Prevalence of contagious caprine pleuropneumonia in the Borana pastoral areas of Ethiopia

The study was conducted in selected districts of Borana pastoral areas of Ethiopia, namely Yabello, Dire, Moyale and Liban, during June–September 2004 to determine the status of contagious caprine pleuropneumonia (CCPP). The study includes a retrospective study, fieldwork, serology, an abattoir investigation and isolation of the causative Mycoplasma. Outbreaks of CCPP were reported from almost all regions of the country, especially from lowland areas, which are known goat-rearing regions, with the highest in 2002. The predominant clinical findings observed in affected flocks were coughing, nasal discharge, weakness, reluctance to move, abduction of elbow and laboured breathing. A total of 35 sheep and goats, of 183 examined, were sero-positive for MCCP F₃₈ antibodies using CFT. From the total of 217 goats examined in the abattoir for the presence of CCPP lesions, 21 (9.7%) showed pathological lesions, inflamed lungs with marbled appearance, fibrous pleuropneumonia, yellowish-coloured pleural fluid and swollen bronchial and mediastinal lymph nodes. The causative agent (Mycoplasma capricolum sp. capripneumoniae) was isolated and identified from tissue samples and thoracic fluid treated bacteriologically. In conclusion, the study indicates that CCPP is becoming a very important goat disease in Borana pastoral areas.     

Environmental and physiological factors affecting the succinate product ratio during carbohydrate fermentation by Actinobacillus sp. 130Z

Actinobacillus sp. 130Z fermented glucose to the major products succinate, acetate, and formate. Ethanol was formed as a minor fermentation product. Under CO₂-limiting conditions, less succinate and more ethanol were formed. The fermentation product ratio remained constant at pH values from 6.0 to 7.4. More succinate was produced when hydrogen was present in the gas phase. Actinobacillus sp. 130Z grew at the expense of fumarate and l-malate reduction, with hydrogen as an electron donor. Other substrates such as more-reduced carbohydrates (e.g., d-sorbitol) resulted in higher succinate and/or ethanol production. Actinobacillus sp. 130Z contained the key enzymes involved in the Embden-Meyerhof-Parnas and the pentose-phosphate pathways and contained high levels of phosphoenolpyruvate (PEP) carboxykinase, malate dehydrogenase, fumarase, fumarate reductase, pyruvate kinase, pyruvate formate-lyase, phosphotransacetylase, acetate kinase, malic enzyme, and oxaloacetate decarboxylase. The levels of PEP carboxykinase, malate dehydrogenase, and fumarase were significantly higher in Actinobacillus sp. 130Z than in Escherichia coli K-12 and accounted for the differences in succinate production. Key enzymes in end product formation in Actinobacillus sp. 130Z were regulated by the energy substrates. Received: 2 September 1996 / Accepted: 10 January 1997     

The epitope structure in lipopolysaccharide recognized by the serotype 2-specific monoclonal antibody of Antinobacillus pleuropneumoniae

Abstract: The polysaccharide structure recognized by a monoclonal antibody specific to serotype 2 lipopolysaccharide of Actinobacillus pleuropneumoniae was investigated using an enzyme-linked immunosorbent assay inhibition test. Lipopolysaccharide obtained from serotype 2, strain SH-15, was hydrolysed with acetic acid to liberate the polysaccharide portion, and the polysaccharide mixture was fractionated by gel filtration. The longer polysaccharide, composed of O -antigenic polysaccharide and core, fully inhibited the binding of monoclonal antibodies to a whole cell antigen of strain SH-15, whereas the core oligosaccharide without O -polysaccharide did not. No inhibition was observed with the monosaccharides which were the components of serotype 2 LPS. Enzyme-linked immunosorbent assay inhibition ability of O -polysaccharide was completely lost only by O -deacetylation. These results demonstrate that the epitope of the serotype-specific monoclonal antibody resided in O -polysaccharide of LPS and that the O -acetyl group was essential for the epitope structure.     

Actinobacillus pleuropneumoniae haemolysin II is secreted from Escherichia coli by A. pleuropneumoniae pleurotoxin secretion gene products

Abstract Actinobacillus pleuropneumoniae serotype 2 secretes type II haemolysin and pleurotoxin activities. Here, the genes for type II haemolysin were cloned in Escherichia coli , but type II haemolysin antigen and haemolysin activity were only detected intracellularly and not exported to culture supernatant. It has been reported that the genes for type II haemolysin are not linked to functional secretion genes, while those for pleurotoxin are. In this report the means of secretion of type II haemolysis was examined by constructing a hybrid plasmid carrying the genes required for type II haemolysin expression, together with determinants which allow secretion of pleurotoxin and are linked to the pleurotoxin toxin genes. These genes facilitated the export of type II haemolysin from E. coli , and may perform this function in A. pleuropneumoniae .     

Loop-mediated isothermal amplification targeting the apxIVA gene for detection of Actinobacillus pleuropneumoniae

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method performed under isothermal conditions with high specificity and efficiency. We developed a diagnostic method based on LAMP for detection of Actinobacillus pleuropneumoniae . Using six specific primers targeting the apxIVA gene, the LAMP assay rapidly amplified the target gene within 30 min, requiring only a laboratory water bath for the reaction to occur. The resulting amplificon was visualized by adding SYBR Green I to the mixture. The results obtained from testing 15 A. pleuropneumoniae reference strains and other seven bacterial species strains showed that the LAMP was as specific as and 10 times more sensitive than nested PCR. Sixty-five tonsil samples were collected from 65 healthy pigs. All the samples were negative for A. pleuropneumoniae by immunomagnetic separation-based (IMS) bacterial isolation, nested PCR and LAMP, respectively. Meanwhile, 115 tonsil samples were also collected from 115 pigs with apparent respiratory problems. Twenty-two were positive by IMS bacterial isolation. All the samples that were positive by IMS bacterial isolation were also positive by nested PCR and LAMP. The LAMP assay demonstrated exceptionally higher sensitivity than nested PCR by picking up 14 additional positive cases (χ² test, P <0.0001); we concluded that LAMP was a highly sensitive and reliable method for detection of A. pleuropneumoniae infection.     

Malate dehydrogenase and glucose-6-phosphate dehydrogenase, key markers for studying the genetic diversity of Actinobacillus actinomycetemcomitans

Abstract Cell-free extracts of strains belonging to the 5 serotypes of A. actinomycetemcomitans were screened for several enzymes. Enzymes representative of the pentose phosphate pathway/hexose monophosphate shunt and the TCA cycle were present. Of these glucose-6-phosphate dehydrogenase (G6PDH) and malate dehydrogenase (MDH) were the most readily detected and stable. MDH and G6PDH retained more than 50% of their activities at alkaline pHs (10–11) for up to 6 h and 3 h at 25°C, respectively, while at pH 6.5, 50% of their activities were lost within 2–3 h. The K _m for malate oxidation catalysed by MDH was 5.8×10⁻⁴ M while that for glucose-6-phosphate oxidation was 2.0×10⁻⁴ M. The pH optima for MDH and G6PDH oxidation activities were 10 and 9.5, respectively. Among the 5 designated serotypes of A. actinomycetemcomitans three groups were delineated by multilocus enzyme electrophoresis using MDH and G6PDH.     

Characterization of the omlA gene from different serotypes of Actinobacillus pleuropneumoniae: A new insight into an old approach

The OmlA protein is a virulence factor of Actinobacillus pleuropneumoniae, an important pathogen in pigs. The polymorphisms present in the omlA gene sequence of 15 reference serotypes of A. pleuropneumoniae and non-serotypable isolates were assessed to determine the possible evolutionary relationship among them and to validate the importance of this gene as a molecular marker for the characterization of this bacterium. Divergence among the 15 serotypes of A. pleuropneumoniae probably resulted initially from two major evolutionary events that led to subsequent differentiation into nine groups. This differentiation makes it possible to characterize most of the serotypes by using bionformatics, thereby avoiding problems with immunological cross-reactivity. A conserved α-helix common to all the serotypes was most likely involved in connecting the protein to the outer membrane and acting as a signal peptide. A previously unknown gene duplication was also identified and could contribute to the genetic variability that makes it difficult to serotype some isolates. Our data support the importance of the omlA gene in the biology of A. pleuropneumoniae and provide a new area of research into the OmlA protein.     

Microaerophilic property of Actinobacillus actinomycetemcomitans in fructose-limited chemostat cultures

Abstract The effect of oxygen on the growth, metabolism, and leukotoxin production of Actinobacillus actinomycetemcomitans 301-b was examined using a chemostat equipped with a redox potential control system. Steady states were obtained with fructose-limited cultures grown at a dilution rate of 0.1 h⁻¹ under strictly anaerobic ( E _h=−460 mV) and microaerobic conditions ( E _h≤ 150 mV) but not under highly aerated conditions ( E _h≥ 100 mV). The optimum growth was recorded at E _h=−300 to − 200 mV and the recorded Y _fructose value was about 1.3 times the Y _fructose of anaerobic cultures. Although the organism contains a respiratory chain, the increased Y _fructose under the microaerobic conditions might result from the increased substrate-level phosphorylation at the site of acetate kinase but not from electron transport phosphorylation. After passing threshold aeration ( E _h=−100 mV), the culture yielded a variant with transparent colony morphology. Under anaerobic conditions, the Y _fructose of the variant was about 1.6 times that of the original opaque colony-forming cells. The optimum growth of the variant was also recorded at E _h=− 300 to − 200 mV. In both types of cells, the production of leukotoxin reached a maximum at E _h=−350 to − 200 mV. These findings suggested the microaerophilic nature of A. actinomycetemcomitans .     

Efficient succinic acid production from high-sugar-content beverages by Actinobacillus succinogenes

This study presents the production of succinic acid (SA) by Actinobacillus succinogenes using high-sugar-content beverages (HSCBs) as feedstock. The aim of this study was the valorization of a by-product stream from the beverage industry for the production of an important building block chemical, such as SA. Three types of commercial beverages were investigated: fruit juices (pineapple and ace), syrups (almond), and soft drinks (cola and lemon). They contained mainly glucose, fructose, and sucrose at high concentration—between 50 and 1,000 g/L. The batch fermentation tests highlighted that A. succinogenes was able to grow on HSCBs supplemented with yeast extract, but also on the unsupplemented fruit juices. Indeed, the bacteria did not grow on the unsupplemented syrup and soft drinks because of the lack of indispensable nutrients. About 30–40 g/L of SA were obtained, depending on the type of HSCB, with yield ranging between 0.75 and 1.00 g_SA/g_S. The prehydrolysis step improved the fermentation performance: SA production was improved by 6–24%, depending on the HSCB, and sugar conversion was improved of about 30–50%.