, or Bartonella bacilliformis using 2 of these primer sets. When the primer set amplifying the 690-bp fragment was used, faint bands were observed with H. felis as the target DNA. No PCR products were amplified from DNA that had been extracted from the blood of a noninfected pig or using PCR reagents without target DNA. The detection limits for E. suis by competitive quantitative PCR were estimated to range from 57 and 800 organisms/assay. This is the first report of the utility of PCR-facilitated diagnosis and quantitation of E. suis based on the 16S rRNA gene. The PCR method developed will be useful in monitoring the progression and significance of E. suis in the disease process in the pig.Eperythrozoon suis is an extracellular, rod-shaped parasite that attaches to and causes deformity and damage to the red blood cells of pigs. 27 The disease, eperythrozoonosis, is characterized by 4 syndromes: 1) decreased reproductive efficiency of sows, 2) weakness and anemia in baby pigs with increased incidence of enteric and respiratory infections, 3) delayed production gains in feeder pigs, and 4) acute hemolytic anemia in feeder pigs. 5,14,35 This disease was first reported in the United States in 1934. 16 Nonetheless, our understanding of this organism and the disease(s) it causes remains largely incomplete.Eperythrozoon suis has not been successfully grown in agar or cell cultures. Diagnosis of acute infection with E. suis is usually based on direct microscopic observation of organisms attached to red blood cells on Giemsa-stained peripheral blood smears. However, this method has limitations because the parasitemia is transient and parasites are not often seen on blood smears until clinical signs develop. 27 The diagnosis of eperythrozoonosis is also complicated by the lack of a readily identifiable parasitemia in latent and chronic infections. 27 variability in antibody response and failure to identify acutely infected pigs. The lack of an efficient test for diagnosing acute and chronic E. suis infection has resulted in tremendous controversy concerning the true impact of this disease in pig populations. The refinement of the polymerase chain reaction (PCR) during the past decade has presented new opportunities for the development of diagnostic tests that have the potential to be highly sensitive and specific. In this respect, there are reports of a PCR protocol for detection of E. suis infection in pigs ba...

Mycoplasma suisAntigens Recognized during Humoral Immune Response in Experimentally Infected Pigs

“…Hitherto, laboratory diagnosis of M. suis usually relies on the microscopic examination of chemically stained peripheral blood smears to directly visualize the microorganisms attached to erythrocytes (15). Recently established PCR assays for M. suis can detect acutely diseased animals and also asymptomatic carrier pigs and are therefore principally suitable tools for the diagnosis of PE (6,10,15).…”

Application of quantitative PCR for detection of Mycoplasma suis in blood samples

“…Among the classic methods of PCR, which allow detection of M. suis, the use of KSU-2 DNA (4), 16S rRNA gene (2,14,16), and 1,8 kb EcoRI (10) was previously reported. Some of these molecular methods have been implemented into a routine diagnostic assay.…”

“…In order to prepare a recombinant plasmid for PCR quantification, the entire M. suis 16S rRNA gene was amplified using a set of primers described previously (14). Further on, the amplicon was cloned in the plasmid TOPO TA Cloning (Invitrogen) according to the manufacturer's guidelines.…”

“…Diagnosis of infection is based on clinical symptoms and the observation of microorganisms on the surface of red blood cells in blood smears. Another recently developed method involves the detection of microorganisms by the use of molecular methods (4,10,12,14). Blood smear methods, however, are too imprecise when compared to the molecular methods used for detecting infections.…”

“…Over the last ten years, molecular methods, especially those based on PCR, have improved diagnostic possibilities in respect of HM diseases. The conventional PCR methods are still successfully used to diagnose M. suis infections in pigs (5,10,14). To minimise the time necessary for conducting the PCR procedure, by eliminating labour-intensive electrophoresis, a real-time PCR was used to replace the conventional PCR technique.…”