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.

Chromogenic in situ hybridization technique for detecting porcine circovirus 3 in lung and lymphoid tissues.

Background and Aim: Porcine circovirus 3 (PCV3) was recently reported in Malaysian commercial pig population in 2020 by conventional polymerase chain reaction (PCR), revealing a molecular prevalence of 17.02% in the sampled domestic pig population. This study aims to describe a chromogenic in situ hybridization (ISH) technique using digoxigenin (DIG)-labeled cloned PCV3 open reading frame 1 (ORF1) fragment DNA to detect and localize the PCV3 antigen in formalin-fixed, paraffin-embedded lung, and lymphoid tissue specimens. Materials and Methods: Since PCV3 was mainly detected in lung and lymphoid tissues, we obtained tissue specimens from these organs from the previous Malaysian PCV3 study. Digoxigenin-labeled ISH probes were designed to target a 69 bp region of PCV3 ORF1 spanning from the nucleotide positions (282-350). Results: Light microscopy analysis revealed that chromogenic staining of PCV3 antigens was visualized within the cytoplasm of pneumocytes and lymphocytes, indicating positive ISH results. The results of molecular detection of PCV3 using PCR and ISH showed a high agreement of 90.91%, including for the negative PCV3 status for all samples. Conclusion: This study reports a chromogenic ISH technique using DIG-labeled probes targeting PCV3 ORF1 to detect PCV3 antigens in lung and lymphoid tissues. Despite the limited availability of PCV3 antibodies, ISH remains relevant for investigating PCV3 replication and pathogenesis and can be used complementarily with PCR for evaluating the localization of antigens in infected tissues.

Serological and molecular surveillance of West Nile virus in domesticated mammals of peninsular Malaysia.

West Nile virus is a mosquito-borne neurotropic pathogen with a wide host range that constitutes a significant risk to public and animal health. There is limited information on WNV infection in domesticated mammals in Malaysia; however, current reports indicate infections in birds, macaques, bats and pigs from Malaysia. In this study, 203 serum samples from cattle, goats, and horses were tested for the presence of anti-WNV IgG using a competitive enzyme-linked immunosorbent assay (c-ELISA). Additionally, using one-step RT-PCR, nasopharyngeal swabs were analyzed for WNV RNA from all 203 animals in this study. The WNV seroprevalence was 32.53% (27/83) at 95% CI (0.2342-0.4319) in cattle, 48.27% (14/29) at 95% CI (0.3139-0.6557) in goats and 53.84% (49/91) at 95% CI (0.4366-0.6373) in horses. Cross-reactive JEV antibodies were detected in two cattle and 34 horses. None of the cattle or goats tested positive for WNV RT-PCR. Seven horses were positive for WNV RT-PCR, a molecular prevalence of 7.69% (7/91) at 95% CI (0.0353-0.1528). This is the first reported detection of WNV in domesticated mammals of Malaysia, a significant addition to the growing evidence that WNV is being transmitted from vectors to susceptible hosts in Malaysia.

Molecular Characterization and Phylogenetic Analysis of Outer membrane protein P2 (OmpP2) of Glaesserella (Haemophilus) parasuis Isolates in Central State of Peninsular Malaysia.

Glaesserella (Haemophilus) parasuis, the etiological agent of Glässer's disease, is an economically significant pathogen commonly associated with serofibrinous polyserositis, arthritis, fibrinous bronchopneumonia and/or meningitis. This study is the first attempt to molecularly characterize and provide a detailed overview of the genetic variants of G. parasuis present in Malaysia, in reference to its serotype, virulence-associated trimeric autotransporters (vtaA) gene and outer membrane protein P2 (OmpP2) gene. The G. parasuis isolates (n = 11) from clinically sick field samples collected from two major pig producing states (Selangor and Perak) were selected for analysis. Upon multiplex PCR, the majority of the isolates (eight out of 11) were identified to be serotype 5 or 12, and interestingly, serotypes 3, 8 and 15 were also detected, which had never been reported in Malaysia prior to this. Generally, virulent vtaA was detected for all isolates, except for one, which displayed a nonvirulent vtaA. A phylogenetic analysis of the OmpP2 gene revealed that the majority of Malaysian isolates were clustered into genotype 1, which could be further divided into Ia and Ib, while only one isolate was clustered into genotype 2.

Genotype Shift of Malaysian Porcine Circovirus 2 (PCV2) from PCV2b to PCV2d within a Decade.

This paper aims to update the molecular status of porcine circovirus 2 (PCV2) in Malaysia. Firstly, the molecular detection rate of PCV2 in farm and sampled pig population were reported to be 83.78% (31/37 farms) and 83.54% (66/79 pigs) positive for PCV2, respectively. PCV2 was detected across all age groups, from fetuses, porkers to sows. Co-detection of PCV2 and PCV3 antigens was also reported at a rate of 28.77% (21/73). Secondly, PCV2 antigen was also detected in Malaysian abattoir lung samples: 18 out of 19 (94.74%) samples originating from clinically healthy finishers were tested positive. Further, this is the first study to confirm the circulation of PCV2 in the wild boar population roaming Peninsular Malaysia, where 28 out of 28 (100%) wild boar lung samples were found positive. One decade earlier, only genotype PCV2b was reported in Malaysia. This most recent update revealed that genotypes PCV2a, PCV2b and PCV2d were present, with PCV2d being the predominant circulating genotype. PCV2 cap gene nucleotide sequences in this study were found to be under negative selection pressure, with an estimated substitution rate of 1.102 × 10-3 substitutions/site/year (ssy).

Mental Model of Malaysian Pig Farmers in Implementing Disease Prevention and Control Practices.

The 1998 Nipah virus outbreak in Malaysia resulted in major financial losses to the multi-million-dollar swine industry. While productivity and biosecurity of pig farms have improved since, biosecurity in some farms remains substandard with farmers struggling to adapt to current national pig farming policies. Farm viability and animal health depends on farmers' role as decision-makers in managing disease threats and other aspects of farm management. This study aimed to describe the mental model of farmers in making decisions about disease prevention and control measures during the 1998 Nipah virus outbreak, and in 2019, 20 years after the last reported Nipah case. Using a qualitative approach, in-depth, semi-structured interviews with 12 pig farmers (mostly small-scale or medium-scale farms) were conducted in three states in Malaysia. Data were analyzed via inductive content analysis. Thirty-six unique dimensions in the mental model were identified, representing six interrelated themes corresponding to participants' decision making related to disease prevention and control: drivers of action to prevent and control disease; perception of practice options; individual determinants of familiar practices; external social factors; external economic factors; and additional external factors. Key drivers of disease control and prevention responses during the Nipah outbreak included heightened perception of risk, emotions, perceived economic loss, and subjective norms whereas key drivers in 2019 included perception of risk, perceived effectiveness, perceived benefits, and other dimensions such as perception of the future, perceived economic cost, barriers, and loss. An unfavorable future outlook, perceived economic factors, and socio-political and personal factors currently hinders farm improvement and adoption of Pig Farming Areas (PFAs) and Modern Pig Farming (MPF) systems. Private sector service providers and veterinarians are highly influential in advocating for good biosecurity, herd health, and animal health intervention practices. Insights gained can inform the development of strategic policies and interventions.

Mycobacterium Tuberculosis and Avium Complex Investigation among Malaysian Free-Ranging Wild Boar and Wild Macaques at Wildlife-Livestock-Human Interface.

Wild animals are considered reservoirs, contributing to the transmission of emerging zoonotic diseases such as tuberculosis (TB). A cross-sectional study was conducted by opportunistic sampling from fresh carcasses of free-ranging wild boar (n = 30), and free-ranging wild macaques (n = 42). Stained smears from these tissues were tested for acid-fast bacilli (AFB) with Ziehl-Neelsen staining. Mycobacterial culture was conducted using Lowenstein-Jensen media and Middlebrook 7H11 agar media. Polymerase chain reaction (PCR) was performed through the detection of the 16S rRNA gene, with multiple sets of primers for the detection of Mycobacterium tuberculosis complex (MTBC) and Mycobacterium avium complex (MAC). In wild boars, 30% (9/30; 95% Confidence Interval: 16.7-47.9%) of examined samples showed gross tuberculosis-like lesions (TBLLs). Multiple nodular lesions that were necrotic/miliary with cavitation were found in the submandibular lymph nodes, tonsils, lungs, kidney and liver, while single nodular lesions were found in the mediastinal lymph nodes, spleen and mesenteric lymph nodes. Conventional PCR on the submandibular lymphoid tissues of wild boar (nine samples with TBLLs and three non-TBLL samples) showed that 75% (9/12) were positive for Mycobacterium bovis (95% CI: 46.8-91.1), and 91% (CI: 64.6-98.5) were positive for Mycobacterium avium. For macaques, 33.3% (10/30) were positive for M. avium (95% CI: 19.2-51.2) but negative for MTBC.

Detection of Mycobacterium tuberculosis complex antibodies in free-ranged wild boar and wild macaques in selected districts in Selangor and reevaluation of tuberculosis serodetection in captive Asian elephants in Pahang, Peninsular Malaysia.

Tuberculosis (TB) is a chronic inflammatory and zoonotic disease caused by Mycobacterium tuberculosis complex (MTBC) members, affecting several domestic animals, wildlife species and humans. The preliminary investigation was aimed to detect antibody against MTBC among indigenous wildlife which are free-ranged wild boar, free-ranged wild macaques and captive Asian elephants in selected areas of Selangor and elephant conservation centre in Pahang, respectively. The results indicate that MTBC serodetection rate in wild boar was 16.7% (7.3-33.5 at 95% confidence interval (CI)) using an in-house ELISA bPPD IgG and 10% (3.5-25.6 at 95% CI) by DPP®VetTB assay, while the wild macaques and Asian elephant were seronegative. The univariate analysis indicates no statistically significant difference in risk factors for sex and age of wild boar but there was a significant positive correlation (P<0.05) between bovine TB in dairy cattle and wild boar seropositivity in the Sepang district.

Animal Coronavirus Diseases: Parallels with COVID-19 in Humans.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel coronavirus in humans, has expanded globally over the past year. COVID-19 remains an important subject of intensive research owing to its huge impact on economic and public health globally. Based on historical archives, the first coronavirus-related disease recorded was possibly animal-related, a case of feline infectious peritonitis described as early as 1912. Despite over a century of documented coronaviruses in animals, the global animal industry still suffers from outbreaks. Knowledge and experience handling animal coronaviruses provide a valuable tool to complement our understanding of the ongoing COVID-19 pandemic. In this review, we present an overview of coronaviruses, clinical signs, COVID-19 in animals, genome organization and recombination, immunopathogenesis, transmission, viral shedding, diagnosis, treatment, and prevention. By drawing parallels between COVID-19 in animals and humans, we provide perspectives on the pathophysiological mechanisms by which coronaviruses cause diseases in both animals and humans, providing a critical basis for the development of effective vaccines and therapeutics against these deadly viruses.

What do we know about porcine circovirus 3 (PCV3) diagnosis so far?: A review.

Porcine circovirus 3 (PCV3) was first discovered in 2016, almost concomitantly by two groups of researchers in the United States. The novel case was reported in a group of sows with chronic reproductive problems with clinical presentation alike porcine dermatitis and nephropathy syndrome (PDNS), where metagenomic sequencing revealed a genetically divergent porcine circovirus designated PCV3. The discovery of PCV3 in a PDNS case, which used to be considered as part of PCVAD attributed to PCV2 (porcine circovirus 2), has garnered attention and effort in further research of the novel virus. Just when an infectious molecular DNA clone of PCV3 has been developed and successfully used in an in vivo pathogenicity study, yet another novel PCV strain surfaced, designated PCV4 (porcine circovirus 4). So far, PCV3 has been reported in domestic swine population globally at low to moderate prevalence, from almost all sample types including organ tissues, faecal, semen and colostrum samples. PCV3 has been associated with a myriad of clinical presentations, from PDNS to porcine respiratory disease complex (PRDC). This review paper summarizes the studies on PCV3 to date, with focus on diagnosis.

Mycobacterium tuberculosis complex in wildlife: Review of current applications of antemortem and postmortem diagnosis.

Tuberculosis (TB) is a chronic inflammatory and zoonotic disease caused by Mycobacterium tuberculosis complex (MTBC) members, which affects various domestic animals, wildlife, and humans. Some wild animals serve as reservoir hosts in the transmission and epidemiology of the disease. Therefore, the monitoring and surveillance of both wild and domestic hosts are critical for prevention and control strategies. For TB diagnosis, the single intradermal tuberculin test or the single comparative intradermal tuberculin test, and the gamma-interferon test, which is regarded as an ancillary test, are used. Postmortem examination can identify granulomatous lesions compatible with a diagnosis of TB. In contrast, smears of the lesions can be stained for acid-fast bacilli, and samples of the affected organs can be subjected to histopathological analyses. Culture is the gold standard test for isolating mycobacterial bacilli because it has high sensitivity and specificity compared with other methods. Serology for antibody detection allows the testing of many samples simply, rapidly, and inexpensively, and the protocol can be standardized in different laboratories. Molecular biological analyses are also applicable to trace the epidemiology of the disease. In conclusion, reviewing the various techniques used in MTBC diagnosis can help establish guidelines for researchers when choosing a particular diagnostic method depending on the situation at hand, be it disease outbreaks in wildlife or for epidemiological studies. This is because a good understanding of various diagnostic techniques will aid in monitoring and managing emerging pandemic threats of infectious diseases from wildlife and also preventing the potential spread of zoonotic TB to livestock and humans. This review aimed to provide up-to-date information on different techniques used for diagnosing TB at the interfaces between wildlife, livestock, and humans.

First molecular detection and complete sequence analysis of porcine circovirus type 3 (PCV3) in Peninsular Malaysia.

Porcine circovirus type 3 (PCV3) is a newly emerging virus in the swine industry, first reported recently in 2016. PCV3 assembles into a 2000 bp circular genome; slightly larger than PCV1 (1758-1760 bp), PCV2 (1766-1769 bp) and PCV4 (1770 bp). Apart from being associated with porcine dermatitis and nephropathy syndrome (PDNS), PCV3 has been isolated from pigs with clinical signs of reproductive failures, myocarditis, porcine respiratory disease complex (PRDC) and neurologic disease. Given that PCV3 is increasingly reported in countries including Thailand and U.S. with whom Malaysia shares trade and geographical relationship; and that PCV3 is associated with several clinical presentations that affect productivity, there is a need to study the presence and molecular characteristics of PCV3 in Malaysian swine farms. Twenty-four commercial swine farms, three abattoirs and retail shops in Peninsular Malaysia were sampled using convenience sampling method. A total of 281 samples from 141 pigs, including 49 lung archive samples were tested for PCV3 by conventional PCR. Twenty-eight lung samples from wild boar population in Peninsular Malaysia were also included. Nucleotide sequences were analyzed for maximum likelihood phylogeny relationship and pairwise distances. Results revealed that PCV3 is present in Peninsular Malaysia at a molecular prevalence of 17.02%, with inguinal lymph nodes and lungs showing the highest molecular detection rates of 81.82% and 71.43% respectively. Despite wide reports of PCV3 in healthy animals and wild boars, no positive samples were detected in clinically healthy finishers and wild boar population of this study. PCV3 strain A1 and A2 were present in Malaysia, and Malaysian PCV3 strains were found to be phylogenetically related to Spanish, U.S. and Mexico strains.

Exploring the Mental Model of Cattle Farmers in Disease Prevention and Control Practices.

Farmers play an integral role in minimizing disease threats and managing ongoing diseases on their farms. Various environmental factors influence the decision-making processes of farmers. Deciphering the mental models of farmers allows us to understand the motivations and reasons behind disease prevention and control choices. This study aimed to explore the mental models of cattle farmers in implementing disease prevention and control practices. Using qualitative in-depth, semi-structured interviews, seven cattle farmers from a university's foster farm extension program were sampled. Interview transcripts were analyzed using inductive content analysis. Results revealed 23 dimensions comprising the mental model of cattle farmers. The dimensions were conceptualized under four major themes. Farmers were most influenced by perceived risk of disease, perceived effectiveness and benefits of disease prevention and control practices, experience, knowledge and emotions, subjective norms and perceived economic loss. The decision-making processes of farmers are complex and are influenced by various factors. While additional research is needed to confirm the findings using quantitative methods and larger sample sizes, insights gained from the study can be used as inputs to tailor communication and training strategies for improved disease prevention and control interventions.

Molecular epidemiology of Clostridium difficile isolated from piglets.

Information on the epidemiology of C. difficile infection (CDI) in South-East Asian countries is limited, as is data on possible animal reservoirs of C. difficile in the region. We investigated the prevalence and molecular epidemiology of C. difficile in piglets and the piggery environment in Thailand and Malaysia. Piglet rectal swabs (n = 224) and piggery environmental specimens (n = 23) were collected between 2015 and 2016 from 11 farms located in Thailand and Malaysia. All specimens were tested for the presence of C. difficile with toxigenic culture. PCR assays were performed on isolates to determine the ribotype (RT), and the presence of toxin genes. Whole genome sequencing was used on a subset of isolates to determine the evolutionary relatedness of RT038 (the most prevalent RT identified) common to pigs and humans from Thailand and Indonesia. C. difficile was recovered from 35% (58/165) and 92% (54/59) of the piglets, and 89% (8/9) and 93% (13/14) of the environmental specimens from Thailand and Malaysia, respectively. All strains from Thailand, and 30 strains from Malaysia (23 piglet and 7 environmental isolates) were non-toxigenic. To our knowledge, this is the first and only report with a complete lack of toxigenic C. difficile among piglets, a feature which could have a protective effect on the host. The most common strain belonged to RT038 (ST48), accounting for 88% (51/58) of piglet and 78% (7/9) of environmental isolates from Thailand, and all 30 isolates tested from Malaysia. Piglet RT038 isolates from Thailand and Malaysia differed by only 18 core-genome single nucleotide variants (cgSNVs) and both were, on average, 30 cgSNVs different from the human strains from Thailand and Indonesia, indicating a common ancestor in the last two decades.

Molecular Characterization of Multidrug-Resistant and Extended-Spectrum Beta-Lactamase-Producing Klebsiella pneumoniae Isolated from Swine Farms in Malaysia.

Aims: The high prevalence of multidrug resistance (MDR) and extended-spectrum ß-lactamase (ESBL)-producing Klebsiella pneumoniae associated with nosocomial infections has caused serious therapeutic challenges. The objectives of this study were to determine the genotypic and phenotypic characteristics of K. pneumoniae strains isolated from Malaysian swine farms and the transferability of ESBL genes by plasmids. Results: A total of 50 K. pneumoniae strains were isolated from 389 samples, which were collected from healthy and unhealthy pigs (swine rectum and oral cavities), healthy farmers (human rectum, urine, and nasal cavities), farm's environment, and animal feeds from seven Malaysian swine farms. Antimicrobial susceptibility analysis of these 50 K. pneumoniae strains showed that the majority (86%) were resistant to tetracycline, while 44% and 36% of these strains were MDR and ESBL producers, respectively. PCR and DNA sequencing of the amplicons showed the occurrence of blaTEM (15/18), blaSHV (15/18), blaCTX-M-1 group (7/18), and blaCTX-M-2 group (2/18), while only class 1 integron-encoded integrase was detected. Conjugation experiments and plasmid analysis indicated that the majority of the ESBL genes were plasmid encoded and the plasmids in 11 strains were conjugative. Genotyping by pulsed-field gel electrophoresis and repetitive extragenic palindrome-polymerase chain reaction (REP-PCR) showed that these 50 strains were genetically diverse with 44 pulsotypes and 43 REP-PCR subtypes. Conclusions: ESBL-producing K. pneumoniae strains showed high resistance to tetracycline as this antibiotic is used for prophylaxis and therapeutic purposes at the swine farms. The findings in this study have drawn attention to the issue of increasing MDR in animal husbandry and it should be taken seriously to prevent the spread and treatment failure due to antimicrobial resistance.

The emergence of colistin-resistant Klebsiella pneumoniae strains from swine in Malaysia.

OBJECTIVE: Colistin is the last line of therapy for infections caused by multidrug-resistant Gram-negative bacteria. The objective of this study was to determine the phenotypic and genotypic characteristics of colistin-resistant Klebsiella pneumoniae (K. pneumoniae) isolated from swine samples in Malaysia. METHODS: A total of 46 swine K. pneumoniae strains isolated from 2013-2015 in Malaysia were analysed for the production of extended-spectrum ß-lactamases and carbapenemase. The resistance traits and genetic diversity of these strains were characterised by polymerase chain reaction, conjugation, plasmid analysis, and pulsed-field gel electrophoresis. RESULTS: Nineteen of 46 strains were multidrug resistant while 13 were resistant to colistin. The majority of colistin-resistant strains harboured blaTEM gene (92.3%), followed by blaSHV (69.23%), blaCTXM-1 (38.46%), and blaMCR-1 (23.08%). All three colistin-resistant strains had transferable plasmids and the colistin resistance gene blaMCR-1. Genotyping by pulsed-field gel electrophoresis showed high genetic diversity among the K. pneumoniae and that the colistin-resistant K. pneumoniae strains were heterogenous. CONCLUSION: It is believed that this is the first report of colistin-resistant K. pneumoniae among swine strains associated with mcr-1 plasmid in Malaysia. Due to the emergence of ß-lactam, carbapenem and colistin resistance, the use of colistin in animal husbandry and agriculture should be avoided to prevent treatment failure.

Chicken feather valorization by thermal alkaline pretreatment followed by enzymatic hydrolysis for protein-rich hydrolysate production.

A huge amount of feathers is generated as a waste every year. Feathers can be a protein source if it is treated with an appropriate method. The present study investigates feasibility of autoclave alkaline and microwave alkaline pretreatments to be combined with enzymatic treatment for feather solubilization and protein production. Hydrolysis of chicken feather by autoclave alkaline pretreatment followed by an enzymatic method (AAS) or microwave alkaline pretreatment followed by an enzymatic method (MAS) was optimized by response surface methodology. Various NaOH concentrations for autoclave alkaline pretreatment (0.01-0.1 M) and microwave-alkaline pretreatment (0.01-0.05 M) were applied. The holding time for both pretreatments ranged from 1 to 10 min. The pretreated feathers were subjected to enzymatic hydrolysis using a commercial enzyme prior to analysis of protein content, feather solubilization, functional groups, and elemental composition (carbon, hydrogen, nitrogen and sulfur) of the treated feathers. The results revealed that both autoclave alkaline pretreatment and microwave alkaline pretreatment under optimized conditions of 0.068 M NaOH, 2 min holding time, 105 °C and 450 W, 0.05 M NaOH for 10 min, respectively, enhanced the subsequent Savinase hydrolysis of chicken feathers to achieve more than 80% degradation and more than 70% protein recovery. Fourier transform infrared spectroscopy results showed that both thermal-alkaline pretreatments weakened the structure of the feather. Reduction of carbon, nitrogen, and sulfur occurred in both thermal-alkaline pretreatments of feathers indicating degradation of the feather as well as protein release. Thermal-alkaline pretreatment may be a promising method for enhancing the enzymatic hydrolysis of chicken feathers and for producing a protein-rich hydrolysate.

Occurrence of virulent multidrug-resistant Enterococcus faecalis and Enterococcus faecium in the pigs, farmers and farm environments in Malaysia.

BACKGROUND: Enterococcus faecalis and Enterococcus faecium are ubiquitous opportunistic pathogens found in the guts of humans and farmed animals. This study aimed to determine the occurrence, antimicrobial resistance, virulence, biofilm-forming ability and genotypes of E. faecalis and E. faecium from swine farms. Correlations between the genotypes, virulotypes, antibiotic resistance, and the environmental factors such as locality of farms and farm hygiene practice were explored. METHODS: E. faecalis and E. faecium strains were isolated from the oral, rectal and fecal samples of 140 pigs; nasal, urine and fecal samples of 34 farmers working in the farms and 42 environmental samples collected from seven swine farms located in Peninsular Malaysia. Antibiotic susceptibility test was performed using the disk diffusion method, and the antibiotic resistance and virulence genes were detected by Polymerase Chain Reaction. Repetitive Extragenic Palindromic-Polymerase Chain Reaction and Pulsed-Field Gel Electrophoresis were performed to determine the clonality of the strains. Crosstab/Chi-square test and DistLM statistical analyses methods were used to determine the correlations between the genotypes, virulence factors, antibiotic resistance, and the environmental factors. RESULTS: A total of 211 E. faecalis and 42 E. faecium were recovered from 140 pigs, 34 farmers and 42 environmental samples collected from seven swine farms in Peninsular Malaysia. Ninety-eight percent of the strains were multidrug-resistant (resistant to chloramphenicol, tetracycline, ciprofloxacin and erythromycin). Fifty-two percent of the strains formed biofilms. Virulence genes efa, asaI, gelE, esp, cyl and ace genes were detected. Virulence genes efa and asaI were most prevalent in E. faecalis (90%) and E. faecium (43%), respectively. Cluster analyses based on REP-PCR and PFGE showed the strains were genetically diverse. Overall, the strains isolated from pigs and farmers were distinct, except for three highly similar strains found in pigs and farmers. The strains were regional- and host-specific. DISCUSSION: This study revealed alarming high frequencies of multidrug-resistant enterococci in pigs and swine farmers. The presence of resistance and virulence genes and the ability to form biofilm further enhance the persistence and pathogenicity of the strains. Although the overall clonality of the strains were regionals and host-specific, strains with high similarity were found in different hosts. This study reiterates a need of a more stringent regulation to ensure the proper use of antibiotics in swine husbandry to reduce the wide spread of multidrug-resistant strains.

First molecular detection of porcine bocavirus in Malaysia.

Several strains of porcine bocaviruses have been reported worldwide since their first detection in Sweden in 2009. Subsequently, the virus has been reported to be associated with gastrointestinal and respiratory signs in weaner and grower pigs. Although Malaysia is host to a self-sufficient swine livestock industry, there is no study that describes porcine bocavirus in the country. This report is the first to describe porcine bocavirus (PBoV) in Malaysian swine herds. PBoV was identified in various tissues from sick and runt pigs using the conventional PCR method with primers targeting conserved regions encoding for the nonstructural protein (NS1) gene. Out of 103 samples tested from 17 pigs, 32 samples from 15 pigs were positive for porcine bocavirus. In addition, a higher detection rate was identified from mesenteric lymph nodes (52.9%), followed by tonsil (37.0%), and lungs (33.3%). Pairwise comparison and phylogenetic analyses based on a 658-bp fragment of NS1 gene revealed that the Malaysian PBoV strains are highly similar to PBoV3 isolated in Minnesota, USA. The presence of porcine bocavirus in Malaysia and their phylogenetic bond was marked for the first time by this study. Further studies will establish the molecular epidemiology of PBoV in Malaysia and clarify pathogenicity of the local isolates.