An experimental oil emulsion Newcastle disease vaccine was evaluated for its efficacy in broiler chickens. A group of chickens vaccinated at one day old with a live lentogenic Newcastle disease vaccine and subsequently revaccinated at three and eight weeks old with the experimental oil emulsion vaccine showed satisfactory haemagglutination inhibition antibody response which persisted for 18 weeks. Between 90 and 100 per cent of the vaccinated chickens were protected when challenged with the velogenic viscerotropic Newcastle disease virus. Although the vaccinated chickens were protected against clinical disease, virus could be isolated from a number of birds. By day 10 to 12 after challenge all the chickens were free from Newcastle disease infection.
This paper reviews the literature on leptospirosis in Malaysia from its first description in 1928 until the present day. Most of the early reports were on investigations of leptospirosis in wildlife and man and up-to-date, thirty-seven leptospiral serovars from thirteen serogroups have been bacteriologically identified. The thirteen serogroups are: Australis, Autumnalis Bataviae, Canicola, Celledoni, Grippotyphosa, Hebdomadis, Icterohaemorrhagiae, Javanica, Pomona, Pyrogenes, Sejroe and Tarassovi. Rats have been ascribed as the principal maintenance host of leptospires in Malaysia. However, serovars from the Pomona, Pyrogenes and Sejroe serogroups have yet to be isolated from rats. It is considered that the majority of leptospirosis cases in man were due to association of man with an environment where rats were plentiful. Recent investigations on domestic animals disclosed a high prevalence of infection in cattle and pigs and they were suspected as being the maintenance host for serovar hardjo and pomona respectively. There is ample scope for research in leptospirosis, particularly in the epidemiology and control of the disease in domestic animals. The strategy to control the infection in domestic animals and man in Malaysia is bound to be different from that of the temperate countries, basically due to the presence of a large number of leptospiral serovars in wildlife, further confounded by geographical and financial constraints.
A leptospiral isolate from a bovine kidney was found to be antigenically different from all previously recognized serovars of Leptospira interrogans based on the cross-agglutinin absorption test. The new serovar belongs to the Sejroe serogroup, and the name Leptospira interrogans serovar unipertama is proposed for it, with strain K2-1 as the reference strain.
A cross-sectional serological survey of domestic animals in West Malaysia revealed that 25.5% of the animals examined had agglutinating antibodies to one or more antigens belonging to Leptospira interrogans. Significant prevalence of infection was observed in cattle (40.5%), buffaloes (31%) and pigs (16%). The Sejroe serogroup was shown to be the principal one involved in cattle and buffaloes, and to a lesser extent the Tarassovi and Pomona serogroups. Evidence of infection in domestic animals by strains bearing the other seven antigens appeared insignificant and was indicative of sporadic infection. A majority of the large (semi-intensive) cattle and buffalo farms demonstrated a high prevalence of leptospiral infection. In both species of domestic animals mentioned above, the prevalence of infection was significantly higher (P = 0.01) in the semi-intensive farms than in the smallholdings. Amongst cattle, the droughtmasters had the highest prevalence whilst the Kedah-Kelantan (an indigenous breed) had the lowest prevalence of leptospiral infection. In general, the temperate breeds of cattle had a significantly (P = 0.01) higher prevalence of infection than local breeds. Leptospiral infection in goats and sheep was shown to be sporadic, and the Pomona serogroup was the principal leptospiral serogroup involved in these small ruminants. The prevalence of infection in pigs was observed to decline during the study period, and it is suspected that pigs in West Malaysia are the maintenance host for serovar pomona whilst cattle are the maintenance host for serovar hardjo. Overall, it appears that domestic animals in Malaysia will play a bigger role in the epidemiology of leptospiral infection with the advent of sophisticated farming.
A case of Newcastle disease virus infection in a female laboratory technician is reported for the first time in Malaysia. Infection was acquired by droplet infection of the eye while grinding infected chicken in the laboratory. The case was confirmed by isolation of Newcastle disease virus from an eye swab taken from the subject on the first day of clinical signs. A four-fold rise of haemagglutination-inhibition titre was shown when sera on the third day of infection and 15 days later were compared.
The Australian, heat-resistant, a virulent V4 strain of Newcastle disease (ND) virus was selected for further heat resistance to give a variant designated V4-UPM. V4-UPM was sprayed on to food pellets which were fed to chickens in amounts calculated to give about 10(6) EID50 per chicken. Chickens vaccinated only once by feeding developed no haemagglutination-inhibition (HI) antibodies and were not protected against challenge with a viscerotropic velogenic strain of ND virus. Chickens given food pellet vaccine at 3 and 6 weeks of age developed HI antibodies and were substantially protected against parenteral and contact challenge with virulent ND virus. Similar protection was achieved when the V4-UPM vaccine was given intranasally on two occasions or when the vaccine virus was allowed to spread by contact from intranasally vaccinated chickens to nonvaccinated chickens. Heat resistant ND vaccine incorporated in food pellets may provide a method for protecting village chickens against ND in tropical countries.
The food pellet vaccine has been shown to be effective in trials conducted under laboratory and simulated field conditions. The village chickens vaccinated with the food pellet vaccine during the field trial were protected against virulent Newcastle disease virus. The efficacy of the food pellet vaccine in the field was evaluated by challenge trial in which 60 per cent protection was obtained, or by monitoring the incidence of Newcastle disease in vaccinated and unvaccinated birds. There was no report of Newcastle disease outbreaks in the vaccinated birds during the two-year period of the field trial. The ease in administering the food pellet vaccine makes it readily accepted by the farmers.
A UV-HPLC method optimized by Box-Behnken design model was developed to determine caffeine in pharmaceutical preparations and urine samples. The chromatographic conditions followed were mobile phase: methanol/water/ citrate buffer (pH 4.6) (40:25:35, v/v/v),AcclaimTMDionex C18 column (ODS 100A˚, 5 µm; 4.6 × 250 mm),flow rate (0.9 mL min-1), column temperature (30 °C) and UV-detection wavelength (204 nm). The chromatographic variables: pH (A), % methanol fraction (B), flow rate(C) and column temperature (D) were optimized at 50 μg mL-1caffeine using BBD model. The chromatogram resulted in the asymmetry factor (1.23), theoretical plate 13,786 and retention time (5.79 min). The proposed HPLC method's greenness point was assessed byAnalytical Eco-scale and found to be 78 (as per guidelines, ranked as excellent). The linearity was ranged from2.0 to 70 µg mL-1 with coefficient of correlation (r = 0.999) and detection limit of 0.19 µg mL-1. The proposedmethod was developed successfully and applied for the assay of active caffeine in pharmaceutical preparations and urine samples. The % recovery obtained by both (proposed and reference) methods ranged from 99.98 to 100.05 % followed the compliance (100 ± 2 %) with Canadian Health Protection regulatory guidelines. The performance of the proposed method was compared with published papers and found to be acceptable and superior. The proposed method was quite effective as the reference method, and hence can be used as an alternative method for the assay of active caffeine in pharmaceutical preparations and urine samples.
The nucleotide sequence of the haemagglutinin-neuraminidase (HN) gene of Newcastle disease virus (NDV) viscerotropic-velogenic strain AF2240 was determined by direct RNA sequencing and by sequencing RT-PCR products. It encodes a single open reading frame of 581 amino acids with a calculated Mr of 63.8 kDa. The predicted sequence contains five asparagine glycosylation sites. Comparison of the AF2240 HN protein sequence with 13 other previously published sequences showed 88% homology. This HN protein is unique because it lacked the Arg 403 residue which is present in all of the other strains and cannot be grouped under the proposed three size classes of HN proteins in NDV.
A cross-sectional bacteriological survey of cattle in West Malaysia revealed 14.4% (32/222) had leptospiral infection. Isolates were obtained from all except one herd with prevalence of infection in herds ranging from 0-44.8%. A small number of buffalo urine samples were examined and all of them were found to be negative. A leptospiral isolate obtained from a bovine kidney proved to be a new serovar of Leptospira interrogans and the name unipertama was assigned to it. Six other leptospiral serovars were isolated, namely canicola, australis, javanica, ballum, pomona and hardjo. All six serovars were isolated for the first time in cattle in Malaysia. Cattle in Malaysia appear to be the maintenance host for serovar hardjo. The presence of the other serovars in cattle was probably due to contact with the maintenance hosts, pigs for serovar pomona and rodents for the other three serovars. It appears that the epidemiology of leptospiral infection in cattle in Malaysia is similar to that reported overseas.
A Rhodococcus sp. UKMP-5M isolate was shown to detoxify cyanide successfully, suggesting the presence of an intrinsic property in the bacterium which required no prior cyanide exposure for induction of this property. However, in order to promote growth, Rhodococcus sp. UKMP-5M was fully acclimatized to cyanide after 7 successive subcultures in 0.1 mM KCN for 30 days. To further shorten the lag phase and simultaneously increase the tolerance towards higher cyanide concentrations, the bacterium was induced with various nitrile compounds sharing a similar degradatory pathway to cyanide. Acetonitrile emerged as the most favored inducer and the induced cells were able to degrade 0.1 mM KCN almost completely within 18 h. With the addition of subsequent aliquots of 0.1 mM KCN a shorter period for complete removal of cyanide was required, which proved to be advantageous economically. Both resting cells and crude enzyme of Rhodococcus sp. UKMP-5M were able to biodegrade cyanide to ammonia and formate without the formation of formamide, implying the identification of a simple hydrolytic cyanide degradation pathway involving the enzyme cyanidase. Further verification with SDS-PAGE revealed that the molecular weight of the active enzyme was estimated to be 38 kDa, which is consistent with previously reported cyanidases. Since the recent advancement in the application of biological methods in treating cyanide-bearing wastewater has been promising, the discovery of this new bacterium will add value by diversifying the existing microbial populations capable of cyanide detoxification.
The nucleotide sequence of the haemagglutinin-neuraminidase (HN) glycoprotein gene of Newcastle disease virus (NDV) variant strain V4(UPM) was determined by direct genomic RNA sequencing and confirmed by cycle sequencing. The gene comprises 1996 nucleotides encoding a 615 amino acid protein of size 67.4 kDa. The nucleotide and amino acid sequences of this strain were compared with those of the parent strain V4(QUE). There are 16 nucleotide substitutions on V4(UPM), eight of which are silent mutations and another eliminated a potential Asn-linked glycosylation site in V4(UPM). In addition, an Arg (403) residue was shown to be absent in the variant strain. This deletion is thought to be significant because of its location in a highly conserved region of the HN protein.
One-day-old chickens were transported from Australia to Malaysia and vaccinated orotracheally with an uninactivated vaccine prepared from avirulent Australian V4 strain of Newcastle disease virus (NDV). The vaccination regimes were as follows: group A, once, at 2 weeks old; group B, once, at 3 weeks old; group C, twice, at 2 and at 3 weeks old; group D, direct contact with groups A, B, and C; and group E, indirect contact with groups A, B, C, and D. Group F was unvaccinated controls. Challenge was with NDV virulent Ipoh AF 2240-226 strain, administered at 4 weeks old intramuscularly to 10 chickens in each group and orotracheally to 10 chickens in each group. The remaining chickens were challenged by contact with the inoculated chickens. Group mortalities following challenge were: A, 1/77; B, 1/34; C, 0/39; D, 0/45; E, 6/43; and F, 60/60.
In the present study, locally isolated Rhodococcus strains were attempted as biological tools for methyl red removal, a mutagenic azo dye posing threat to the environment if left untreated. Rhodococcus strain UCC 0016 demonstrated superior methyl red-decolourizing activity of 100% after 24 h at static condition in comparison to Rhodococcus strain UCC 0008 which recorded 65% decolourization after 72 h. Optimization of physicochemical parameters at 30°C, pH 7 and supplementing glucose as the carbon source resulted in improved methyl red-decolourizing activity at static condition and reduced the time taken to achieve complete decolourization by 80%. Higher concentration of methyl red (5 g/L) was able to be decolourized completely within 10 h by adopting the technology of immobilization. The encapsulated cells of Rhodococcus strain UCC 0016 demonstrated higher substrate affinity (Km = 0.6995 g/L) and an accelerated rate of disappearance of methyl red (Vmax = 0.3203 g/L/h) compared to the free cells. Furthermore, the gellan gum beads could be reused up to nine batches without substantial loss in the catalytic activity indicating the economic importance of this protocol. Analysis of methyl red degradation products revealed no germination inhibition on Triticum aestivum and Vigna radiata demonstrating complete toxicity removal of the parent dye after biological treatment. The occurrence of new and altered peaks (UV-Vis and FTIR) further supported the notion that the removal of methyl red by Rhodococcus strain UCC 0016 was indeed through biodegradation. Therefore, this strain has a huge potential as a candidate for efficient bioremediation of wastewater containing methyl red.
The capability of the crude extract of Rhodococcus UKMP-5M was enhanced by adopting the technology of immobilization. Among the matrices screened to encapsulate the crude extract, gellan gum emerged as the most suitable immobilization material, exceeding the activity of cyanide-degrading enzyme by 61% and 361% in comparison to alginate carrier and non-immobilized crude extract, respectively. Improved bead mechanical strength which supported higher biocatalyst activity by 63% was observed when concentration of gellan gum, concentration of calcium chloride, number of beads and bead size were optimized. The immobilized crude extract demonstrated higher tolerance towards broad range of pH (5-10) and temperature (30°C-40°C), superior cyanide-degrading activity over time and improved storage stability by maintaining 76% of its initial activity after 30 days at 4°C. Furthermore, repeated use of the gellan gum beads up to 20 batches without substantial loss in the catalytic activity was documented in the present study, indicating that the durability of the beads and the stability of the enzyme are both above adequate. Collectively, the findings reported here revealed that the utilization of the encapsulated crude extract of Rhodococcus UKMP-5M can be considered as a novel attempt to develop an environmentally favourable and financially viable method in cyanide biodegradation.
The essential oil derived from Citrus plants has long been used for medicinal purposes, due to its broad spectrum of therapeutic characteristics. To date, approximately 162 Citrus species have been identified, and many investigational studies have been conducted to explore the pharmacological potential of Citrus spp. oils. This study investigated the volatile constituents of essential oil distilled from the leaves of C. hystrix, C. limon, C. pyriformis, and C. microcarpa, using gas chromatography-quadrupole mass spectrometry. A total of 80 secondary compounds were tentatively identified, representing 84.88-97.99% of the total ion count and mainly comprising monoterpene (5.20-76.15%) and sesquiterpene (1.36-27.14%) hydrocarbons, oxygenated monoterpenes (3.91-89.52%) and sesquiterpenes (0.21-38.87%), and other minor chemical classes (0.10-0.52%). In particular, 27 compounds (1.19-39.06%) were detected across all Citrus species. Principal component analysis of the identified phytoconstituents and their relative quantities enabled differentiation of the Citrus leaf oils according to their species, with the loading variables contributing to these metabolic differences being identified. The Citrus leaf oils were tested for their antioxidant and antiproliferative activities using 2,2-diphenyl-1-picryl-hydrazylhydrate (DPPH) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. The results indicated that C. limon displayed the highest DPPH radical scavenging ability (IC50 value of 29.14 ± 1.97 mg/mL), while C. hystrix exhibited the lowest activity (IC50 value of 279.03 ± 10.37 mg/mL). On the other hand, all the Citrus oils exhibit potent antiproliferative activities against the HeLa cervical cancer cell line, with IC50 values of 11.66 μg/mL (C. limon), 20.41 μg/mL (C. microcarpa), 25.91 μg/mL (C. hystrix), and 87.17 μg/mL (C. pyriformis).
Hemagglutination activity, structural protein profiles and neutralization assays were used in a comparative study of bovine herpesvirus 1 strains from the U.S.A., Canada, Great Britain, Denmark and Malaysia with equine, feline and human herpesviruses in order to further characterize the bovine herpesvirus 1 hemagglutinin. Bovine herpesvirus 1 strains of different geographical origins all showed hemagglutinating activity for mouse erythrocytes; furthermore, feline herpesvirus 1 was also shown to hemagglutinate mouse erythrocytes. Analyses of partly purified viruses showed that a distinctive and specific polypeptides profile is associated with each species of herpesviruses used in our study; strains of bovine herpesvirus 1 from North America, Europe and Southeast Asia however, presented a remarkable similarity as to their electrophoretic protein patterns. A protein similar to the 97-kDa bovine viral hemagglutinin was not identified with the hemagglutinating feline herpesvirus. An important neutralization epitope on the bovine viral hemagglutinin was also not found on feline, equine and human herpesviruses but was identified on all bovine strains tested from North America, Europe and Southeast Asia stressing the importance of the bovine hemagglutinin for eventual prophylactic purposes.