III Methods of diagnosis of pasteurella microorganisms :
I- Biochemical methods of identification^
4- Random Amplified Polymorphic DNA C RAPD ) analysis for Past, multocida isolates
Williams et al., (1990) revealed that genetic maps consisting of RAPD markers can be obtained more efficiently and with greater marker density than by RFLP or targeted PCR based methods .
Williams et al., (1992) mentioned that the presence of more than one amplification band per reaction gave the random amplified polymorphic DNA (RAPD) technique a distinct advantage over iso- enzymes or restriction fragment length polymorphism (RFLP) for differentiation among the isolates .
Dancla et al., [1996] studied 41 'strains of Past. mulrocida recovered from rabbits for epidemiological purposes by ribotyping and random amplified polymorphic DNA (RAPD) assays. They found that the results of RAPD assays were in accordance with those of ribotyping and validate the use of RAPD assays for epidemiological studies of Past. multocida strains.
Schuur et al., [1997] isolated Past. multocida from the cerebrospinal fluids ( CSF ) of a - 4 month old infant who presented with meningitis. The patient had been scratched on the head by a cat. Culture of the cat's claws also yielded Past. multocida. The isolates had identical biochemical patterns. Analysis of both strains by random amplification of polymorphic DNA (RAPD) and comparison of these strains with Past.
multocida strains isolated from other cats showed that the two strains were identical and completely different from the unrelated isolates. They concluded that RAPD analysis provided strong evidence for the causal relationship between the cat scratch and patient's meningitis. They added that application of this technique may eventually shed more light on the mechanisms by which Past. rnultocida is able to invade the CNS in humans.
Koeleman et al., (1998) compared thirty-one strains of Acinetobacter species , including type strain of the 18 genomic species and 13 clinical isolates by amplified ribosomal DNA restriction analysis (ARDRA), random amplified polymorphic DNA [RAPD] analysis and amplified fragment length polymorphism (AFLP) fingerprinting. They found that the standardized commercially available RAPD kit clearly enabled the discrimination of all Acinetobacter genomic species but showed great polymorphism between isolates of Acinetobacter baumannii.
AL-Haddawi et al., [1999] applied random amplified polymorphic DNA - polymerase chain reaction ( RAPD - PCR ) to subtype forty isolates of Past. multocida from healthy (17 isolates ) and diseased (23 isolates) rabbits. Two single primers were tested for their abilities to generate individual fingerprints by using PCR. They revealed that primer 1 grouped the isolates into 7 profiles, and primer 2 grouped them into 15. They recorded that RAPD - PCR results showed the presence of a wide heterogenicity within Past. multocida isolates. They concluded that RAPD - PCR is an efficient technique to detect the DNA polymorphism and could be used to discriminate Past. multocida of rabbit isolates together with serologic typing .
Charlton et al., [1999] used randomly amplified polymorphic DNA (RAPD) to investigate the molecular epidemiology of 26 Mycoplasma gallisepticum ( MG ) isolates obtained from turkeys. MG isolates were recovered from 5 different companies and 13 ranches. They stated that RAPD analysis of MG isolates within a ranch during an outbreak revealed only a single strain involved in each outbreak. They noticed that RAPD analysis identified an isolate from one ranch with a banding pattern identical to that of the 6/85 vaccine strain, which had been used on that particular ranch . They added that similar RAPD banding patterns of isolates from different ranches within the same company suggested horizontal spread of MG between ranches concluding that the use of two primer sets in RAPD analysis was critical to prevent misinterpretation of relationships between different isolates .
Warner and Oliver [1999] used RAPD - PCR for detecting V. vulnificus , as well as other members of the genus Vibrio. They found that RAPD method clearly differentiated between members of genus Vibrio and between isolates of V.vulnlficus. They added that each V.vulnificus strain produced a unique band pattern, indicating that the members of this species are genetically quite heterogeneous. They noticed that all of Vibrios were found to have amplification products whose sizes were within four common molecular weight ranges, while V. vulnificus strains had an additional two molecular weight range bands in common. Also they recorded that band pattern differences were observed between encapsulated and non-encapsulated isogenic morphotypes of the same strain of V.vulnificus .
Abbas et al., [2000] used random amplified polymorphic DNA (RAPD) markers, as a genetic markers to monitor genetic variability between Past. muttocida vaccinal strains. A set of single , short generic five primers of arbitrary sequence was used in this study. They revealed that a total of 111 reproducible polymorphic DNA fragments were produced by two arbitrary primers. Three common species specific amplified DNA bands were detected in all Past. multocida vaccinal strains. They found that ninety - four percent ( 94% ) and 100% of the total DNA bands, amplified by primer OP-G4 and OP-E3 respectively showed polymorphism. So these two primers can differentiate between Past. multocida vaccinal strains. They concluded that RAPD markers generated sufficient genetic data exploiting sequence differences within Past. multocida vaccinal strains.
Martinez et al., [2000] evaluate the genetic diversity of S. agalactia strains isolated from bovine milk and from asymptomatic women vaginas in Quebec, Canada, by randomly amplified polymorphic DNA ( RAPD ) analysis. They indicated that a total of 185 bovine isolates and 38 human isolates were firstly serotyped then studied by RAPD using 3 primers, designated OPS 11, OPB 17, and OPB 18. They showed that all isolates (of bovine and human origin ) shared 58% similarity. Ninety - four percent of these isolates were clustered in four groups ( I , II , III and IV ) with 70% similarity among them. Three clusters, A ( 48 isolates ), B ( 14 isolates ) and C[32 isolates], with 79 to 80 % similarity were identified within group IV, whereas the three other groups did not present any clusters. Despite some clustering of human isolates, relatively high diversity was seen among them. They recorded that high heterogenicity was observed with the RAPD profiles, not only for field strains belonging to different serotypes but also for those within a given serotype.
Ramasoota et al., [2000] used a standardized- reagents commercial kit for random amplified polymorphic DNA (RAPD) analysis for typing 58 E-coli strains that were recovered from the milk of sows, having coliform mastitis, within a single swine herd in Sweden. They stated that the RAPD analysis was fast, easily performed and required only a nanogram of DNA. They recorded that RAPD analysis is a technique that provided reproducible results for typing of E. coli strains that cause mastitis in sows. They added that all 58 strains can be differentiated by means of the RAPD technique .
Dziva et al., [2001] subjected eighty-one isolates presumptively identified as Past. multocida from a variety of diseases in animals in Zimbabwe to biochemical characterization, capsular typing and RAPD analysis. They stated that although capsular typing offers a relatively fast analysis of Past. multocida, it is shortfalls are well recognized and one of the commonly encountered obstacles is the occurrence of a capsular forms of the bacteria, which consequently become nontypeable. Such problems were not encountered when genotypic methods are employed in the typing of this organism. They showed that no relationship between RAPD pattern and capsular serogroup. They suggested that RAPD analysis is a useful tool for strain differentiation.
Huber et al., [2002] analyzed and compared 61 clinical isolates of Past. multocida recovered from turkeys that died of fowl cholera to M-9 vaccine strain. Genetic analysis of the isolates were done by random amplified polymorphic DNA[RAPD] analysis and amplified fragment length polymorphism [AFLP] fingerprinting. They found that both genetic techniques effectively identified similar subtle genomic differences. They showed that pathogenic isolates from vaccinated
turkeys were more genetically similar to the M-9 vaccine strain than isolates from non vaccinated turkeys. Statistical analysis revealed that this relationship could not have been determined by serotyping alone, demonstrating the value of RAPD and AFLP analysis in the characterization of disease causing strains.
Khoodoo et al. [2002] assessed the genetic diversity of 24 isolates of Salmonella enterica [five chicken and 19 human] by RAPD fingerprinting. They confirmed that some strains of Salmonella isolated from chicken were genetically similar to those isolated from human.
Lainson et al., [2002 ] isolated Past. multocida strains from 15 pigs with PDNS and 51 pigs without PDND. They characterized the isolates by capsule and somatic antigen typing, RAPD typing, and restriction analysis of genomic DNA using pulsed field gel electrophoresis [PFGE], They concluded that capsular, somatic and RAPD typing did not discriminate PDNS isolates. They added that all of the isolates from PDNS cases showed an identical ApaI PFGE restriction pattern. This pattern was also found in a high proportion [36%] of Past. multocida strains isolated from non PDNS cases .
Xia et al., [2002] used 94 RAPD primers of different nucleotide composition to probe the genomic differences between a highly virulent Past. mulocida strain and an attenuated vaccine strain derived from the virulent strain after culturing the latter under increasing temperature for approximately 14,400 generations. They found that the GC content of the vaccine strain was significantly lower than that of the virulent strain. They added that the frequencies of AA,TA and TT dinucleotides were
higher, and those of AT, GC and CG dinucleotides were lower in the vaccine strain than in the virulent strain .
Katsuda et al., [2003] applied RAPD method for molecular typing of 130 Past. haemolytica serotype Al isolates obtained from 13 prefectures in Japan. RAPD analysis revealed four banding patterns [types I-IV] and among 130 isolates 60.7% [79/130] of isolates were RAPD type I. They found that all of the RAPD type I isolates were grouped into clusters A-C by PFGE. They noticed that there was no relationship between molecular typing and geographic origin of these isolates. They indicated that isolates of Past. haemolytica Al strain with various profiles have already spread in Japan and may have caused sporadic infection .