Molecular Characterization of Chicken Anemia Virus (CAV) and Expression of the CAV VP3 Protein

Chicken anemia virus (CAV) from non-attenuated and attenuated isolates were characterized based on sequence and phylogenetic analysis. The CAV BL-5 isolate, isolated from UPM was propagated and attenuated in MSB-1 cells until passage 90. The whole genome of non-attenuated isolate, BL-5P5 and attenuated isolate, BL-5P90 were amplified, cloned and subjected for sequencing. The sequences were analyzed and compared with other 25 isolates from local and foreign countries. Sequence analysis of VP1, VP2 and VP3 coding regions revealed that most of the variations were at the VP1 region. Sequence analysis of VP1 revealed that the BL-5P5 isolate was closely related to BL-5P90, CAF475 (China), AF313 (USA), C140 and A2 (Japan) and 3-1/P60 (Malaysia) isolates between 98% to 99% homology and distantly related to CAU269/7 (Australia) and SMSC-1 (Malaysia) isolates with 95% homology. However, analysis based on amino acid sequence indicated that the BL-5P5 isolate was closely related (98% to 99%) to all the above isolates, including the CAU269/7 isolate. It was found that the CAU269/7 has a very unusual low nonsynonymous/synonymous (NS/S) ratio of 0.188 when compared to the BL-5P5. Similarly, phylogenetic analysis based on the VP1 nucleotide sequences revealed that the BL-5P5 was closely related to BL-5P90, CAF475 (China) and AF313 (USA) and distantly related to CAU269/7 (Australia) and SMSC-1 (Malaysia) isolates . Analysis based on amino acid sequences revealed that the BL-5P5 was closely related to BL-5P90, CAU269/7 (Australia), Cf.F475 (China) and AF313 (USA) and distantly related to ConnB (USA), SMSC-1 (Malaysia) and P3102A9-resist isolates. The BL-5P90 showed only 15 nucleotide differences compared to BL-5P5 isolates. However, these differences associated with 11 amino acid changes which were found mainly in the hypervariable region of VP1. Thus, the NS/S ratio (2.75) is significantly higher than the SINS ratio (0.36). The BL-5P90 isolate has an amino acid substitution at position 144 from glutamic acid (E) to lysine (K) in VP1 hypervariable region. This amino acid substitution might play an important role in viral attenuation. The CAV VP3 gene from nonattenuated BL-5P5 isolate was expressed as a fusion protein in prokaryotic system. The SOS-PAGE and Western blot analysis indicated that the expressed VP3 protein of a pproximately 18 kOa was observed from the cell lysate sample after 4 hours post induction with isopropyl-I3-0-thiogalactosidase (IPTG). However, the protein was expressed in insoluble form and was relatively non-immunogenic since hyperimmune serum against the expressed protein showed non-specific reactions following Western blot and indirect immunofluorescence anti body test (IFAT) assay. Thus, the expressed VP3 protein in the present form is not suitable for use as antigen in production of antibody for the development of VP3 protein as diagnostic marker. Further studies on the application of the VP3 as diagnostic protein of CAV remains to be confirmed

Determination of concentration boundaries for the toxicity of Terminalia catappa Linn. leaves extract on healthy Carassius auratus

Terminalia catappa from Family Combretaceae or locally known as Ketapang is naturally grows plant and abundantly available in many tropical areas. The medicinal properties such as anti-parasitic and antibacterial of the leaves have been proven repeatedly in many scientific studies of aquaculture to treat infected fish. In the present study, toxicity level of different leaves extract concentrations on the healthy Carassius auratus or goldfish was investigated before the next study on its efficacy to treat the unhealthy C. auratus could be initiated. Ten C. auratus in 30-L tank aquarium were tested with five different concentrations of leaves extract; 400 mg/L, 500 mg/L, 600 mg/L, 800 mg/L and 1000 mg/L respectively and pH of bath extracts were determined before performing the tests. The results of pH are reduced or became acidic when the concentrations of leaves extract are increased significantly. During the test, toxicological signs were detected by physiological changes such as increased respiration frequency or erratic behavior. Overall results showed the methanolic extract of T. catappa leaves demonstrated a significantly high safety margin for the host of C. auratus. The C. auratus were well-tolerated with methanolic extract of T. catappa at the concentration of 400 mg/L for 3 hours without any visible effect. At 500 mg/L concentration, C. auratus also managed to survive for 3 hours but number of survived fish is reduced significantly different at (P<0.05) throughout 3 hours exposure time compared to control. Exposure to 600 mg/L, C. auratus were managed to survive up to 2 hours; meanwhile at 800 mg/L only up to 1 hour survived. At the highest concentration of 1000 mg/L, C. auratus have demonstrated an increased operculum movement and inconsistent behavior within few minutes of exposure time. However, no lethality of C. auratus was observed in the experimental period

Evaluation of ampicillin, vancomycin, and gentamycin antibiotic resistant among lactobacillus isolates / Rozila Alias... [et al.]

Antibiotic resistance is an ever-increasing in worldwide problem nowadays. It is happened when the exten- sive use of antibiotics which will creates the selective pressure resulting from mutation of normal genes and spreading of a variety of an antimicrobial resistance. Uses of Lactobacillus as probiotics or in starter cultures may serve as hosts of antibiotic resistance genes, which could be transfer or resistance to multiple antibiotics. Therefore, it is important to screen the susceptible or resistance towards antibiotics which will not transferable resistance genes. In the recent study, a total of 37 strains of Lactobacillus species isolated from various milk and dairy products such as goat’s milk, cattle’s milk, human’s milk, homemade yogurt and commercial yogurt were examined for the antibiotic profile using Kirby-Bauer method. The results of this study show that human’s milk has a high number of Lactobacillus isolates resistant to ampicillin (75%), vancomycin (62.5%) and gentamycin (62.5%). Whereas Lactobacillus isolated from cattle’s milk have resistant to ampicillin (42.1%), vancomycin (42.1%) and susceptible to gentamycin (0%). But however, goat’s milk still has lower percentage number of resistant to ampicillin (28.0%), vancomycin (14.3%) and are susceptible to gentamycin (0%). These results indicate that ampicillin and vancomycin resistant seems to be very common among Lactobacillus isolates but gentamycin are still susceptible used. Vancomycin resistant are more concerned because of the emerging problem in hospital and often described as one of the last resorts against the infection caused by multidrug-resistant pathogens. Therefore, lactobacilli as probiot- ics would be more monitoring when applied in food industry and clinical especially for immune compromised patients or during anti-biotherapy. Acquisition and retransfer of resistance genes should be addressed in the new safety aspects of probiotics uses.addressed in the new safety aspects of probiotics uses

Responses of tropical fruit bats to monoculture and polyculture farming in oil palm smallholdings

The oil palm industry is one of the main economic drivers in Southeast Asia. The industry has caused tropical deforestation on a massive scale in producing countries, and this forest conversion to oil palm agriculture has decimated the habitat of numerous native species. Monoculture and polyculture practices are two distinctive oil palm production systems. We hypothesize that polyculture farming hosts a greater diversity of species than monoculture farming. Habitat complexity in smallholdings is influenced by multiple farming practices (i.e. polyculture and monoculture). However, little is known about the effects of such farming practices in smallholdings on mammalian biodiversity, and particularly frugivorous bats. Our study aimed to find the best farming practice to reconcile oil palm production with biodiversity conservation. Mist-nets were used to trap frugivorous bats at 120 smallholdings in Peninsular Malaysia. We compared species richness and the abundance of frugivorous bats between monoculture and poly-culture smallholdings. We investigated their relationships with vegetation structure characteristics. Our results revealed that species richness and abundance of frugivorous bats were significantly greater in polyculture smallholdings than monoculture smallholdings. We also found that 28.21% of the variation in species richness was explained by in situ habitat characteristics, including the number of dead standing oil palms and immature oil palms, non-grass cover, height of non-grass cover, and farming practices. Thein situ habitat quality was closely associated with oil palm farming management. Commercial growers should implement polyculture rather than monoculture farming because polyculture farming has positive effects on the abundance and species richness of bats in oil palm production landscapes

Immunological cross-reactivity between anti-vitellogenin antibodies and vitellogenin in three species of catfish

Vitellogenin (Vtg) is a specific biomarker of egg yolk precursor protein that is abundant in circulatory blood and indicates reproductive status in female fish and endocrine disruption in male fish. The protein highly conserved in various forms of Vtg in specific species. The aims of this study was to evaluate the cross-reactivity between Hemibagrus nemurus Vtg polyclonal antibody and vitellogenin from other catfish, Hemibagrus wyckiodes, Pangasius pangasius and Clarias gariepinus by enzyme-linked immunosorbent assay (ELISA). The plasma of three species of catfish was characterized using 7.5% SDS-PAGE and revealed a major polypeptide band corresponding to 100-175 kDa in H. wyckiodes, 70-160 kDa in Pangasius pangasius and 95-160 kDa in Clarias gariepinus and the existence of Vtg has been confirmed. The optimal detection of the H. nemurus Vtg was at dilution of 1/1000 polyclonal antibody. Furthermore, cross-reactivity tests showed that polyclonal antibody specific to H. nemurus Vtg reveals a cross-reaction with the plasma Vtg from three species of catfish. Thus, the polyclonal antibody can be used to detect and quantify Vtg in the three species of catfish that are closely related in order to examine the vitellogenesis periods and oocyte maturation