Study On Infection Of Mycoplasma Suis To PIGS And Mice

Porcine eperythrozoonosis (PE) caused by Mycoplasma suis (M. suis) infection and results in a febrile acute icteroanemia in pigs and human. Symptoms of chronic low-grade M. suis infection may vary from asymptomatic infection to a wide range of clinical conditions, including anaemia and mild icterus in newborns, growth retardation in feeder pigs, poor reproductive performance in sows. As the acute and chronic M. suis infection increases the likelihood of the animals contracting other infectious agents that cause porcine respiratory and enteric disease, thus leading to a significant economic loss in pig industry. To study the M. suis pathogenic mechanism, a real-time PCR was developed to quantity the M. suis loads and reveal the correlations between the M. suis loads and hematological parameters. Moreover, according to an infection to pigs, clinical symptoms, M. suis loads, hematological parameters, histopathology, cellular and humoral immunity were studied. In addition, a real-time PCR for detecting mice cytokine was developed and mouse infection by M. suis was studied. All the experiments and results are as follows:1. Establishment of SYBR Green-based real-time PCRPrimers target to the nucleotides sequence of16S rRNA gene of Mycoplasma. suis were designed to develop the SYBR Green-based real-time PCR. Plasmid containing16S rRNA gene was used as template to optimize the conditions of the SYBR Green-based real-time PCR. Fluorescence melting curve analysis, agarose gel electrophoresis and DNA sequencing were done to verify the specificity of the real time PCR. The standard curves were created according to the threshold cycle of diluted plasmid to measure the detection limit of this assay. Intra-and inter-assay repeatability of the real-time PCR was carried out. The detection limit of the real-time PCR was15copies of M. suis and specificity, sensitivity and reproducibility are excellent.2. Infection study of porcine eperythrozoonosisPorcine eperythrozoonosis (PE) caused by Mycoplasma. suis (M. suis) infection and results in a febrile acute icteroanemia in pigs and human. In order to study PE, ten M. suis negative pigs were randomly divided into three groups, Four and3pigs in splenectomized (B group) and non-splenectomized group (A group), respectively, were infected with M. suis and3pigs as control (C group). After infection with M.suis, these three groups were isolated at different houses. The clinical symptoms were monitored daily and body weight was weighted twice a week. M. suis loads, hematological parameters, cellular and humoral immunity were studied on day7,14,21,28. All the pigs were euthanised to study the gross pathology and histopathology on day28. The pigs in B group and A group showed clinical symptoms of PE and the pigs in C group had no visible clinical symptoms of PE during the first week. Compared with A group, the B group had almost the same quantity of M. suis loads (around1.0×105copies per1mL) in blood at7dpi, and which increased sharply to about1.0×108copies per1mL in blood at14dpi and fluctuated in this level till the end of experiment. M. suis loads in A group rose slowly before14dpi and increased to the same point as B group at21dpi and maintain this level till the end of the experiment. The blood routine examinations showed that the RBC number in B group was significantly higher than that in C group at7dpi, whereas the RBC number, hemoglobin, hematocrit and the concentration of Fe in B group was significantly lower than that of C group at21dpi. Similarly, the RBC number and hemoglobin in B group was also significantly lower than that of C group at21dpi. Correlations between M. suis loads and the values of WBC, hematocrit, RBC and Fe were significant. The antibody against MSG1was only detected in a pig of B group at day21and percentage inhibition of this pig was increased during the following time, while no antibody against MSG1was detected in other groups. The IL-4and IFN-γ were not detected in all of the groups during the whole experiment. In this study, PE was reproduced and the process of this disease was recorded, which will play a fundamental role in revealing the pathomechanism, providing the guideline for prevention and diagnosis of this disease.3. Establishment of the real-time PCR for detecting cytokine of miceTotal mRNA was extracted from lymphocytes in mice spleen and reverse transcripted to cDNA which subjected to real time PCR using the specific primers designed for IL-2, IL-4, IL-6, IL-10, IFN-y and IFN-βrespectively. Fluorescence melting curve analysis, agarose gel electrophoresis and DNA sequencing were done to verify the specificity of the real time PCR. The standard curves were created according to the threshold cycle of diluted plasmid to measure the detection limit of this assay. Intra-and inter-assay repeatability of the real-time PCR was carried out. This assay has a good performance in its specificity, sensitivity and reproducibility. The detection limit of IL-2, IL-4, IL-6, IL-10, IFN-γ and IFN-β were50,50,20,68,50and13target copies. This assay for detecting the IL-2, IL-4, IL-6, IL-10, IFN-y and IFN-βin mice providing a powerful tool in evaluation of mice as model to study Mycoplasma suis infection.