Detection of Mycoplasma hyopneumoniae viability using a PCR-based assay.

Mycoplasma hyopneumoniae detection in clinical specimens is accomplished by PCR targeting bacterial DNA. However, the high stability of DNA and the lack of relationship between bacterial viability and DNA detection by PCR can lead to diagnostic interpretation issues. Bacterial messenger RNA is rapidly degraded after cell death, and consequently, assays targeting mRNA detection can be used for the exclusive detection of viable bacterial cells. Therefore, this study aimed at developing a PCR-based assay for the detection of M. hyopneumoniae mRNA and at validating its applicability to differentiate viable from inert bacteria. Development of the RNA-based PCR encompassed studies to determine its analytical sensitivity, specificity, and repeatability, as well as its diagnostic accuracy. Comparisons between DNA and mRNA detection for the same target gene were performed to evaluate the ability of the RNA-based PCR to detect exclusively viable M. hyopneumoniae after bacterial inactivation using various methods. The RNA-based PCR was also compared to the DNA-based PCR as a tool to monitor the growth of M. hyopneumoniae in vitro. Under the conditions of this study, the developed RNA-based PCR assay detected only viable or very recently inactivated M. hyopneumoniae, while the DNA-based PCR consistently detected cells irrespective of their viability status. Changes in growth activity over time were only observable via RNA-based PCR. This viability PCR assay could be directly applied to evaluate the clearance of M. hyopneumoniae or to determine the viability of the bacterium at late stages of eradication programs.

Characterizing the detection of inactivated Mycoplasma hyopneumoniae DNA in the respiratory tract of pigs.

A positive Mycoplasma hyopneumoniae PCR result in a clinical specimen may eventually represent the mere detection of non-viable bacteria, complicating the diagnostic interpretation. Thus, the objective of this study was to evaluate the PCR detection of non-viable M. hyopneumoniae and its residual cell-free DNA in live pigs. Pigs were inoculated with either active or inactivated M. hyopneumoniae and were sampled for up to 14 days. Mycoplasma hyopneumoniae was not detected by PCR at any timepoint in pigs inoculated with the inactivated bacterium, suggesting that in healthy pigs, the non-viable M. hyopneumoniae DNA was rapidly sensed and cleared.

Improving Mycoplasma hyopneumoniae diagnostic capabilities by harnessing the infection dynamics.

Mycoplasma hyopneumoniae remains one of the most problematic bacterial pathogens for pig production. Despite an abundance of observational and laboratory testing capabilities for this organism, diagnostic interpretation of test results can be challenging and ambiguous. This is partly explained by the chronic nature of M. hyopneumoniae infection and its tropism for lower respiratory tract epithelium, which affects diagnostic sensitivities associated with sampling location and stage of infection. A thorough knowledge of the available tools for routine M. hyopneumoniae diagnostic testing, together with a detailed understanding of infection dynamics, are essential for optimizing sampling strategies and providing confidence in the diagnostic process. This study reviewed known information on sampling and diagnostic tools for M. hyopneumoniae and summarized literature reports of the dynamics of key infection outcomes, including clinical signs, lung lesions, pathogen detection, and humoral immune responses. The information gathethered in this manuscript can facilitate better understanding of the performance of different diagnostic approaches at various stages of infection with Mycoplasma hyopneumoniae.

Causes of cetacean stranding and death on the Catalonian coast (western Mediterranean Sea), 2012-2019.

The causes of cetacean stranding and death along the Catalan coast between 2012 and 2019 were systematically investigated. Necropsies and detailed pathological investigations were performed on 89 well-preserved stranded cetaceans, including 72 striped dolphins Stenella coeruleoalba, 9 Risso's dolphins Grampus griseus, 5 bottlenose dolphins Tursiops truncatus, 1 common dolphin Delphinus delphis, 1 Cuvier's beaked whale Ziphius cavirostris and 1 fin whale Balaenoptera physalus. The cause of death was determined for 89.9% of the stranded cetaceans. Fisheries interaction was the most frequent cause of death in striped dolphins (27.8%) and bottlenose dolphins (60%). Cetacean morbillivirus (CeMV) was detected on the Catalan coast from 2016 to 2017, causing systemic disease and death in 8 of the 72 (11.1%) striped dolphins. Chronic CeMV infection of the central nervous system was observed from 2018-2019 in a further 5 striped dolphins. Thus, acute and chronic CeMV disease caused mortality in 18% of striped dolphins and 14.6% of all 89 cetaceans. Brucella ceti was isolated in 6 striped dolphins and 1 bottlenose dolphin with typical brucellosis lesions and in 1 striped dolphin with systemic CeMV. Sinusitis due to severe infestation by the nematode parasite Crassicauda grampicola caused the death of 4 out of 6 adult Risso's dolphins. Maternal separation, in some cases complicated with septicemia, was a frequent cause of death in 13 of 14 calves. Other less common causes of death were encephalomalacia of unknown origin, septicemia, peritonitis due to gastric perforation by parasites and hepatitis caused by Sarcocystis spp.

Efficacy of parenteral vaccination against tuberculosis with heat-inactivated Mycobacterium bovis in experimentally challenged goats.

Tuberculosis (TB) in animals is a re-emerging disease with a wide range of hosts that causes large economic losses in livestock. Goats are particularly susceptible to TB and, in endemic areas, vaccination may be a valuable measure to control the disease. The main aim of this study was to evaluate the efficacy of parenteral vaccination of goats with a heat-inactivated Mycobacterium bovis (HIMB) vaccine, and compare it to M. bovis Bacille Calmette-Guérin (BCG) vaccine. Twenty-four goat kids were divided in 3 groups as following: HIMB vaccinated group (n = 8), BCG vaccinated group (n = 8) and unvaccinated group (n = 8). Afterwards, goats were experimentally challenged with Mycobacterium caprae by the endobronchial route. Antigen specific interferon-γ release assays and serology were performed after vaccination and challenge. Pathological and bacteriological parameters were evaluated after necropsy at 9 weeks post-challenge (p.c.). HIMB vaccine showed similar levels of protection to BCG in terms of volume reduction of thoracic TB lesions, presence of extra-pulmonary lesions, as well as a slight reduction of bacterial load in pulmonary lymph nodes. Moreover, HIMB vaccine did not induce interferences on the interferon-γ release assay based on reagents previously developed to differentiate infected from BCG vaccinated individuals. The results indicate that HIMB is a suitable vaccine candidate for further larger-scale trials under field conditions in goats.

Corrigendum to "Efficacy of E2 glycoprotein fused to porcine CD154 as a novel chimeric subunit vaccine to prevent classical swine fever virus vertical transmission in pregnant sows".

Here we evaluated the effect of double vaccination with a novel subunit marker vaccine candidate based in the CSFV E2 glycoprotein fused to the porcine CD154 to prevent CSFV vertical transmission. A lentivirus-based gene delivery system was used to obtain a stable recombinant HEK 293 cell line for the expression of E2 fused to porcine CD154 molecule. Six pregnant sows were distributed in two groups and at 64days of gestation animals numbered 1-4 (group 1) were vaccinated via intramuscular inoculation with 50µg of E2-CD154 subunit vaccine. Animals from group 2 (numbered 5 and 6, control animals) were injected with PBS. Seventeen days later sows from group 1 were boosted with the same vaccine dose. Twenty-seven days after the first immunization, the sows were challenged with a virulent CSFV Margarita strain and clinical signs were registered. Samples were collected during the experiment and at necropsy to evaluate immune response and virological protection. Between 14 and 18days after challenge, the sows were euthanized, the foetuses were obtained and samples of sera and tissues were collected. E2-CD154 vaccinated animals remained clinically healthy until the end of the study; also, no adverse reaction was shown after vaccination. An effective boost effect in the neutralizing antibody response after the second immunization and viral challenge was observed and supports the virological protection detected in these animals after vaccination. Protection against CSFV vertical transmission was found in the 100% of serums samples from foetus of vaccinated sows. Only two out of 208 samples (0.96%) were positive with Ct value about 36 corresponding to one tonsil and one thymus, which may be non-infective viral particles. Besides, its DIVA potential and protection from vertical transmission, the novel CSFV E2 bound to CD154 subunit vaccine, is a promising alternative to the live-attenuated vaccine for developing countries.

Extreme Tetralogy of Fallot With Polycythemia in a Ferret (Mustela putorius furo).

A 5-month-old, intact male ferret (Mustela putorius furo) was presented with apathy, tachypnea and exercise intolerance. On initial physical examination, tachypnea and cyanosis were the two primary clinical signs detected. The complete blood count also revealed a severe polycythemia, with lymphopenia, neutrophilia and eosinophilia. Further diagnostic imaging tests were performed. The thoracic radiographs revealed cardiomegaly and a diffuse alveolar pulmonary pattern. Consequently, an echocardiography was performed and showed an interventricular septal defect, pulmonic artery occlusion, overriding of the aorta and right ventricle hypertrophy. All abnormalities were compatible with a tetralogy of Fallot (TOF). As a treatment plan, a phlebotomy was performed and the ferret was posteriorly maintained with oxygen and fluid therapy. The following day, the hematocrit decreased 11% and the respiratory distress improved. Four days later, the ferret got clinically worse and was euthanized. A necropsy was performed and confirmed the presence of an extreme TOF with a diffused interstitial pneumonia secondary to Pneumocystis carinii. To the author's knowledge, this is the first report of an extreme tetralogy of Fallot with polycythemia in a ferret.

Efficacy of E2 glycoprotein fused to porcine CD154 as a novel chimeric subunit vaccine to prevent classical swine fever virus vertical transmission in pregnant sows.

Here we evaluated the effect of double vaccination with a novel subunit marker vaccine candidate based in the CSFV E2 glycoprotein fused to the porcine CD154 to prevent CSFV vertical transmission. A lentivirus-based gene delivery system was used to obtain a stable recombinant HEK 293 cell line for the expression of E2 fused to porcine CD154 molecule. Six pregnant sows were distributed in two groups and at 64days of gestation animals numbered 1-4 (group 1) were vaccinated via intramuscular inoculation with 50µg of E2-CD154 subunit vaccine. Animals from group 2 (numbered 5 and 6, control animals) were injected with PBS. Seventeen days later sows from group 1 were boosted with the same vaccine dose. Twenty-seven days after the first immunization, the sows were challenged with a virulent CSFV Margarita strain and clinical signs were registered. Samples were collected during the experiment and at necropsy to evaluate immune response and virological protection. Between 14 and 18days after challenge, the sows were euthanized, the foetuses were obtained and samples of sera and tissues were collected. E2-CD154 vaccinated animals remained clinically healthy until the end of the study; also, no adverse reaction was shown after vaccination. An effective boost effect in the neutralizing antibody response after the second immunization and viral challenge was observed and support the virological protection detected in these animals after vaccination. Protection against CSFV vertical transmission was found in the 100% of serums samples from foetus of vaccinated sows. Only two out of 208 samples (0.96%) were positive with Ct value about 36 corresponding to one tonsil and one thymus, which may be non-infective viral particles. Besides, its DIVA potential and protection from vertical transmission, the novel CSFV E2 bound to CD154 subunit vaccine, is a promising alternative to the live-attenuated vaccine for developing countries.