Multiclonal human origin and global expansion of an endemic bacterial pathogen of livestock.

Most new pathogens of humans and animals arise via switching events from distinct host species. However, our understanding of the evolutionary and ecological drivers of successful host adaptation, expansion, and dissemination are limited. Staphylococcus aureus is a major bacterial pathogen of humans and a leading cause of mastitis in dairy cows worldwide. Here we trace the evolutionary history of bovine S. aureus using a global dataset of 10,254 S. aureus genomes including 1,896 bovine isolates from 32 countries in 6 continents. We identified 7 major contemporary endemic clones of S. aureus causing bovine mastitis around the world and traced them back to 4 independent host-jump events from humans that occurred up to 2,500 y ago. Individual clones emerged and underwent clonal expansion from the mid-19th to late 20th century coinciding with the commercialization and industrialization of dairy farming, and older lineages have become globally distributed via established cattle trade links. Importantly, we identified lineage-dependent differences in the frequency of host transmission events between humans and cows in both directions revealing high risk clones threatening veterinary and human health. Finally, pangenome network analysis revealed that some bovine S. aureus lineages contained distinct sets of bovine-associated genes, consistent with multiple trajectories to host adaptation via gene acquisition. Taken together, we have dissected the evolutionary history of a major endemic pathogen of livestock providing a comprehensive temporal, geographic, and gene-level perspective of its remarkable success.

Phenotypic and genotypic characterization of colistin-resistant Escherichia Coli with mcr-4, mcr-5, mcr-6, and mcr-9 genes from broiler chicken and farm environment.

BACKGROUND: Colistin is an antibiotic used as a last-resort to treat multidrug-resistant Gram-negative bacterial infections. Colistin had been used for a long time in veterinary medicine for disease control and as a growth promoter in food-producing animals. This excessive use of colistin in food animals causes an increase in colistin resistance. This study aimed to determine molecular characteristics of colistin-resistant Escherichia coli in broiler chicken and chicken farm environments. RESULTS: Four hundred fifty-three cloacal and farm environment samples were collected from six different commercial chicken farms in Kelantan, Malaysia. E. coli was isolated using standard bacteriological methods, and the isolates were tested for antimicrobial susceptibility using disc diffusion and colistin minimum inhibitory concentration (MIC) by broth microdilution. Multiplex PCR was used to detect mcr genes, and DNA sequencing was used to confirm the resistance genes. Virulence gene detection, phylogroup, and multilocus sequence typing (MLST) were done to further characterize the E. coli isolates. Out of the 425 (94%; 425/453) E. coli isolated from the chicken and farm environment samples, 10.8% (48/425) isolates were carrying one or more colistin-resistance encoding genes. Of the 48 colistin-resistant isolates, 54.2% (26/48) of the mcr positive isolates were genotypically and phenotypically resistant to colistin with MIC of colistin ≥ 4 µg/ml. The most prominent mcr gene detected was mcr-1 (47.9%; 23/48), followed by mcr-8 (18.8%; 9/48), mcr-7 (14.5%; 7/48), mcr-6 (12.5%; 6/48), mcr-4 (2.1%; 1/48), mcr-5 (2.1%; 1/48), and mcr-9 (2.1%; 1/48) genes. One E. coli isolate originating from the fecal sample was found to harbor both mcr-4 and mcr-6 genes and another isolate from the drinking water sample was carrying mcr-1 and mcr-8 genes. The majority of the mcr positive isolates were categorized under phylogroup A followed by phylogroup B1. The most prevalent sequence typing (ST) was ST1771 (n = 4) followed by ST206 (n = 3). 100% of the mcr positive E. coli isolates were multidrug resistant. The most frequently detected virulence genes among mcr positive E. coli isolates were ast (38%; 18/48) followed by iss (23%; 11/48). This is the first research to report the prevalence of mcr-4, mcr-5, mcr-6, mcr-7, and mcr-8 genes in E. coli from broiler chickens and farm environments in Malaysia. CONCLUSION: Our findings suggest that broiler chickens and broiler farm environments could be reservoirs of colistin-resistant E. coli, posing a risk to public health and food safety.

Occurrence of antibiotic resistant C. jejuni and E. coli in wild birds, chickens, humans, and the environment in Malay villages, Kedah, Malaysia.

Foodborne pathogens have become a major concern not only due to the diseases they cause, but also because of the rise of antibiotic resistant strains in human and animals. The purposes of this study were to determine the occurrence of Campylobacter jejuni and Escherichia coli and their antibiotic resistance profiles in wild birds, chickens, humans, and the environment in Malay villages in Malaysia. Three Malay villages in Kota Setar, Kedah were chosen. Three hundred nine (309) samples were collected in this study including wild birds (38), chickens (71), humans (47), and the environment (153). Subsequently, the C. jejuni and E coli isolates were tested against antibiotics using the disc diffusion method. Campylobacter jejuni was found positive in 17 (37.8%) flies and 8 (11.3%) chickens. Also, E. coli was found positive in 89.4% of human, 47.4% of bird, 44 62% of chicken and in 71.2% of the environmental samples. Ten antibiotics were used to determine the susceptibility of the isolates. Eighty four percent (84%) of C. jejuni and 100% of E. coli isolates were found to show resistance towards at least one antibiotic. The isolates showed high resistance to cefpodoxime and tetracycline.

Isolation of Mycobacterium avium and other nontuberculous mycobacteria in chickens and captive birds in peninsular Malaysia.

BACKGROUND: Mycobacterium avium complex (MAC) causes a chronic infectious in the birds known as avian mycobacteriosis. Almost all species of the birds are susceptible to MAC which consists of two closely related species of mycobacteria, that is, M. avium and M. intracellulare. This study aimed to determine the occurrence of Mycobacterium avium subsp. avium (MAA) in chickens and captive birds in selected states of Peninsular Malaysia. RESULTS: A 300 fecal samples were collected from village chickens (n = 100), layer chickens (n = 100) and captive birds (n = 100). Fecal samples were split into two aliquots for microbiological and molecular detection of MAA. Microbiology detection consisted of microscopy (Ziehl-Neelsen staining) and culture of samples decontaminated with 1% Cetylperidinium chloride and vancomycin, nalidixic acid and amphotericin B (VNA) antibiotic cocktail [vancomycin (VAN) 100 µg/ml, nalidixic acid (NAL) 100 µg/ml and amphotericin B (AMB) 50 µg/ml] onto Löwenstein-Jensen (L-J). Molecular detection (PCR-IS901) was performed to detect MAA DNA from the feces and PCR-16S rRNA and IS901 for identification of genus Mycobacterium and Mycobacterium avium sub species avium isolated onto L-J. All samples (296) were AFB negative smear. M. avium was isolated in 0.3% (1/296) samples by culture and detected in 2.5% (6/242) samples by PCR (IS901). Other mycobacteria were found in 1.7% (5/296) chickens. Of five isolates, two were identified as Mycobacterium terrae and M. engbaekii and remaining isolates were not sequenced. Birds positive for M. avium included White Pelican (n = 1) Black Hornbill (n = 1), Macaw (n = 2), Cockatoo (n = 2) and village chicken (n = 1). CONCLUSION: It is concluded that chickens and birds were infected with M. avium in selected areas of Peninsular Malaysia. Although, PCR is rapid, reliable and cost effective method for detection of M. avium in a subclinical stage, the culture of the avian feces should still be used as a reference test for the diagnosis of avian tuberculosis.

Analysis of Salmonella enterica serovar Enteritidis isolates from chickens and chicken meat products in Malaysia using PFGE, and MLST.

BACKGROUND: Salmonella is a very important foodborne pathogen causing illness in humans. The emergence of drug-resistant strains also constitutes a serious worry to global health and livestock productivity. This study investigated Salmonella isolates from chicken and chicken meat products using the phenotypic antimicrobial screening as well as the molecular characteristics of Salmonella isolates. Upon serotyping of the isolates, the antimicrobial susceptibility profiling using a panel of 9 commonly used antimicrobials was done. Subsequently, the molecular profiles of all the isolates were further determined using Pulsed Field Gel Electrophoresis (PFGE) and the Whole Genome Multi-Locus Sequence Type (wgMLST) analysis in order to obtain the sequence types. RESULTS: The PFGE data was input into FPQuest software, and the dendrogram generated was studied for possible genetic relatedness among the isolates. All the isolates were found to belong to the Salmonella Enteritidis serotype with notable resistance to tetracycline, gentamycin, streptomycin, and sulfadimidine. The S. Enteritidis isolates tested predominantly subtyped into the ST11 and ST1925, which was found to be a single cell variant of ST11. The STs were found to occur in chicken meats, foods, and live chicken cloacal swabs, which may indicate the persistence of the bacteria in multiple foci. CONCLUSION: The data demonstrate the presence of S. Enteritidis among chickens, indicating its preference and reservoir status for enteric Salmonella pathogens.

Antigenic potential of a recombinant polyvalent DNA vaccine against pathogenic leptospiral infection.

Leptospirosis is a serious epidemic disease caused by pathogenic Leptospira species. The disease is endemic in most tropical and sub-tropical regions of the world. Currently, there is no effective polyvalent vaccine for prevention against most of the circulating serovars. Moreover, development of an efficient leptospiral vaccine capable of stimulating cross-protective immune responses against a wide range of serovars remains a daunting challenge. This, in part, is associated with the extensive diversity and variation of leptospiral serovars from region to region. In this study, a multi-epitope DNA vaccine encoding highly immunogenic epitopes from LipL32 and LipL41 was designed using in-silico approach. The DNA encoding antigenic epitopes was constructed from conserved pathogenic Leptospira genes (LipL32 and LipL41). Immunization of golden Syrian hamsters with the multi-epitope chimeric DNA vaccine resulted in the production of both agglutinating and neutralizing antibodies as evidence by MAT and in-vitro growth inhibition tests respectively. The antibodies produced reacted against eight different serovars and significantly reduced renal colonization following in vivo challenge. The vaccine was also able to significantly reduce renal colonization which is a very important factor responsible for persistence of leptospires among susceptible and reservoir animal hosts. In conclusion, the leptospiral multi-epitope chimeric DNA vaccine can serve as a potentially effective and safe vaccine against infection with different pathogenic leptospiral serovars.

Physicochemical properties associated with the presence of Burkholderia pseudomallei in small ruminant farm water supplies in Peninsular Malaysia.

Burkholderia pseudomallei causes melioidosis, a life-threatening infection in both humans and animals. Water is an important reservoir of the bacteria and may serve as a source of environmental contamination leading to infection. B. pseudomallei has an unusual ability to survive in water for a long period. This paper investigates physicochemical properties of water associated with the presence of B. pseudomallei in water supply in small ruminant farms in Peninsular Malaysia. Physicochemical properties of water samples taken from small ruminant farms that included temperature, pH, dissolved oxygen (DO2), optical density (OD), and chemical oxygen demand (COD) were measured after which the samples were cultured for B. pseudomallei. Multivariable logistic regression model revealed that slightly acidic water pH and higher COD level were significantly associated with the likelihood of the B. pseudomallei presence in the water.

Screening of Streptococcus suis in swine workers of selected states in Peninsular Malaysia.

Background and Aim: Streptococcus suis is a zoonotic pathogen that is highly associated with contact between live pigs and raw pig material. In view of the recent reports of human infections in Malaysia, epidemiological data on the status of S. suis in the human population, especially among people working closely with pigs and/or raw pork, should be provided. The aim of this study was to detect S. suis among individuals working in the swine industry in several major pig production areas in Peninsular Malaysia. Materials and Methods: Demographic information, exposure determinants, and oral swabs were collected from swine personnel, including farmers, butchers, and veterinarians. Oral swabs were subjected to bacterial isolation and conventional polymerase chain reaction (PCR) assays for S. suis detection. Results: The study included 40 participants working in the swine industry, with a predominant representation of males (62.5%) and Malaysian Chinese individuals (60.0%) who consumed pork (92.5%). Notably, none of the participants reported consuming raw or partially cooked pork. In spite of their occupational exposure risk, none of the oral swabs showed positive results for S. suis infection. Conclusion: To the best of our knowledge, this is the first report and detection study of S. suis using oral swabs obtained from swine personnel in Peninsular Malaysia.

Genetic variability of vancomycin-resistant Enterococcus faecium and Enterococcus faecalis isolates from humans, chickens, and pigs in Malaysia.

Vancomycin-resistant enterococci (VRE) have been reported to be present in humans, chickens, and pigs in Malaysia. In the present study, representative samples of VRE isolated from these populations were examined for similarities and differences by using the multilocus sequence typing (MLST) method. Housekeeping genes of Enterococcus faecium (n = 14) and Enterococcus faecalis (n = 11) isolates were sequenced and analyzed using the MLST databases eBURST and goeBURST. We found five sequence types (STs) of E. faecium and six STs of E. faecalis existing in Malaysia. Enterococcus faecium isolates belonging to ST203, ST17, ST55, ST79, and ST29 were identified, and E. faecium ST203 was the most common among humans. The MLST profiles of E. faecium from humans in this study were similar to the globally reported nosocomial-related strain lineage belonging to clonal complex 17 (CC17). Isolates from chickens and pigs have few similarities to those from humans, except for one isolate from a chicken, which was identified as ST203. E. faecalis isolates were more diverse and were identified as ST4, ST6, ST87, ST108, ST274, and ST244, which were grouped as specific to the three hosts. E. faecalis, belonging to the high-risk CC2 and CC87, were detected among isolates from humans. In conclusion, even though one isolate from a chicken was found clonal to that of humans, the MLST analysis of E. faecium and E. faecalis supports the findings of others who suggest VRE to be predominantly host specific and that clinically important strains are found mainly among humans. The infrequent detection of a human VRE clone in a chicken may in fact suggest a reverse transmission of VRE from humans to animals.

Virulence gene profiles and antimicrobial susceptibility of Salmonella Brancaster from chicken.

BACKGROUND: The current conventional serotyping based on antigen-antisera agglutination could not provide a better understanding of the potential pathogenicity of Salmonella enterica subsp. enterica serovar Brancaster. Surveillance data from Malaysian poultry farms indicated an increase in its presence over the years. OBJECTIVE: This study aims to investigate the virulence determinants and antimicrobial resistance in S. Brancaster isolated from chickens in Malaysia. METHODS: One hundred strains of archived S. Brancaster isolated from chicken cloacal swabs and raw chicken meat from 2017 to 2022 were studied. Two sets of multiplex polymerase chain reaction (PCR) were conducted to identify eight virulence genes associated with pathogenicity in Salmonella (invasion protein gene [invA], Salmonella invasion protein gene [sipB], Salmonella-induced filament gene [sifA], cytolethal-distending toxin B gene [cdtB], Salmonella iron transporter gene [sitC], Salmonella pathogenicity islands gene [spiA], Salmonella plasmid virulence gene [spvB], and inositol phosphate phosphatase gene [sopB]). Antimicrobial susceptibility assessment was conducted by disc diffusion method on nine selected antibiotics for the S. Brancaster isolates. S. Brancaster, with the phenotypic ACSSuT-resistance pattern (ampicillin, chloramphenicol, streptomycin, sulphonamides, and tetracycline), was subjected to PCR to detect the corresponding resistance gene(s). RESULTS: Virulence genes detected in S. Brancaster in this study were invA, sitC, spiA, sipB, sopB, sifA, cdtB, and spvB. A total of 36 antibiogram patterns of S. Brancaster with a high level of multidrug resistance were observed, with ampicillin exhibiting the highest resistance. Over a third of the isolates displayed ACSSuT-resistance, and seven resistance genes (ß-lactamase temoneira [blaTEM], florfenicol/chloramphenicol resistance gene [floR], streptomycin resistance gene [strA], aminoglycoside nucleotidyltransferase gene [ant(3″)-Ia], sulfonamides resistance gene [sul-1, sul-2], and tetracycline resistance gene [tetA]) were detected. CONCLUSION: Multidrug-resistant S. Brancaster from chickens harbored an array of virulence-associated genes similar to other clinically significant and invasive non-typhoidal Salmonella serovars, placing it as another significant foodborne zoonosis.

The global profile of antibiotic resistance in bacteria isolated from goats and sheep: A systematic review.

Background and Aim: Antibiotic resistance has become an issue of global importance due to increasing levels of bacterial infections worldwide. Farm management and usage of antibiotics in livestock are known risk factors associated with the increase in global levels of antibiotic resistance. Goats and sheep are examples of livestock with large populations. Although antibiotic resistance in bacteria from livestock negatively affects both human health and the economy, the global data regarding this issue in goats and sheep are limited. Therefore, this study aimed to provide information on the antibiotic-resistance profile of bacteria isolated from goats and sheep worldwide (Asia, Europe, and Africa). Materials and Methods: We performed a systematic review of articles published on this topic without any restriction on the year of publication. We searched the Directory of Open Access Journals, PubMed, Google Scholar, and Scopus using Boolean logic through various keywords. The search generated a total of 1325 articles, and after screening for duplicates and implementing inclusion and exclusion criteria, qualitative synthesis (i.e., qualitative systematic review) was performed on 37 articles. Results: The synthesized information indicated that 18 Gram-positive and 13 Gram-negative bacterial species from goats and sheep were resistant to ten antibiotics, namely penicillin, ampicillin, amoxicillin, chloramphenicol, streptomycin, tetracycline, cephalothin, gentamicin, ciprofloxacin (CIP), and sulfamethoxazole. The prevalence of antibiotic resistance ranged from 0.4% to 100%. However, up to 100% of some bacteria, namely, Salmonella Dublin, Aeromonas caviae, and Aeromonas sobria, were susceptible to CIP. Staphylococcus aureus and Escherichia coli were highly resistant to all antibiotics tested. Moreover, eight of the ten antibiotics tested were critically important antibiotics for humans. Conclusion: Antibiotic-resistant bacteria in goats and sheep are a potential risk to animal and human health. Collaboration between all stakeholders and further research is needed to prevent the negative impacts of antibiotic resistance.

Prevalence and multidrug-resistant profile of methicillin-resistant Staphylococcus aureus and methicillin-resistant Staphylococcus pseudintermedius in dogs, cats, and pet owners in Malaysia.

Background and Aim: Staphylococcus aureus and Staphylococcus pseudintermedius are widespread skin and mucous membrane colonizers and may cause opportunistic infections in humans and animals. This study aimed to identify and characterize methicillin-resistant S. aureus (MRSA) and methicillin-resistant S. pseudintermedius (MRSP) isolates from domestic and stray dogs and cats and pet owners in Malaysia using molecular epidemiology and antimicrobial profiling. Materials and Methods: Three hundred and fifty oral and nasal swabs were taken from pet and stray dogs and cats and pet owners; all samples were subjected to culture and biochemical tests and polymerase chain reaction; the selected isolates were put through disk diffusion test and multilocus sequence typing. Results: One S. aureus isolate and three S. pseudintermedius isolates were identified as MRSA and MRSP, respectively, of which the MRSA isolate and one of the MRSP isolates showed multidrug resistance and the remaining two MRSP isolates were resistant to one or two antimicrobials. Multilocus sequence typing showed that the MRSA isolate belongs to clonal complex (CC) 789, while for the MRSP isolates, two were in CC45 and one was a singleton. Conclusion: This study is the first study in Malaysia to perform molecular characterization of MRSP isolated from pet dogs and cats and pet owners. The outcomes of this study reveal that even healthy pet dogs and cats and their owners can be carriers of drug-resistant staphylococci, highlighting the role of pets and pet owners as carriers of MRSA and MRSP in Malaysia.

The occurrence and molecular detection of mcr-1 and mcr-5 genes in Enterobacteriaceae isolated from poultry and poultry meats in Malaysia.

The advent of antimicrobials-resistant (AMR), including colistin-resistant bacteria, poses a significant challenge to animal and human health, food safety, socio-economic growth, and the global environment. This study aimed to ascertain the colistin resistance prevalence and molecular mechanisms of colistin resistance in Enterobacteriaceae. The colistin resistance was determined using broth microdilution assay, PCR; and Sanger sequencing of mcr genes responsible for colistin resistance in Enterobacteriaceae (n = 627), including Escherichia coli (436), Salmonella spp. (n = 140), and Klebsiella pneumoniae (n = 51), obtained from chicken and chicken meats. Out of 627 Enterobacteriaceae, 8.6% of isolates exhibited colistin resistance phenotypically. Among these colistin resistant isolates, 9.3% (n = 37) were isolated from chicken meat, 7.2% (n = 11) from the cloacal swab of chicken and 7.9% (n = 6) from the litter samples. Overall, 12.96% of colistin-resistant isolates were positive with mcr genes, in which mcr-1 and mcr-5 genes were determined in 11.11% and 1.85% of colistin-resistant isolates, respectively. The E. coli isolates obtained from chicken meats, cloacal swabs and litter samples were found positive for mcr-1, and Salmonella spp. originated from the chicken meat sample was observed with mcr-5, whereas no mcr genes were observed in K. pneumoniae strains isolated from any of the collected samples. The other colistin resistance genes, including mcr-2, mcr-3, mcr-4, mcr-6, mcr-7, mcr-8, mcr-9, and mcr-10 were not detected in the studied samples. The mcr-1 and mcr-5 genes were sequenced and found to be 100% identical to the mcr-1 and mcr-5 gene sequences available in the NCBI database. This is the first report of colistin resistance mcr-5 gene in Malaysia which could portend the emergence of mcr-5 harboring bacterial strains for infection. Further studies are needed to characterize the mr-5 harbouring bacteria for the determination of plasmid associated with mcr-5 gene.

Antimicrobial Resistance Profiles and Co-Existence of Multiple Antimicrobial Resistance Genes in mcr-Harbouring Colistin-Resistant Enterobacteriaceae Isolates Recovered from Poultry and Poultry Meats in Malaysia.

The co-existence of the colistin resistance (mcr) gene with multiple drug-resistance genes has raised concerns about the possibility of the development of pan-drug-resistant bacteria that will complicate treatment. This study aimed to investigate the antibiotic resistance profiles and co-existence of antibiotic resistance genes among the colistin-resistant Enterobacteriaceae isolates recovered from poultry and poultry meats. The antibiotic susceptibility to various classes of antibiotics was performed using the Kirby-Bauer disk diffusion method and selected antimicrobial resistance genes were detected using PCR in a total of 54 colistin-resistant Enterobacteriaceae isolates including Escherichia coli (E. coli) (n = 32), Salmonella spp. (n = 16) and Klebsiella pneumoniae (K. pneumoniae) (n = 6) isolates. Most of the isolates had multi-drug resistance (MDR), with antibiotic resistance against up to seven classes of antibiotics. All mcr-harbouring, colistin-resistant Enterobacteriaceae isolates showed this MDR (100%) phenotype. The mcr-1 harbouring E. coli isolates were co-harbouring multiple antibiotic resistance genes. The seven most commonly identified resistance genes (blaTEM, tetA, floR, aac-3-IV, aadA1, fosA, aac(6_)-lb) were detected in an mcr-1-harbouring E. coli isolate recovered from a cloacal swab. The mcr-5 harbouring Salmonella spp. isolate recovered from poultry meats was positive for blaTEM, tetA, floR, aac-3-IV, fosA and aac(6_)-lb genes. In conclusion, the colistin-resistant Enterobacteriaceae with mcr genes co-existing multiple clinically important antimicrobial resistance genes in poultry and poultry meats may cause potential future threats to infection treatment choices in humans and animals.

Molecular detection and antimicrobial resistance profiles of Extended-Spectrum Beta-Lactamase (ESBL) producing Escherichia coli in broiler chicken farms in Malaysia.

Antimicrobial resistance is one of the major public health threats globally. This challenge has been aggravated with the overuse and misuse of antibiotics in food animals and humans. The present study aimed to investigate the prevalence of Extended-Spectrum ß-lactamase (ESBL) genes in Escherichia coli (E. coli) isolated from broiler chickens in Kelantan, Malaysia. A total of 320 cloacal swabs were collected from farms in different districts of Kelantan and were analyzed using routine bacteriology, antimicrobial susceptibility test, and molecular techniques for further identification and characterization of ESBL encoding genes. Based on PCR detection for the E. coli species-specific Pho gene, 30.3% (97/320) of isolates were confirmed as E. coli, and 84.5% (82/97) of the isolates were positive for at least one ESBL gene. Majority of the isolates, 62.9% (61/97) were harboring blaCTX-M followed by 45.4% (44/97) of blaTEM genes, while 16.5% (16/97) of the isolates were positive for both mcr-1 and ESBL genes. Overall, 93.8% (90/97) of the E. coli were resistant to three or more antimicrobials; indicating that the isolates were multi-drug resistance. 90.7% of multiple antibiotic resistance (MAR) index value greater than 0.2, would also suggest the isolates were from high-risk sources of contamination. The MLST result shows that the isolates are widely diverse. Our findings provide insight into the alarmingly high distribution of antimicrobial resistant bacteria, mainly ESBL producing E. coli in apparently healthy chickens indicating the role of food animals in the emergence and spread of antimicrobial resistance, and the potential public health threats it may pose.

Seroprevalence and Factors Associated with Toxoplasma gondii, Neospora caninum, and Besnoitia besnoiti Infections in Cattle and Goats in Selangor, Malaysia.

Apicomplexan parasites such as Toxoplasma gondii, Neospora caninum, and Besnoitia besnoiti are widely recognized as causes of production diseases in ruminants. This study aimed to investigate the serological occurrence of T. gondii, N. caninum, and B. besnoiti in cattle and goats from smallholder farms in Selangor, Malaysia. A cross-sectional study was conducted on 19 farms by collecting 404 bovine (n = 225) and caprine (n = 179) serum samples, which were then essayed for T. gondii, N. caninum, and B. besnoiti antibodies using commercially available ELISA test kits. Farm data and animal characteristics were documented, and the data were analyzed using descriptive statistics and logistic regression models. The seroprevalence of T. gondii at animal and farm levels in cattle was 5.3% (95% CI 1.2-7.4%) and 36.8% (95% CI 22.4-58.0%), respectively. Animal-level seropositivity for N. caninum was 2.7% (95% CI 0.4-4.2%) and 5.7% for B. besnoiti (95% CI 1.3-9.4%) with corresponding farm-level seropositivity of 21.0% and 31.5%, respectively. For the goat samples, a high animal- (69.8%; 95% CI 34.1-82.0%) and farm-level (92.3%) seropositivity was recorded for T. gondii, but was relatively lower for N. caninum antibodies, at 3.9% (95% CI 1.5-6.2%) and 38.4% (5/13). The factors associated with T. gondii seropositivity were older animals (above 12 months) (OR = 5.3; 95% CI 1.7-16.6), semi-intensive farms (OR = 2.2; 95% CI 1.3-6.2), the presence of either dogs or cats (OR = 3.6; 95% CI 1.1-12.3), a large herd size (>100 animals) (OR = 3.7; 95% CI 1.4-10.0), and a single source of replacement animals (OR = 3.9; 95% CI 1.6-9.6). These findings are vital in developing effective control measures against these parasites in ruminant farms in Selangor, Malaysia. More national epidemiological research is required to elucidate the spatial distribution of these infections and their potential impact on Malaysia's livestock industry.

Attenuated Salmonella typhimurium SV4089 as a potential carrier of oral DNA vaccine in chickens.

Attenuated Salmonella has been used as a carrier for DNA vaccine. However, in vitro and in vivo studies on the bacteria following transfection of plasmid DNA were poorly studied. In this paper, eukaryotic expression plasmids encoding avian influenza virus (AIV) subtype H5N1 genes, pcDNA3.1/HA, NA, and NP, were transfected into an attenuated Salmonella enteric typhimurium SV4089. In vitro stability of the transfected plasmids into Salmonella were over 90% after 100 generations. The attenuated Salmonella were able to invade MCF-7 (1.2%) and MCF-10A (0.5%) human breast cancer cells. Newly hatched specific-pathogen-free (SPF) chicks were inoculated once by oral gavage with 10(9) colony-forming unit (CFU) of the attenuated Salmonella. No abnormal clinical signs or deaths were recorded after inoculation. Viable bacteria were detected 3 days after inoculation by plating from spleen, liver, and cecum. Fluorescent in situ hybridization (FISH) and polymerase chain reaction (PCR) were carried out for confirmation. Salmonella was not detected in blood cultures although serum antibody immune responses to Salmonella O antiserum group D1 factor 1, 9, and 12 antigens were observed in all the inoculated chickens after 7 days up to 35 days. Our results showed that live attenuated S. typhimurium SV4089 harboring pcDNA3.1/HA, NA, and NP may provide a unique alternative as a carrier for DNA oral vaccine in chickens.

Prevalence and risk factors of feline leukaemia virus and feline immunodeficiency virus in peninsular Malaysia.

BACKGROUND: Feline leukaemia virus (FeLV) and feline immunodeficiency virus (FIV) are major causes of morbidity and mortality in domestic and wild felids. Despite the clinical importance of feline retroviruses and the growing interest in cats as pets, information about FeLV and FIV in Malaysia is presently insufficient to properly advise veterinarians and pet owners. A cross-sectional study was carried out from January 2010 to December 2010 to determine the prevalence and risk factors associated with FeLV and FIV among domestic cats in peninsular Malaysia. Plasma samples were harvested from the blood of 368 domestic cats and screened for evidence of FeLV p27 antigen and FIV antibodies, using an immunochromatographic kit. Additionally, data on cat demographics and health were collected using a structured questionnaire, and were evaluated as potential risk factors for FeLV or FIV status. RESULTS: Of the 368 cats that were evaluated in this study, 12.2% (45/368; 95% CI = 8.88 - 15.58) were positive for FeLV p27 antigen, 31.3%, (115/368; 95% CI = 26.51 - 35.99) were seropositive to FIV antibodies, and 4.3% (16/368; 95% CI = 2.27 - 6.43) had evidence of both viruses. Factors found to significantly increase the risk for FeLV seropositivity include sex, age, behaviour, sickness, and living in a multi-cat household. Seropositive response to FIV was significantly associated with sex, neuter status, age, behaviour, and health status. CONCLUSIONS: The present study indicates that FeLV and FIV are common among domestic cats in peninsular Malaysia, and that factors related to cat demographics and health such as age, sex, behaviour, health status and type of household are important predictors for seropositive status to FeLV or FIV in peninsular Malaysia. High prevalence of FeLV or FIV observed in our study is of concern, in view of the immunosuppressive potentials of the two pathogens. Specific measures for control and prevention such as screening and routine vaccination are needed to ensure that FeLV and FIV are controlled in the cat population of peninsular Malaysia.

Prevalence and Antimicrobial Resistance of Escherichia coli, Salmonella and Vibrio Derived from Farm-Raised Red Hybrid Tilapia (Oreochromis spp.) and Asian Sea Bass (Lates calcarifer, Bloch 1970) on the West Coast of Peninsular Malaysia.

Antibiotics are widely used in intensive fish farming, which in turn increases the emergence of antimicrobial-resistant (AMR) bacteria in the aquatic environment. The current study investigates the prevalence and determines the antimicrobial susceptibility of E. coli, Salmonella, and Vibrio in farmed fishes on the west coast of Peninsular Malaysia. Over a period of 12 months, 32 aquaculture farms from the Malaysian states of Selangor, Negeri Sembilan, Melaka, and Perak were sampled. Both E. coli and Salmonella were highly resistant to erythromycin, ampicillin, tetracycline, and trimethoprim, while Vibrio was highly resistant to ampicillin and streptomycin. Resistance to the antibiotics listed as the highest priority and critically important for human therapy, such as colistin in E. coli (18.1%) and Salmonella (20%) in fish, is a growing public health concern. The multi-drug resistance (MDR) levels of E. coli and Salmonella in tilapia were 46.5% and 77.8%, respectively. Meanwhile, the MDR levels of E. coli, Salmonella, V. parahaemolyticus, V. vulnificus and V. cholerae in Asian seabass were 34%, 100%, 21.6%, 8.3% and 16.7%, respectively. Our findings provide much-needed information on AMR in aquaculture settings that can be used to tailor better strategies for the use of antibiotics in aquaculture production at the local and regional levels.

Pathogenicity of Salmonella enterica subspecies enterica serovar Enteritidis phage type 1 in one-day-old specific pathogen-free chicks.

Background: The studies about Salmonella infection in newly hatched chicks were not extensive. Aim: The objective of this study was to determine the pathogenicity of Salmonella enterica subspecies enterica serovar Enteritidis (SE) phage type (PT) 1 in one-day-old specific pathogen-free (SPF) chicks. Methods: Seventy, one-day-old SPF chicks, were divided into SE group (30 chicks), mortality group (10 chicks), both orally inoculated (1.0 ml) with SE PT1 (1 × 108 colony-forming unit per 1.0 ml), and one control group (30 chicks). The chicks were sacrificed at 6 and 12 hours, and days 1, 2, 3, 5, 7, 10, 14, and 21 post-inoculation (pi). Samples were collected for bacterial isolation, histological examination, and ultrastructural examination. Results: Starting from day 2 pi, the body weight in the SE group significantly (p < 0.05) decreased. The SE isolation percentages from the liver, spleen, mid-intestinal content, cecal content, cecal tonsil, blood, and cloacal swab were 0.73, 0.77, 0.33, 0.33, 0.36, 0.40, and 0.30, respectively. The isolation percentage in the liver was significantly (p < 0.05) higher than the blood and cloacal swab. The villi heights and crypt depths in the SE group were significantly (p < 0.05) greater and smaller, respectively. Ultrastructurally, erosion and necrosis were observed in the microvilli of the cecal tonsil. The bacteria were engulfed by macrophages at the interepithelial clefts of the M-like M cells. Conclusion: It was concluded that the inoculation of SE PT 1 in one-day-old chicks caused a systemic infection with diarrhea, a decrease in the body weight and villi height in the duodenum, jejunum, and ileum, and high bacterial loading in the liver with mild gross and histological lesions of organs, erosion, and necrosis of microvilli and low mortality. The bacteria entered the body system from the intestinal tract through the interepithelial clefts of the M-like M cells of the cecal tonsil.