| Mycoplasma hyopneumoniae (Mhyo) is the undisputed agent responsible for the occurrence of Enzootic porcine pneumonia (EPP) and contributes greatly to the development of porcine respiratory disease complex (PRDC) that is detrimental to swine herds globally by decreasing the productivity of pigs.In order mitigate and reduce the impact of Mhyo infection, several diagnostic assays based on previous studies have been adopted to help establish the’real time’health status of swine herds in the course of a production system with varying degree of success. The complexity of infection epidemiology makes the complete elimination of Mhyo impossible meaning reliable diagnostics as well as good management practices are the best means of infection control and reduce potential disease outbreak. The goal of this study was to compare existing diagnostic assays in determining the health status of pig herds to increase the knowledge of transmission and infection studies of Mhyo in China. We investigated the following objectives to achieve the above goal;i) Assess the detection capacity of Nested PCR and Real time PCR to Mhyo from swine herds exposed to field conditionsii) Assess the detection capacity of Real time PCR, ELISA and Culture to Mhyo from swine herds exposed to field conditionsiii)Assess the presence and levels of antibodies in using different samples notably serum and oral fluids.We first assessed the rate of Mhyo infection in field swine herds by sampling by way of nasal swabs. The pigs were of different ages i.e.7,14,21,28,30 and 35 days respectively and the assays used namely nested and Real time PCR were to determine the degree of early Mhyo infections and to determine the age at which the piglets are most likely to be infected. The second study of comparing Real time PCR, ELISA and culture was conducted on field pigs that were 8 weeks old with the most interesting aspect under investigation being the time frame after which the animals underwent sero-conversion after an active Mhyo infection. Also under analysis was the suitability of using these three assays together to give a true health status of pig herds. The final study involved the analysis of antibody levels in three different samples i.e. nasal swabs, serum and oral fluids. Samples were collected once a week for 3 weeks and antibody levels determined by indirect ELISA in P97 Rl antigen coated plates. The results of the studies are as follows;1) Real time PCR has a superior detection capacity than Nested PCRReal time PCR assay proved to be much more superior in its detection capacity with 152 (50.2%) out of 303 samples testing positive for Mhyo across all the ages of the piglets’. Its sensitivity in this study was noted in its ability to detect a minute sample of 10 copies of Mhyo DNA/μl. Nested PCR on the other hand was able to positively detect 38 (12.5%) samples with the two assays matching in positively detecting 22 (7.3%) samples and again matching in 127 (41.9%) samples negative for Mhyo infection. The pattern of infection in using both assays was similar where 7 and 35 day old piglets in both assays had the highest rates of infection i.e.15.6 and 18.4% for n-PCR and 53.1 and 56.6% for Real time PCR for 7 and 35 day old piglets respectively2) Real time PCR has a superior detection capacity than ELISA and culturingReal time PCR assay had a superior detection capacity where it was able to positively detect Mhyo in 81(79.4%) out of 102 samples with culture detecting 51%(52/102) samples to be positive for Mhyo with the most potent sample having a CCU at dilution 10-7.The time taken for an acid colour shift ranged from between 4-11 days. The ELISA assay had all the samples as negative and this matched in only 11/102 samples (10.7%) with real time PCR that was much lower when compared to matching culture with real time PCR which agreed in 45/102 samples(44.1%) highlighting their superior detection capacity in this study.3) Existence of correlation between oral fluids and serum in ELISAIn oral fluid ELISA, absorbance values in all the groups except group 4 that decreased by 16.7% had a marked increase between week 3 and week 4. The same pattern emerged with serum absorbance readings where all absorbance readings increased except in group 4 that decreased by 17.2% between week 3 and week 4. The correlation between the two sample types was calculated (Fig 4-4) where in week 3 the correlation was r2=0.8400 (p<0.0005) and in week 4 r2=0.3615 (p< 0.0868)... |
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