Molecular Analysis of Dichelobacter Nodosus Isolated From Footrot Infected Sheep in Malaysia

Footrot has become an increasingly important disease of sheep in Malaysia. Therefore, the molecular analysis of the causative agent of footrot, Dichelobader nodosus isolated from footrot infected sheep was undertaken. Fifteen D. nodosus isolates were recovered from 38 sheep showing clinical signs of footrot in two government sheep farms located approximately 200km apart The isolates were studied and results analysed. Preliminary identification of the organism was carried out by the Gram-stain method while the polymerase chain reaction (PCR) method using species-specific primers, A and Ac, was employed for species confirmation. All 15 isolates produced a single product of approximately 780 basepairs. Although obtained from two different locations, all isolates were found to be of serogroup B. Two conventional methods, namely the elastase and gelatingel tests, were used to assess the virulence of the isolates. Generally, the isolates exhibited variations in the laboratory characteristics. Based on the virulence assessment, some of the isolates appeared to have the capability for causing virulent footrot but were isolated from sheep that did not show clinical signs of the virulent form of footrot. This was probably due to the constant topical treatment regime and the vaccination programme practised by the farm management which may have caused the bacteria to not fully express its virulence characteristics. Analysis of the fimbrial subunit gene sequence revealed the local strains had sequences that are distinct from the prototype strains. There were 94 to 97 percent amino acid similarities (identities and conserved changes) between the local isolates and the prototype strains. The expression of D. nodosus fimbriae serotype B2 in an easily grown aerobe, Pseudamonas aeruginosa, were carried out successfully. Dichelobader nodosus fimbrial subunit gene was cloned in an expression vector, pUCpKS downstream the lac promoter to construct the recombinant plasmid pMAL99. Recombinant P. aeruginosa cells containing this construct were able to produce a high yield of fimbriae. The fimbriae were physically, structurally and antigenically indistinguishable from those produced by the D. nodosus isolates from which the fimbrial subunit gene was originally derived. This was shown and confirmed by Western blot analysis. When the fimbriae produced by the P. aeruginosa harbouring pMAL99 were extracted, purified and used as vaccines in sheep, the results conclusively showed that these vaccines were equally effective as either the native whole cells or isolated fimbriae from D. nodosus in eliciting the antibody response. The vaccinated sheep were found protected against homologous serogroup challenge. The recombinant fimbriae also produced crossprotective antibodies to heterologous serotypes B3 and B4 infections. Therefore, the monovalent serogroup specific recombinant vaccine has a good potential for use in farms in this country to protect sheep against footrot

Isolation and Characterisation of Dichelobacter Nodosus from Footrot Infected Sheep in Malaysia

Twelve Dichelobacter nodosus were isolated from 12 sheep with footrot with lesion score 2. The isolates were studied and the results analysed. Diagnosis was done successfully by Gram-stain method while polymerase chain reaction (PCR) method with species specific primers, A and Ac were employed for species confirmation. All 12 isolates reacted positively in the peR method by producing a single product of approximately 780 basepairs. All isolates, although obtained from distant locations, were from serogroup B (10 isolates were B2 serotype, 2 isolates were B1 serotype). The E-test method was used to determine the minimum inhibition concentration (MIC) values of nine antimicrobial agents against all 12 isolates. Penicillin G proved to be the most effective antibiotic with MIC90% of 0.023 ug/ml. Two standard conventional methods, the elastase and gelatin gel tests, were used in assessing the virulence of the isolates. Generally, the isolates exhibited variation in the laboratory characteristics although they had been isolated from similar lesion score. Some of the isolates which appeared to have the capability of causing virulent footrot in-vitro, failed to show clinical signs of virulent form of footrot. This was probably due to the frequent topical regimen adhered to and result of the vaccination programme by the farm management. All isolates were found not to contain plasmid by standard plasmid extracting method. This indicates that the genes coding for virulence of the isolates were not plasmidmediated. Molecular typing of the isolates was successfully carried out by pulsed field gel electrophoresis (PFGE) analysis. Significant patterns were generated by three GC-rich enzymes (ApaI, SfiI and SmaI) discriminating the isolates into eight genome types. Isolates from the same flock were also shown to possess variation in their PFGE profiles. These results demonstrate the diversity of D. nodosus strains infecting sheep in Malaysia and also indicated that the isolates were from diverse sources

Bacterial analysis of Australian Jade Perch frys.

Fifty-two Jade Perch frys from Australia were sampled for bacterial analysis to determine if any bacteria of pathogenic significance could be cultured. The frys were supplied in two groups: the first batch comprised of 32 frys obtained directly from the hatchery in Queensland, Australia and the second batch comprised of 20 frys from the same source that had been at a farm in the Klang Valley for one week. The kidneys of the fish and accompanying water were sampled for bacterial growth on Tryptic Soya agar (TSA) and Blood agar. Bacteria were identified using conventional biochemical tests and DNA sequencing. Seven known species of bacteria were identified through conventional and sequencing methods. Three of these are known bacterial pathogens of fish, namely Edwardsiella tarda, Vibrio spp. and Photobacterium damsela. Four of the identified bacteria namely Pleisiomonas shigelloides, Vibrio spp., Acinetobacter spp., and Pseudomonas aeruginosa are of public health significance. In addition, two relatively unknown species of bacteria, Aquitalea magnusonii and Hydrogenophaga spp., were successfully identified using the sequencing method

Evaluation of the survival of vancomycin-resistant enterococci (VRE) isolated from chickens and possible inactivation by in-use concentration of Lindores®, Ecos Timsen® and Omnicide®

Vancomycin-resistant enterococci (VRE) are well-known ascendant nosocomial pathogens. The recent detection of epidemiologic strain carrying vanA gene in the community of people working with animals and in chickens has brought to the forefront the potential public health danger posed by these organism. The farm environment is a major source of VRE persistence in poultry farms. We carried out survival test to test the survival of the VRE isolates on dry condition and surface test to evaluate the inactivation of the isolates by in-use concentration of commonly used disinfectants. In the survival test, all isolates survived for at least 4 weeks in colony counts of (1.00 × 103 – 3.86 × 103 CFU/ml) under clean condition and (1.00 × 103 – 2.02 × 104) for soiled condition. Those that were suspended in 5% BSA solution to mimic organic matter load as obtainable on farms survived for at least 8 weeks at (1.54 × 102 – 1.34 × 103 CFU/ml). In the surface test, inactivation of VRE isolates by in-use concentration of Lindores, Omnicide and Ecos Timsen was tested using the European surface test (EST). All the tested disinfectants were active against the VRE isolates on both the standard test surface (stainless steel) and our test surface (wooden). The results shows microbiocidal effects (ME)for test disinfectants, i.e. the log10 CFU of micro-organisms compared between test biocide and control treated with distilled water, after 7 min of exposure as follows; Lindores® active on both surfaces 5.24 and 3.17, Ecos Timsen® active significantly on steel 4.90 than wood 2.98 and Omnicide® significantly less active on stainless steel 2.40 than on wood 3.50

Occurrence and antibiotic resistance of Salmonella sp. in snakes in Singapore

Snakes are known to be asymptomatic Salmonella carriers that can be a source of infections to humans. Treatment of salmonellosis may become complicated with the rise of the antibiotic resistance among the Salmonella strains. A study was conducted to determine the prevalence of Salmonella and its resistance towards antibiotics in snakes at the Singapore Zoo. Cloacal samples were collected from snakes at the zoo and were subjected to isolation and identification of Salmonella sp. and its resistance to commonly used antimicrobial agents. Salmonella sp. was present in 20 (65%) of 31 snakes sampled. A total of 9 different serovars were found, and the predominant serovars were S. Mountpleasani (15%) and S. Cerro (15%); followed by S. Rissen (10%), S. Lohbruegge (10%), S. Lansing (5%), S. Hvittingfoss (5%), S. Sachsenwald (5%), S. Pomona (5%) and S. Lindern (5%). Twenty percent were unknown Salmonella serovars. The antibiotic susceptibility test revealed that all serotypes except for one were susceptible to six different antibiotics tested which included enrofloxacin (100%), marbofloxacin (100%), ceftiofur (100%), cephalexin (100%), chloramphenicol (95%) and amoxicillin (95%). The only serotype that was not susceptible to antibiotics was S. Lohbruegge. It was isolated from a corn snake and has an intermediate sensitivity towards chloramphenicol while resistant towards amoxicillin. The study showed snakes are infected with bacteria that could potentially transmit the infection to handlers and visitors. Thus precaution is advised when handling snakes

Presence of vancomycin resistance among enterococcus isolates from stray cats in Universiti Putra Malaysia and selected neighbourhood in Sri Serdang, Selangor, Malaysia

The present study was undertaken to determine the distribution of Enterococcus species from stray cats in Universiti Putra Malaysia and Seri Serdang and the presence of vancomycin resistance among the isolates. Fifty-five rectal swabs were collected from stray cats found in UPM and around the area of Sri Serdang. The Enterococcus species isolated were inoculated onto vancomycin resistant enterococci (VRE) agar supplemented with 8 μg/mL of vancomycin. Biochemical tests such as catalase, bile-aesculin and 6.5% NaCI were conducted to further confirm VRE isolates. Multiplex polymerase chain reaction assay (PCR) were performed for Enterococcus genus and species identification and vancomycin-resistant gene detection. Presence of Enterococcus spp. were demonstrated in every rectal sample tested. Two species were identified: Enterococcus faecalis (E. faecalis) and Enterococcus faecium (E. faecium). Among 55 isolates of Enterococcus tested, none was resistant to vancomycin at 8 μg/mL

A preliminary study of methicillin-resistant Staphylococcus aureus and antimicrobial resistance profile of bacteria in selected pig farms in Peninsular Malaysia

Staphylococcus aureus is one of the most common and devastating human and animal pathogens. In this study, conventional technique of isolation and identification was used to isolate Staphylococus aureus, Staphylococcus hyicus, Streptococcus spp., and E. coli. Detection and confirmation of MRSA was done using Staphytect® Kit and Oxacillin Resistance Screening Agar Base (ORSAB). Two farms from each pig industry rearing state namely Selangor, Perak, Johor and Penang were selected. Nasal and rectal swab samples were taken from 64 piglets and nasal swab samples were taken from 16 farm workers. Nineteen (13.2%) MRSA were isolated. Fourteen (21.9%) isolates were from pigs while 5 (31.3%) isolates originated from humans. These alarming findings from the present study indicated that MRSA is an emerging pathogen as well as a zoonotic potential in pigs and humans in Malaysia. This study also determined the antimicrobial sensitivity profile of some isolates towards commonly used antimicrobials in pig farms. Results showed that lincomycin is no longer effective for treatment in the farms while spectinomycin, florfenicol and enrofloxacin are starting to be less effective in controlling pathogens. Both colistin and ceftiofur are still effective as the bacteria tested are sensitive towards them. The findings from this study warrant that suitable measures must be undertaken to prevent the spread of MRSA as well as to control the rise of antibiotic resistant bacteria in the farms

Efficacy of a commercial probiotic in protecting mice against Salmonella infection

It is believed that probiotics are able to inhibit pathogenic bacteria from colonizing the gastrointestinal tract and thereby prevent infection and even mortality. On this basis, this project was undertaken to examine the efficacy of a commercial probiotic in preventing infection in the mouse model. This probiotic is made up of 8 bacterial species from 3 genera. Forty 6-week old white ICR mice were used in this project. The mice were divided into 7 groups consisting of positive and negative controls, 2 preventive groups and 3 treatment groups. The infective inocula were made up of a Salmonella typhimurium isolate. All mice in the positive control and treatment groups were severely affected when inoculated with the Salmonella isolate. Eleven (73%) of the 15 mice in the treatment groups died from the Salmonella infection. Salmonella was recovered from the internal organs of the mice in the positive control group and the treatment groups. No Salmonella was isolated from the internal organs of the mice in the negative control and the preventive groups. This showed that the probiotic was not able to prevent serious infection if given during or after infection. When the probiotic was given earlier as a prophylaxis, it was able to prevent serious infection. In this project, it is seen that none of the mice from the preventive groups succumbed to the Salmonella infection. It was clearly shown that probiotics were able to prevent adverse infection if given earlier as a prophylaxis