Onderstepoort Journal of Veterinary Research

Construction of an artificial recombinant bicistronic plasmid DNA vaccine against porcine rotavirus

The attenuated Salmonella typhimurium χ4550 strain was used to harbour a reconstructed bicistronic DNA vaccine against porcine rotavirus, which carried the rotavirus nonstructural protein 4 (NSP4) and VP7 genes simultaneously. Using a balanced lethal system, the kanamycin resistance gene of expressing eukaryotic plasmids pVAX1 and pVAXD were replaced by the aspartate β-semialdehyde dehydrogenase (asd) gene. The NSP4 cleavage product (259-525) of rotavirus OSU strain and VP7 full-length genes were amplified by reverse transcription polymerase chain reaction and then inserted into the eukaryotic single-expression plasmid, pVAX1-asd, and the eukaryotic dual-expression plasmid, pVAXD-asd, respectively. The recombinant plasmids pVAX1-asd-NSP4, pVAX1-asd-VP7 and pVAXD-asd-NSP4-VP7 were transformed into the attenuated S. typhimurium χ4550 strain by electrotransformation. An indirect immunofluorescence assay of the expressed COS-7 cell suggested that the recombinant S. typhimurium χ4550 strain was constructed successfully. The recombinant S. typhimurium χ4550 strain was orally administered to BALB/c mice. The group immunised with dual-expression plasmids produced a significantly higher level of serum Immunoglobulin G (IgG) and intestinal Immunoglobulin A (IgA) than the group immunised with single-expression plasmids. These results indicated that eukaryotic bicistronic plasmid DNA vaccines could be successfully constructed to enhance humoural, mucosal and cellular immune response against rotavirus infection.

Temporal and spatial history of Rift Valley fever in South Africa: 1950 to 2011

Several outbreaks of Rift Valley fever (RVF) have been documented in South Africa since it first occurred in the country in 1950. However, there is no comprehensive account of the timing, location and extent of all known outbreaks. As part of a study investigating the epidemiology of RVF in South Africa, a full history of outbreaks was compiled using references to the disease in South Africa from scientific literature, annual reports, disease reports and animal disease databases. The geographic location and temporal occurrence of each outbreak were recorded as accurately as allowed by the available records. The result was a better and more complete picture than has hitherto been available of the spatial and temporal distribution of RVF in South Africa for the period between 1950 and 2011. Several smaller outbreaks which had not been described previously in literature were documented. Extensive outbreaks occurred in the central interior of the country (Free State, Eastern Cape and Northern Cape provinces), interspersed with smaller outbreaks or long intervening periods of absence, whilst smaller outbreaks occurred in the eastern part of the country (KwaZulu-Natal, Mpumalanga and Gauteng).

Construction and immunogenicity of a ΔapxIC/ompP2 mutant of Actinobacillus pleuropneumonias and Haemophilus parasuis

The apxIC genes of the Actinobacillus pleuropneumoniae serovar 5 (SC-1), encoding the ApxI-activating proteins, was deleted by a method involving sucrose counter-selection. In this study, a mutant strain of A. pleuropneumoniae (SC-1) was constructed and named AapxIC/ ompP2. The mutant strain contained foreign DNA in the deletion site of ompP2 gene of Haemophilus parasuis. It showed no haemolytic activity and lower virulence of cytotoxicity in mice compared with the parent strain, and its safety and immunogenicity were also evaluated in mice. The LD50 data shown that the mutant strain was attenuated 30-fold, compared with the parent strain (LD50 of the mutant strain and parent strain in mice were determined to be 1.0 x 10(7) CFU and 3.5 x 10(5) CFU respectively). The mutant strain that was attenuated could secrete inactivated ApxIA RTX toxins with complete antigenicity and could be used as a candidate live vaccine strain against infections of A. pleuropneumoniae and H. parasuis.

**Occurrence of Tetracampos ciliotheca and Proteocephalus glanduligerus in Clarias gariepinus (Burchell, 1822) collected from the Vaal Dam, South Africa**

Cestodes are parasitic flatworms that live in the digestive tract of vertebrates as adults and often in the liver, muscle, haemocoel, mesentery and brain of various animals as larval stages. To identify the cestodes infecting Clarias gariepinus Burchell, 1822 (sharptooth catfish) in the Vaal Dam, a total of 45 host specimens were collected with the aid of gill nets between October 2011, January and April 2012. The fish were sacrificed and examined for cestode parasites. Two adult cestodes, Tetracampos ciliotheca Wedl, 1861 (prevalence 86.7%, mean intensity = 15, n = 45) and Proteocephalus glanduligerus (Janicki, 1928) (prevalence 51.1%, mean intensity = 5, n = 45) were found in the intestines of the catfish. Both T. ciliotheca and P. glanduligerus are new locality records. There were statistically insignificant differences in the infection of the male and female C. gariepinu. Fish with standard length ranging from 40 cm - 54 cm (>3 years) had the highest prevalence and mean intensity while those ranging from 10 cm - 24 cm (< 1 year) had the lowest prevalence and mean intensity for both cestodes. The study highlights the importance of changing feeding habits of C. gariepinus with age on the prevalence and mean intensity of the two gastrointestinal cestode parasites.

Comparison of pathogenic domains of rabies and African rabies-related lyssaviruses and pathogenicity observed in mice

Several lyssavirus species occur in Africa (Rabies virus, Lagos bat virus, Mokola virus, Duvenhage virus, Shimoni bat virus and Ikoma lyssavirus), displaying a high sequence diversity between isolates belonging to the same species. There is limited information about comparative pathogenesis of these African lyssaviruses and this precludes authoritative opinion on the potential public and veterinary health impact. In this study, an analysis of representative African lyssaviruses attempted to correlate viral genomic sequence similarities and differences with the corresponding pathogenic profiles observed in mice. The study demonstrated that the virus isolates evaluated could be lethal to mice when introduced intramuscularly and that different isolates of the same lyssavirus species differ in their virulence. Using real-time polymerase chain reaction (PCR), viral RNA was detected in brain tissue, but no viral RNA was detected in the salivary glands or blood of mice that succumbed to infection. Comparison of known pathogenic domains indicated that pathogenicity is likely to be dependent on multiple domains. Cumulatively, our results re-emphasised the realisation that the pathogenicity of a lyssavirus species cannot be deduced based on studies of only a single isolate of the species or a single pathogenic domain.

Immunohistochemical studies of the enteric nervous system and interstitial cells of Cajal in the canine stomach

The distribution of interstitial cells of Cajal (ICC), the probable pacemakers in gastrointestinal motility, was investigated using an antigenic marker of gastric ICC known as C-Kit. Antiserum raised against the general neuronal marker protein gene peptide 9.5 (PGP) as well as the nitrergic neuronal marker neuronal nitric oxide synthase (nNOS) were used to investigate the distribution of gastric nerves. Polyclonal goat anti-human C-Kit was reliable in labelling ICC in the stomach. Two classes of ICC were identified according to their distribution: ICC-MY distributed around the periphery of myenteric ganglia and ICC-IM in the circular and longitudinal muscle layers. The neuronal marker PGP was reliably consistent in revealing the density and distribution of the enteric nervous system. Density of nerve fibres was higher in circular smooth muscle than in longitudinal smooth muscle. From nNOS immunohistochemistry, it is evident that inhibitory (nitrergic) nerves constitute a substantial fraction of the enteric nervous system.