The reporting of a Bacillus anthracis B-clade strain in South Africa after more than 20 years.

OBJECTIVES: Anthrax is a disease with an age old history in Africa caused by the Gram-positive endospore forming soil bacterium Bacillus anthracis. Epizootics of wild ungulates occur annually in the enzootic region of Pafuri, Kruger National Park (KNP) in the Limpopo Province of South Africa. Rigorous routine surveillance and diagnostics in KNP, has not revealed these rare isolates since the 1990s, despite unabated annual outbreaks. In 2011 a cheetah was diagnosed as anthrax positive from a private game reserve in Limpopo Province and reported to State Veterinary Services for further investigation. Isolation, molecular diagnostics, whole genome sequencing and comparative genomics were carried out for B. anthracis KC2011. RESULTS: Bacteriological and molecular diagnostics confirmed the isolate as B. anthracis. Subsequent typing and whole genome single nucleotide polymorphisms analysis indicated it clustered alongside B. anthracis SA A0091 in the B.Br.010 SNP branch. Unlike B. anthracis KrugerB strain, KC2011 strain has unique SNPs and represents a new branch in the B-clade. The isolation and genotypic characterisation of KC2011 demonstrates a gap in the reporting of anthrax outbreaks in the greater Limpopo province area. The identification of vulnerable and susceptible cheetah mortalities due to this strain has implications for conservation measures and disease control.

Comparative Biochemistry and In Vitro Pathway Reconstruction as Powerful Partners in Studies of Metabolic Diversity.

There are estimated to be >300,000 plant species, producing >200,000 metabolites. Many of these metabolites are restricted to specific plant lineages and are referred to as "specialized" metabolites. These serve varied functions in plants including defense against biotic and abiotic stresses, plant-plant and plant-microbe communication, and pollinator attraction. These compounds also have important applications in agriculture, medicine, skin care, and in diverse aspects of human culture. The specialized metabolic repertoire of plants can vary even within and between closely related species, in terms of the number and classes of specialized metabolites as well as their structural variants. This phenotypic variation can be exploited to discover the underlying variation in the metabolic enzymes. We describe approaches for using the diversity of specialized metabolites and variation in enzyme structure and function to identify novel enzymatic activities and understand the structural basis for these differences. The knowledge obtained from these studies will provide new modules for the synthetic biology toolbox.

Malignant Catarrhal Fever: An Emerging Disease in the African Buffalo (Syncerus caffer).

Within the tribe Bovini in the subfamily Bovinae, the water buffalo (Bubalus Bubalis), American bison (Bison bison), European bison (Bubalus bonasus) and yak (Bos grunniens) are recognized as species highly susceptible to malignant catarrhal fever (MCF). In contrast, the lack of reports describing clinical MCF in the African buffalo (Syncerus caffer) whether free ranging or captive has led to a perception that African buffaloes are resistant to MCF. During the last decade, several cases of MCF in African buffaloes were confirmed in South Africa and experience with seven of these cases is described in this report. Detection of viral nucleic acid in blood or tissues was successful in six African buffaloes that suffered from clinical signs compatible with MCF. Four were positive for infection with ovine herpesvirus type 2 (the causative virus of sheep-associated MCF), and two were positive for alcelaphine herpesvirus type 1 (causative virus of wildebeest-associated MCF). Histopathological examination of tissue samples from all the animals yielded typical lesions that were consistent with those described for MCF in domestic cattle. Developments in the management of African buffaloes translocated from their traditional habitats have likely contributed to the identification of another susceptible host in the subfamily Bovinae.

A field trial evaluation of the prophylactic efficacy of amitraz-impregnated collars against canine babesiosis (Babesia canis rossi) in South Africa.

South African canine babesiosis caused by Babesia canis rossi is a common clinical disease in dogs in South Africa and remains a significant cause of domestic dog mortality. To determine whether tick-repellent, 9% amitraz-impregnated tick collars (Preventic-Virbac) could prevent tick-borne exposure to B. canis rossi, 50 dogs were assigned to two groups. Group 1 (20 dogs), polymerase chain reaction (PCR)--and reverse line blot (RLB)-negative for B. canis rossi, were fitted with amitraz collars and blood samples collected monthly, over a 6-month period, and analysed for B. canis rossi. Group 2 (30 dogs) included 5 dogs selected on a month-by-month basis from a population of dogs from the same geographical area as the group 1 dogs, but with no history of previous tick control, which were blood-sampled together with the treatment group and analysed for B. canis rossi by PCR and RLB, to serve as the control group. Eight of the 30 control dogs (26.6%) were PCR/RLB positive for B. canis rossi, indicating high pathogen exposure during the trial period. All twenty of the treatment group dogs remained negative for B. canis rossi throughout the 6 months of the trial. These results suggest that the use of amitraz-impregnated collars had a significant effect on reducing infection with B. canis rossi.

An outbreak of perirenal oedema syndrome in cattle associated with ingestion of pigweed (Amaranthus hybridus L.).

Forty seven of 150, 15-month-old long weaners died of an acute renal disease syndrome following introduction into an old maize field with a heavy stand of Amaranthus spp. The clinical syndrome was characterised by sudden onset neurological disease with ataxia and recumbency. Subcutaneous oedema, ascites and perirenal oedema with urine odour were the major gross necropsy findings. Renal histopathology revealed marked coagulative renal tubular necrosis of the proximal and distal straight tubules with intertubular haemorrhage. Acute renal failure and perirenal oedema has been described in cattle, pigs, horses and sheep associated with the ingestion of A. hybridus L. and A. retroflexus L. This perirenal oedema syndrome has been widely reported in the Americas, while in South Africa intoxication with the amaranths has only previously been associated with nitrate and possibly oxalate poisoning in cattle.

Congenital dilatation of the large and segmental intrahepatic bile ducts (Caroli's disease) in two Golden retriever littermates.

Two, sibling, male Golden retriever puppies, 13 weeks of age, were presented with congenital biliary cysts of the liver involving both hepatic and segmental bile ducts, as well as bilateral polycystic kidney disease. Ultrasonography of the livers of both pups demonstrated segmental cystic lesions that were contiguous with the bile ducts. Histopathology revealed cystic ectatic bile duct hyperplasia and dysplasia with variable portal fibrosis in the liver, while in the kidneys there were radially arranged, cylindrically dilated cysts of the collecting ducts, which extended through the medulla and cortex. This pathology was compatible with that of congenital dilatation of the large and segmental bile ducts (Caroli's disease) described in humans, dogs and rats. In humans Caroli's disease has an autosomal recessive inheritance pattern, while in rats activation of the MEK5/ERK cascade initiates the biliary dysgenesis of Caroli's disease in this species. However, the exact mode of inheritance and pathogenesis of Caroli's disease in dogs is as yet unknown. Previous reports on congenital hepatic cystic diseases of the dog have described Caroli's disease like lesions in various breeds, but these are believed to be the 1st reported cases in the Golden retriever breed.

Possible death of a buffalo calf (Syncercus caffer) due to suspected heartwater (Ehrlichia ruminantium).

Rickettsial organisms resembling Ehrlichia ruminantium (the causative organism of heartwater) were demonstrated in brain smears and formalin-fixed brain sections derived from a buffalo calf that died on a private game reserve in northern KwaZulu-Natal. The possibility that the tick-free environment of a quarantine boma may have affected the calf's immunity, is discussed. These findings suggest that monitoring heartwater in wild ruminants and making brain smears as a routine during post mortem evaluations of wild ruminants, should be encouraged.

Isolation of Haemophilus somnus from dairy cattle in kwaZulu-Natal. An emerging cause of 'dirty cow syndrome' and infertility?

Haemophilus somnus was consistently isolated from vaginal discharges of dairy cows submitted from field cases of vaginitis, cervicitis and/or metritis in the KwaZulu-Natal Midlands during the period July 1995 - December 2000 and from the East Griqualand area in November/December 2000. The purulent vaginal discharges, red granular vaginitis and cervicitis, and pain on palpation described in these cases was very similar to that reported in outbreaks of H.somnus endometritis syndrome in Australia, Europe and North America. In all the herds involved in these outbreaks, natural breeding with bulls was employed. Although there was a good cure rate in clinically-affected animals treated with tetracyclines, culling rates for chronic infertility were unacceptably high. Employment of artificial insemination in these herds improved pregnancy rates in cows that had calved previously, but many cows that had formerly been infected failed to conceive.

Arabidopsis cyt1 mutants are deficient in a mannose-1-phosphate guanylyltransferase and point to a requirement of N-linked glycosylation for cellulose biosynthesis.

Arabidopsis cyt1 mutants have a complex phenotype indicative of a severe defect in cell wall biogenesis. Mutant embryos arrest as wide, heart-shaped structures characterized by ectopic accumulation of callose and the occurrence of incomplete cell walls. Texture and thickness of the cell walls are irregular, and unesterified pectins show an abnormally diffuse distribution. To determine the molecular basis of these defects, we have cloned the CYT1 gene by a map-based approach and found that it encodes mannose-1-phosphate guanylyltransferase. A weak mutation in the same gene, called vtc1, has previously been identified on the basis of ozone sensitivity due to reduced levels of ascorbic acid. Mutant cyt1 embryos are deficient in N-glycosylation and have an altered composition of cell wall polysaccharides. Most notably, they show a 5-fold decrease in cellulose content. Characteristic aspects of the cyt1 phenotype, including radial swelling and accumulation of callose, can be mimicked with the inhibitor of N-glycosylation, tunicamycin. Our results suggest that N-glycosylation is required for cellulose biosynthesis and that a deficiency in this process can account for most phenotypic features of cyt1 embryos.

Identification of ascorbic acid-deficient Arabidopsis thaliana mutants.

Vitamin C (l-ascorbic acid) is a potent antioxidant and cellular reductant present at millimolar concentrations in plants. This small molecule has roles in the reduction of prosthetic metal ions, cell wall expansion, cell division, and in the detoxification of reactive oxygen generated by photosynthesis and adverse environmental conditions. However, unlike in animals, the biosynthesis of ascorbic acid (AsA) in plants is only beginning to be unraveled. The previously described AsA-deficient Arabidopsis mutant vtc1 (vitamin c-1) was recently shown to have a defect in GDP-mannose pyrophosphorylase, providing strong evidence for the recently proposed role of GDP-mannose in AsA biosynthesis. To genetically define other AsA biosynthetic loci, we have used a novel AsA assay to isolate four vtc mutants that define three additional VTC loci. We have also isolated a second mutant allele of VTC1. The four loci represented by the vtc mutant collection have been genetically characterized and mapped onto the Arabidopsis genome. The vtc mutants have differing ozone sensitivities. In addition, two of the mutants, vtc2-1 and vtc2-2, have unusually low levels of AsA in the leaf tissue of mature plants.

LSD1 regulates salicylic acid induction of copper zinc superoxide dismutase in Arabidopsis thaliana.

We characterized the accumulation patterns of Arabidopsis thaliana proteins, two CuZnSODs, FeSOD, MnSOD, PR1, PR5, and GST1, in response to various pathogen-associated treatments. These treatments included inoculation with virulent and avirulent Pseudomonas syringae strains, spontaneous lesion formation in the lsd1 mutant, and treatment with the salicylic acid (SA) analogs INA (2,6-dichloroisonicotinic acid) and BTH (benzothiadiazole). The PR1, PR5, and GST1 proteins were inducible by all treatments tested, as expected from previous mRNA blot analysis. The two CuZnSOD proteins were induced by SA analogs and in conjunction with lsd1-mediated spreading cell death. Additionally, LSD1 is a part of a signaling pathway for the induction of the CuZnSOD proteins in response to SA but not in lsd1-mediated cell death. We suggest that the spreading lesion phenotype of lsd1 results from a lack of up-regulation of a CuZnSOD responsible for detoxification of accumulating superoxide before the reactive oxygen species can trigger a cell death cascade.

Genetic evidence for the role of GDP-mannose in plant ascorbic acid (vitamin C) biosynthesis.

Vitamin C (L-ascorbic acid; AsA) acts as a potent antioxidant and cellular reductant in plants and animals. AsA has long been known to have many critical physiological roles in plants, yet its biosynthesis is only currently being defined. A pathway for AsA biosynthesis that features GDP-mannose and L-galactose has recently been proposed for plants. We have isolated a collection of AsA-deficient mutants of Arabidopsis thaliana that are valuable tools for testing of an AsA biosynthetic pathway. The best-characterized of these mutants (vtc1) contains approximately 25% of wild-type AsA and is defective in AsA biosynthesis. By using a combination of biochemical, molecular, and genetic techniques, we have demonstrated that the VTC1 locus encodes a GDP-mannose pyrophosphorylase (mannose-1-P guanyltransferase). This enzyme provides GDP-mannose, which is used for cell wall carbohydrate biosynthesis and protein glycosylation as well as for AsA biosynthesis. In addition to genetically defining the first locus involved in AsA biosynthesis, this work highlights the power of using traditional mutagenesis techniques coupled with the Arabidopsis Genome Initiative to rapidly clone physiologically important genes.

UV-B-induced photomorphogenesis in Arabidopsis thaliana.

Relatively little is known about the types of photomorphogenic responses and signal transduction pathways that plants employ in response to ultraviolet-B (UV-B, 290-320 nm) radiation. In wild-type Arabidopsis seedlings, hypocotyl growth inhibition and cotyledon expansion were both reproducibly promoted by continuous UV-B. The fluence rate response of hypocotyl elongation was examined and showed a biphasic response. Whereas photomorphogenic responses were observed at low doses, higher fluences resulted in damage symptoms. In support of our theory that photomorphogenesis, but not damage, occurs at low doses of UV-B, photomorphogenic responses of UV-B sensitive mutants were indistinguishable from wild-type plants at the low dose. This allowed us to examine UV-B-induced photomorphogenesis in photoreceptor deficient plants and constitutive photomorphogenic mutants. The cry1 cryptochrome structural gene mutant, and phytochrome deficient hy1, phyA and phyB mutant seedlings resembled wild-type seedlings, while phyA/phyB double mutants were less sensitive to the photomorphogenic effects of UV-B. These results suggest that either phyA or phyB is required for UV-B-induced photomorphogenesis. The constitutive photomorphogenic mutants cop1 and det1 did not show significant inhibition of hypocotyl growth in response to UV-B, while det2 was strongly affected by UV-B irradiation. This suggests that COP1 and DET1 work downstream of the UV-B signaling pathway.

Superoxide dismutase in Arabidopsis: an eclectic enzyme family with disparate regulation and protein localization.

A number of environmental stresses can lead to enhanced production of superoxide within plant tissues, and plants are believed to rely on the enzyme superoxide dismutase (SOD) to detoxify this reactive oxygen species. We have identified seven cDNAs and genes for SOD in Arabidopsis. These consist of three CuZnSODs (CSD1, CSD2, and CSD3), three FeSODs (FSD1, FSD2, and FSD3), and one MnSOD (MSD1). The chromosomal location of these seven SOD genes has been established. To study this enzyme family, antibodies were generated against five proteins: CSD1, CSD2, CSD3, FSD1, and MSD1. Using these antisera and nondenaturing-polyacrylamide gel electrophoresis enzyme assays, we identified protein and activity for two CuZnSODs and for FeSOD and MnSOD in Arabidopsis rosette tissue. Additionally, subcellular fractionation studies revealed the presence of CSD2 and FeSOD protein within Arabidopsis chloroplasts. The seven SOD mRNAs and the four proteins identified were differentially regulated in response to various light regimes, ozone fumigation, and ultraviolet-B irradiation. To our knowledge, this is the first report of a large-scale analysis of the regulation of multiple SOD proteins in a plant species.

Induction of Arabidopsis tryptophan pathway enzymes and camalexin by amino acid starvation, oxidative stress, and an abiotic elicitor.

The tryptophan (Trp) biosynthetic pathway leads to the production of many secondary metabolites with diverse functions, and its regulation is predicted to respond to the needs for both protein synthesis and secondary metabolism. We have tested the response of the Trp pathway enzymes and three other amino acid biosynthetic enzymes to starvation for aromatic amino acids, branched-chain amino acids, or methionine. The Trp pathway enzymes and cytosolic glutamine synthetase were induced under all of the amino acid starvation test conditions, whereas methionine synthase and acetolactate synthase were not. The mRNAs for two stress-inducible enzymes unrelated to amino acid biosynthesis and accumulation of the indolic phytoalexin camalexin were also induced by amino acid starvation. These results suggest that regulation of the Trp pathway enzymes under amino acid deprivation conditions is largely a stress response to allow for increased biosynthesis of secondary metabolites. Consistent with this hypothesis, treatments with the oxidative stress-inducing herbicide acifluorfen and the abiotic elicitor alpha-amino butyric acid induced responses similar to those induced by the amino acid starvation treatments. The role of salicylic acid in herbicide-mediated Trp and camalexin induction was investigated.

L-ascorbic acid metabolism in the ascorbate-deficient arabidopsis mutant vtc1.

The biosynthesis of L-ascorbic acid (vitamin C) is not well understood in plants. The ozone-sensitive Arabidopsis thaliana mutant vitamin c-1 (vtc1; formerly known as soz1) is deficient in ascorbic acid, accumulating approximately 30% of wild-type levels. This deficiency could result from elevated catabolism or decreased biosynthesis. No differences that could account for the deficiency were found in the activities of enzymes that catalyze the oxidation or reduction of ascorbic acid. The absolute rate of ascorbic acid turnover is actually less in vtc1 than in wild type; however, the turnover rate relative to the pool of ascorbic acid is not significantly different. The results from [U-14C]Glc labeling experiments suggest that the deficiency is the result of a biosynthetic defect: less L-[14C]ascorbic acid as a percentage of total soluble 14C accumulates in vtc1 than in wild type. The feeding of two putative biosynthetic intermediates, D-glucosone and L-sorbosone, had no positive effect on ascorbic acid levels in either genotype. The vtc1 defect does not appear to be the result of a deficiency in L-galactono-1,4-lactone dehydrogenase, an enzyme able to convert L-galactono-1,4-lactone to ascorbic acid.

Introns act post-transcriptionally to increase expression of the Arabidopsis thaliana tryptophan pathway gene PAT1.

The expression of the Arabidopsis thaliana PAT1 gene, which encodes the tryptophan biosynthetic enzyme phosphoribosylanthranilate transferase, was investigated using translational fusions of the PAT1 promoter to the GUS reporter gene. Independent stably transformed A. thaliana lines containing a single copy of a fusion that includes the entire plastid transit peptide and the first two introns of PAT1 had on average 30 times more GUS enzyme activity than plants transformed with a construct in which GUS was fused a short distance downstream of the PAT1 start codon. Plants containing the construct without introns or leader peptide accumulated undetectable amounts of PAT1-GUS fusion protein and mRNA, even though the transcriptional rate of both fusion constructs was comparable. Fusions containing the entire transit peptide and either of the first two introns yield as much GUS activity as constructs containing both introns, but constructs containing the transit peptide but no introns give rise to much lower levels. Therefore, introns greatly enhance the expression of PAT1-GUS fusions, and they act post-transcriptionally to increase the steady-state level of mRNA.

An enzyme similar to animal type II photolyases mediates photoreactivation in Arabidopsis.

The important issue of photoreactivation DNA repair in plants has become even more interesting in recent years because a family of genes that are highly homologous to photoreactivating DNA repair enzymes but that function as blue light photoreceptors has been isolated. Here, we report the isolation of a novel photolyase-like sequence from Arabidopsis designated PHR1 (for photoreactivating enzyme). It shares little sequence similarity with either type I photolyases or the cryptochrome family of blue light photoreceptors. Instead, the PHR1 gene encodes an amino acid sequence with significant homology to the recently characterized type II photolyases identified in a number of prokaryotic and animal systems. PHR1 is a single-copy gene and is not expressed in dark-grown etiolated seedlings: the message is light inducible, which is similar to the expression profile for photoreactivation activity in plants. The PHR1 protein complements a photolyase-deficient mutant of Escherichia coli and thus confers photoreactivation activity. In addition, an Arabidopsis mutant that is entirely lacking in photolyase activity has been found to contain a lesion within this Arabidopsis type II photolyase sequence. We conclude that PHR1 represents a genuine plant photolyase gene and that the plant genes with homology to type I photolyases (the cryptochrome family of blue light photoreceptors) do not contribute to photoreactivation repair, at least in the case of Arabidopsis.