Talaromyces marneffei isolated from nasal swabs in Portugal.

Mycobiota are essential to the health of any living being, creating a balanced and complex interaction between bacteria, the immune system, and the tissue cells of the host. Talaromyces marneffei (also known as Penicillium marneffei) is a dimorphic fungus, endemic in South Asia, which often causes a life-threatening systemic fungal infection (called penicilliosis), particularly in immunocompromised hosts. Nasal swabs from 73 healthy volunteers were analysed to characterize their mycobiota, through its cultural characteristics, morphology, and molecular methods (PCR). All volunteers were also asked to answer to an anonymous questionnaire. Three women were positive (and asymptomatic) for T. marneffei. One of them was reported to have lupus. This study contributes to improving our knowledge about human normal mycobiota, identifying mycotic agents that may cause complicated systemic infections (as T. marneffei), especially in immunosuppressed patients, as well as other possible risk factors of exposure or prognosis.

• Talaromyces marneffei is a zoonotic fungus that may be responsible for life-threatening systemic infections in immune-comprised patients. • Talaromyces marneffei was identified in nasal swabs from asymptomatic volunteers. • This suggests that this fungus may be part of the nasal normal mycobiota of some humans.

Environmental implications of stored cattle slurry treatment with sulphuric acid and biochar: A life cycle assessment approach.

With the increase of animal slurry produced from livestock production, the monitoring and mitigation of greenhouse gas (GHG) and ammonia (NH3) emissions represent a major issue. Life cycle assessment (LCA) has been used to evaluate the long-term environmental effects of applied strategies and technologies on cattle slurry management for mitigation of environmental harmful gases. This study was carried on two main aims: first, the effect of the addition of sulphuric acid (SA), biochar (SBi) or A + Bi to liquid cattle-slurry (treated systems) on gas emissions during storage compared to the untreated system (S) was investigated in a laboratory-controlled experiment; second, the environmental implications of each treated or untreated system were assessed through a LCA approach according to ISO 14040/44. Five CML 2001 impact categories were used: eutrophication potential (EP), acidification potential (AP), global warming potential (GWP), human toxicity potential (HTP) and Ozone Layer Depletion Potential (ODP). Comparisons were based on 1 ton fresh dairy cattle slurry. The environmental profile of untreated system showed lower efficiency in mitigation of total GHG and NH3 emissions (0.0312 and 0.0001 kg CO2-eq respectively), during storage period and greater impact on GWP and HTP categories. The electricity consumption in mechanical separation dominated the environmental impacts. From the three proposed treated systems, SA showed the highest efficiency on mitigation of gas emissions compared to the other treatments, reducing NH3, CH4 and CO2 emissions respectively in 61%, 98% and 15%, when compared to the SBi system. In all categories, acidified slurry also showed the lowest environmental impact relative to other treated systems and the benefit was more evident when the impacts were expressed per kilogram of nitrogen in the slurry. Therefore, LCA methodology was of great importance to assess the environmental implications of the treatments and acidification can be considered as an effective technique on the mitigation of environmental implications of livestock production and cattle-effluent valorization. Optimization and uniformity of performed studies are essential to validate new strategies to improve the sustainability of this sector in the management of animal wastewater.

A serosurvey of Mycobacterium avium subsp. paratuberculosis infection of goats in the North of Portugal.

To better understand the epizootiology of caprine paratuberculosis in the North of Portugal, a cross-sectional study was conducted from 2014 to 2015. The seroprevalence and risk factors for Mycobacterium avium subsp. paratuberculosis (Map) seropositivity were evaluated. Antibodies against Map were determined by a commercial ELISA. In 936 sera tested from 56 goat herds, 120 (12.8%, 95% CI: 10.8-15.1%) goats and 34 (60.7%, 95% CI: 47.6-72.4%) herds were positive. Risk factors for seropositivity were investigated by logistic regression models. The odds of Map seropositivity were found to be higher for animals with clinical signs, OR = 5.1 (95% CI: 2.7-9.6%), animals belonging to herds with previous wasting disease, OR = 2.3 (95% CI: 1.1-4.8%), and accumulation of manure in the herd, OR = 3.1 (95% CI: 1.7-5.7%). The potential risk factors identified in this study support the current recommendations for the control of paratuberculosis in these and other animals.

Impact of different treatments on Escherichia coli during storage of cattle slurry.

There are different types of effluents from farming operations including untreated slurry (a mixture of manure, urine, split feed, and water), and treated slurry that normally is filtered to separate the solid fraction from the liquid fraction. With the amount of slurry applied on the soils as fertiliser every year, there are necessary to measure the leaching of microbial capable of transmitting infective agents that can be normally on slurry, because slurry can be a potential biohazard capable of transmitting infective agents. The aim of this study was to investigate the presence and survival of Escherichia coli (E. coli) on liquid fraction of dairy slurry, with the addition of different treatments during storage, such as addition of Biochar, beneficial microorganisms, sulphuric acid and the combinations of them. All the applied treatments to slurry show statistically significant differences (P < 0.001), according to the different sampling dates. Results showed that there are conditions and treatments that benefit the survival probability of E. coli, the treatments that include the acidification of slurry have the highest averages of CFU/ml (243288.3 at 37 °C; 136584 at 44 °C). These results can contribute to the improvement of the quality of slurry applied on soils, beneficiating agriculture but also public health.

Survey of Mycobacterium avium subspecies paratuberculosis in road-killed wild carnivores in Portugal.

A survey to determine the occurrence of Mycobacterium avium subsp. paratuberculosis (MAP) in wild carnivores in Portugal was conducted by testing samples from road-killed animals between 2009 and 2012. Postmortem examinations were performed and tissues were collected from wild carnivores representing four families and six different species, with a total of 74 animals analyzed. Cultures were performed by using Löwenstein-Jensen and Middlebrook 7H11 solid media and acid-fast isolates were identified by polymerase chain reaction (PCR) and mycobactin dependency characteristics. Tissues were also screened for MAP by directly extracting DNA and testing for the MAP-specific sequences. The occurrence of infected animals (an animal had at least one tissue that was positive for culture or direct PCR) was 27.0% (n = 20). MAP was isolated from culture of 25 tissue samples (3.8%) and was detected by direct PCR in 40 (6.0%) samples. Infection was recorded in 5/6 studied species: 7/49 (14.3%) red foxes (Vulpes vulpes), 3/3 (100%) beech martens (Martes foina), 2/4 (50.0%) Eurasian otters (Lutra lutra), 7/15 (46.7%) Egyptian mongooses (Herpestes ichneumon), and 1/1 (100%) European badger (Meles meles). These species represent three different taxonomic families: Canidae (14.3% were positive), Mustelidae (75.0% were positive), and Herpestidae (46.7% were positive). The results of this study confirm the presence of MAP infection in wild carnivores in Portugal.

Ehrlichia canis and Rickettsia conorii Infections in Shelter Dogs: Seropositivity and Implications for Public Health.

A cross-sectional study was conducted to gain insight into the epidemiology of canine ehrlichiosis and rickettsiosis in northern Portugal. Specific IgG antibodies to Ehrlichia canis were analysed using a commercial enzyme-linked immunosorbent assay (ELISA), and antibodies to Rickettsia conorii were analysed using a commercial indirect immunofluorescence antibody test (IFAT). A total of 113 dogs from two different shelters were sampled, and seroprevalence values of 0.9% (95% confidence (CI): 0.2-4.8%) for E. canis and 9.7 (95% CI: 5.5-16.6%) for R. conorii were found. Multiple logistic regression investigated risk factors for seropositivity. The odds ratios (ORs) of R. conorii seropositivity were higher for female dogs (OR = 6.429; 95% CI: 1.201-34.407). Dogs seropositive for co-infection (E. canis + R. conorii) were more frequently observed among females (OR = 7.606; CI 95%: 1.478-39.132) and in Shelter 2 (OR = 18.229; 95% CI: 2.190-151.756). These findings show that shelter dogs in northern Portugal are exposed to E. canis and R. conorii, which can affect both canines and humans. It is imperative to adopt a One Health approach to educate the public about the hazards of canine zoonoses and develop legislation and procedures to control their spread and preserve public health.

A Genome-Wide Association Study for Resistance to Tropical Theileriosis in Two Bovine Portuguese Autochthonous Breeds.

The control of Tropical Theileriosis, a tick-borne disease with a strong impact on cattle breeding, can be facilitated using marker-assisted selection in breeding programs. Genome-wide association studies (GWAS) using high-density arrays are extremely important for the ongoing process of identifying genomic variants associated with resistance to Theileria annulata infection. In this work, single-nucleotide polymorphisms (SNPs) were analyzed in the Portuguese autochthonous cattle breeds Alentejana and Mertolenga. In total, 24 SNPs suggestive of significance (p ≤ 10-4) were identified for Alentejana cattle and 20 SNPs were identified for Mertolenga cattle. The genomic regions around these SNPs were further investigated for annotated genes and quantitative trait loci (QTLs) previously described by other authors. Regarding the Alentejana breed, the MAP3K1, CMTM7, SSFA2, and ATG13 genes are located near suggestive SNPs and appear as candidate genes for resistance to Tropical Theileriosis, considering its action in the immune response and resistance to other diseases. On the other hand, in the Mertolenga breed, the UOX gene is also a candidate gene due to its apparent link to the pathogenesis of the disease. These results may represent a first step toward the possibility of including genetic markers for resistance to Tropical Theileriosis in current breed selection programs.

A study of knowledge, attitudes, and practices on ticks and tick-borne diseases of cattle among breeders of two bovine Portuguese autochthonous breeds.

Beef cattle production in Portugal is an important sector of national agricultural production, with half of the herd being in the Alentejo region. Despite this, animal health is essential for its productivity, which may be compromised by ticks and tick-borne diseases. So far, no study has been conducted in Portugal to assess knowledge, attitudes, and practices (KAP) on ticks and tick-borne diseases in cattle, which the authors are aware of. This type of questionnaire is a very useful tool in the development and application of effective and sustainable prevention and control measures. Therefore, a KAP questionnaire was applied to 44 cattle breeders of autochthonous Portuguese breeds, namely 14 breeders of the Alentejana breed and 30 of the Mertolenga breed, between January 1 and May 9, 2023. Based on the analysis criteria of these surveys, 64% of the Alentejana breeders and 63% of the Mertolenga breeders have an average level of knowledge about ticks and tick-borne diseases, and 21% of the Alentejana breeders and 33% of the Mertolenga breeders have a high level of knowledge. Although only 21.4% of the Alentejana and 36.7% of the Mertolenga breeders consider tick infestation as a major animal health problem, 71.4% of the Alentejana and 63.3% of breeders of the Mertolenga state that one of the main reasons for veterinary consultations on their farm is deworming of animals, and 92.9% of breeders of the Alentejana and 96.7% of breeders of the Mertolenga refer the use of dewormers as a strategy to control tick infestation. The results of this study contribute to highlighting the importance of correcting some identified knowledge gaps and improving knowledge, especially on the life cycle of this parasite, its local distribution and seasonality, resistance to acaricides, and alternative control strategies.

Mycobacterium avium subsp. paratuberculosis in Wild Boar (Sus scrofa) in Portugal.

Paratuberculosis, or Johne's disease, caused by Mycobacterium avium subsp. paratuberculosis (MAP), is a chronic granulomatous enteritis affecting both domestic and wild ruminants. The agent was also found in wild mammals such as wild boar (Sus scrofa); however, the role of wild mammals in the epidemiology of MAP is unclear. During the research period, 941 free-ranging wild boar (S. scrofa) legally hunted in two locations in the central-eastern region of Portugal were examined. Ninety-seven wild boars exhibited one or more gross lesions and were tested for the presence of Mycobacterium avium subsp. paratuberculosis using acid-fast staining, mycobacterial culture, polymerase chain reaction (PCR), and histopathological examination. Forty-five animals (46.4%, 95% CI: 36.5-56.3%) were identified as infected, as indicated by positive results in culture and/or PCR. The findings revealed that the most significant risk factor was being a juvenile compared to yearlings and adults (OR = 10.2, 95% CI: 2.2-48.0). Based on our results, 37.9% (n = 11) of the infected animals were considered suitable for human consumption. Our findings offer novel insights into mycobacterial infections in wild boar populations in Portugal and suggest that wild boar could be a source of human infection if zoonotic potential is considered.

Hunting for Answers: Assessing Brucella spp. Seroprevalence and Risks in Red Deer and Wild Boar in Central Portugal.

Between 2016 and 2023, a cross-sectional study was conducted in the central region of Portugal in order to better understand the epidemiology and public health risks resulting from the handling and consumption of game animals infected with Brucella spp. The seroprevalence and risk factors for Brucella spp. seropositivity were evaluated. Antibodies against Brucella spp. were determined using a commercial enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions. Results showed that in the 650 serum samples collected from red deer (n = 298) and wild boars (n = 352) in Portugal, 21.7% (n = 141; 95% CI: 18.6-25.1%) tested positive. Wild boar had a significantly higher prevalence (35.5%; 95% CI: 30.5-40.8%) than red deer (5.4%, 95% CI: 3.1-8.6%; p ≤ 0.001). Risk factors for seropositivity were investigated using multivariable logistic regression models. The odds of being seropositive was 8.39 (95% CI: 4.75-14.84; p ≤ 0.001) times higher in wild boar than in red deer. Correlations between sex, age, body condition, and seropositivity could not be observed. The higher seroprevalence in wild boar suggests that this species may primarily contribute to the Brucella spp. ecology in central Portugal.

Molecular evidence of sporadic Coxiella burnetii excretion in sheep milk, central Portugal.

Coxiella burnetii is the etiologic agent of Q fever, a worldwide zoonosis. Cattle, sheep and goats are considered the main reservoirs of the disease. Transmission to humans occurs mainly through the inhalation of infectious aerosols from milk, faeces, urine, and birth products from infected ruminants. In this study, a 2-year longitudinal approach was performed to ascertain the excretion of C. burnetii in bulk tank milk samples of sheep from a mountain plateau in central Portugal, with sampling conducted during the years 2015 and 2016. From a total of 156 bulk tank milk samples tested by qPCR, only one showed to be positive for C. burnetii (1.28% [95%CI: 0.03-6.94]), from 2015, the first year of collection. Bidirectional sequencing and phylogenetic analysis of IS1111 transposase partial region confirmed the presence of C. burnetii DNA. The presence of C. burnetii in raw milk samples highlights the necessity for additional research to determine if raw milk is a potential source for human infection. Animal health surveillance and prevention measures against this zoonotic disease should be considered.

Delayed diagnosis and associated factors among new pulmonary tuberculosis patients diagnosed at the emergency department of a tertiary care hospital in Porto Alegre, South Brazil: a prospective patient recruitment study.

BACKGROUND: Control of tuberculosis (TB) depends on early diagnosis and treatment at the primary health care level. However, many patients are still diagnosed late with TB at hospitals. The present study aimed to investigate the delay in diagnosis of TB patients at the emergency department. METHODS: This was a prospective study in a general, tertiary care, university-affiliated hospital of a city with a high prevalence of TB in Brazil. New TB patients ≥ 14 years diagnosed with pulmonary TB at the emergency department of Hospital de Clínicas de Porto Alegre were prospectively recruited between February 2010 and January 2012. The consenting patients meeting our inclusion criteria were interviewed using a pre-tested questionnaire. We evaluated the delay in time until diagnosis and identified factors associated with delayed diagnosis (patient and health care system delays). RESULTS: We included 153 patients. The median total time of delay, patient delay, and health care system delay were 60 (interquartile range [IQR]: 30-90.5 days), 30 (lQR: 7-60 days), and 18 (IQR: 9-39.5 days) days, respectively. The factors that were independently associated with patient delay (time ≥ 30 days) were crack (odds ratio [OR] = 4.88, p = 0.043) and cocaine (OR = 6.68, p = 0.011) use. The factors that were independently associated with health care system delay (time ≥ 18 days) were weight loss (OR = 2.76, p = 0.025), miliary pattern (OR = 5.33, p = 0.032), and fibrotic changes (OR = 0.12, p = 0.013) on chest X-ray. CONCLUSIONS: Patient delay appears to be the main problem in this city with a high prevalence of TB in Brazil. The main factor associated with patient delay is drug abuse (crack and cocaine). Our study shows substance abuse programs need to be aware of control of TB, with health interventions focusing on TB education programs.

Disseminated Mycobacterium avium subsp. paratuberculosis infection in two wild Eurasian otters (Lutra lutra L.) from Portugal.

Disseminated Mycobacterium avium subsp. paratuberculosis (MAP) infections were found in two Eurasian otters (Lutra lutra, L. 1758) killed by vehicular trauma in February and March 2010 in Castelo Branco, Portugal. At postmortem examination, the organs showed no significant gross alterations; however, microscopically, both animals had diffuse lymphadenitis with macrophage infiltration and deposition of hyaline material in the center of the lymphoid follicles. Acid-fast organisms were isolated from gastrointestinal tissue samples via bacteriologic culture. These organisms were identified as M. avium subsp. paratuberculosis by IS900 polymerase chain reaction (PCR). Additionally, direct IS900 PCR-positive results were obtained for multiple organs of both animals. This is the first report of MAP infection of otters in Portugal.

Leishmania Seroprevalence in Dogs: Comparing Shelter and Domestic Communities.

Canine leishmaniosis (CanL) is a chronic, systemic, and often severe disease. The main causative agent of CanL is a protozoan parasite, Leishmania infantum, with phlebotomine sand flies acting as vectors. In Europe and other continents, L. infantum is also responsible for leishmaniosis in other animals, such as cats, horses, and humans. In Portugal, animal and human leishmaniosis is endemic, and high prevalence levels of infections and disease have been reported in dogs. There is a prejudice against stray animals and also those housed in shelters, assuming they have higher levels of infection with vector-borne pathogens, including L. infantum, when compared to domestic animals. In northeastern Portugal, serum samples were obtained from March to May 2022 in three shelters (n = 179) and thirteen veterinary clinics (n = 164), resulting in 343 dogs being analyzed for antibodies to Leishmania spp. by the direct agglutination test (DAT). The overall seroprevalence was 9.9%, with 15.2% seroprevalence in domestic dogs and 5.0% in the shelter ones (p = 0.003). The fact that shelter dogs had a lower seroprevalence could be explained by more regular veterinary care provided in shelters regarding preventive measures, including insecticides with an antifeeding effect, in comparison with domestic dogs.

Small Ruminant Lentivirus Infection in Sheep and Goats in North Portugal: Seroprevalence and Risk Factors.

Small ruminant lentiviruses (SRLVs) are transmitted among ovine and caprine species. This disease is a severe problem for small ruminant production, not only for animals' well-being but also for flocks' efficiency. The main aim of this research was to quantify the seroprevalence and associated risk factors for SRLV infection in the northern region of Portugal. Samples were collected from a total of 150 flocks, of which 129 (86.0%; 95% CI: 80.67%-91.33%) had at least one seropositive animal. Out of 2607 individual blood samples, 1074 (41.2%) were positive for SRLVs. Risk factors associated with SRLV infection were species (caprine), age (>2 years old), flock size (>100 animals), production system (intensive), food production system (milk), type of activity (professional), participation in livestock competitions (yes), replacement young ewe bought (yes), and natural feeding management (yes). This knowledge empowers the implementation of effective preventive measures. Overall, biosecurity measures should be promoted and implemented with the main aim of reducing viral transmission and reducing the prevalence of this disease. We recognise that government authorities should promote and audit voluntary control and eradication programs in small ruminant flocks in the region studied.

Prevalence and Risk Factors Associated with Theileria annulata Infection in Two Bovine Portuguese Autochthonous Breeds.

Tropical Bovine Theileriosis is an important tick-borne disease. This study aims to assess the occurrence of Theileria annulata infection in two indigenous Portuguese cattle breeds. A total of 843 blood samples collected from animals of Alentejana (n = 420) and Mertolenga (n = 423) breeds were analyzed. The detection of Theileria annulata was determined by amplification of a fragment of the merozoite-pyroplasm surface antigen gene with 319 base pairs (bp). The prevalence found (10.8%) is lower than that reported in previous studies (21.3%). A statistically significant difference was found for positivity between breeds (p < 0.05). There is also a higher probability of older animals being positive compared to younger ones (p < 0.05). The region where Mertolenga animals are located is shown to have a significant impact on positivity (p < 0.05). Thus, the development of sustainable T. annulata control strategies and their implementation, adapted to the epidemiological conditions of higher risk, will be extremely important.

First morphological and molecular isolation of Talaromyces marneffei in beech marten (Martes foina) in Portugal.

Talaromyces marneffei is a zoonotic fungus that mostly infects immunocompromised individuals. For the first time, this fungus was isolated in an adult beech marten (Martes foina) hit by a car, found dead in Penamacor, Portugal. During the necropsy, different samples (skin, fur, lymph nodes, lung, spleen, kidneys, and brain) were collected and processed for microbiology (including mycology) and molecular biology. T. marneffei was identified through its mycological characteristics and confirmed by PCR in hair samples. No other lesions or alterations were reported, except a concomitant presence of M. avium subsp. paratuberculosis in lung, kidney and brain samples. To the authors' knowledge, this is the first description of this fungus beech marten, as well as the first case of co-infection with M. avium subsp. paratuberculosis in wildlife fauna. These results suggest a sylvatic life-cycle of T. marneffei, involving beech martens, in Portugal.

Morphologic and Genetic Analysis of Synhimantus (Synhimantus) laticeps from a Long-Eared Owl (Asio otus).

The long-eared owl (Asio otus) is a medium-sized owl species that is well-distributed in almost all of the territories in Portugal. Nematodes were found in the oral cavity of a long-eared owl (A. otus) admitted to CRASSA (Wildlife Rehabilitation Centre of Santo André). During a physical exam and stabilization of the bird, five nematodes were collected. The worms were examined and measured under light microscopy, and photos were taken. After a morphological analysis was conducted, all the nematodes (five females) were identified as Synhimantus (Synhimantus) laticeps. Two specimens were subjected to molecular analysis, which confirmed the result. This study provides a combined morphological and genetic approach to S. laticeps. To the authors' best knowledge, this is the first report including genetic sequencing of S. laticeps in a long-eared owl (A. otus) from Portugal.

Seropositivity for Coxiella burnetii in Wild Boar (Sus scrofa) and Red Deer (Cervus elaphus) in Portugal.

Q fever is caused by the pathogen Coxiella burnetii and is a zoonosis that naturally infects goats, sheep, and cats, but can also infect humans, birds, reptiles, or arthropods. A survey was conducted for the detection of antibodies against C. burnetii in a sample of 617 free-ranging wild ruminants, 358 wild boar (Sus scrofa) and 259 red deer (Cervus elaphus), in east-central Portugal during the 2016-2022 hunting seasons. Only adult animals were sampled in this study. Antibodies specific to C. burnetii were detected using a commercial enzyme-linked immunosorbent assay (ELISA; IDVet®, Montpellier, France) according to the manufacturer's instructions. The seroprevalence of C. burnetii infection was 1.5% (n = 9; 95% confidence interval [CI]: 0.7-2.8%). Antibodies against C. burnetii were detected in 4/358 wild boar (1.1%; 95% CI: CI: 0.3-2.8%) and 5/259 red deer (1.9%; 0.6-4.5%). Results of the present study indicate that antibodies against C. burnetii were present in wild boar and red deer in Portugal. These findings can help local health authorities to focus on the problem of C. burnetii in wildlife and facilitate the application of a One Health approach to its prevention and control.

Prevalence and Risk Factors for Hepatitis E Virus in Wild Boar and Red Deer in Portugal.

Hepatitis E virus (HEV) is a zoonotic foodborne virus with an annual infection prevalence of 20 million human cases, which seriously affects public health and economic development in both developed and developing countries. To better understand the epidemiology of HEV in Central Portugal, a cross-sectional study was conducted from 2016 to 2023 with sera samples from wild ungulates. The seroprevalence and risk factors for HEV seropositivity were evaluated in the present study. Specifically, antibodies against HEV were determined by a commercial enzyme-linked immune-sorbent assay (ELISA). Our results show that in the 650 sera samples collected from 298 wild red deer and 352 wild boars in Portugal, 9.1% red deer and 1.7% wild boar were positive for antibodies to HEV. Regarding age, the seropositivity in juvenile wild ungulates was 1.3%, whereas it was 7.2% in adults. Logistic regression models investigated risk factors for seropositivity. The odds of being seropositive was 3.6 times higher in adults than in juveniles, and the risk was 4.2 times higher in red deer than in wild boar. Both wild ungulate species were exposed to HEV. The higher seroprevalence in red deer suggests that this species may make a major contribution to the ecology of HEV in Central Portugal. Further research is needed to understand how wildlife affects the epidemiology of HEV infections in Portugal.