Genetic characterization and diversity of porcine circovirus type 2 in non‐vaccinated South African swine herds

The porcine circovirus type 2 (PCV2) is a swine infectious viral pathogen of great significance in global swine herds. It was recently detected at another Province of South Africa sequel to the first detection of North American‐like strain (PCV2a) at Gauteng about two decades ago, but there is a dearth of information about the genomic features and diversity of the viral strains in circulation within the country and the entire sub‐Saharan Africa region. To date, only one complete genome of the virus from South Africa is available on global data base. This current effort is therefore geared towards the full‐genome characterization of the circulating PCV2 strains in the pigs of Eastern Cape Province. With the use of conventional polymerase chain reaction method, fifteen complete PCV2 genomes were successfully amplified, sequenced and assembled from field samples obtained from non‐vaccinated pigs in the region. Neighbor Joining and Maximum Likelihood phylogenetic analyses of the ORF2 gene and full genomes unanimously showed that most of the assembled genomes (11) belong to genotype PCV2b. Furthermore, three of the characterized sequences formed clade with other reference mutant PCV2b and PCV2b subtype 1C (i.e. PCV2d) strains from the USA, China and South Korea. The last sequence, however, clustered with other reference strains belonging to PCV2 intermediate clade 2 (PCV2‐IM2), recently identified in a global PCV2 strains phylogenetic analysis. This study reports the first complete genome sequences of PCV2b, PCV2d and PCV2‐IM2 in pigs from South Africa, and it gives a possible insight into the genetic characteristics and variability of the viral strains presently in circulation within the country. It further emphasizes the need for more stringent measures in curtailing the introduction and spread of transboundary swine pathogens in the country and entire Southern African region.     

Toll-like receptor signaling inhibits structural development of the distal fetal mouse lung.

We tested the hypothesis that innate immune signaling in utero could disrupt the structural development of the fetal lung, contributing to the pathogenesis of bronchopulmonary dysplasia. Injection of Escherichia coli lipopolysaccharide (LPS) into the amniotic fluid of E15 BALB/cJ mice increased the luminal volume density of fetal mouse lungs at embryonic day (E) 17 and E18. LPS also increased luminal volume and decreased distal lung branching in fetal mouse lung explants. This effect required NF-kappaB activation and functional Toll-Like Receptor 4. Airway branching may require fibronectin-dependent epithelial-mesenchymal interactions, representing a potential target for innate immune signaling. Anti-fibronectin antibodies and LPS both blocked distal lung branching. By immunofluorescence, fibronectin localized to the clefts between newly formed airways but was restricted to peripheral mesenchymal cells in LPS-exposed explants. These data suggest that LPS may alter the expression pattern of mesenchymal fibronectin, potentially disrupting epithelial-mesenchymal interactions and inhibiting distal airway branching and alveolarization. This mechanism may link innate immune signaling with defects in structural development of the fetal lung.     

Vaccination challenge studies with variants of street rabies virus isolated in Nigeria

In a preliminary study it was observed that adult ICR mice immunized with serial dilutions of an inactivated experimental human rabies vaccine from the Pitman-Moore (PM) vaccine virus were well protected against challenge with homologous virulent PM virus and challenge virus standard (CVS). However only one of five variant representatives in five of seven groups of 41 isolates of street rabies virus from Nigeria characterized by hybridoma monoclonal antibodies specific for the nucleocapsid and glycoprotein antigens of rabies virion was protected for by the vaccine. Guinea pigs immunized with a live attenuated low egg passage (LEP, Flury strain) vaccine currently used in canine vaccination in Nigeria protected against challenge with all five variants. The LEP vaccine protected against the variants and CVS quite well even when 1:125 dilution of the vaccine was used.     

Differential expression and biodistribution of cytokeratin 18 and desmoplakins in non-small cell lung carcinoma subtypes

Adenocarcinoma (AC), squamous cell carcinoma (SCC) and adenosquamous carcinoma (ASC) of the lung are morphologically distinguished in part by cyto-architectural features. However, little is known about the relative expression and distribution of cyto-architectural proteins among AC, SCC and ASC. Initial microarray analysis revealed significant differences in expression of two cyto-architectural genes in AC, SCC and ASC. Desmoplakin (DP) 1 and 2, which link desmosomes to intermediate filaments, was strongly expressed in SCC relative to AC and ASC. Cytokeratin 18 (CK18), an intermediate filament that is commonly linked to desmoplakin, was strongly expressed in AC and ASC relative to SCC. Western blot analysis demonstrated that AC and ASC had abundant CK18 protein, whereas CK18 was weakly detected in SCC. DP 1 and 2 are strongly expressed in SCC and minimally expressed in AC and ASC. However, the ratio of one to the other is the same in SCC and AC, but DP2 is lost in ASC. Microscopic analysis with fluorescence-labeled antibodies for CK18 and DP 1 and 2 revealed abundant membrane localization of DP and minimal perinuclear localization of CK18 in SCC. In contrast, in both AC and ASC, the CK18 protein was diffusely distributed within the cytoplasm, and DP showed both membranous and cytoplasmic localization. In conclusion, the data here shows that AC, SCC and ASC each have specific patterns of DP 1 and 2 and CK18 gene expression, protein content and biodistribution.     

Effects of Chronic Ethanol on Enzymes Regulating Sialylation and Desialylation of Transferrin in Rats

Transferrin is a N-glycosylated glycoprotein and plays an important role in iron transport from sites of absorption and storage to sites of utilization. Chronic ethanol alters the normal microheterogeneity pattern of transferrin as a consequence of changes in the sialic acid content. However the underlying basis of this change in sialic acid contents of transferrin in alcohol abuse remains unclear. We have undertaken this study in order to investigate the effects of chronic ethanol in rats with respect to the hepatic rate of (i) transferrin synthesis based on labeled leucine incorporation, (ii) the incorporation of labeled N-acetyl mannosamine (NAM) into sialic acid residues of transferrin, and (iii) roles of specific sialyltransferase and sialidase at hepatic subcellular level. The results showed no significant difference in the incorporation of labeled leucine into transferrin at all levels between the control and ethanol group, whereas the incorporation of NAM into transferrin was significantly decreased by 84% (p < 0.001) both at the whole cell and Golgi level. Thus, the incorporation of labeled NAM relative to the incorporation of labeled leucine into hepatic transferrin was significantly decreased by 86% (p < 0.001) in chronic ethanol-treated animals as compared with the controls both at the whole cell and Golgi levels. These data are further supported by our finding of concomitant decrease in the activity of β-galactoside α2,6-sialyltransferase by 58% (p < 0.01) in ethanol-treated rats as compared with control animals. In contrast, both the plasma membrane and plasma sialidase activities were increased by 95% (p < 0.01) and 85% (p < 0.01), respectively, in ethanol-fed rats as compared with their respective controls. We conclude that decreased activity of sialyltransferase and increased activity of sialidase sequentially at the plasma membrane and plasma compartment may be responsible for decreased incorporation of sialic acid residues in serum transferrin molecules after chronic ethanol treatment.     

Redox signalling to nuclear regulatory proteins by reactive oxygen species contributes to oestrogen-induced growth of breast cancer cells

Background:

17β-Oestradiol (E2)-induced reactive oxygen species (ROS) have been implicated in regulating the growth of breast cancer cells. However, the underlying mechanism of this is not clear. Here we show how ROS through a novel redox signalling pathway involving nuclear respiratory factor-1 (NRF-1) and p27 contribute to E2-induced growth of MCF-7 breast cancer cells.

Methods:

Chromatin immunoprecipitation, qPCR, mass spectrometry, redox western blot, colony formation, cell proliferation, ROS assay, and immunofluorescence microscopy were used to study the role of NRF-1.

Results:

The major novel finding of this study is the demonstration of oxidative modification of phosphatases PTEN and CDC25A by E2-generated ROS along with the subsequent activation of AKT and ERK pathways that culminated in the activation of NRF-1 leading to the upregulation of cell cycle genes. 17β-Oestradiol-induced ROS by influencing nuclear proteins p27 and Jab1 also contributed to the growth of MCF-7 cells.

Conclusions:

Taken together, our results present evidence in the support of E2-induced ROS-mediated AKT signalling leading to the activation of NRF-1-regulated cell cycle genes as well as the impairment of p27 activity, which is presumably necessary for the growth of MCF-7 cells. These observations are important because they provide a new paradigm by which oestrogen may contribute to the growth of breast cancer.     

Commensal pseudomonas species isolated from wastewater and freshwater milieus in the eastern cape province, South Africa, as reservoir of antibiotic resistant determinants

Pseudomonas species are opportunistic pathogens with implications in a wide range of diseases including cystic fibrosis and sickle cell anaemia. Because of their status as multidrug resistant (MDR) and extremely drug resistant (XDR) bacteria Pseudomonas species represent a threat to public health. Prevalence, antibiogram and associated antibiotic resistant genes of Pseudomonas species isolated from freshwater and mixed liquor environments in the Eastern Cape Province of South Africa were assessed. Polymerase chain reaction (PCR) based technique was used to identify the isolates and screen for antibiotic resistant genes. The result shows occurrence of Pseudomonas spp. in freshwater and mixed liquor as follows: 71.42% and 37.5% (P. putida), 14.28% and 31.25% (P. flourescens), 7.14% and 6.25% (P. aeruginosa) and 7.14% and 25% for other Pseudomonas species respectively. Disk diffusion antibiogram of the Pseudomonas isolates from the two locations showed 100% resistance to penicillin, oxacillin, clindamycin, rifampicin and 100% susceptibility to ciprofloxacin and gentamicin with varied percentage resistances to cephalothin, nalidixic acid, tetracycline, and ampicillin. The bla(TEM) antibiotic resistant gene was detected in 12.5% of P. putida, 57.14% of P. fluorescens, 100% P. aeruginosa and 40% in other Pseudomonas species. Similarly, Integrons conserved segment were detected in 12.5% of P. putida, 57.14% of P. fluorescens, 100% of P. aeruginosa and 40% of other Pseudomonas species. The presence of bla(TEM) gene and integrons conserved segment in some of the isolates is worrisome and suggest Pseudomonas species as important reservoirs of multidrug resistance genes in the Eastern Cape Province environment.     

Prevalence of multiple antibiotics resistant (MAR) Pseudomonas species in the final effluents of three municipal wastewater treatment facilities in South Africa

The final effluents of three (Alice, Dimbaza, and East London) wastewater treatment plants (WWTPs) were evaluated to determine their physicochemical quality and prevalence of multiple antibiotics resistant (MAR) Pseudomonas species, between August 2007 and July 2008. The annual mean total Pseudomonas count (TPC) was 1.20 × 10(4) (cfu/100 mL), 1.08 × 10(4) (cfu/100 mL), and 2.66 × 10(4) (cfu/100 mL), for the Alice, Dimbaza, and East London WWTPs respectively. The effluents were generally compliant with recommended limits for pH, temperature, TDS, DO, nitrite and nitrate; but fell short of target standards for turbidity, COD, and phosphate. The tested isolates were highly sensitive to gentamicin (100%), ofloxacin (100%), clindamycin (90%), erythromycin (90%) and nitrofurantoin (80%); whereas high resistance was observed against the penicillins (90-100%), rifampin (90%), sulphamethoxazole (90%) and the cephems (70%). MAR index ranged between 0.26 and 0.58. The study demonstrated that MAR Pseudomonas species were quite prevalent in the final effluents of WWTPs in South Africa; and this can lead to serious health risk for communities that depend on the effluent-receiving waters for sundry purposes.