R.R. Bragg

Global genetic diversity and geographical and host-species distribution of beak and feather disease virus isolates

Psittacine beak and feather disease (PBFD) has a broad host range and is widespread in wild and captive psittacine populations in Asia, Africa, the Americas, Europe and Australasia. Beak and feather disease circovirus (BFDV) is the causative agent. BFDV has an ∼2 kb single stranded circular DNA genome encoding just two proteins (Rep and CP). In this study we provide support for demarcation of BFDV strains by phylogenetic analysis of 65 complete genomes from databases and 22 new BFDV sequences isolated from infected psittacines in South Africa. We propose 94% genome-wide sequence identity as a strain demarcation threshold, with isolates sharing >94% identity belonging to the same strain, and strain subtypes sharing >98% identity. Currently, BFDV diversity falls within 14 strains, with five highly divergent isolates from budgerigars probably representing a new species of circovirus with three strains (budgerigar circovirus; BCV-A, -B and -C). The geographical distribution of BFDV and BCV strains is strongly linked to the international trade in exotic birds; strains with more than one host are generally located in the same geographical area. Lastly, we examined BFDV and BCV sequences for evidence of recombination, and determined that recombination had occurred in most BFDV and BCV strains. We established that there were two globally significant recombination hotspots in the viral genome: the first is along the entire intergenic region and the second is in the C-terminal portion of the CP ORF. The implications of our results for the taxonomy and classification of circoviruses are discussed.

A unique isolate of beak and feather disease virus isolated from budgerigars (Melopsittacus undulatus) in South Africa.

Beak and feather disease virus (BFDV), the causative agent of psittacine beak and feather disease (PBFD) infects psittaciformes worldwide. We provide an annotated sequence record of three full-length unique genomes of BFDV isolates from budgerigars (Melopsittacus undulatus) from a breeding farm in South Africa. The isolates share >99% nucleotide sequence identity with each other and ~96% nucleotide sequence identity to two recent isolates (Melopsittacus undulatus) from Thailand but only between 91.6 and 86.6% identity with all other full-length BFDV sequences. Maximum-likelihood analysis and recombination analysis suggest that the South African budgerigar BFDV isolates are unique to budgerigars, are non-recombinant in origin, and represent a new genotype of BFDV.

Bacterial resistance to Quaternary Ammonium Compounds (QAC) disinfectants.

Control of bacterial diseases has, for many years, been dependent on the use of antibiotics. Due to the high levels of efficacy of antibiotics in the past other disease control options have, to a large extent, been neglected. Mankind is now facing an increasing problem with antibiotic resistance. In an effort to retain some antibiotics for human use, there are moves afoot to limit or even ban the use of antibiotics in animal production. The use of antibiotics as growth promoters have been banned in the European Union and the USA. The potential ban on the use of antibiotics to treat diseases in production animals creates a dilemma for man-suffer significant problem with bacterial infection or suffer from a severe shortage of food! There are other options for the control of bacterial diseases. These include vaccine development, bacteriophage therapy, and improved biosecurity. Vaccine development against bacterial pathogens, particularly opportunistic pathogens, is often very challenging, as in many cases the molecular basis of the virulence is not always clearly understood. This is particularly true for Escherichia coli. Biosecurity (disinfection) has been a highly neglected area in disease control. With the ever-increasing problems with antibiotic resistance-the focus should return to improvements in biosecurity. As with antibiotics, bacteria also have mechanisms for resistance to disinfectants. To ensure that we do not replace one set of problems (increasing antibiotic resistance) with another (increasing resistance to disinfectants) we need to fully understand the modes of action of disinfectants and how the bacteria develop resistance to these disinfectants. Molecular studies have been undertaken to relate the presence of QAC resistance genes in bacteria to their levels of sensitivity to different generations of QAC-based products. The mode of action of QAC on bacteria has been studied using NanoSAM technology, where it was revealed that the QAC causes disruption of the bacterial cell wall and leaking of the cytoplasm out of the cells. Our main focus is on the control of bacterial and viral diseases in the poultry industry in a post-antibiotic era, but the principles remain similar for disease control in any veterinary field as well as in human medicine.

Genetic diversity of the Rep gene of beak and feather disease virus in South Africa

Summary.A study on the genetic variation of Beak and feather disease virus (BFDV) isolates in South Africa was performed by amplifying and sequencing a region within the ORF1 of the genome. Six different BFDV isolates were found in 15 psittacine species from 6 regions within South Africa, representing three unique isolates and three isolates that clustered into a budgerigar lineage (BG) previously described.

Multiplex polymerase chain reaction for screening avian pathogenic Escherichia coli for virulence genes.

Colibacillosis is a disease in poultry caused by avian pathogenic Escherichia coli (APEC) strains which leads to great economic losses in the poultry industry. These E. coli strains contain various virulence genes which grant the bacteria the ability to proliferate in the poultry host and cause disease. Many genes which can contribute to virulence have been identified and can be used to screen E. coli strains to infer pathogenicity and aid in the identification and classification of APEC. Multiplex polymerase chain reaction methods were designed and optimized to rapidly detect 18 different virulence genes in E. coli strains that were isolated in South Africa and Zimbabwe from various sources, including from chickens showing signs of colibacillosis. Virulence gene profiles were constructed for each E. coli isolate from the multiplex data for the comparison of the colibacillosis isolates with the other isolates. The South African E. coli isolated from chickens with signs of colibacillosis showed higher virulence gene prevalence in comparison with the Zimbabwean and other samples except those isolated from chicken faeces. The multiplex polymerase chain reaction designed in the present study successfully screened E. coli isolates for various APEC-related virulence genes, including genes recently described in the literature.

Bacteriophages as Potential Treatment Option for Antibiotic Resistant Bacteria

The world is facing an ever-increasing problem with antibiotic resistant bacteria and we are rapidly heading for a post-antibiotic era. There is an urgent need to investigate alterative treatment options while there are still a few antibiotics left. Bacteriophages are viruses that specifically target bacteria. Before the development of antibiotics, some efforts were made to use bacteriophages as a treatment option, but most of this research stopped soon after the discovery of antibiotics. There are two different replication options which bacteriophages employ. These are the lytic and lysogenic life cycles. Both these life cycles have potential as treatment options. There are various advantages and disadvantages to the use of bacteriophages as treatment options. The main advantage is the specificity of bacteriophages and treatments can be designed to specifically target pathogenic bacteria while not negatively affecting the normal microbiota. There are various advantages to this. However, the high level of specificity also creates potential problems, the main being the requirement of highly specific diagnostic procedures. Another potential problem with phage therapy includes the development of immunity and limitations with the registration of phage therapy options. The latter is driving research toward the expression of phage genes which break the bacterial cell wall, which could then be used as a treatment option. Various aspects of phage therapy have been investigated in studies undertaken by our research group. We have investigated specificity of phages to various avian pathogenic E. coli isolates. Furthermore, the exciting NanoSAM technology has been employed to investigate bacteriophage replication and aspects of this will be discussed.

Pathogenic Gram-positive cocci in South African rainbow trout, Oncorhynchus mykiss (Walbaum)

Fish cultured in aquaculture systems are continuously exposed to pathogens present in the water, soil, air or in the fish (Rottmann, Francis-Floyd & Durborow 1992). The fish are weakened by stress conditions including increased fish density and poor water quality, injury during handling, inadequate nutrition, poor sanitation and increased water temperatures. During the last decade, Gram-positive cocci have become important pathogens causing disease in fish (Eldar & Ghittino 1999). Diseases have been reported from Japan (Hoshina, Sanu & Marimoto 1958), Singapore (Foo, Ho & Lam 1985), Australia (Carson, Gudkovs & Austin 1993), Israel (Eldar, Frealier, Asanta, Varner, Lawhon & Bercovier 1995), Italy (Ghittino & Praero 1992), Spain (Toranzo, Curtin, Romalde, Nunez & Barja 1995; Domenech, Fernandez-Garayzabal, Pasqual, Garcia, Cutuli, Moreno, Collins & Dominguez 1996), France (Michel, Nougayrède, Eldar, Sochon & De Kinkelin 1997), South Africa (Bragg & Broere 1986) and the United States (Perera, Johnson, Collins & Lewis 1994). This includes species from streptococci, lactococci and vagococci (Eldar & Ghittino 1999). In South Africa, a Streptococcus infection caused large numbers of deaths in rainbow trout, Oncorhynchus mykiss (Walbaum), (Bragg & Broere 1986) in the former Eastern Transvaal area. Clinical signs seen in the fish included extreme exophthalmus, often with haemorrhages in the eye chamber, and often resulting in the rupture of one or both eyes. Darkening of the skin pigmentation also occurred. Internal examination showed enlargement of the spleen, and a haemorrhagic intestine filled with a yellowish fluid, while both the stomach and intestine contained food. Only adult fish were affected and the largest, fast growing fish showed the disease first in most cases. These Streptococcus isolates could, however, not be classified (Bragg & Broere 1986). A number of the isolates from the study done by Bragg & Broere (1986) which were preserved by freeze drying were used in the current study. The aims were to identify some of these isolates using phenotypic tests as well as 16S rRNA sequencing. The twelve isolates used in this study are given in Table 1. The freeze-dried isolates were revived in brain heart infusion (BHI) broth (Biolab C114) and incubated under anaerobic conditions at 25 C for 3 days using an anaerobic jar with a gasgenerating kit (Oxoid BR0038). Anaerobic conditions enhanced the growth of the organisms. The purity of the cultures was confirmed by Gram staining. For the phenotypic tests, growth from a 24-h BHI agar slant culture was standardized in 5 mL sterile 1 N phosphate buffer to a density comparable to a McFarland 2 standard (Difco 0691326). Journal of Fish Diseases 2011, 34, 483–487 doi:10.1111/j.1365-2761.2011.01259.x

Nanomedicine and infectious diseases

First International Conference on Infectious Diseases and Nanomedicine – 2012 Kathmandu, Nepal, 15–18 December 2012. The First International Conference on Infectious Diseases and Nanomedicine congress facilitated the mixing of researchers in various fields of nanomedicine and infectious diseases, bringing together researchers from the fields of physics and chemistry, on the production of nanoparticles and researchers from various fields of microbiology where these nanoparticles have practical applications. The manufacture and applications of nanoparticles was one of the main themes of the congress, with much emphasis on the use of nanoparticles for the treatment of cancer. The ever increasing problems and concerns around antibiotic resistance also featured prominently in the congress. Various interesting presentations on human viruses were also presented during this congress.

The cloning and sequencing of the UDP-galactose 4-epimerase gene (galE) from Avibacterium paragallinarum

The putative uridine diphosphate (UDP)-galactose 4-epimerase encoding gene, galE, was isolated from Avibacterium paragallinarum with the use of degenerate primers, colony hybridization and inverse PCR. The data revealed an open reading frame of 1017 bp encoding a protein of 338 amino acids with a molecular weight of 37 kDa and an isoelectric point of 5.5. High sequence homology was obtained with an 87, 91 and 89% sequence identity on protein level towards the galE genes from Actinobacillus pleuropneumoniae, Haemophilus influenza and Pasteurella multocida, respectively. To verify that the cloned galE gene encodes for a UDP-galactose 4-epimeras, this gene was cloned into the pYES- 2 expression vector, followed by transformation in a Saccharomyces cerevisiae gal10 deletion strain. Complementation of the gal10 deletion mutant with the galE gene confirmed that this gene encodes a UDP-galactose 4-epimerase.

The Potential Use of Bacteriophage Therapy as a Treatment Option in a Post-Antibiotic Era

The impending postantibiotic era creates an urgent requirement for alternative treatments of infectious diseases in humans and animals. Bacteriophages are viruses that infect and kill bacteria. The application of bacteriophages as a treatment option was investigated before the development of antibiotics. However, the initial success of antibiotic therapy soon shifted the focus from bacteriophage research. The revitalization of phage therapy has received increased global attention since the appearance of multidrug-resistant bacteria. Bacteriophages replicate via either the lytic or lysogenic cycle. While both life cycles have potential applications in bacteriophage therapy, the lytic cycle seems most suited to antibacterial therapy. The most striking advantage of bacteriophage therapy is the high degree of host specificity exhibited by these viruses, which enables the formulation of tailored treatments that kill only pathogenic bacteria. However, the high specificity of such treatments requires highly accurate diagnostic procedures in order to succeed. Other restrictions of bacteriophage therapy, such as limitations with the registration of phage therapy options, may possibly be overcome by the expression and engineering of phage lytic enzymes, which break the bacterial cell wall. The problem of bacterial immunity to phage infection also cannot be ignored, although it is more solvable than resistance to antibiotics. Considering the available information, phage therapy holds promise as an alternative treatment option, although the road ahead is not without obstacles.