Methicillin-resistant Staphylococcus aureus (MRSA) of sequence type 398 (ST398) has frequently been detected in pigs and pig handlers. However, in Malaysia, sampling 360 pigs and 90 pig handlers from 30 farms identified novel ST9-spa type t4358-staphylococcal cassette chromosome mec type V MRSA strains that were found to transiently colonize more than 1% of pigs and 5.5% of pig handlers.
In some laboratories, mosquitoes' direct blood-feeding on live animals has been replaced with various membrane blood-feeding systems. The selection of blood meal sources used in membrane feeding is crucial in vector mass rearing as it influences the mosquitoes' development and reproductive fitness. Therefore, this scoping review aimed to evaluate the existing literature on the use of different blood sources and components in artificial membrane feeding systems and their effects on blood-feeding and the fecundity rate of Ae. aegypti. A literature review search was conducted by using PubMed, Scopus, and Web of Science databases according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA-ScR). The EndNote version 20 software was used to import all searched articles. Relevant information was retrieved for analysis into a Microsoft Excel Spreadsheet. A total of 104 full-text articles were assessed for eligibility criteria, whereby the articles should include the comparison between different types of blood source by using the membrane feeding systems. Only 16 articles were finally included in the analysis. Several studies had reported that human blood was superior in blood-feeding Ae. aegypti as compared to sheep blood which resulted in lower fecundity due to accumulation of free fatty acids (FFA) in the cuticles. In contrast, cattle whole blood and pig whole blood showed no significant differences in the blood-feeding and fecundity rate as compared to human blood. This review also indicated that bovine whole blood and pig whole blood enhanced Ae. aegypti's vitellogenesis and egg production as compared to plasma and blood cells. In addition, human blood of up to 10 days after the expiration date could still be used to establish Ae. aegypti colonies with good blood-feeding rates and number of eggs produced. Thus, future studies must consider the importance of selecting suitable blood sources and components for membrane blood feeding especially in mosquito colonisation and control measure studies.
In vitro blood-brain barrier (BBB) models from primary brain endothelial cells can closely resemble the in vivo BBB, offering valuable models to assay BBB functions and to screen potential central nervous system drugs. We have recently developed an in vitro BBB model using primary porcine brain endothelial cells. The model shows expression of tight junction proteins and high transendothelial electrical resistance, evidence for a restrictive paracellular pathway. Validation studies using small drug-like compounds demonstrated functional uptake and efflux transporters, showing the suitability of the model to assay drug permeability. However, one limitation of in vitro model permeability measurement is the presence of the aqueous boundary layer (ABL) resulting from inefficient stirring during the permeability assay. The ABL can be a rate-limiting step in permeation, particularly for lipophilic compounds, causing underestimation of the permeability. If the ABL effect is ignored, the permeability measured in vitro will not reflect the permeability in vivo. To address the issue, we explored the combination of in vitro permeability measurement using our porcine model with the pKa(FLUX) method in pCEL-X software to correct for the ABL effect and allow a detailed analysis of in vitro (transendothelial) permeability data, Papp. Published Papp using porcine models generated by our group and other groups are also analyzed. From the Papp, intrinsic transcellular permeability (P0) is derived by simultaneous refinement using a weighted nonlinear regression, taking into account permeability through the ABL, paracellular permeability and filter restrictions on permeation. The in vitro P0 derived for 22 compounds (35 measurements) showed good correlation with P0 derived from in situ brain perfusion data (r(2)=0.61). The analysis also gave evidence for carrier-mediated uptake of naloxone, propranolol and vinblastine. The combination of the in vitro porcine model and the software analysis provides a useful tool to better predict BBB permeability in vivo and gain better mechanistic information about BBB permeation.
In recent years, zoonotic RNA viruses such as Nipah, SARS coronavirus, avian influenza (H5N1) and Chikungunya have emerged with global impact. The latest has now been designated by World Health Organization (WHO) as pandemic (H1N1) 2009 virus. It was first reported as an outbreak in Mexico in April, and has now caused the first influenza pandemic since 1968. By July 11, 2009, there were 105,304 confirmed cases and 463 deaths in 143 countries, including 627 cases in Malaysia1 . The rapid spread of the disease has been matched by the speed of dissemination of information and protocols, co-ordinated by WHO. The experiences of SARS and H5N1 have been enormously beneficial in preparing the world for a pandemic.
The Nipah virus outbreak in Malaysia (September 1998 to May 1999) resulted in 265 cases of acute encephalitis with 105 deaths, and near collapse of the billion-dollar pig-farming industry. Because it was initially attributed to Japanese encephalitis, early control measures were ineffective, and the outbreak spread to other parts of Malaysia and nearby Singapore. The isolation of the novel aetiological agent, the Nipah virus (NiV), from the cerebrospinal fluid of an outbreak victim was the turning point which led to outbreak control 2 months later. Together with the Hendra virus, NiV is now recognised as a new genus, Henipavirus (Hendra + Nipah), in the Paramyxoviridae family. Efforts of the local and international scientific community have since elucidated the epidemiology, clinico-pathophysiology and pathogenesis of this new disease. Humans contracted the infection from close contact with infected pigs, and formed the basis for pig-culling that eventually stopped the outbreak. NiV targeted medium-sized and small blood vessels resulting in endothelial multinucleated syncytia and fibrinoid necrosis. Autopsies revealed disseminated cerebral microinfarctions resulting from vasculitis-induced thrombosis and direct neuronal involvement. The discovery of NiV in the urine and saliva of Malaysian Island flying foxes (Pteropus hypomelanus and Petropus vampyrus) implicated these as natural reservoir hosts of NiV. It is probable that initial transmission of NiV from bats to pigs occurred in late 1997/early 1998 through contamination of pig swill by bat excretions, as a result of migration of these forest fruitbats to cultivated orchards and pig-farms, driven by fruiting failure of forest trees during the El Nino-related drought and anthropogenic fires in Indonesia in 1997-1998. This outbreak emphasizes the need for sharing information of any unusual illnesses in animals and humans, an open-minded approach and close collaboration and co-ordination between the medical profession, veterinarians and wildlife specialists in the investigation of such illnesses. Environmental mismanagement (such as deforestation and haze) has far-reaching effects, including encroachment of wildlife into human habitats and the introduction of zoonotic infections into domestic animals and humans.
Nipah virus infection of porcine stable kidney cells (PS), human neuronal cells (SK-N-MC), human lung fibroblasts cells (MRC-5), and human monocytes (THP-1) were examined. Rapid progression of cytopathic effects (CPE) and cell death were noted in PS cell cultures treated with Nipah virus, followed by MRC-5, SK-N-MC, and THP-1 cell cultures, in descending order of rapidity. Significant increase in the intracellular Nipah virus RNA occurred beginning at 24 hr PI in all the infected cells. Whereas, the extracellular release of Nipah virus RNA increased significantly beginning at 48 and 72 hr PI for the infected MRC-5 cells and PS cells, respectively. No significant release of extracellular Nipah virus RNA was detected from the Nipah virus-infected SK-N-MC and THP-1 cells. At its peak, approximately 6.6 log PFU/microl of extracellular Nipah virus RNA was released from the Nipah virus-infected PS cells, with at least a 100-fold less virus RNA was recorded in the Nipah virus-infected SK-N-MC and THP-1. Approximately 15.2% (+/-0.1%) of the released virus from the infected PS cell cultures was infectious in contrast to approximately 5.5% (+/-0.7%) from the infected SK-N-MC cells. The findings suggest that there are no differences in the capacity to support Nipah virus replication between pigs and humans in fully susceptible PS and MRC-5 cells. However, there are differences between these cells and human neuronal cells and monocytes in the ability to support Nipah virus replication and virus release.
Between September 1998 and May 1999, 265 cases of encephalitis were reported from among those involved in pig rearing. A few cases were also reported among abattoir workers. This raised questions of the risk of transmission among those who handled raw pork. A serosurvey was conducted among pork sellers in Seremban town, which is about 20 km from one of the pig rearing areas which had reported cases of encephalitis. It was found that out of the 28 pork sellers tested, only one tested positive for Nipah virus antibodies and that this pork seller also worked in an abattoir in the same district, removing the urinary bladders from slaughtered pigs. Based on these findings, it was concluded that the risk of transmission of the virus from handling raw pork appeared to be low.
A hospital-based case-control study of viral encephalitis was carried out at Port Dickson Hospital, in the state of Negeri Sembilan, Malaysia. Between March and May 1999, 69 clinically diagnosed viral encephalitis cases and 31 controls were interviewed. Job histories on pig farming activities were assessed by a group of epidemiologists and veterinary surgeons. Results show that among clinical cases of viral encephalitis, 52 (75.4%) cases were diagnosed to have Nipah virus infection based on positive serology for antibodies to the cross-reacting Hendra virus antigen. The Nipah virus encephalitis was significantly associated with a history of working in pig farms (p < 0.001, OR = 196.0, 95% CI = 20.4-4741.6), history of contact with animals (p < 0.001, OR = 38.3, 95% CI = 8.2-209.0) and with history of direct contact with pigs (p = 0.002, OR = 34.4, 95% CI = 2.6-1,024.4). The Nipah virus infection was also significantly associated with history of feeding/cleaning pigs (p < 0.001, OR = 102, 95% CI = 11.9-2,271.5). These results provide evidence that involvement in pig farming activities is significantly associated with the risk of getting Nipah virus infection. They are potential risk factors for Nipah virus transmission in the major pig-producing area of Bukit Pelandok, Port Dickson Negeri Sembilan.
A test for assessing pork adulteration in meatballs, using TaqMan probe real-time polymerase chain reaction, was developed. The assay combined porcine-specific primers and TaqMan probe for the detection of a 109 bp fragment of porcine cytochrome b gene. Specificity test with 10 ng DNA of eleven different species yielded a threshold cycle (Ct) of 15.5 ± 0.20 for the pork and negative results for the others. Analysis of beef meatballs with spiked pork showed the assay can determine 100-0.01% contaminated pork with 102% PCR efficiency, high linear regression (r(2) = 0.994) and ≤ 6% relative errors. Residuals analysis revealed a high precision in all determinations. Random analysis of commercial meatballs from pork, beef, chicken, mutton and goat, yielded a Ct between 15.89 ± 0.16 and 16.37 ± 0.22 from pork meatballs and negative results from the others, showing the suitability of the assay to determine pork in commercial meatballs with a high accuracy and precision.
Formalin-fixed, paraffin wax-embedded tissues of three Malaysian farm pigs naturally infected with Nipah virus were used to investigate the value of anti-Nipah virus mouse monoclonal antibodies (Mabs) and rabbit polyclonal antibody for immunohistochemical diagnosis. Mabs 11F6 and 12A5 gave intense immunolabelling in lung tissue that had been fixed in 10% neutral buffered formalin for about 4 years, whereas the reactivity of Mabs 13A5 and 18C4 and polyclonal antibody was reduced significantly by long-term formalin fixation. Immunohistochemical examination of Malaysian farm pig samples with Mab 11F6 confirmed the affinity of Nipah virus for respiratory epithelium, renal glomerular and tubular epithelium, meningeal arachnoidal cells, and systemic vascular endothelium and smooth muscle. In addition, Nipah virus antigens were identified in laryngeal epithelial cells, Schwann cells of peripheral nerve fascicles in the spleen, and endothelial cells in the atrioventricular valve. The study demonstrated the value of Mabs 11F6 and 12A5 for the immunohistochemical diagnosis of Nipah virus infection in pigs.
Antibodies to the protozoan parasite, Toxoplasma gondii were assayed in sera of 200 goats, 100 pigs, 126 cattle from various states of Malaysia, and 135 dogs and 55 cats around Ipoh region using an indirect fluorescence antibody test (IFAT, cut-off titer 1:200); antibodies were found in 35.5% of goats, 14.5% cats, 9.6% dogs, 7.9% local cattle and 4% yellow cattle but not in pigs. Results indicate that infection is most prevalent in goats.
Cattle, buffalo, and porcine materials are widely adulterated, and their quantification might safeguard health, religious, economic, and social sanctity. Recently, conventional polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) assays have been documented but they are just suitable for identification, cannot quantify adulterations. We described here a quantitative tetraplex real-time PCR assay with TaqMan Probes to quantify contributions from cattle, buffalo, and porcine materials simultaneously. Amplicon-sizes were very short (106-, 90-, and 146-bp for cattle, buffalo, and porcine) because longer targets could be broken down, bringing serious ambiguity in molecular diagnostics. False negative detection was eliminated through an endogenous control (141-bp site of eukaryotic 18S rRNA). Analysis of 27 frankfurters and 27 meatballs reflected 84-115% target recovery at 0.1-10% adulterations. Finally, a test of 36 commercial products revealed 71% beef frankfurters, 100% meatballs, and 85% burgers contained buffalo adulteration, but no porcine was found in beef products.
The possible role of pigs as arbovirus maintenance hosts and their importance as amplifier hosts was studied. Blood samples from 464 pigs of all ages collected in 1962 and 1964 were tested against 10 arboviruses. Antibodies to Japanese encephalitis and Getah viruses were particularly prevalent and their calculated monthly infection rates were 19-5% and 13-3% respectively. In 1969, 447 pigs were bled monthly throughout the year and the infection rates for Japanese encephalitis virus were calculated in pigs during the first year of life. Infection rates were not uniform throughout the year; the rate increases as the pig grew older and there was a marked seasonal increase in the infection rate in the period from November to January. This coincided with the seasonal major population peak of Culex tritaeniorhynchus following intense breeding of this mosquito prior to rice planting. It is suggested that, in Sarawak, the pig acts as a maintenance host of Japanese encephalitis in a cycle involving C. gelidus mosquitoes and also acts as an important amplifier host towards the end of the year in a cycle involving C. tritaeniorhynchus. It is further suggested that Getah virus is maintained in a similar cycle between C. tritaeniorhynchus and pigs.
Probiotics have become highly recognized as supplements for humans and animals because of their beneficial effects on health and well-being. The present review aims to provide an overview of different steps through which microbial strains become applicable probiotics in food and/or feed industries. Isolation of potential probiotic strains is the first step. Lactic acid bacteria are the most frequently used microorganisms as probiotics, which can be isolated from human, animal, plant, and environment. The next steps are identification of the isolates and characterization of them based on the main selection criteria for any potential probiotic microorganism, including resistance to gastric acidity and bile salt, adherence to mucus and/or intestinal epithelial cells and cell lines, and antimicrobial and antagonism activity against potentially pathogenic microbes. There are additional probiotic properties that may be considered for selection of probiotic strains with specific effects, such as cholesterol reduction ability, antioxidant activity, or cytotoxic effect against cancer cells. However, a potential probiotic does not need to fulfill all such selection criteria. As the last step, safety status of probiotics for humans is verified by taxonomy clarification, in vitro and in vivo tests, human trials, and genome sequencing.
Beef, buffalo, and pork adulteration in the food chain is an emerging and sensitive issue. Current molecular techniques to authenticate these species depend on polymerase chain reaction (PCR) assays involving long and single targets which break down under natural decomposition and/or processing treatments. This novel multiplex polymerase chain reaction-restriction fragment length polymorphism assay targeted two different gene sites for each of the bovine, buffalo, and porcine materials. This authentication ensured better security, first through a complementation approach because it is highly unlikely that both sites will be missing under compromised states, and second through molecular fingerprints. Mitochondrial cytochrome b and ND5 genes were targeted, and all targets (73, 90, 106, 120, 138, and 146 bp) were stable under extreme boiling and autoclaving treatments. Target specificity and authenticity were ensured through cross-amplification reaction and restriction digestion of PCR products with AluI, EciI, FatI, and CviKI-1 enzymes. A survey of Malaysian frankfurter products revealed rampant substitution of beef with buffalo but purity in porcine materials.
Using manure collected from swine fed with diet containing antibiotics and antibiotic-free swine manure spiked with antibiotics are the two common methods of studying the degradation behavior of veterinary antibiotic in manure in the environment. However, few studies had been conducted to co-compare these two different antibiotic addition methods. This study used oxytetracycline (OTC) as a model antibiotic to study antibiotic degradation behavior in manure under the above two OTC addition methods. In addition, the role of microorganisms present in the manure on degradation behavior was also examined. The results showed that degradation half-life of OTC in manure from swine fed OTC (9.04 days) was significantly shorter than that of the manure directly treated with OTC (9.65 days). Concentration of 4-epi-OTC in manure from swine fed OTC peaked earlier than that in manure spiked with OTC, and the degradation rates of 4-epi-OTC and α-apo-OTC in the manure from swine fed OTC were faster, but the peak concentrations were lower, than those in manure spiked with OTC. Bacterial diversity and relative abundance of Bacillus cereus data demonstrated that sterilization of the manure before experiment significantly decreased OTC degradation rate in both of the addition methods. Results of the present study demonstrated that the presence of the metabolites (especially 4-epi-OTC) and microorganisms had significant influence on OTC degradation.
Nipah virus, a novel paramyxovirus, closely related to Hendra virus emerged in northern part of Peninsular Malaysia in 1998. The virus caused an outbreak of severe febrile encephalitis in humans with a high mortality rate, whereas, in pigs, encephalitis and respiratory diseases but with a relatively low mortality rate. The outbreak subsequently spread to various regions of the country and Singapore in the south due to the movement of infected pigs. Nipah virus caused systemic infections in humans, pigs and other mammals. Histopathological and radiological findings were characteristic of the disease. Fruitbats of Pteropid species were identified as the natural reservoir hosts. Evidence suggested that climatic and anthropogenic driven ecological changes coupled with the location of piggeries in orchard and the design of pigsties allowed the spill-over of this novel paramyxovirus from its reservoir host into the domestic pigs and ultimately to humans and other animals.
A human isolate of Nipah virus from an outbreak of febrile encephalitis in Malaysia that coincided with a field outbreak of disease in pigs was used to infect eight 6-week-old pigs orally or subcutaneously and two cats oronasally. In pigs, the virus induced a respiratory and neurological syndrome consistent with that observed in the Malaysian pigs. Not all the pigs showed clinical signs, but Nipah virus was recovered from the nose and oropharynx of both clinically and sub-clinically infected animals. Natural infection of in-contact pigs, which was readily demonstrated, appeared to be acute and self-limiting. Subclinical infections occurred in both inoculated and in-contact pigs. Respiratory and neurological disease was also produced in the cats, with recovery of virus from urine as well as from the oropharynx. The clinical and pathological syndrome induced by Nipah virus in cats was comparable with that associated with Hendra virus infection in this species, except that in fatal infection with Nipah virus there was extensive inflammation of the respiratory epithelium, associated with the presence of viral antigen. Viral shedding via the nasopharynx, as observed in pigs and cats in the present study, was not a regular feature of earlier reports of experimental Hendra virus infection in cats and horses. The findings indicate the possibility of field transmission of Nipah virus between pigs via respiratory and oropharyngeal secretions.
The primary objective of this study was to investigate the effect of dietary fiber on methanogenic diversity and community composition in the hindgut of indigenous Chinese Lantang gilts to explain the unexpected findings reported earlier that Lantang gilts fed low-fiber diet (LFD) produced more methane than those fed high-fiber diet (HFD). In total, 12 Lantang gilts (58.7±0.37 kg) were randomly divided into two dietary groups (six replicates (pigs) per group) and fed either LFD (NDF=201.46 g/kg) or HFD (NDF=329.70 g/kg). Wheat bran was the main source of fiber for the LFD, whereas ground rice hull (mixture of rice hull and rice bran) was used for the HFD. Results showed that the methanogens in the hindgut of Lantang gilts belonged to four known species (Methanobrevibacter ruminantium, Methanobrevibacter wolinii, Methanosphaera stadtmanae and Methanobrevibacter smithii), with about 89% of the methanogens belonging to the genus Methanobrevibacter. The 16S ribosomal RNA (rRNA) gene copies of Methanobrevibacter were more than three times higher (P0.05) was observed in 16S rRNA gene copies of Fibrobacter succinogenes between the two dietary groups, and 18S rRNA gene copies of anaerobic fungi in gilts fed LFD were lower than (P<0.05) those fed HFD. To better explain the effect of different fiber source on the methanogen community, a follow-up in vitro fermentation using a factorial design comprised of two inocula (prepared from hindgut content of gilts fed two diets differing in their dietary fiber)×four substrates (LFD, HFD, wheat bran, ground rice hull) was conducted. Results of the in vitro fermentation confirmed that the predominant methanogens belonged to the genus of Methanobrevibacter, and about 23% methanogens was found to be distantly related (90%) to Thermogymnomonas acidicola. In vitro fermentation also seems to suggest that fiber source did change the methanogens community. Although the density of Methanobrevibacter species was positively correlated with CH4 production in both in vivo (P<0.01, r=0.737) and in vitro trials (P<0.05, r=0.854), which could partly explain the higher methane production from gilts fed LFD compared with those in the HFD group. Further investigation is needed to explain how the rice hull affected the methanogens and inhibited CH4 emission from gilts fed HFD.