Actinobacillus pleuropneumoniae induces SJPL cell cycle arrest in G2/M-phase and inhibits porcine reproductive and respiratory syndrome virus replication

Background: Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important pathogens in the swine industry and causes important economic losses. No effective antiviral drugs against it are commercially available. We recently reported that the culture supernatant of Actinobacillus pleuropneumoniae, the porcine pleuropneumonia causative agent, has an antiviral activity in vitro against PRRSV in SJPL cells. Objectives of this study were (i) to identify the mechanism behind the antiviral activity displayed by A. pleuropneumoniae and (ii) to characterize the active molecules present in the bacterial culture supernatant. Methods: Antibody microarray analysis was used in order to point out cellular pathways modulated by the A. pleuropneumoniae supernatant. Subsequent, flow cytometry analysis and cell cycle inhibitors were used to confirm antibody microarray data and to link them to the antiviral activity of the A. pleuropneumoniae supernatant. Finally, A. pleuropneumoniae supernatant characterization was partially achieved using mass spectrometry. Results: Using antibody microarray, we observed modulations in G2/M-phase cell cycle regulation pathway when SJPL cells were treated with A. pleuropneumoniae culture supernatant. These modulations were confirmed by a cell cycle arrest at the G2/M-phase when cells were treated with the A. pleuropneumoniae culture supernatant. Furthermore, two G2/M-phase cell cycle inhibitors demonstrated the ability to inhibit PRRSV infection, indicating a potential key role for PRRSV infection. Finally, mass spectrometry lead to identify two molecules (m/z 515.2 and m/z 663.6) present only in the culture supernatant. Conclusions: We demonstrated for the first time that A. pleuropneumoniae is able to disrupt SJPL cell cycle resulting in inhibitory activity against PRRSV. Furthermore, two putative molecules were identified from the culture supernatant. This study highlighted the cell cycle importance for PRRSV and will allow the development of new prophylactic or therapeutic approaches against PRRSV.

Genotipificação de amostras sorotipificáveis e não sorotipificáveis de Actinobacillus pleuropneumoniae através de RAPD.

Actinobacillus pleuropneumoniae é o agente etiológico da pleuropneumonia suína, enfermidade amplamente distribuída no rebanho suíno mundial, responsável por prejuízos econômicos relevantes. Possui 12 sorotipos, determinados por técnicas de sorotipificação. Além disso é descrita a ocorrência de amostras não sorotipificáveis. O conhecimento do sorotipo prevalente nos surtos da enfermidade é necessário aos programas de profilaxia. Procurando contornar as dificuldades normalmente encontradas na sorotipificação de A. pleuropneumpnoae, a técnica de RAPD foi avaliada na genotipificação de amostras sorotipificáveis e não sorotipificáveis do agente. Foram utilizados amostras ATCC dos 12 sorotipos e amostras dos sorotipos, 1, 3, 5a, 5b, 7, 11 e 12 isolados no Brasil. Os primers OPG e OPG-19, utilizados individualmente nas reações, foram mais adequados para a diferenciação dos sorotipos. O primer OPG-19 detectou polimorfismos semelhantes entrer os sorotipos 1, 3, 4, 5 e 11; e sorotipos 7 e 12. O perfil de RAPD detectado pelo primier OPGF-10 diferenciou os isolados de campo dos sorotipos 1, 7, 11 e 12. Os sorotipos 3 e 5 apresentaram padrão de RAPD semelhantes, sendo diferenciados pelo perfil de exotoxinas característico, determinado previamente através de PCR. Este primer identificou quatro diferentes perfis de RAPD no sorotipo 3. Um destes foi semelhante ao obtido como sorotipo 11. Neste isolado, foi detecta a presença dos genes para ApxI e ApxII, características do sorotipo 11. As amostras do sorotipo 4 apresentaram perfil de RAPD semelhante ao identificado nos sorotipos 3 ou 5 com o primeir OPG-10, sendo identificada, por PCR, a presença dos genes para ApxI e ApxI, os quais não são característicos do sorotipo. Estas amostras foram isoladas em anos posteriores à amostras dos sorotipos 3 e 5 analisadas. Foi possível caracterizar 14 das 14 amostras não sorotipificáveis de A.pleuropneumpniae obtidas de suínos com sinais da doença. Entre as 4 amostras não sorotipificáveis isoladas de leitões sem sinais clínicos, apenas uma foi caracterizada através de RAPD. É possível que as demais amostras sejam outrtas bactérias NAD-dependente isoladas do trato respiratório de suínos. Amostras caracterizadas como A. minor e A. indolicus apresentaram perfis de RAPD divergentes dos identificados em isolados puros de A. pleuropneumoniae, comprovando a capacidade da técnica na caracterização do agente. Diferentes amostras do mesmo sorotipo de A. pleuropneumoniae apresentaram polimorfismos de RAPD idênticos, demonstrando reprodutividade da técnica. Os resultados comprovam a capacidade de tipificação de A. Pleuropneumoniae através de RAPD. A pesquisa de primers adequados para a diferenciação dos sorotipos 3, 4 e 5 aprimorar sua caracterização, o que pode vir a contribuir com as técnicas de sorotipificação tradicionalmente utilizados, ou permitir o uso como método de confirmação nas amostras cuja sorotipificação é problemática.

Padronização de três ELISAs polivalentes com lipopolissacarídeos de cadeia longa dos sorotipos 1 e 5, 2, 3 e 7 ou 10 e 12 de Actinobacillus pleuropneumoniae

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Doença de Chagas aguda pós-transfusional sem miocardite

Trata-se de paciente do sexo feminino, com 59 anos de idade, procedente de Itaporanga (SP), diabética e nefropata crônica, internada em virtude de surtos de pielonefnte e insuficiência renal aguda. Dentre outras medidas terapêuticas, recebeu transfusão de sangue. Cerca de dois dias após a última transfusão (sangue oriundo de doador, posteriormente identificado como chagásico) encontraram-se formas tripomastigotas de Trypanosoma cruzi em lâmina preparada para execução de hemograma. Iniciou-se tratamento com Benzonidazol. A paciente cursou para, pleuropneumonia e de secreção purulenta cirúrgica isolou-se Klebsiella spp. A septicemia conduziu a paciente ao êxito letal. Nenhuma lesão tecidual foi observada no miocárdio, no sistema nervoso central, adrenal ou nos demais órgãos examinados.

Atualidades no tratamento da laminite em equinos

Laminitis is still a major cause of lameness in horses, causing damage to many interested in it. It has been long studied, but there are several points that are not yet fully understood. Doubts about its pathogenesis end up extending at the treatments. Just as there are several theories that attempt to explain the mechanisms of its development, also appear treatments that are sometimes contradictory and inconsistent. Mostly times, laminitis is a consequence of another disease process that occurs systemically in the body. Among them are the main grain overload and processes that cause the animal to the table of endotoxemia, strangled colic, metritis with retained placenta and pleuropneumonia. The proposed treatments include the use of anti-inflammatory drugs, cryotherapy, use of vasodilators and correction of Starling forces, and other measures to support the frog. The stall should be bedded deeply with sand or other material that provides support to the frog

Hyperacute necrotizing pneumonia and septicaemia caused by to Clostridium perfringens in Cervus timorensis: case report

Clostridia are uncommon causes of pleuropneumonia in wildlife In human and domestic animals,different hemorrhagic pneumonia with involvement of the pleura. In livestock, most cases are associated with sudden changes of diet, iatrogenic lesionscaused by invasive procedures such as thoracente thoracotomy, or traumatic percutaneous introduction of the microorganism.The clinical course of pleuropneumonia by clostridia infections may be very variable, although usually are associated with hyperacute or acute course and high mortality. The pr necrotizing pneumonia and sepsis caused by hyperacute fatal course, highlighting clinical, epidemiological, microbiological, and histopathological aspects.

[Intrathoracic esophageal perforation of unknown cause in four horses] Intrathorakale Oesophagusperforation ungeklärter Genese bei vier Pferden

Three horses (age 17 - 23 years) were referred to the equine clinic of the University of Berne due to colic, fever, tachycardia and tachypnea. All horses showed pleural effusion. Clinical findings in 2 of the horses were highly suggestive of an intra-thoracic esophageal perforation. Severe septic pleuropneumonia without suspicion of an esophageal lesion was diagnosed in the 3rd horse. In addition, an 11 year old stallion was referred to the equine clinic for treatment of a presumptive large colon impaction. The horse was given laxatives after nasogastric intubation. Subsequent dramatic clinical deterioration and signs consistent with severe pleuropneumonia suggest that esophageal perforation had occurred when passing the nasogastric tube. All 4 horses were euthanized due to a poor prognosis. Esophageal perforation was diagnosed or confirmed post mortem in all cases. A hypertrophy of the tunica muscularis of the intra-thoracic esophagus was found in 3 of 4 horses.

A new variant of Actinobacillus pleuropneumoniae serotype 3 lacking the entire apxII operon

Actinobacillus pleuropneumoniae is an important respiratory pathogen causing pleuropneumonia in pig. The species is genetically characterized by the presence of 4 RTX (Repeats in the Structural ToXin) toxin genes: apxI, apxII, and apxIII genes are differentially present in various combinations among the different serotypes, thereby defining pathogenicity; the apxIV gene is present in all serotypes. Polymerase chain reaction (PCR)-based apx gene typing is done in many veterinary diagnostic laboratories, especially reference laboratories. The present report describes the isolation of atypical A. pleuropneumoniae from 4 independent cases from 2 countries. All isolates were beta-nicotinamide adenine dinucleotide (beta-NAD) dependent and nonhemolytic but showed strong co-hemolysis with the sphingomyelinase of Staphylococcus aureus on sheep blood agar. Classical biochemical tests as well as Matrix-assisted laser desorption ionization time-of-flight mass spectrometry and sequence-based analysis (16S ribosomal RNA [rRNA] and rpoB genes) identified them as A. pleuropneumoniae. Apx-toxin gene typing using 2 different PCR systems showed the presence of apxIV and only the apxIII operon (apxIIICABD). None of the apxI or apxII genes were present as confirmed by Southern blot analysis. The 16S rRNA and rpoB gene analyses as well as serotype-specific PCR indicate that the isolates are variants of serotype 3. Strains harboring only apxIV and the apxIII operon are possibly emerging types of A. pleuropneumoniae and should therefore be carefully monitored for epidemiological reasons.

Comparative analysis of four lipoproteins from Mycoplasma mycoides subsp. mycoides Small Colony identifies LppA as a major T-cell antigen

Control of contagious bovine pleuropneumonia (CBPP), caused by Mycoplasma mycoides subsp. mycoides Small Colony (MmmSC), remains an important goal in Africa. Subunit vaccines triggering B and T-cell responses could represent a promising approach. To this aim, the T-cell immunogenicity of four MmmSC lipoproteins (LppA, LppB, LppC and LppQ), present in African strains and able to elicit humoral response, was evaluated. In vitro assays revealed that only LppA was recognized by lymph node lymphocytes taken from three cattle, 3 weeks after MmmSC exposure. Maintenance of the LppA-specific response, relying on CD4 T-cells and IFN gamma production, was then demonstrated 1 year after infection. LppA is thus an important target for the CD4 T-cells generated early after MmmSC infection and persisting in the lymph nodes of recovered cattle. Its role as a protective antigen and ability to in vivo trigger both arms of the host immune response remain to be evaluated.

Detection of Mycoplasma mycoides subsp. mycoides SC in bronchoalveolar lavage fluids of cows based on a TaqMan real-time PCR discriminating wild type strains from an lppQ(-) mutant vaccine strain used for DIVA-strategies

Contagious bovine pleuropneumonia (CBPP) is the most serious cattle disease in Africa, caused by Mycoplasma mycoides subsp. mycoides small-colony type (SC). CBPP control strategies currently rely on vaccination with a vaccine based on live attenuated strains of the organism. Recently, an lppQ(-) mutant of the existing vaccine strain T1/44 has been developed (Janis et al., 2008). This T1lppQ(-) mutant strain is devoid of lipoprotein LppQ, a potential virulence attribute of M. mycoides subsp. mycoides SC. It is designated as a potential live DIVA (Differentiating Infected from Vaccinated Animals) vaccine strain allowing both serological and etiological differentiation. The present paper reports on the validation of a control strategy for CBPP in cattle, whereby a TaqMan real-time PCR based on the lppQ gene has been developed for the direct detection of M. mycoides subsp. mycoides SC in ex vivo bronchoalveolar lavage fluids of cows and for the discrimination of wild type strains from the lppQ(-) mutant vaccine strain.

Thoraxschmerzen bei Kindern und Jugendlichen - das Herz?

Chest pain in children and adolescents is a frequent observation, although potentially relevant disease is rather rare and then found in situations with acute presentation. In children with an inflammatory/infectious clinical context the differential diagnosis is oesophagitis, pleuropneumonia or pericarditis. Potentially dangerous complications may be found in youth with predisposing conditions for aortic dissection, pneumothorax or pulmonary embolism, or even in rare instances for an acute coronary complication. In these cases aggressive diagnostic work-up is mandatory. In the frequent elective outpatient evaluation of teenagers with long-lasting episodes of chest pain, relevant underlying cardiovascular disease only rarely can be found as the cause. In the elective outpatient evaluation for chest pain, usually patient history and clinical examination may be enough to track the problem, the main role of the physician is to provide reassurance with minimal but appropriate testing.

Molecular mechanisms of pathogenicity of Mycoplasma mycoides subsp. mycoides SC

Mycoplasma mycoides subsp. mycoides SC, the aetiological agent of contagious bovine pleuropneumonia (CBPP), is considered the most pathogenic of the Mycoplasma species. Its virulence is probably the result of a coordinated action of various components of an antigenically and functionally dynamic surface architecture. The different virulence attributes allow the pathogen to evade the host's immune defence, adhere tightly to the host cell surface, persist and disseminate in the host causing mycoplasmaemia, efficiently import energetically valuable nutrients present in the environment, and release and simultaneously translocate toxic metabolic pathway products to the host cell where they cause cytotoxic effects that are known to induce inflammatory processes and disease. This strategy enables the mycoplasma to exploit the minimal genetic information in its small genome, not only to fulfil the basic functions for its replication but also to damage host cells in intimate proximity thereby acquiring the necessary bio-molecules, such as amino acids and nucleic acid precursors, for its own biosynthesis and survival.

Beta-D-glucoside utilization by Mycoplasma mycoides subsp. mycoides SC: possible involvement in the control of cytotoxicity towards bovine lung cells

BACKGROUND: Contagious bovine pleuropneumonia (CBPP) caused by Mycoplasma mycoides subsp. mycoides small-colony type (SC) is among the most serious threats for livestock producers in Africa. Glycerol metabolism-associated H2O2 production seems to play a crucial role in virulence of this mycoplasma. A wide number of attenuated strains of M. mycoides subsp. mycoides SC are currently used in Africa as live vaccines. Glycerol metabolism is not affected in these vaccine strains and therefore it does not seem to be the determinant of their attenuation. A non-synonymous single nucleotide polymorphism (SNP) in the bgl gene coding for the 6-phospho-beta-glucosidase (Bgl) has been described recently. The SNP differentiates virulent African strains isolated from outbreaks with severe CBPP, which express the Bgl isoform Val204, from strains to be considered less virulent isolated from CBPP outbreaks with low mortality and vaccine strains, which express the Bgl isoform Ala204. RESULTS: Strains of M. mycoides subsp. mycoides SC considered virulent and possessing the Bgl isoform Val204, but not strains with the Bgl isoform Ala204, do trigger elevated levels of damage to embryonic bovine lung (EBL) cells upon incubation with the disaccharides (i.e., beta-D-glucosides) sucrose and lactose. However, strains expressing the Bgl isoform Val204 show a lower hydrolysing activity on the chromogenic substrate p-nitrophenyl-beta-D-glucopyranoside (pNPbG) when compared to strains that possess the Bgl isoform Ala204. Defective activity of Bgl in M. mycoides subsp. mycoides SC does not lead to H2O2 production. Rather, the viability during addition of beta-D-glucosides in medium-free buffers is higher for strains harbouring the Bgl isoform Val204 than for those with the isoform Ala204. CONCLUSION: Our results indicate that the studied SNP in the bgl gene is one possible cause of the difference in bacterial virulence among strains of M. mycoides subsp. mycoides SC. Bgl does not act as a direct virulence factor, but strains possessing the Bgl isoform Val204 with low hydrolysing activity are more prone to survive in environments that contain high levels of beta-D-glucosides, thus contributing in some extent to mycoplasmaemia.

Domain analysis of lipoprotein LppQ in Mycoplasma mycoides subsp. mycoides SC

The lipoprotein LppQ is the most prominent antigen of Mycoplasma mycoides subsp. mycoides small colony type (SC) during infection of cattle. This pathogen causes contagious bovine pleuropneumonia (CBPP), a devastating disease of considerable socio-economic importance in many countries worldwide. The dominant antigenicity and high specificity for M. mycoides subsp. mycoides SC of lipoprotein LppQ have been exploited for serological diagnosis and for epidemiological investigations of CBPP. Scanning electron microscopy and immunogold labelling were used to provide ultrastructural evidence that LppQ is located to the cell membrane at the outer surface of M. mycoides subsp. mycoides SC. The selectivity and specificity of this method were demonstrated through discriminating localization of extracellular (i.e., in the zone of contact with host cells) vs. integral membrane domains of LppQ. Thus, our findings support the suggestion that the accessible N-terminal domain of LppQ is surface exposed and such surface localization may be implicated in the pathogenesis of CBPP.

Functional and antigenic properties of GlpO from Mycoplasma mycoides subsp. mycoides SC: characterization of a flavin adenine dinucleotide-binding site deletion mutant

L-alpha-glycerophosphate oxidase (GlpO) plays a central role in virulence of Mycoplasma mycoides subsp. mycoides SC, a severe bacterial pathogen causing contagious bovine pleuropneumonia (CBPP). It is involved in production and translocation of toxic H(2)O(2) into the host cell, causing inflammation and cell death. The binding site on GlpO for the cofactor flavin adenine dinucleotide (FAD) has been identified as Gly(12)-Gly(13)-Gly(14)-Ile(15)-Ile(16)-Gly(17). Recombinant GlpO lacking these six amino acids (GlpODeltaFAD) was unable to bind FAD and was also devoid of glycerophosphate oxidase activity, in contrast to non-modified recombinant GlpO that binds FAD and is enzymatically active. Polyclonal monospecific antibodies directed against GlpODeltaFAD, similarly to anti-GlpO antibodies, neutralised H(2)O(2) production of M. mycoides subsp. mycoides SC grown in the presence of glycerol, as well as cytotoxicity towards embryonic calf nasal epithelial (ECaNEp) cells. The FAD-binding site of GlpO is therefore suggested as a valuable target site for the future construction of deletion mutants to yield attenuated live vaccines of M. mycoides subsp. mycoides SC necessary to efficiently combat CBPP.