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Abstract
SUMMARY
Mycobacterial infection is difficult to diagnose particularly in live birds. This is the primary reason that a new laboratory techniques need to be developed and tested to enable the detection M. avium infection. This was one of the major reasons for undertaking the purpose study, that is, determination of the sensitivity of three techniques, culture, PCR and Truant stain to detect the M. avium in live birds.
In this study, experiments were designed by using Rock pigeons (total number 50) and Sential chickens (total number 10) to get more knowledge about following points; the sensitivity of different laboratory techniques to detect MA in feces of live birds; bird immune response and shedding of MA in feces after oral inoculation and vaccination; in addition, to study the role of Rock pigeons in transmission the M. avium infection horizontally to Sential chickens.
A field strain of Mycobacterium avium was isolated and propagated in vitro from liver of an infected BHD. MA from liver of BHD was detected on M7H10 containing 0.5 %(v/v) glycerol and 10% (v/v) OADC Supplement after two weeks of incubation at 37 ºC and applied for all experiment work . In the pre-experimental work fresh feces (pooled, urates removed) and cloacal swabs obtained from 8 healthy MA free chickens were spiked with known quantities of MA (50 µl of 101-108 CFU / ml / gram of feces; 50 μl of 101-108 CFU / ml / swab) which was isolated from BHD. Truant stain Culture and polymerase chain reaction (PCR) techniques were evaluated to determine their limits of detection the original serial dilutions (50 µl) as well as negative controls (PBS-spiked) were also tested. Negative controls were omitted for cloacal swabs tested using the Truant stain method and culturing. Original serial dilutions were positive using culture and PCR at concentrations of 102 - 108 CFU / ml for the former and 101-108 CFU / ml for the latter. Truant stain clearly detected organisms at 106 -108, moderate numbers were seen on 104-105, and MA cells were rarely seen at 101-103. Spiked fecal materials while swabs were found to be positive on culture at 102 -108, although the absolute counts were lower than the original serial dilutions for both and of the spiking feces, swabs were the lowest. PCR detected MA in spiked fecal material at 106 -108 and from swabs at 107-108. Truant stain appeared to show numerous organisms in spiked fecal material at106 -108 and moderate numbers at 101-105. Spiked swabs appeared to show numerous organisms at 108, moderate numbers at 103-107 and rare organisms at 101 -102. In conclusion, Culture is the gold standard and has the highest level of sensitivity, PCR can be used to confirm that the isolates are MA; PCR is useful when MA numbers are high (106/ gram of feces, 107 for cloacal swabs). Truant stain may be value for pre-screening colonies. Although Truant stain may be a less expensive method than PCR for screening samples but positives must be confirmed by culture due to the potentially high false positive
In experiment one; adult Rock pigeons (Columba livia) were orally inoculated with 1ml of 102-108 CFU/ml of MA isolate. Adult pigeons (7 dilutions, 3 birds per dilution, 21 total) were inoculated orally to determine MA infectious dose could induce oral infection in Rock pigeons and to determine the sensitivity of (Truant stain, culture and PCR) to detect MA in feces post oral inoculation. In this study, MA could be detected in feces from 1st week post infection mainly by using truant stain and culture techniques where PCR was sensitive to MA on 1 , 4, 5 and 6 weeks post infection where volume of MA in feces more than 106 CFU/ml while the PCR was insensitive in 2nd and 3rd week post oral infection. In conclusion, MA concentration doses between 102-108 CFU/ml could induce M. avium oral infection in Rock pigeons; Truant stain and culture could detect MA in pigeon’s feces starting by one week post oral infection. The sensitivity of PCR to detect MA in live bird feces mainly depends on MA concentration in sample which most likely start to be increasing at beginning of four weeks post infection. All used techniques, truant stain, culturing and PCR, were less sensitive in detection of MA infection from fecal swabs than fecal matter.
In experiment two, for vaccine trail, 29 used rock pigeons were divided into three groups; vaccinated/ unchallenged group (no =9) (control group), vaccinated/ challenged (no.=10) and unvaccinated/ challenged group (no =10). The challenge was via both routes PO& IV by using 0.1 ml of 105 CFU/ml MA field isolates obtained from liver of bleeding heart dove. Vaccination was intramuscular by using 1ml of 108/CFU/ml of killed mixed boiled and autoclaved ones, the vaccination was proceed with 0.5 IU injection of Cortrosyn IM to induce short term stress before vaccination to insure the vaccination process. The used vaccine succeeded to reduce the shedding of MA in feces with both challenges routs either PO or IV route. In study of Natural (horizontal) infection was evaluated using unvaccinated chickens placed in the seperate cages beneath the pigeons -1 week post challenge. Ten chickens, divided into 6 groups. Each group contained 2 chickens except the controls which contained 1 chicken and corresponded to the pigeon control groups. Chickens were orally inoculated with 1 ml of a pooled fecal suspension in PBS from the corresponding pigeon group above once for three sucessive weeks. Our results, confirmed that, the wild bird could be played a very important and vital role in MA natural infection for chickens where we proved that, the detection of MA by using culture and Truant stain were of highly significant results x2= 42.00 (p<0.01) for both culture (p<0.01) and stain (p<0.01) of infected fecal matter. In conclusion, M. avium killed vaccine could be reducing M. avium infection in Rock pigeons and M. avium oral fecal infection could be transferred from feces of infected Rock pigeons to domestic chickens.