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

Leptospirosis in Dairy Cattle from Southern Brazil - Risk Factors

Background: Cattle are susceptible to chronic leptospirosis infection, that results in reduced milk production and reproductive disorders such as abortions, stillbirths, fetal malformation, and mummified fetuses, causing significant economic losses. Commercially available vaccines against leptospirosis offer limited protection to cattle because they contain only the most prevalent serovars worldwide, even though they are not prevalent in the specific region. This study aimed to evaluate the prevalence of specific antibodies against Leptospira serogroups, reproductive disorders and the risk factors in dairy herds from different mesoregions of Rio Grande do Sul State, Southern Brazil.Materials, Methods & Results: An epidemiological survey was conducted, and serum samples from the bovine population representative of three mesoregions (MR1, MR2, and MR3) were studied; the samples were collected and tested for leptospirosis using the microscopic agglutination test (MAT) for 12 serogroups checking for the presence of agglutination. A total of 442 blood samples were collected from dairy cattle from November to December 2019 (MR1, 187; MR2, 88; and MR3, 167), including cows vaccinated with different commercial vaccines during the three months before sample collection (n = 295) and non-vaccinated against leptospirosis (n = 147). At the time of collection, an interview was conducted with the owners with questions about the health of the animals, management, habitat, feeding and reproduction. Chi-square tests univariate analysis with the SPSS® version 20.0 were performed to estimate the association of serogroup Djasiman seroreactivity with the occurrence of reproductive problems and related risk factors. The mean prevalence of antibodies against leptospires was 78.7% (MR1, 74.9 %; MR2, 84.1 %; and MR3, 80.2 %). Serogroup prevalence was different in each mesoregion evaluated and varied with vaccination status tested of the animals. The most prevalent serogroups in MR1 were Djasiman and Icterohaemorrhagiae. In MR2 and MR3, Djasiman was the most prevalent serogroup, regardless of vaccination status. Other prevalent serogroups in vaccinated animals were Icterohaemorrhagiae (MR1), Sejroe (MR2), Pomona, Sejroe, and Icterohaemorrhagiae (MR3). The other serovars tested had a prevalence of less or equal than 2%. The occurrence of reproductive problems with abortions and estrus repetition, was associated with reactivity to the serogroup Djasiman (P > 0.05). The results showed that the access of animals to flooded areas and the presence of rodents were significant risk factors, according to the literature.Discussion: The prevalence of antibodies against Leptospira in the studied areas was higher than expected, with a high prevalence of Djasiman serogroup. It is important to note that this serogroup is not present in current vaccine formulations. The presence of wetlands and rodents as risk factors in association with Djasiman is consistent with the scientific literature, since wild rodents are natural hosts, and swampy areas may allow Leptospira to survive for up to 180 days in the environment. Due to the presence and high prevalence found of the serogroup Djasiman in the experiment, new studies are being carried out to improve our knowledge about this serogroup and its possible inclusion in a commercial vaccine. Prophylaxis and control measures were recommended to rural producers in the analyzed properties.Keywords: bovine leptospirosis, herds, zoonosis, antibodies, reproductive disorders, leptospires, Djasiman

Auxotrophic Mycobacterium bovis BCG: Updates and Perspectives

Mycobacterium bovis BCG has been used for a century as the only licensed vaccine against tuberculosis. Owing to its strong adjuvant properties, BCG has also been employed as an oncological immunotherapeutic as well as a live vaccine vector against other pathogens. However, BCG vaccination has limited efficacy in protecting against adult forms of tuberculosis (TB), raises concerns about its safety in immunocompromised populations, compromises the diagnosis of TB through the tuberculin test and lacks predictability for successful antigen expression and immune responses to heterologous antigens. Together, these factors propelled the construction and evaluation of auxotrophic BCG strains. Auxotrophs of BCG have been developed from mutations in the genes required for their growth using different approaches and have shown the potential to provide a model to study M. tuberculosis, a more stable, safe, and effective alternative to BCG and a vector for the development of recombinant live vaccines, especially against HIV infection. In this review, we provide an overview of the strategies for developing and using the auxotrophic BCG strains in different scenarios