N. Sarangi¹, A.B. Mandal^1*, A.K. Bandyopadhyay¹, T. Venugopal², S. Mathavan² and T.J. Pandian²

¹Biotechnology Laboratory, Central Agricultural Research Institute, Port Blair 744 101, India.
² School of Biological Sciences, Madurai Kamaraj University, Madurai, Tamil Nadu 625 014, India.

(Received 10 April 1999 / Accepted 19 October 1999)

Abstract.
The rainbow trout growth hormone (rtGH) gene under the transcription control of long terminal repeat of Rous sarcoma virus was successfully introduced into three Indian major carps (IMC) viz. rohu (Labeo rohita) catla (Catla catla) and mrigal (Cirrihinus mrigala), en route electroporated sperms. The present communication enumerates the results of an array of electroporation experiments aimed at standardising diverse variables like voltage, capacitance, resistance and pulse constant to achieve maximum transformation efficiency. Stable genomic integration of the intruded alien gene was demonstrated through slot blot hybridization in all the three species. Per cent transgenic individuals were largely varied in the different species, maximum of 25% being observed in rohu followed by 23 and 13% in mrigal and catla, respectively. This indicates that electroporated sperm mediated gene transfer may emerge as a convenient means in fish transgenic development specially in IMC. Optimisation of electroporation condition for rtGH transformation to produce transgenic IMC are discussed hereunder in detail.

Keywords: rtGH, electroporation, sperm mediation, Indian major carps

C.C. Sieo¹, N. Abdullah^2*, S. Jalaludin³ and Y.W. Ho¹

¹Institute of Bioscience, ²Department of Biochemistry and Microbiology, ³Department of Animal Science,
Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia.

(Received 7 June 1999 / Accepted 14 October 1999)

Abstract.
Electron microscopy, both scanning electron microscopy and transmission electron microscopy, was used to study the mode of attachment and morphological characteristic of structures involved in the attachment of Ruminococcus albus to avicel. The cell of Ruminococcus albus was found to be enclosed by a thick layer (0.06-0.08 um) of extracellular material called the glycocalyx. Fine fibril like structures (0.18-0.23 Put long) projecting from the glycocalyx facilitated the attachment of R. albus to avicel. Initial attachment by these fine fibril like structures, which were found to be permanent structures of the bacteria, were observed after 10 min of incubation with avicel. Multiple points of attachment were observed initially. At longer incubation period (18 h), the Surface layer of the avicel was eroded and at the point of contact between the bacteria and the substrate, the glycocalyx of the bacteria was irregular and less defined than the part which was not in contact with the substrate. The glycocalyx also seemed to diffuse into the substrate. After 30-36 h of incubation, digestion was particularly evident at the point of contact with formation of pits in the avicel corresponding to the bacterial shape.

Keywords: Attachment, avicel, cellulolytic bacteria, Ruminococcus albus, glycocalyx, microcrystalline cellulose

M.Y Sabri, M. Zamri Saad*, A.R. Mutalib, D.A. Israf and N. Muniandy¹

Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia.
Veterinary Research Institute, 59 Jalan Sultan AzIan Shah, 13800 Ipoh, Perak, Malaysia

(Received 16 April 1999 / Accepted 15 September 1999)

Abstract.
The outer membrane proteins of Pasteurella haemolytica A2, A7 and A9 were subjected to SDS-PAGE and immunoblotting. The molecular weights of the polypeptide bands ranged between 33 to 97 kDa. The major polypeptide bands for P.haemolytica A2 were 33.4, 39.2 and 45 kDa while the minor polypeptide bands were 50, 58.7, 66.2, 84.7 and 97.4 kDa. Analysis of the outer membrane proteins of P. haemolytica A7 revealed two major protein bands of 33.4 and 45 kDa and three minor polypeptide of 40, 50 and 66.2 kDa. There were three major (33.4, 37.5 and 45 kDa) and one minor protein band (50 kDa) in the outer membrane proteins of P haemolytica A9. There was one major protein band from each of the P.haemolytica A2, A7 and A9, which was unique to the respective serotype and appeared to represent the respective serotype. These were the 39.2 kDa band for P.haemolytica A2, the 40 kDa band for P.haemolytica A7 and the 37.5 kDa band for P haemolytica A9. Following homologous immunoblot, all the serotypes showed pronounced antigenicity at the 30 kDa band. Heterologous immunoblot using the antiserum of P.haemolytica A2 did not reveal any antigenic band of P.haemolytica A9 but revealed antigenic bands at 30 and 31 kDa of P.haemolytica A7. Heterologous immunoblot using the antiserum of P.haemolytica A7 revealed antigenic band at 30 kDa of all the three serotypes while the antiserum of P.haemolytica A9 failed to reveal any common antigenic band between all three serotypes. Thus, the 30 kDa band of P.haemolytica A7 may be a suitable candidate for a sub unit vaccine against pneumonic pasteurellosis of sheep and goats.

Keywords: Antigenicity, cross reaction, outer membrane proteins, Pasteurella haemolytica

S. Lihan¹, S. Radu^1,*, G. Rusul ², M.I.A. Karim¹, O.W. Ling¹ and N. Maisin³

¹Department of Biotechnology and ²Department of Food Science, Faculty of Food Science and Biotechnology;
3Department of Biology, Faculty of Science and Environmental Studies, Universiti Putra Malaysia,
43400 UPM Serdang, Selangor, Malaysia.

(Received 16 April 1999 / Accepted 15 September 1999)

Abstract.
Ninety five Escherichia coli strains isolated from raw milk were analyzed for plasmid profile and antimicrobial resistance, and typed by RAPD fingerprints. All the E. coli strains were resistant to four or more of the antibiotics tested. However, none were resistant to ceftazidime, gentamicin and norfloxacin. The multiple antibiotic resistance (MAR) index values of the E. coli strains ranged from 0.25 to 0.81, indicating that all were isolated from raw milk samples that were exposed to high risk sources. Plasmids of 1.4 to 68 MDa were detected in 69 (72.6%) of the E. coli strains examined. The detection of a single plasmid of 68 MDa in 58 of 69 plasmid containing strains limits the usefulness of plasmid profiles as an epidemiological marker. RAPD fingerprints of the 95 E. coli strains were obtained using two 10 mer primers, GEN15009 and GEN15010, to differentiate or identify clonal relationship among the strains examined. The dendrogram constructed from the combined results of the RAPD patterns obtained from both primers showed that the 95 E. coli strains were grouped into six major clusters, indicating that five major clonal lineages (clusters 1, 2, 3, 4 and 5) of E. coli strains were predominant in the raw milk samples tested.

Keywords: Escherichia coli, RAPD, plasmid, antibiotic resistance, MAR