Biosecurity in dairy cattle farms in the North and Centre of Portugal

Dissertação de Mestrado Integrado em Medicina VeterináriaThere is an increasing understanding that lack of biosecurity and antimicrobial resistance are crucial points in animal production and the Animal Health Law emphasizes these two aspects. By implementing a questionnaire based on Biocheck from Ghent University‟s biosecurity assessment, this project aimed to make a characterization of dairy farms‟ biosecurity in North and Centre of Portugal, it aimed to create a biosecurity index, evaluate possible risk factors, characterize antibiotics use and create a tool that allows veterinarians to help and advise farmers. The questionnaire was implemented in 151 dairy farms through personal interview and through Googleforms; a report was built and sent to the veterinarians that participated in the project. The biosecurity aspects present in both questionnaire and final report are related to animal purchase, reproduction management, hygiene and disinfection, people and animal movements, health, calving, calf, adult animals, dairy and feed and drinking water management, with one group also added for antibiotic and vaccine use. A difference was observed between total biosecurity scores that went from 42 to 80.4 (in 100 points) and external and internal biosecurity mean scores of 71.3 and 67.7 (in 100 points) respectively. Variables with bigger influence in biosecurity scores were the ones related to the use of individual protective equipment, hands hygiene, cleaning of calving and sick pens and putting animals in quarantine. Regarding antibiotics‟ use, every farm treated animals with antibiotics, but only 47.4% had an antibiotic protocol for its responsible use. This means that there is still a lot to improve regarding biosecurity and awareness regarding the use of antibiotics, although a bigger sample should be taken to make a more reliable and significant characterization.RESUMO - Biossegurança em explorações de bovinos leiteiros no Norte e Centro de Portugal - Existe um entendimento crescente de que a falta de biossegurança e a resistência aos antimicrobianos são pontos cruciais na produção animal e a Lei de Saúde Animal enfatiza esses dois aspetos. Ao implementar um questionário baseado na avaliação de biossegurança do Biocheck da universidade de Ghent, este projeto teve como objetivos caracterizar a biossegurança em explorações leiteiras no Norte e Centro de Portugal, criar um índice de biossegurança, avaliar possíveis fatores de risco, caracterizar o uso de antibióticos e criar uma ferramenta que permitisse aos veterinários ajudar e aconselhar os produtores. Foram amostradas 151 explorações leiteiras, implementando o questionário através de entrevista pessoal e online através do Googleforms, foi criado um relatório em html, o qual foi enviado aos veterinários que participaram no projeto. Os aspetos de biossegurança contidos no questionário e no relatório estão relacionados com compra de animais, maneio reprodutivo, higiene e desinfeção, movimento de pessoas e animais na exploração, controlo de pragas e contacto com outros animais, maneio sanitário, maneio de partos, de vitelos e de animais adultos, maneio de alimentação e abeberamento, tendo-se adicionado também um ponto referente à utilização de antibióticos e vacinas. Observou-se que havia grandes variações nas pontuações de biossegurança total, que iam do mínimo de 42 até máximo de 80,4 pontos (em 100 pontos), com pontuações médias de biossegurança externa e interna de 71,3 e 67,7 (em 100 pontos) respetivamente. As variáveis com maior influência nas pontuações de biossegurança foram as relacionadas com o uso de equipamentos de proteção individual, higiene das mãos, limpeza de maternidades e enfermarias e colocação de animais em quarentena. Em relação ao uso de antibióticos, apesar de os mesmos serem administrados em todas as explorações, apenas 47,4% possuíam um protocolo de utilização de antibióticos. Isto significa que ainda há muito a melhorar em relação à biossegurança e na sensibilização em relação ao uso de antibióticos, no entanto para obter uma caracterização mais confiável e representativa da realidade, uma amostra maior deveria ser coletada.N/

Assessment of temperature and time on the survivability of porcine reproductive and respiratory syndrome virus (PRRSV) and porcine epidemic diarrhea virus (PEDV) on experimentally contaminated surfaces

Fomites might be responsible for virus introduction in swine farms, highlighting the importance of implementing practices to minimize the probability of virus introduction. The study’s objective was to assess the efficacy of different combinations of temperatures and holding-times on detecting live PRRSV and PEDV on surfaces commonly found in supply entry rooms in swine farms. Two PRRSV isolates (MN 184 and 1-4-4 L1C variant) and one PEDV isolate (NC 49469/2013) were inoculated on cardboard and aluminum. An experimental study tested combinations of four temperatures (20°C, 30°C, 40°C, and 50°C) and six holding-times (15 minutes, 60 minutes, 6 hours, 12 hours, 24 hours, and 36 hours) for the presence of the viruses on each surface type. After virus titration, virus presence was assessed by assessing the cytopathic effects and immunofluorescence staining. The titers were expressed as log10 TCID50/ml, and regression models; half-lives equations were calculated to assess differences between treatments and time to not detect the live virus. The results suggest that the minimum time that surfaces should be held to not detect the virus at 30°C was 24 hours, 40°C required 12 hours, and 50°C required 6 hours; aluminum surfaces took longer to reach the desired temperature compared to cardboard. The results suggest that PRRSV 1-4-4 L1C variant had higher half-lives at higher temperatures than PRRSV MN 184. In conclusion, time and temperature combinations effectively decrease the concentration of PRRSV and PEDV on different surfaces found in supply entry rooms in swine farms.This article is published as Mil-Homens, Mafalda, Ethan Aljets, Rodrigo C. Paiva, Isadora Machado, Guilherme Cezar, Onyekachukwu Osemeke, Daniel Moraes et al. "Assessment of temperature and time on the survivability of porcine reproductive and respiratory syndrome virus (PRRSV) and porcine epidemic diarrhea virus (PEDV) on experimentally contaminated surfaces." Plos one 19, no. 1 (2024): e0291181. doi: https://doi.org/10.1371/journal.pone.0291181. © 2024 Mil-Homens et al. This is an open access article distributed under the terms of the Creative Commons Attribution License

Assessment of temperature and holding times to inactivate PRRSV and PEDV on contaminated surfaces commonly found in supply entry rooms in swine farms

Fomites can be responsible for virus introduction in swine farms, which highlights the importance of implementing practices such as cleaning and disinfecting, using ultraviolet C light chambers or applying combinations of time and temperature to minimize the probability of virus introduction. The objective of the study was to assess the efficacy of different combinations of temperatures and holding times to inactivate PRRSV and PEDV on cardboard and diamond plate aluminum, surfaces that are commonly found at supply entry rooms in swine farms. An experimental study was conducted to assess the effect of time and temperature on two PRRSV strains and PEDV inactivation on cardboard and diamond plate aluminum. A total of 2 negative controls and 144 treatments with PRRSV MN 184, PRRSV 144 L1C Variant and PEDV were subjected to four different temperatures (20ºC, 30ºC, 40ºC, and 50ºC), and six time-settings (15 minutes, 60 minutes, 6 hours, 12 hours, 24 hours, and 36 hours). Virus titration followed by immunofluorescent staining were implemented after the virus recovery, and the cytopathic effect was observed. The time that it took for the virus to stop being detected, Weibull curves were calculated. Virus was detected at 20ºC after 36 hours, after 15 minutes and after 60 minutes, virus was not detected on aluminum surfaces at 50ºC. The results from the least-squared means suggest that the minimum time that surfaces should be held to stop getting virus detection at 30ºC was 24 hours, at 40ºC was 12 hours and at 50ºC was 6 hours, and that aluminum surfaces take longer to reach the desired temperature

A New Determinant of Candida glabrata Virulence: The Acetate Exporter CgDtr1

Persistence and virulence of Candida glabrata infections are multifactorial phenomena, whose understanding is crucial to design more suitable therapeutic strategies. In this study, the putative multidrug transporter CgDtr1, encoded by ORF CAGL0M06281g, is identified as a determinant of C. glabrata virulence in the infection model Galleria mellonella. CgDTR1 deletion is shown to decrease the ability to kill G. mellonella larvae by decreasing C. glabrata ability to proliferate in G. mellonella hemolymph, and to tolerate the action of hemocytes. The possible role of CgDtr1 in the resistance to several stress factors that underlie death induced by phagocytosis was assessed. CgDTR1 was found to confer resistance to oxidative and acetic acid stress. Consistently, CgDtr1 was found to be a plasma membrane acetic acid exporter, relieving the stress induced upon C. glabrata cells within hemocytes, and thus enabling increased proliferation and virulence against G. mellonella larvae

Spatial distribution and temporal trends of butyltin compounds (TBT, DBT & MBT) in short sediment cores of the SW Portuguese Shelf (western Iberian Margin, NE Atlantic)

Spatial patterns and temporal trends of the butyltin compounds tributyltin (TBT), dibutyltin (DBT), and mon-obutyltin (MBT) were investigated in a set of sediment samples collected along the SW Portuguese continental shelf. This region did not reach the Good Environmental Status (GES) in accordance with the Marine Strategy Framework Directive (MSFD) during a first evaluation carried out in 2012. Overall, MBT and DBT were the predominant organotin species detected, but high concentrations of TBT were found in and around disposal sites for dredge sludge derived from the dredging in navigation channels, harbours, and shipyard facilities of the Tagus and Sado estuaries. Although Portuguese regulations for monitoring sediment quality in relation to dredging activities consider only PAH, PCB and HCB, they also dictate that other organic contaminants such as butyltin compounds (BTs) should be monitored if suspicion of high values exists, but no action limits are defined for these (MAOTDR, 2007). Without action limits, the monitoring recommendation given in the regulations is not put into practice. Considering their toxicity, BT derivates should be integrated in the legislation, because they represent an environmental threat in the relocation of dredged material, especially when derived from harbour and shipyards areas. Based on this study, we recommend giving more attention to the amounts and impacts of BTs in sediments at dredged material disposal sites (DMDS) and their surroundings. Or even better, in order to be more efficient, monitoring should be done at the source of the dredged materials and not at the sink. In case it is not done, the monitoring of concentrations of TBT (and other BTs) in sediments and organisms, including imposex studies, at all Portuguese sites for disposal of dredged material receiving slightly to strongly contami-nated dredged material must be developed.MAR-01.04.02-FEAMP-0013; ALT20-03-0145-FEDER-000013; PINFRA/22157/2016-EMSO-PT; LA/P/0101/2020info:eu-repo/semantics/publishedVersio

Role of CgTpo4 in Polyamine and Antimicrobial Peptide Resistance: Determining Virulence in <i>Candida glabrata</i>

Candida glabrata is an emerging fungal pathogen whose success depends on its ability to resist antifungal drugs but also to thrive against host defenses. In this study, the predicted multidrug transporter CgTpo4 (encoded by ORF CAGL0L10912g) is described as a new determinant of virulence in C. glabrata, using the infection model Galleria mellonella. The CgTPO4 gene was found to be required for the C. glabrata ability to kill G. mellonella. The transporter encoded by this gene is also necessary for antimicrobial peptide (AMP) resistance, specifically against histatin-5. Interestingly, G. mellonella’s AMP expression was found to be strongly activated in response to C. glabrata infection, suggesting AMPs are a key antifungal defense. CgTpo4 was also found to be a plasma membrane exporter of polyamines, especially spermidine, suggesting that CgTpo4 is able to export polyamines and AMPs, thus conferring resistance to both stress agents. Altogether, this study presents the polyamine exporter CgTpo4 as a determinant of C. glabrata virulence, which acts by protecting the yeast cells from the overexpression of AMPs, deployed as a host defense mechanism

Porcine reproductive and respiratory syndrome virus RNA detection in tongue tips from dead animals

The control of porcine reproductive and respiratory syndrome virus (PRRSV) hinges on monitoring and surveillance. The objective of this study was to assess PRRSV RNA detection by RT-PCR in tongue tips from dead suckling piglets compared to serum samples, processing fluids, and family oral fluids. Tongue tips and serum samples were collected from three PRRSV-positive breeding herd farms (farms A, B, and C) of three different age groups: newborns (<24h), processing (2 to 7 days of age), and weaning (18 to 22 days of age). Additionally, processing fluids and family oral fluids were collected from 2-7 days of age and weaning age respectively. In farms A and B, PRRSV RNA was detected in tongue tips from all age groups (100% and 95%, respectively). In addition, PRRSV RNA was detected in pooled serum samples (42% and 27%), processing fluids (100% and 50%), and family oral fluids (11% and 22%). Interestingly, the average Ct value from tongue tips was numerically lower than the average Ct value from serum samples in the newborn age. In farm C, PRRSV RNA was only detected in serum samples (60%) and family oral fluids (43%), both from the weaning age. Further, no PRRSV RNA was detected in tongue tips when pooled serum samples from the same age group tested PRRSV RNA-negative. Taken together, these results demonstrate the potential value of tongue tips for PRRSV monitoring and surveillance.This is a pre-print of the article Isadora F. Machado, Edison S. Magalhães, Ana Paula S. Poeta Silva, et al. Porcine reproductive and respiratory syndrome virus RNA detection in tongue tips from dead animals. Authorea. June 24, 2022. DOI: 10.22541/au.165609902.27121343/v1. Copyright 2022 The Authors. Posted with permission

A cross-sectional assessment of PRRSV nucleic acid detection by RT-qPCR in serum, ear-vein blood swabs, nasal swabs, and oral swabs from weaning-age pigs under field conditions

Introduction: The porcine reproductive and respiratory syndrome virus (PRRSV) continues to challenge swine production in the US and most parts of the world. Effective PRRSV surveillance in swine herds can be challenging, especially because the virus can persist and sustain a very low prevalence. Although weaning-age pigs are a strategic subpopulation in the surveillance of PRRSV in breeding herds, very few sample types have been validated and characterized for surveillance of this subpopulation. The objectives of this study, therefore, were to compare PRRSV RNA detection rates in serum, oral swabs (OS), nasal swabs (NS), ear-vein blood swabs (ES), and family oral fluids (FOF) obtained from weaning-age pigs and to assess the effect of litter-level pooling on the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) detection of PRRSV RNA. Methods: Three eligible PRRSV-positive herds in the Midwestern USA were selected for this study. 666 pigs across 55 litters were sampled for serum, NS, ES, OS, and FOF. RT-qPCR tests were done on these samples individually and on the litter-level pools of the swabs. Litter-level pools of each swab sample type were made by combining equal volumes of each swab taken from the pigs within a litter. Results: Ninety-six piglets distributed across 22 litters were positive by PRRSV RT-qPCR on serum, 80 piglets distributed across 15 litters were positive on ES, 80 piglets distributed across 17 litters were positive on OS, and 72 piglets distributed across 14 litters were positive on NS. Cohen's kappa analyses showed near-perfect agreement between all paired ES, OS, NS, and serum comparisons (). The serum RT-qPCR cycle threshold values (Ct) strongly predicted PRRSV detection in swab samples. There was a ≥ 95% probability of PRRSV detection in ES-, OS-, and NS pools when the proportion of positive swab samples was ≥ 23%, ≥ 27%, and ≥ 26%, respectively. Discussion: ES, NS, and OS can be used as surveillance samples for detecting PRRSV RNA by RT-qPCR in weaning-age pigs. The minimum number of piglets to be sampled by serum, ES, OS, and NS to be 95% confident of detecting ≥ 1 infected piglet when PRRSV prevalence is ≥ 10% is 30, 36, 36, and 40, respectively.This article is published as Osemeke OH, Cezar GA, Paiva RC, Moraes DCA, Machado IF, Magalhaes ES, Poeta Silva APS, Mil-Homens M, Peng L, Jayaraman S, Trevisan G, Silva GS, Gauger PC and Linhares DCL (2023) A cross-sectional assessment of PRRSV nucleic acid detection by RT-qPCR in serum, ear-vein blood swabs, nasal swabs, and oral swabs from weaning-age pigs under field conditions. Front. Vet. Sci. 10:1200376. doi: 10.3389/fvets.2023.1200376. Posted with permission.This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms

Assessment of temperature and time on the survivability of porcine reproductive and respiratory syndrome virus (PRRSV) and porcine epidemic diarrhea virus (PEDV) on experimentally contaminated surfaces.

Fomites might be responsible for virus introduction in swine farms, highlighting the importance of implementing practices to minimize the probability of virus introduction. The study's objective was to assess the efficacy of different combinations of temperatures and holding-times on detecting live PRRSV and PEDV on surfaces commonly found in supply entry rooms in swine farms. Two PRRSV isolates (MN 184 and 1-4-4 L1C variant) and one PEDV isolate (NC 49469/2013) were inoculated on cardboard and aluminum. An experimental study tested combinations of four temperatures (20°C, 30°C, 40°C, and 50°C) and six holding-times (15 minutes, 60 minutes, 6 hours, 12 hours, 24 hours, and 36 hours) for the presence of the viruses on each surface type. After virus titration, virus presence was assessed by assessing the cytopathic effects and immunofluorescence staining. The titers were expressed as log10 TCID50/ml, and regression models; half-lives equations were calculated to assess differences between treatments and time to not detect the live virus. The results suggest that the minimum time that surfaces should be held to not detect the virus at 30°C was 24 hours, 40°C required 12 hours, and 50°C required 6 hours; aluminum surfaces took longer to reach the desired temperature compared to cardboard. The results suggest that PRRSV 1-4-4 L1C variant had higher half-lives at higher temperatures than PRRSV MN 184. In conclusion, time and temperature combinations effectively decrease the concentration of PRRSV and PEDV on different surfaces found in supply entry rooms in swine farms