Serological prevalence of Mycoplasma bovis infection in suckling beef cattle in France.

The prevalence of Mycoplasma bovis infection in France was assessed by means of a serological survey of suckling beef cattle, using an ELISA. The survey included 824 randomly selected herds in eight French counties and a total of 32,197 animals more than one year old. In each county, the number of herds tested was determined statistically on the basis of the hypothesis that about 40 per cent of herds are infected. The proportion of herds containing at least one infected animal ranged from 28 to 90 per cent depending on the county. Among the 32,197 sera tested, the animal infection rate ranged between 2 per cent and 13 per cent. In infected herds, the average number of positive animals per herd was between 10 and 20 per cent, and the infection was unevenly distributed among the areas tested.

Host-pathogen interactions in mycoplasma pathogenesis: virulence and survival strategies of minimalist prokaryotes.

Despite their very small genomes mycoplasmas are successful pathogens of man and a wide range of animal hosts. Because of the lack of effective therapeutics and vaccines, mycoplasma diseases continue to be a significant problem for public health as well as livestock production with major socio-economic consequences worldwide. Recent outbreaks and epidemiological studies predict that the incidence of human and animal mycoplasma diseases might increase which indicates the urgent need to develop new approaches for prevention and therapy. Development of such reagents, however, requires a solid understanding of the molecular biology of mycoplasma infections. Knowledge in this field has considerably increased during the past decade since new techniques have been developed and adapted to mycoplasmas that allow these organisms to be studied at the molecular level. Research on the two human pathogens Mycoplasma pneumoniae and Mycoplasma genitalium of which the genome sequences have recently been completed as well as the substantial number of studies carried out on the AIDS-associated mycoplasmas, Mycoplasma penetrans and Mycoplasma fermentans, has led the way, but a number of animal mycoplasmas are becoming increasingly appreciated as models for the study of the molecular basis of mycoplasma diseases. This review summarizes and highlights some of the recent findings concerning the molecular interactions that occur between pathogenic mycoplasmas and their hosts, both the common strategies as well as some unique approaches evolved by particular mycoplasma pathogens, including adherence to and uptake into non-phagocytic host cells, as well as mechanisms of escaping the host immune system.

Development of a recombinant antigen for antibody-based diagnosis of Mycoplasma bovis infection in cattle.

Mycoplasma bovis induces various clinical manifestations in cattle, such as mastitis, arthritis, and pneumonia. We have evaluated the immunoreactivity of three variable surface proteins (Vsps) of M. bovis, namely VspA, VspB, and VspC, with sera collected from herds with mycoplasmosis or from cattle experimentally infected with M. bovis. Western blot analysis revealed that the Vsps are the predominant antigens recognized by the host humoral response during M. bovis infection. The immunoreactivity of VspA, VspB, and VspC with host antibodies was independent of the clinical manifestations, the geographical origin of the M. bovis isolates, the mode of infection, and the animal's history. Moreover, the results showed that Vsp-specific host antibodies can be detected about 10 days after experimental infection and for up to several months. The full-length or truncated versions of the VspA product were overexpressed in Escherichia coli as fusion proteins (FP-VspA). Recombinant products showed strong immunoreactivity with the Vsp-specific monoclonal antibodies 1A1 and 1E5, with the corresponding epitopes localized at the VspA N-terminal and C-terminal ends, respectively. Anti-M. bovis sera of cattle naturally or experimentally infected also strongly recognized the full-length FP-VspA. The seroreactivity of sera collected from cattle between 6 and 10 days after experimental infection was weaker with truncated versions of VspA lacking the 1E5 epitope than with the full-length VspA or the truncated versions lacking the 1A1 epitope. Overall, the results indicate that the Vsps, despite their inter- and intraclonal variability, may be applied as target antigens in serodiagnostic assays for epidemiological studies.

Control levels of acetylcholinesterase expression in the mammalian skeletal muscle.

Protein expression can be controled at different levels. Understanding acetylcholinesterase (EC. 3.1.1.7, AChE) expression in the living organisms therefore necessitates: (1) determination and mapping of control levels of AChE metabolism; (2) identification of the regulatory factors acting at these levels; and (3) detailed insight into the mechanisms of action of these factors. Here we summarize the results of our studies on the regulation of AChE expression in the mammalian skeletal muscle. Three experimental models were employed: in vitro innervated human muscle, mechanically denervated adult fast rat muscle, and the glucocorticoid treated fast rat muscle. In situ hybridization of AChE mRNA, combined with AChE histochemistry, revealed that different distribution patterns of AChE, observed during in vitro ontogenesis and synaptogenesis of human skeletal muscle, reflect alterations in the distribution of AChE mRNA (Z. Grubic, R. Komel, W.F. Walker, A.F. Miranda, Myoblast fusion and innervation with rat motor nerve alter the distribution of acetylcholinesterase and its mRNA in human muscle cultures, Neuron 14 (1995) 317-327). To study the mechanisms of AChE mRNA loss in denervated adult rat skeletal muscle, we exposed deproteinated AChE mRNA to various subcellular fractions in vitro. Fractions were isolated from the normal and denervated rat sternomastoideus muscle. We found significantly increased, but non-specific AChE mRNA degradation capacities in the three fractions studied, suggesting that increased susceptibility of muscle mRNA to degradation might be at least partly responsible for the decreased AChE mRNA observed under such conditions (K. Zajc-Kreft, S. Kreft, Z. Grubic, Degradation of AChE mRNA in the normal and denervated rat skeletal muscle, Book of Abstracts, The Sixth International Meeting on Cholinesterases, La Jolla, CA, March 20-24, 1998, p. A3.). In adult fast rat muscle, treated chronically with glucocorticoids, we found the fraction of early synthesized AChE molecular forms to be reduced and AChE mRNA unchanged. This observation is consistent with the explanation that translation and/or early post-translational processes are impaired under such conditions (M. Brank, K. Zajc-Kreft, S. Kreft, R. Komel, Z. Grubic, Biogenesis of acetylcholinesterase is impaired, although its mRNA level remains normal, in the glucocorticoid-treated rat skeletal muscle, Eur. J. Biochem. 251 (1998) 374-381). The AChE mRNA level is therefore important but not the only control level of AChE expression in the mammalian skeletal muscle.

Glucocorticoids differentially control synthesis of acetylcholinesterase and butyrylcholinesterase in rat liver and brain.

Mammalian organisms possess two cholinesterases: acetylcholinesterase (AChE, EC 3.1.1.7.) and butyrylcholinesterase (BuChE, EC 3.1.1.8.). A clear explanation for this dual expression of acetylcholine-hydrolyzing enzymes is still missing. Better knowledge on how these two enzymes respond to various physiological or pharmacological factors would importantly contribute to the understanding of their function. The aim of the present study is to elucidate glucocorticoid (GC) influences on the synthesis of AChE and BuChE in rat liver and brain. Female Wistar rats were treated with dexamethasone until body weight loss was greater than 15%, signaling full expression of a GC response. At this stage, liver and brain were isolated and AChE and BuChE activities were determined in their homogenates. A new approach, based on precise radiometric measurements of AChE and BuChE activities in the polysomal fractions, prepared under non-denaturing conditions, was used to study GC influences on the early stages of biosynthesis of both enzymes. We found a differential GC influence on AChE and BuChE. In brain, only BuChE activity was affected (-30%), while AChE remained practically unchanged. In liver, BuChE activity fell by 60%, while AChE lost only 18% of its control activity. In case of BuChE, decreased activities in the whole homogenates correlated with decreased activities in the polysomal fractions, suggesting that early stages of enzyme biosynthesis were primarily affected. On the other hand, decreased AChE activity in liver homogenates was not paralleled by a significant change at the level of polysomal AChE activity in this organ, suggesting that higher AChE turn-over is primarily responsible for the decreased activity in homogenate. These results, together with the GC-mediated elimination of the correlation between brain and liver BuChE activities, strongly support the proposal of Edwards and Brimijoin (J.A. Edwards, S. Brimijoin, Effects of hypophysectomy on acetylcholinesterase and butyrylcholinesterase in the rat, Biochem. Pharmacol. 32 (1983) 1183-1189) that BuChE is regulated by systemically acting factors, including various hormones, while regulation of AChE is primarily tissue-specific.

Biogenesis of acetylcholinesterase is impaired, although its mRNA level remains normal, in the glucocorticoid-treated rat skeletal muscle.

Acetylcholinesterase (AChE) is responsible for the hydrolysis of acetylcholine in the neuromuscular junction and other cholinergic synapses. Insight into the mechanisms controlling AChE expression in skeletal muscle is important for understanding formation, plasticity, and various dysfunctions of the neuromuscular junction. We have investigated the mechanisms responsible for the decreased AChE activity in the fast rat sternomastoideus muscle after chronic glucocorticoid treatment. Under such conditions fast skeletal muscles become atrophic and loose 30-40% of their AChE activity. In order to establish at which level synthesis of AChE is affected by glucocorticoids, we studied the effects of chronic dexamethasone treatment at both AChE mRNA and mature enzyme levels. Reduced rate of AChE recovery after subtotal irreversible AChE inhibition was observed during the first week of dexamethasone treatment, but not later. Statistical analyses of four independent northern blots revealed unchanged AChE mRNA levels. At the same time, we observed more than 60% decrease in the (G1+G2)/A12 ratio of molecular forms at the expense of G forms. It has been generally accepted that globular G1 and G2 molecular forms are synthesized in the rough endoplasmic reticulum as precursors of asymmetric (A) AChE forms, assembled in the Golgi apparatus. Reduced levels of G1 and G2 AChE forms, in combination with unchanged AChE mRNA, are therefore consistent with the reports demonstrating that glucocorticoids downregulate muscle protein synthesis at the translational level. Our findings support but not entirely prove the concept that impaired translation and/or posttranslational control are the primary cause of decreased AChE activity in the glucocorticoid-treated muscle.

The influence of chronic treatment with dexamethasone on the acetylcholinesterase activity in rat skeletal muscle.

In the conditions of chronically elevated glucocorticoid agents in plasma, a drop in AChE activity of about 45% was reported. This data suggests the possibility that among other factors glucocorticoids also control AChE activity in the skeletal muscles. The question addressed in the present investigation was if AChE activity was reduced uniformly or selectively in the rat skeletal muscles after chronic application of dexamethasone? Selective effects of glucocorticoids on the AChE activity in different muscles and/or different types or regions of muscles would suggest the potential of these agents to regulate AChE metabolism in the skeletal muscle according to the environmental demands. Specific activity of skeletal muscle AChE was reduced in sternomastoideus (SM), extensor digitorum longus (EDL) and diaphragm (D) but not in soleus (SOL) after chronic dexamethasone treatment. Axial SM (white part) was more affected than distal white muscle EDL. AChE was better preserved in red rather than in white parts of muscles. The endplate-free region lost twice as much of specific AChE activity than the endplate-rich region. Our results suggest, but do not prove that glucocorticoid agents act in a selective way on the AChE metabolism of the skeletal muscles.

The effects of pretreatment with soman simulator in the skeletal muscle: direct interactions with acetylcholinesterase.

Soman simulator PDP is a compound that has a chemical structure identical to soman, except that the fluorine atom is replaced by a methyl group which makes PDP unable to bind covalently to the AChE active center. In rats, late mortality observed after treatment with high doses of soman could be prevented by PDP pretreatment. Such pretreatment has been much less efficient in primates. The effect of PDP in rats has been explained by blocking the deposition of soman in so-called soman depots in which soman is stored intact and subsequently released. In this paper we demonstrate that in the presence of PDP, inhibition of rat muscle AChE by soman is reduced in rat but not in human muscle homogenates. This result suggests that at least part of the beneficial effects of PDP pretreatment in rat might be due to the direct interaction of PDP with AChE resulting in reduced AChE phosphorylation by soman.

The influence of soman simulator on reactivation by HI-6 of soman-inhibited acetylcholinesterase in preparations of rat and human skeletal muscle.

The aim of our study was to elucidate the phenomenon called "soman depot". Our investigations were focused on the depot formed in the skeletal muscle and on the effects of 1,2,2-trimethylpropyl dimethylphosphonate (PDP), a reported blocker of soman depot formation. The following questions were addressed: (1) how much of acetylcholinesterase (EC 3.1.1.7, AChE) activity can additionally be recovered by Hagedorn bispiridinium oxime reactivator 2-hydroxyimino-methylpyridinium-1-methyl-4'-carbamoyl-pyridinium-1 '-methylether dichloride monohydrate (HI-6) in the skeletal muscle preparations if they are pretreated by PDP prior to incubation in soman (1,2,2-trimethylpropyl methylphosphonofluoridate)? (2) Is this effect uniform along the muscle fibre or different in the endplate in comparison to the endplate-free region? (3) Is the effect of PDP species specific, i.e. does it differ between rat and human muscle? (4) What are the molecular mechanisms of the effects of PDP? PDP pretreatment increased the reactivation of soman-inhibited AChE by HI-6 in both regions of rat skeletal muscle. This increase was smaller in human skeletal muscle. The PDP-mediated increase in HI-6 reactivation was most efficient in the endplate-rich region of rat diaphragm as demonstrated biochemically and histochemically, but it could not be explained by the blockade of soman depot alone since it was also observed at low soman concentrations, at which soman depot is not supposed to form. This PDP effect could be better explained by the direct interactions of PDP with AChE resulting in decreased AChE phosphorylation. Soman concentration-dependent increase in HI-6 reactivation by PDP, which was more efficient at a high than a low soman concentration and could therefore originate from blockade of soman depot, was observed in the endplate-free region of rat diaphragm. It was also found in human muscle but was again smaller in this species. According to our EPR study, solubilization of soman in the lipophilic cell membrane compartment can be excluded as a mechanism producing significant soman depot. In general, our results suggest a more complex mechanism of PDP action than reported previously.