Ocorrência de Mycoplasma hyopneumoniae em amostras de pulmão de suínos obtidas de frigorífico do sul do Brasil

O Mycoplasma hyopneumoniae é o agente causador da Pneumonia Enzoótica Suína (PES), doença altamente prevalente e mundialmente distribuída, causando grandes perdas econômicas para a indústria suinícola. A progressão da doença é caracterizada pela redução das taxas de conversão alimentar e o desenvolvimento de lesões pulmonares. Visto que há informação limitada sobre a epidemiologia da PES no sul do Brasil, o objetivo do presente trabalho foi determinar a prevalência de M. hyopneumoniae em amostras de pulmão suíno e avaliar o score de lesões pulmonares causadas pelas cepas locais. Um total de 120 amostras foram coletadas aleatoriamente, processadas e analisadas. O DNA foi extraído do tecido pulmonar para realização de Nested-PCR e os pulmões foram inspecionados para presença de lesões macroscópicas sugestivas de M. hyopneumoniae. Os resultados demonstraram 95,8% das amostras positivas para o patógeno. A análise do score pulmonar mostrou lesões sugestivas da PES em 60% das amostras. A detecção de amostras positivas no Nested‑PCR foi associada com a presença de lesões sugestivas (P < 0.01). Os dados obtidos neste trabalhodemonstram a alta prevalência da PES em granjas do RS.Mycoplasma hyopneumoniae is the causative agent of enzootic pneumonia (EP), a disease that is highly prevalent and globally distributed, causing significant economic losses to the swine industry. Disease progression is characterized by reduced feed conversion and the development of lung lesions. Considering the limited information about the epidemiology of EP in Southern Brazil, the main objective of this study was to determine the occurrence of M. hyopneumoniae in swine lung samples and to evaluate the scores of lung lesions caused by local strains. A total of 120 samples was randomly collected and processed. DNA was extracted from lung tissue to perform nested-PCR and lungs were inspected to evaluate the presence of the pneumonia-like gross lesions of M. hyopneumoniae. The results showed 95.8% positive samples, while the lung lesion score analysis showed suggestive lesions in 60% of samples. The detection of positive samples in nested-PCR was associated with the presence of pneumonia-like gross lesions (P < 0.01). The results demonstrate a high occurrence of EP in slaughter pigs from southern Brazil

An improved method for characterization of the mutation associated to porcine stress syndrome by PCR amplification followed by restriction analysis

A mutation in the gene coding for the ryanodine receptor 1 (RYR1), also known as halothane (hal) gene or swine stress gene, is associated to the porcine stress syndrome (PSS). Detection of the mutation is normally accomplished by PCR amplification of an 81bp fragment of the hal gene, followed by digestion with the HhaI restriction endonuclease. Wild-type allele (N) is cut in two fragments, whereas the mutant allele (n) is not digested by the restriction enzyme. Electrophoresis of the digested DNA on agarose gel and ethidium bromide staining allows the reading of the result. The correct interpretation is difficult due to the small size of the DNA fragments. In this study we designed a new set of primers for amplification of a 144bp fragment that facilitates the reading of the result. In addition, we optimized the PCR reaction to allow amplification from a single hair bulb, added directly into the PCR mix without previous treatment. This improved method was used to genotype 165 sows and boars used in a breeding program. Forty-nine percent of the animals had the NN genotype, whereas 50% were Nn and only 1% was nn

Challenges on the Follow-Up Experimental Leptospiral Infection in Sheep

Background: Leptospirosis is currently a source of significant economic losses in the agribusiness; as such, experimental studies on this infection are required to develop a better understanding of the pathogenesis, treatment, and immunoprophylaxis of the disease. Sheep may represent a good model for ruminants in such models. Despite the extent of the studies that has been conducted thus far, researchers have yet to reach a consensus on the experimental practices to apply for leptospirosis in this animal species, and several gaps in understanding remain. To bridge these gaps, the present study aimed to assess the usage of several tools for the monitoring of experimental leptospirosis in sheep.Material, Methods & Results: Twelve Santa Ines sheep of different ages were each allocated to one of four groups (A, B, C, and D). The subjects in groups A, B, and C received different doses of Leptospira interrogans serogroup Icterohemorrhagiae by intraperitoneal route, 1x102, 1x105, and 1x108 respectively. Group D was the control. Hematological, biochemical and clinical parameters were evaluated daily. Serology by microscopic agglutination test (MAT) and PCR were performed to evaluate the infection status. The most remarkable clinical signs were fever (41ºC) and dehydration, and acute pain (cub). Two animals from Group C presented leukocytosis. Only those in Group C exhibited positive results according to serology, while positivity in PCR was observed in animals in groups A and C. The results of the experiment indicated that sheep may be experimentally infected and can, therefore, be used as a model for leptospirosis in ruminants. Clinical signs cannot be considered to represent a reliable parameter for evaluating the development of leptospirosis in experimentally infected sheep. We recommend the use of urine PCR and serology to confirm the infection in experimentally infected animals and daily complete blood count (CBC) as a follow-up tool.Discussion: It was observed that the clinical signs cannot be considered as a reliable parameter to evaluate the pathogenesis in experimentally infected ewes, being important to emphasize that the age of the animals does not seem to alter their susceptibility to the infection. This finding is in agreement with other experimental studies, which report that leptospirosis infection in ruminants occurs asymptomatic and subclinical. Hematological and biochemical tests proved to be adequate tools to monitor the experimental infection. Studies have shown that the complete blood count has been used to monitor the acute phase of leptospirosis and is effective in detecting anemia and leukocytosis with neutrophilia in ruminants. Despite the lack of clinical signs, the serological and molecular results confirmed the experimental infection. PCR has been used as an important tool in the diagnosis of leptospirosis. In addition, the current study is the first of its kind to use PCR to detect the carrier status in experimentally infected ewes. Despite this limitation, PCR was very effective in confirming the infection and should be considered for use in experimental studies. Sheep have been used as a good experimental model in several studies, sheep are relatively small compared to other ruminants and can be easily allocated in smaller pens and pens, facilitating the management of research and minimizing the costs of experimentation. In this context, we suggest that sheep represent a good model for the study of leptospirosis in ruminants and therefore a reliable protocol for experimental infection by leptospirosis is necessary

Development of polyclonal antibodies for the detection of recombinant human erythropoietin

Recombinant human erythropoietin (rHuEPO) is detected by using direct pharmacological assays and indirect haematological assays. However, both methods have several limitations including technical challenges and  cost-related issues. The aim of this study was to develop polyclonal antibodies against rHuEPO (anti-rHuEPO pAb) that can be used in immunoassays. In this study, we purified anti-rHuEPO pAb that could be used in immunoblotting assays to efficiently detect rHuEPO. Furthermore, these antirHuEPO pAb which could also detect rHuEPO that was expressed in a eukaryotic expression system (CHO cells). Thus, the anti-rHuEPO pAb developed in this study may be useful for rHuEPO detection.Keywords: Antibodies, rHuEPO, immunoassays, pAb.African Journal of Biotechnology Vol. 12(37), pp. 5595-559

Harnessing Mycobacterium bovis BCG Trained Immunity to Control Human and Bovine Babesiosis

Babesiosis is a disease caused by tickborne hemoprotozoan apicomplexan parasites of the genus Babesia that negatively impacts public health and food security worldwide. Development of effective and sustainable vaccines against babesiosis is currently hindered in part by the absence of definitive host correlates of protection. Despite that, studies in Babesia microti and Babesia bovis, major causative agents of human and bovine babesiosis, respectively, suggest that early activation of innate immune responses is crucial for vertebrates to survive acute infection. Trained immunity (TI) is defined as the development of memory in vertebrate innate immune cells, allowing more efficient responses to subsequent specific and non-specific challenges. Considering that Mycobacterium bovis bacillus Calmette-Guerin (BCG), a widely used anti-tuberculosis attenuated vaccine, induces strong TI pro-inflammatory responses, we hypothesize that BCG TI may protect vertebrates against acute babesiosis. This premise is supported by early investigations demonstrating that BCG inoculation protects mice against experimental B. microti infection and recent observations that BCG vaccination decreases the severity of malaria in children infected with Plasmodium falciparum, a Babesia-related parasite. We also discuss the potential use of TI in conjunction with recombinant BCG vaccines expressing Babesia immunogens. In conclusion, by concentrating on human and bovine babesiosis, herein we intend to raise awareness of BCG TI as a strategy to efficiently control Babesia infection

Substituted diaryl diselenides: Cytotoxic and apoptotic effect in human colon adenocarcinoma cells

AbstractAimsTo investigate the effects and study the underlying cell death mechanisms of diaryl diselenides, including: diphenyl diselenide (C6H5Se)2; 4-chlorodiphenyl diselenide (4-ClC6H4Se)2; 3-(trifluoromethyl)-diphenyl diselenide (3-CF3C6H4Se)2 and 4-methoxydiphenyl diselenide (4-MeOC6H4Se)2, on the human colon adenocarcinoma cell line HT-29.Main methodsThe viability of HT-29 cells after exposure to the diaryl diselenides and its substituted structures was based on the MTT assay. To verify if cell death was mediated throughout apoptosis mechanisms, flow cytometry and real-time PCR (qPCR) analyses were conducted.Key findingsThe MTT assay and flow cytometry analyses showed that (3-CF3C6H4Se)2 and (4-MeOC6H4Se)2 induced cytotoxicity through apoptosis mechanisms in HT-29 cells. qPCR revealed there was an up-regulation of pro-apoptotic (Bax, casapase-9, caspase-8, apoptosis-inducing factor (AIF) and Endonuclease G (EndoG)) and cell-cycle arrest genes (p53 and p21) and down-regulation of anti-apoptotic (Bcl-2 and survivin) and Myc genes.SignificanceThese results demonstrate that (3-CF3C6H4Se)₂ and (4-MeOC6H4Se)2 have the potential to induce apoptosis in HT-29 cells through the activation of caspase-dependent and independent pathways and through cell-cycle arrest

Harnessing Mycobacterium bovis BCG Trained Immunity to Control Human and Bovine Babesiosis

Babesiosis is a disease caused by tickborne hemoprotozoan apicomplexan parasites of the genus Babesia that negatively impacts public health and food security worldwide. Development of effective and sustainable vaccines against babesiosis is currently hindered in part by the absence of definitive host correlates of protection. Despite that, studies in Babesia microti and Babesia bovis, major causative agents of human and bovine babesiosis, respectively, suggest that early activation of innate immune responses is crucial for vertebrates to survive acute infection. Trained immunity (TI) is defined as the development of memory in vertebrate innate immune cells, allowing more efficient responses to subsequent specific and non-specific challenges. Considering that Mycobacterium bovis bacillus Calmette-Guerin (BCG), a widely used anti-tuberculosis attenuated vaccine, induces strong TI pro-inflammatory responses, we hypothesize that BCG TI may protect vertebrates against acute babesiosis. This premise is supported by early investigations demonstrating that BCG inoculation protects mice against experimental B. microti infection and recent observations that BCG vaccination decreases the severity of malaria in children infected with Plasmodium falciparum, a Babesia-related parasite. We also discuss the potential use of TI in conjunction with recombinant BCG vaccines expressing Babesia immunogens. In conclusion, by concentrating on human and bovine babesiosis, herein we intend to raise awareness of BCG TI as a strategy to efficiently control Babesia infection

High yield expression of leptospirosis vaccine candidates LigA and LipL32 in the methylotrophic yeast Pichia pastoris

<p>Abstract</p> <p>Background</p> <p>Leptospirosis, a zoonosis caused by <it>Leptospira </it>spp., is recognized as an emergent infectious disease. Due to the lack of adequate diagnostic tools, vaccines are an attractive intervention strategy. Recombinant proteins produced in <it>Escherichia coli </it>have demonstrated promising results, albeit with variable efficacy. <it>Pichia pastoris </it>is an alternative host with several advantages for the production of recombinant proteins.</p> <p>Results</p> <p>The vaccine candidates LigANI and LipL32 were cloned and expressed in <it>P. pastoris </it>as secreted proteins. Large-scale expression resulted in a yield of 276 mg/L for LigANI and 285 mg/L for LipL32. The recombinant proteins were glycosylated and were recognized by antibodies present in the sera of patients with severe leptospirosis.</p> <p>Conclusions</p> <p>The expression of LigANI and LipL32 in <it>P. pastoris </it>resulted in a significant increase in yield compared to expression in <it>E. coli</it>. In addition, the proteins were secreted, allowing for easy purification, and retained the antigenic characteristics of the native proteins, demonstrating their potential application as subunit vaccine candidates.</p