Cellular and molecular characterisation of porcine congenital splayleg and the involvement of P311 and SPARCL-1 as candidate genes

Porcine congenital splayleg (PCS) is the most important congenital condition of piglets, associated with lameness and immobility, of unknown aetiology and pathogenesis. The aim of this study is to investigate the cellular and molecular changes in skeletal muscles of PCS, thereby gaining new molecular insights into this clinical condition. Based on immunohistochemistry and histological image analyses on 4 sets of 2-day- old splayleg piglets, each with a corresponding normal litter mate, a consistent discovery has been that PCS muscles [semitendinosus (ST), longissimus dorsi (LD) and gastrocnemius (G)] showed extensive fibre atrophy without apparent tissue damage. At present, it is not certain if PCS-associated fibre atrophy is accompanied by fibre hypoplasia. Both normal and PCS muscle fibres showed similar widespread distribution of lipid- and oxidative-positive fibres. Although there was no significant difference in fibre type composition, several structural myosin heavy chain (MyHC) genes were significantly down-regulated in PCS affected muscles. Interestingly, MAFbx, a major atrophy marker, was highly up-regulated in almost all PCS muscles, when compared with controls from normal litter mates. In contrast, P311, a novel 8- kDa protein, was relatively down-regulated in all PCS muscles examined. To further investigate the functional role of P311 in skeletal muscle, its full-length cDNA was sequenced (Accession. N0. EF416570) and over-expressed in murine C2C12 muscle cells. P311 over-expression enhanced cell proliferation and reduced myotube formation in C2C12 cells. The over-expression of calcineurin, a key intracellular calcium-dependent signalling factor of muscle differentiation, down- regulated P311 expression. Reduced P311 expression in PCS piglets might contribute to atrophy through reduced myotube contribution. To investigate the functional role of SPARCL-1, a matricellular secreted glycoprotein that belongs to SPARC family, its full-length cDNA was sequenced (Accession. N0. EF416571) and over-expressed in murine C2C12 muscle cells. SPARCL-1 overexpression led to reduced cell proliferation and down-regulation of MyHC genes during late differentiation. SPARCL-1 might be associated as a negative regulator of skeletal muscle cell proliferation and cell differentiation. However, endogenous SPARCL-1 expression was similar between PCS and normal muscles. Hence, although SPARCL-1 could play a role in muscle development, it is unlikely to be a main factor in the development of PCS. In summary, PCS is shown to be a condition characterised by extensive fibre atrophy and raised fibre density, and it is proposed that the combined differential expression of MAFbx and P311 is of potential value in the diagnosis of sub-clinical PCS

Detection of mycoplasma hyopneumoniae and porcine reproductive and respiratory syndrome in clinical samples by polymerase chain reaction

The aim of this study was detect Swine Enzootic Pneumonia (SEP) and Porcine Reproductive and Respiratory Syndrome (PRRS) from clinical affected lung samples with PCR technique. In this technique three primers set used for M. hyopneumonieae detection were Cai (Forward: GAG CCT TCA AGC TIC ACC AAG A: Reverse: TGT GTT AGT GAC TTT TGC CAC C), Baumeister (Forward: TAG AAA TGA CTG GCA GAC AA: Reverse: GAG GCT TGA TTT TGG AGT C) and Caron (Forward: GAC CCG ATG AAA CCT ATT AAA ATA GAC: Reverse; GAA GCG AAA TTA AAT ATT TTT AAT TCG ATA CTG). For the detection of PRRS virus the primer sets used Oleksievicz (Forward: GAA CCT GCC CAI CAC G: Reverse: TAC CCC TAA TTG AAT AGG GGA) amd Suàrez )Forward: GGG AAT GGC CAG CCA GTC AAT CAA CTG T: Reverse: TGT AGA AGT CAC GCG AAT CAG GCG CAC T). Nine pigs aged betwee 6-10 weeks were collected from farms in Selangor, Malaysia. One healthy pig and 8 other pigs with clinical signs of respiratory distress problem were sacrificed and lung bronchoalveolar lavage samples were obtained. Healthy pigs were selected as negative control while samples were harvested from 4 pigs with suspected SEP and 4 with respiratory problems for M. hyoneumoniae and PRRS virus detection. Base on the result, the Caronand Caiprimer sets were able to detect SEP from the affected lungs. For PRRS virus, RNA was extracted using easy-BLUE™ Total RNA Extraction Kit and converted it to cDNA with Maxime RT PreMix Kit. The oleksiewicz primer set was ideally suited for the detection of PRRS virus

Detection of Glasser's Disease in clinical samples using Polymerase chain reaction.

Glasser’s Disease is one of the porcine common respiratory problems in out country. Glasser’s disease is caused by Haemophilus parasuis (HP). The HP has a wide range of antibiotics sensitivity and can be treated during early infection; therefore early diagnosis is very important. In the past, diagnosis was based on history, clinical signs and postmortem lesions, then confirm by bacteria isolation and identification. These traditional microbiology methods are time-consuming and labourious. Polymerase chain reaction (PCR) is one of the methods that provide rapid and accurate diagnosis. The procedure can be completed within 1 to 2 days and only require one trained personal to perform. Currently, there is limited publish articles on PCR test for the diagnosis of Glasser’s Disease in Malaysia. For the analysis 2 pairs of primers set for HP were selected; HPS1-forward (5’AGT AGG AAG GGT GGT GT 3’) and HPSI-reverse (5’ CGT TTC GTC ACC CTC TGT GT 3’) and HPS2- forward (5’ TAG AAA AAA TCT TTA ATT G 3’) and HPS2-reverse (5’ CAC CAT AGA AAC TTC TTT TC 3’). Lung tissues, pericardial swabs and thoracic swabs samples were collected form farms in Sepang, Selangor, Malaysia. The PCR test was carried after some modifications and optimization. Both HP primers able to detect positive clinical lung samples and can be further developed as PCR diagnosis tool in out country

Characterisation of porcine reproductive and respiratory syndrome virus strains in selected farms in Malaysia

Porcine reproductive and respiratory syndrome (PRRS) is a disease characterised by late-term reproductive failure in sows and gilts, and respiratory problems in piglets and growing pigs. The PRRS virus can be divided into two antigenically and genetically different strains: Type I (European) and Type II (North American). In this study, 120 sera were collected from 12 farms in 6 states in Malaysia for the seroprevalence study. Ten sera from apparently healthy sows/gilts, finishers and growers were collected from each farm and tested using IDEXX HerdChek ELISA for both strains. All the farms tested were seropositive with an overall seroprevalence of 89.2%. Tissue samples were collected and PRRSV isolated were genotyped using nested-PCR (with reported primer pairs targeting ORF7) that enabled the differentiation of Type I and Type II PRRSV by producing different sizes of PCR products. Out of 27 tissue samples collected from 11 farms, 12 were positive for PRRSV. All the PRRSV genomes from the 12 PRRSV-positive tissue homogenates were of Type II PRRSV, whereas no Type I PRRSV was detected. These data indicate that PRRS is endemic in the farms tested with a high possibility of subclinical infections

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

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

Detection of the Japanese encephalitis virus in wild boars and comparison of blood profiles between wild boars and domestic pigs in Selangor, Malaysia

The Japanese encephalitis virus (JEV) RNA was detected in mosquito vectors in Selangor, Malaysia almost two decades ago. However, the JEV status in wild boars, a potential reservoir, has yet to be investigated. Blood profiles can be used for health monitoring however data on the wild boar blood profile is limited. This study was performed to detect the presence of JEV RNA in wild boars in Selangor, Malaysia and to determine the haematological and serum biochemical values of the wild boars. The blood profiles of domestic pigs were also determined for comparison. Thirty-five wild boar tonsils were collected for RNA extraction while 21 wild boar and 40 domestic pig blood were collected for analyses. The RNA extraction was performed using the QIAamp RNA Blood Mini Kit (Qiagen, USA) while reverse transcription and PCR amplification were performed using the i-JEV Detection Kit (iNtron Biotechnology, Korea). The blood analyses were performed using standard protocols in the Haematology and Clinical Biochemistry Laboratory, Faculty of Veterinary Medicine, Universiti Putra Malaysia. The JEV RNA (genotype I and/or III) was detected in one of 21 (4.8%) tonsils; gene sequencing can be done to plot a phylogenetic tree to identify the origin of virus. The positivity may be higher if a vigourously optimized multiplex RT-PCR was used in the detection JEV genotypes. The leucocyte, segmented neutrophil counts and serum globulin concentration of the wild boars were significantly (p<0.05) lower than in domestic pigs and the existing reference data. Serum AST and CK concentration of the wild boars were significantly higher than that of domestic pigs, which might be attributed the physically more active wild boars

Effect of simultaneous injection of classical swine fever virus vaccine and Mycoplasma hyopneumoniae vaccine on immune response of swine

Objectives of this study were (1) to compare sero-conversion in pigs following simultaneous and separate vaccination against Classical Swine Fever (CSF) and Mycoplasma hyopneumoniae and (2) to determine safety of CSF and M. hyopneumoniae vaccines when given simultaneously. Twenty-four weaned pigs were divided into 3 groups of 8 heads. Groups were designated as non-simultaneous vaccinated group, simultaneous vaccinated group and negative control, respectively. Vaccines used in study were M.hyopneumoniae vaccine (SPRINTVAC®MH) and CSF vaccine (PESTIFFA®). IDEXX ELISA test kit (HerdChek M hyo) and LSIVET SUIS HC/PPC Blocking ELISA test kit were used to detect antibody titre on weekly basis. Sero-conversion rate of CSF antibody titre and M.hyo antibody titre were calculated. Result showed both simultaneous vaccination and non-simultaneous vaccination for CSF antibody titre reached 100% sero-conversion rate at 5 weeks post vaccination. Therefore, simultaneous vaccination was able to accomplish similar results as in non-simultaneous vaccination. Sero-conversion rate for CSF antibody titre in non-simultaneous group was slower before it reached 5 weeks post vaccination. 12.5% of animal from negative control group sero-converted at 5 weeks post vaccination due to false-positive result or field infections. M. hyopneumoniae antibody titre sero-conversion rate in both simultaneous vaccination and non-simultaneous vaccination reached 100% sero-conversion rate after 6 weeks post vaccination. Control group showed negative result for M. hyopneumoniae antibody titre throughout whole experiment. Vaccines used in trial did not cause any adverse effect after post vaccination when given simultaneously

Effects of ß-Glucan on growth performance and immunomodulation in weaned piglets

Seventy-four weaned piglets were used in a 6-week experiment to determine the effects of ß-Glucan on growth performance and immunomodulation of weaned piglets. Piglets were randomly chosen and divided into control and treatment groups. Body weight gain and feed consumption were recorded at weeks 2, 3 and 6. The immunomodulatory effects of ß-Glucan were determined by gross examination of lung lesions during post mortem. Weaned piglets from the treatment group had overall greater growth performance compared to the control group. This was also evident from the overall higher body weight and percentage of body weight gain as well as a lower feed conversion ratio. The fecal coliform count also implied that fecal coliform count in the treatment group was lower than the control group. Piglets treated with ß-Glucan were observed to have positive immunomodulatory effects on piglets. This was shown by an overall lower lung lesion score in the treatment group. The post mortem revealed 2 piglets with fibrinous pneumonia (APP) and 1 pig with severe atrophic rhinitis (Grade 5). In conclusion, treatment with ß-Glucan may lessen inflammatory response towards Gram negative bacteria via the inhibition of inflammatory cytokines and promote the production of anti-inflammatory cytokines. Further studies are needed to determine the efficacy of ß-Glucan in reducing total coliform count and its effect on immunomodulation

Porcine congenital splayleg: a review

Porcine congenital splayleg (PCS) is a clinical condition of newborn piglets, characterised by muscle weakness, resulting in the inability to properly stand and walk, with affected limbs extended sideways or forwards. It is arguably the most important congenital defect of commercial piglets and causes significant economic loss to pig farmers. The aetiology and pathogenetic mechanisms for PCS are still not well understood. Various management, nutrition and genetic factors have been found associated with PCS problems, but the actual cause remain obscure. Proper management and good farm practise are essential to control this multi-factorials PCS problem

Effect of medium-chain triglycerides on piglets in three farms in Selangor and Penang, Malaysia

The aims of this study were to evaluate effect of medium-chain triglycerides (MCT) between treatment and control groups on growth performance, mortality rate and fecal coliform count in piglets. In addition, the effect of MCT on weaning body weight was evaluated. This study was separated into Phase I and Phase II. In phase I, 18 litters were selected from three farms in Selangor and Penang. Piglets in each litter was equally divided into control group and treatment group where only treatment group piglets were fed twice with 2 ml MCT on Day 1. Body weight was taken on day one, three, five and seven. Phase II involved 12 litters from a farm. The same procedure as in phase I was done, except the body weight was taken on day one, fourteen and twenty eight. MCT treatment group piglets were found to have higher growth performance and lower mortality rate thAn control group piglets. No difference in fecal coliform count was observed between treatment and control group. MCT showed more prominent effect on growth performance during weaning period of the piglets. In conclusion, MCT supplementation had positive effect on the growth performance of piglets as a result of increased body weight and average daily gain. Besides, MCT were able to reduce mortality in piglets in all the farms