Suis DNA Research Articles

Prevalence of Mycoplasma suis (Eperythrozoon suis) infection in swine and swine-farm workers in Shanghai, China

To determine the prevalence of Mycoplasma suis infection in swine, swine-farm workers, and swine veterinarians in Shanghai, China. 172 swine and 65 workers and veterinarians from 19 commercial swine farms. Blood samples were collected from all study subjects. Blood samples were examined for the presence of M suis by means of compound and scanning electron microscopy. A species-specific PCR assay was developed for detection of M suis DNA extracted from blood samples. Relationships between infection status of swine and sex, age, geographic location, and clinical signs of disease were evaluated by use of a C(2) test. The phylogenetic relationship between partial 16S ribosomal RNA (rRNA) sequences from swine and human isolates of M suis was determined. 86% (148/172) of swine and 49% (32/65) of humans had positive PCR assay results for M suis infection. Swine infection status was not associated with any variable, with the exception of pyrexia and subcutaneous bleeding. The partial 16S rRNA sequences from human and swine isolates of M suis were 98% homologous and in the same phylogenetic cluster as a previously identified swine isolate of M suis. A large proportion of swine and humans in close contact with those swine were infected with M suis in Shanghai, China. The close phylogenetic relationship between swine and human isolates of M suis suggested possible interspecies transmission; however, additional research is required to better assess that possibility.

Protective immunization against “ Candidatus Helicobacter suis” with heterologous antigens of H. pylori and H. felis

“ Helicobacter ( H.) heilmannii” type 1 colonizes the human stomach. It has been shown to be identical to “ Candidatus H. suis”, a Helicobacter species colonizing the stomach of more than 60% of slaughter pigs. This bacterium is, until now, not isolated in vitro. The effect of vaccination on “ Candidatus H. suis” infection was studied in a mouse model. Mice were vaccinated intranasally or subcutaneously with whole bacterial cell lysate of Helicobacter pylori or Helicobacter felis and subsequently challenge infected with “ Candidatus H. suis”. Intranasal and subcutaneous immunisation caused a decrease in faecal excretion of “ Candidatus H. suis” DNA. Urease tests on stomach tissue samples at 16 weeks after challenge infection were negative in all H. felis intranasally immunized animals and in the majority of the animals of the other immunisation groups. Since PCR on stomach tissue samples at 16 weeks after challenge infection could still detect “ Candidatus H. suis DNA” in all immunisation-challenge groups, complete clearance of challenge bacteria was not achieved.

Development of a Brucella suis specific hybridisation probe and PCR which distinguishes B. suis from Brucella abortus

A genomic library was prepared from Brucella suis DNA ( MboI digested) and cloned into the BamHI site of pUC18. Colony hybridisation using a probe prepared from purified B. suis DNA labelled with α 32P was carried out to identify colonies of interest. About 20 colonies, which gave an intense signal upon hybridisation with whole B. suis genomic DNA as a probe, were selected. Because of the high degree of DNA homology between B. suis and Brucella abortus, a short probe was chosen as it would more likely give species specificity. Of seven fragments selected to probe whole B. suis, B. abortus, and Yersinia enterocolitica DNA, one was found to hybridise with B. suis only. The probe was sequenced in two directions and sense and anti sense primers of 25 bp in length were chosen to yield a product of 421 bp. After optimisation of the PCR, a product of 420 bp was obtained with B. suis template DNA and two bands of 420 and 650 bp were detected with B. abortus template DNA. This is the first reported PCR of the Brucella genome where a single pair of primers will discriminate between B. suis and B. abortus. No band was observed when the two primers were used to amplify E. coli, Y. enterocolitica, Enterobacter cloacae, Staphylococcus aureus, Streptococcus uberis, Corynebacterium bovis, or Serratia marcescens template DNA.

In situ hybridizations of Eperythrozoon suis visualized by electron microscopy

Eperythrozoon suis is an extracellular red blood cell parasite that causes icteroanemia and poor growth performance in feeder pigs and has been associated with anemias in baby pigs and reproductive failures in sows. At present, few efficient tests are available for the diagnosis and study of E. suis infection in swine. This report discusses how a recently developed recombinant DNA probe (KSU-2) specific to E. suis DNA was utilized in in situ DNA hybridizations that couple biotinylated TagI digested products of KSU-2 DNA with an immunogold detection system allowing the visualization by transmission electron microscopy of the gold particles within eperythrozoon organisms. Specific labelling by the probe of eperythrozoon organisms demonstrated a pattern that changed with the various stages of the eperythrozoon life cycle.

Experimental Infections and Natural Outbreaks of Eperythrozoonosis in Pigs Identified by PCR-DNA Hybridizations

Eperythrozoon-specific DNA amplification reactions and subsequent hybridizations with an eperythrozoon DNA probe (KSU-2) were used in experimental infection studies to identify Eperythrozoon suis DNA in the blood of splenectomized and nonsplenectomized pigs. The results indicate that E. suis DNA is present in nonsplenectomized pigs at levels that can be amplified by polymerase chain reaction (PCR) and identified in DNA hybridizations within 24 hours after infection. The ability of the E. suis PCR/hybridization assay to detect eperythrozoonosis was further demonstrated in blood samples collected from pigs in 2 separate natural outbreaks in Oklahoma. Results from these initial samplings indicate that pigs infected with E. suis from geographically distinct locations can be identified using the eperythrozoon-specific PCR/hybridization assay, which offers many advantages over conventional laboratory procedures for diagnosing eperythrozoonosis in pigs.

Detection of Eperythrozoon suis using the polymerase chain reaction.

Eperythrozoon suis is an extracellular red blood cell parasite that causes icteroanemia in acutely ill pigs and a variety of syndromes in chronically infected pigs. Current techniques to detect E. suis infection are limited by variability of parasitemias and antibody responses in infected animals. The polymerase chain reaction (PCR) was investigated to determine its potential as a means of detecting E. suis infection in pigs. With DNA samples extracted from either purified E. suis organisms or E. suis-infected pig blood, PCR produced an amplification product 492 base pairs in length. This amplification product hybridized successfully with the fragment of the DNA probe from which the primer sequences had been selected. Sensitivity studies indicated that the PCR protocol was capable of amplifying total genomic E. suis DNA in quantities as low as 450 pg. When PCR was used with DNA from blood samples from a splenectomized pig that had been infected with E. suis, amplification products were detectable as early as 24 hours postinfection. This preliminary analysis indicates that PCR shows promise as a means of efficiently detecting E. suis infection in pigs.

Detection of Eperythrozoon suis DNA from swine blood by whole organism DNA hybridizations

A procedure is described for isolating Eperyhrozoon suis DNA of sufficient quantity and purity to serve as a probe in whole-organism DNA hybridizations for detecting parasitized wine. The E. suis organisms were isolated from the blood of infected swine; the DNA was recovered and digested with restriction endonucleases and resolved on agarose gels. In DNA hybridizations using recovered E. suis DNA, blood samples from parasitized swine could be differentiated from uninfected, control samples. A high salt lysate recovery technique was used in sampling swine whole blood for E. suis DNA and found to offer many advantages in the collection and recovery process.