Marinda C. Oosthuizen

Proteomic analysis reveals differential protein expression by Bacillus cereus during biofilm formation.

ABSTRACT Bacillus cereus, a dairy-associated toxigenic bacterium, readily forms biofilms on various surfaces and was used to gain a better understanding of biofilm development by gram-positive aerobic rods. B. cereus DL5 was shown to readily adapt to an attached mode of growth, with dense biofilm structures developing within 18 h after inoculation when glass wool was used as a surface. Two-dimensional gel electrophoresis (2DE) revealed distinct and reproducible phenotypic differences between 2- and 18-h-old biofilm and planktonic cells (grown both in the presence and in the absence of glass wool). Whereas the 2-h-old biofilm proteome indicated expression of 15 unique proteins, the 18-h-old biofilm proteome contained 7 uniquely expressed proteins. Differences between the microcolony (2-h) proteome and the more developed biofilm (18-h) proteome were largely due to up- and down-regulation of the expression of a multitude of proteins. Selected protein spots excised from 2DE gels were subjected to N-terminal sequencing and identified with high confidence. Among the proteins were catabolic ornithine carbamoyltransferase and l-lactate dehydrogenase. Interestingly, increased levels of YhbH, a member of the sigma 54 modulation protein family which is strongly induced in response to environmental stresses and energy depletion via both σB and σH, could be observed within 2 h in both attached cells and planktonic cultures growing in the presence of glass wool, indicating that this protein plays an important role in regulation of the biofilm phenotype. Distinct band differences were also found between the extracellular proteins of 18-h-old cultures grown in the presence and in the absence of glass wool.

Sequence heterogeneity in the 18S rRNA gene within Theileria equi and Babesia caballi from horses in South Africa

A molecular epidemiological survey of the protozoal parasites that cause equine piroplasmosis was conducted using samples collected from horses and zebra from different geographical locations in South Africa. A total of 488 samples were tested for the presence of Theileria equi and/or Babesia caballi using the reverse line blot hybridization assay. Ten percent of the samples hybridized to the Theileria/Babesia genus-specific probe and not to the B. caballi or T. equi species-specific probes, suggesting the presence of a novel species or genotype. The small subunit of rRNA gene (18S; approximately 1600bp) was amplified and sequenced from 33 of these 488 samples. Sequences were compared with published sequences from the public sequence databases. Twelve distinct T. equi and six B. caballi 18S rRNA sequences were identified. Alignments demonstrated extensive sequence variation in the V4 hypervariable region of the 18S rRNA gene within T. equi. Sequence variation was also found in B. caballi 18S rRNA genes, although there was less variation than observed for T. equi. Phylogenetic analysis based on 18S rRNA gene sequences revealed three T. equi clades and two B. caballi clades in South Africa. The extent of sequence heterogeneity detected within T. equi and B. caballi 18S rRNA genes was unexpected since concerted evolution is thought to maintain homogeneity within repeated gene families, including rRNA genes, in eukaryotes. The findings reported here show that careful examination of variants of the 18S rRNA gene of T. equi and B. caballi is required prior to the development of molecular diagnostic tests to detect these parasites in horses. Species-specific probes must be in designed in regions of the gene that are both conserved within and unique to each species.

Identification of a Novel Babesia sp. from a Sable Antelope (Hippotragus niger Harris, 1838)

ABSTRACT Babesiosis in a sable antelope (Hippotragus niger Harris, 1838) was first reported in 1930; the parasite was named Babesia irvinesmithi. Recently, specimens from an adult sable that presented with a sudden onset of disease and that subsequently died during immobilization were submitted for molecular characterization. Microscopic examination of thin blood smears revealed the presence of small piroplasms. DNA was extracted from blood samples; the V4 variable region of the 18S rRNA gene was amplified and analyzed using the reverse line blot (RLB) assay. Amplicons did not hybridize with any of the Babesia or Theileria species-specific probes present on the blot and hybridized only with a Babesia or Theileria genus-specific probe, suggesting the presence of a novel species. The full-length 18S rRNA gene sequence was obtained and aligned with published sequences of related genera, and phylogenetic trees were constructed. Sequence similarity analyses indicated that a Babesia species, designated Babesia sp. (sable), was present. The sequence showed its highest similarity to B. orientalis and to an unnamed Babesia species previously detected in bovine samples. The latter was later established to be Babesia occultans. A Babesia sp. (sable)-specific RLB oligonucleotide probe was designed and used to screen 200 South African sable samples, but so far, no other sample has been found to be positive for the presence of Babesia sp. (sable) DNA. In summary, we identified a novel piroplasm parasite from a sable antelope that died from an unknown illness. While the parasite was observed in blood smears, there is no direct evidence that it was the cause of death.

Development and evaluation of a real-time polymerase chain reaction test for the detection of Theileria parva infections in Cape buffalo (Syncerus caffer) and cattle.

Corridor disease, caused by the tick-borne protozoan parasite Theileria parva, is a controlled disease in South Africa. The Cape buffalo is the reservoir host and uninfected buffalo have become sought-after by the game industry in South Africa, particularly for introduction into Corridor disease-free areas. A real-time polymerase chain reaction (PCR) test for detection of T. parva DNA in buffalo and cattle was developed to improve the sensitivity and specificity of the official diagnostic test package in South Africa. Oligonucleotide primers and hybridization probes were designed based on the 18S ribosomal RNA (rRNA) gene. Amplification of control DNA using Theileria genus-specific primers resulted in detection of T. taurotragi and T. annulata, in addition to T. parva. A T. parva-specific forward primer was designed which eliminated amplification of all other Theileria species, except for Theileria sp. (buffalo); however only the T. parva product was detected by the T. parva-specific hybridization probe set. The real-time PCR assay requires less time to perform, is more sensitive than the other molecular assays previously used in T. parva diagnostics and can reliably detect the parasite in carrier animals with a piroplasm parasitaemia as low as 8.79 x 10(-4)%.

Detection of a Theileria species in dogs in South Africa.

A Theileria species was detected by PCR in blood samples collected from dogs in the Pietermaritzburg area and was also found in dogs presented at the Outpatients Clinic of the Onderstepoort Veterinary Academic Hospital (OVAH), in the Pretoria area, South Africa. In the Pietermaritzburg area, 79 of the 192 samples were positive, while 3 out of 1137 of the Onderstepoort samples were positive. Three positive samples from Pietermaritzburg were co-infected with Ehrlichia canis. PCR positive samples were further analysed by the Reverse Line Blot (RLB) and sequence analysis. Phylogenetic analysis of the 18S rRNA full-length gene sequences of one sample (VT12) from Pietermaritzburg and two samples from OVAH (BC281 and BC295) revealed a close relationship with sequences of Theileria species (sable). Clinical signs of the dogs that were examined at Pietermaritzburg and OVAH included an immune-mediated condition with severe thrombocytopenia. These findings identify a Theileria sp. in dogs for the first time in South Africa and add yet another microorganism to the growing list of haemoprotozoan parasites infecting dogs worldwide. The clinical significance of this infection in dogs is poorly resolved.

Identification of novel Babesia and Theileria species in South African giraffe (Giraffa camelopardalis, Linnaeus, 1758) and roan antelope (Hippotragus equinus, Desmarest 1804).

Blood specimens were received from five cases in which young adult giraffe, from different geographic origins in South Africa, showed sudden onset of disease and subsequently died. Additional specimens from two translocated giraffe, as well as one specimen from a roan antelope, were also included in the study. Blood slides from some of these animals showed the presence of piroplasms. DNA was extracted; the V4 hypervariable region of the 18S rRNA gene amplified and analyzed using the Reverse Line Blot (RLB) hybridization assay. PCR products failed to hybridize with any of the Babesia or Theileria species-specific probes, and only hybridized with the Babesia/Theileria genus-specific probe suggesting the presence of a novel species or variant of a species. Full-length 18S rDNA was amplified, cloned and the recombinants were sequenced. 18S rRNA gene sequence similarity analysis revealed the presence of novel piroplasm species in both healthy giraffe and a roan antelope and clinically sick or dead giraffe. Phylogenetic analysis grouped five of these organisms in the Babesia sensu stricto clade and three in the Theileria sensu stricto clade. Although parasites were observed in blood smears, there is no direct evidence that piroplasmosis caused the death of five giraffe, although it certainly seems to be likely.

Occurrence of Theileria and Babesia species in water buffalo (Bubalus babalis, Linnaeus, 1758) in the Hubei province, South China.

The presence and prevalence of tick-borne haemoparasites in water buffalo from the Hubei province, south China was investigated using the reverse line blot (RLB) hybridization assay and phylogenetic analysis of the parasite 18S rRNA gene. Theileria buffeli (19.1%) was the most frequently found species in all of the locations, followed by Babesia orientalis (8.9%), Babesia bovis (1.0%) and Babesia bigemina (0.7%). Only 12 (3.9%) of the samples had mixed infections. Eleven samples with single infections were selected for further characterization using 18S rRNA gene sequence analysis. Phylogenetic analysis showed that the eight T. buffeli 18S rRNA gene sequences obtained grouped into four clusters, of which three grouped with the known T. buffeli types B and D. The remaining five grouped separately from the previously describe T. buffeli types, constituting new T. buffeli types. The two B. bigemina 18S rRNA gene sequences obtained grouped closely with B. bigemina Kunming; this serves as the first report of B. bigemina in the Hubei province. The B. orientalis Daye 18S rRNA gene sequence obtained grouped closely with the previously reported B. orientalis Wuhan strain and with Babesia sp. Kashi 1 and Kashi 2.

Identification of Theileria parva and Theileria sp. (buffalo) 18S rRNA gene sequence variants in the African Buffalo (Syncerus caffer) in Southern Africa

Theileria parva is the causative agent of Corridor disease in cattle in South Africa. The African buffalo (Syncerus caffer) is the reservoir host, and, as these animals are important for eco-tourism in South Africa, it is compulsory to test and certify them disease free prior to translocation. A T. parva-specific real-time polymerase chain reaction (PCR) test based on the small subunit ribosomal RNA (18S rRNA) gene is one of the tests used for the diagnosis of the parasite in buffalo and cattle in South Africa. However, because of the high similarity between the 18S rRNA gene sequences of T. parva and Theileria sp. (buffalo), the latter is also amplified by the real-time PCR primers, although it is not detected by the T. parva-specific hybridization probes. Preliminary sequencing studies have revealed a small number of sequence differences within the 18S rRNA gene in both species but the extent of this sequence variation is unknown. The aim of the current study was to sequence the 18S rRNA genes of T. parva and Theileria sp. (buffalo), and to determine whether all identified genotypes can be correctly detected by the real-time PCR assay. The reverse line blot (RLB) hybridization assay was used to identify T. parva and Theileria sp. (buffalo) positive samples from buffalo blood samples originating from the Kruger National Park, Hluhluwe-iMfolozi Park, the Greater Limpopo Transfrontier Park, and a private game ranch in the Hoedspruit area. T. parva and Theileria sp. (buffalo) were identified in 42% and 28%, respectively, of 252 samples, mainly as mixed infections. The full-length 18S rRNA gene of selected samples was amplified, cloned and sequenced. From a total of 20 sequences obtained, 10 grouped with previously published T. parva sequences from GenBank while 10 sequences grouped with a previously published Theileria sp. (buffalo) sequence. All these formed a monophyletic group with known pathogenic Theileria species. Our phylogenetic analyses confirm the distinction between Theileria sp. (buffalo) and T. parva and indicate the existence of a single group of T. parva and two Theileria sp. (buffalo) 18S rRNA gene variants in the African buffalo. Despite the observed variation in the full-length parasite 18S rRNA gene sequences, the area in the V4 hypervariable region where the RLB and real-time PCR hybridization probes were developed was relatively conserved. The T. parva specific real-time PCR assay was able to successfully detect all T. parva variants and, although amplicons were obtained from Theileria sp. (buffalo) DNA, none of the Theileria sp. (buffalo) 18S rRNA sequence variants were detected by the T. parva-specific hybridization probes.

Detection of bovine papillomavirus DNA in sarcoid-affected and healthy free-roaming zebra (Equus zebra) populations in South Africa

The endangered Cape mountain zebra (Equus zebra zebra) is protected in small numbers in a few isolated populations in South African game parks. Since 1995, sarcoid lesions appeared in zebras in two of the parks. This study was undertaken to investigate if bovine papillomavirus (BPV) is associated with sarcoids in these zebras. A conventional PCR, targeting the E5 ORF of BPV, and subsequent RFLP analysis were initially used to demonstrate the presence of BPV-1 and -2 DNAs in zebra sarcoid tumours. A rapid, sensitive and reliable real-time PCR to detect and distinguish between BPV-1 and -2 infections in zebras was developed. With this assay it was demonstrated that BPV-1 and -2 DNA (either single or mixed infections) are present in sarcoid tumour, healthy skin and blood of sarcoid-affected and healthy zebras from sarcoid-affected parks as well as in the blood of zebras from parks where no sarcoid has been observed before.

Co-infections determine patterns of mortality in a population exposed to parasite infection.

Highly protective effect of co-infections on mortality due to East Coast fever and consequences for disease epidemiology and control. Many individual hosts are infected with multiple parasite species, and this may increase or decrease the pathogenicity of the infections. This phenomenon is termed heterologous reactivity and is potentially an important determinant of both patterns of morbidity and mortality and of the impact of disease control measures at the population level. Using infections with Theileria parva (a tick-borne protozoan, related to Plasmodium) in indigenous African cattle [where it causes East Coast fever (ECF)] as a model system, we obtain the first quantitative estimate of the effects of heterologous reactivity for any parasitic disease. In individual calves, concurrent co-infection with less pathogenic species of Theileria resulted in an 89% reduction in mortality associated with T. parva infection. Across our study population, this corresponds to a net reduction in mortality due to ECF of greater than 40%. Using a mathematical model, we demonstrate that this degree of heterologous protection provides a unifying explanation for apparently disparate epidemiological patterns: variable disease-induced mortality rates, age-mortality profiles, weak correlations between the incidence of infection and disease (known as endemic stability), and poor efficacy of interventions that reduce exposure to multiple parasite species. These findings can be generalized to many other infectious diseases, including human malaria, and illustrate how co-infections can play a key role in determining population-level patterns of morbidity and mortality due to parasite infections.