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.

Pulmonary and Gastrointestinal Parasitic Infections in Small Ruminant Autochthonous Breeds from Centre Region of Portugal-A Cross Sectional Study.

The production of small ruminant autochthonous breeds in the Centre region of Portugal is practiced in a semi-extensive husbandry system, exposing animals to parasitic infections. The main objective of this study was to estimate the prevalence of lungworm infection and identify risk factors. Fecal samples of 203 goats and 208 sheep from 30 herds were collected per rectum and subjected to the modified Baermann test. The overall prevalence of infection was 57.7%, significantly higher in goats (95.6%) than in sheep (20.7%) (p < 0.001). According to the binary logistic regression model, sheep dewormed with albendazole, mebendazole plus closantel, or ivermectin plus clorsulon presented a risk of Protostrongylidae infection 29.702, 7.426, or 8.720 times higher, respectively, than those dewormed with eprinomectin. Additionally, the presence of gastrointestinal parasites was investigated in 307 fecal samples using Mini-FLOTAC®. The overall prevalence of infection was 86.3%, also significantly higher in goats (93.2%) than in sheep (79.9%) (p < 0.001). Strongyle-type eggs were the most frequently identified, both in sheep (69.8%) and goats (87.8%), followed by Eimeria oocysts (40.3% in sheep and 68.9% in goats). Considering the high prevalence and the burden of lungworm parasitic infection, it is urgent to determine its economic impact and the repercussions in animal health in the Centre region of Portugal to establish appropriate therapeutic guidelines.

Mosaic sarbecovirus vaccination elicits cross-reactive responses in pre-immunized animals.

Immunization with mosaic-8b [60-mer nanoparticles presenting 8 SARS-like betacoronavirus (sarbecovirus) receptor-binding domains (RBDs)] elicits more broadly cross-reactive antibodies than homotypic SARS-CoV-2 RBD-only nanoparticles and protects against sarbecoviruses. To investigate original antigenic sin (OAS) effects on mosaic-8b efficacy, we evaluated effects of prior COVID-19 vaccinations in non-human primates and mice on sarbecovirus response breadths elicited by mosaic-8b, admix-8b (8 homotypics), and homotypic SARS-CoV-2, finding greatest cross-reactivity for mosaic-8b. As demonstrated by molecular fate-mapping in which antibodies derived from specific cohorts of B cells are differentially detected, B cells primed by WA1 spike mRNA-LNP dominated antibody responses after RBD-nanoparticle boosting. While mosaic-8b- and homotypic-nanoparticles boosted cross-reactive antibodies, de novo antibodies were predominantly induced with mosaic-8b boosting, and these were specific for variant RBDs with increased identity to RBDs on mosaic-8b. These results inform OAS mechanisms and support using mosaic-8b to protect COVID-19 vaccinated/infected humans against as-yet-unknown SARS-CoV-2 variants and animal sarbecoviruses with human spillover potential.

An Antibody-Based Survey of Toxoplasma gondii and Neospora caninum Infection in Client-Owned Cats from Portugal.

Toxoplasma gondii and Neospora caninum are obligate intracellular protozoan parasites infecting a wide range of hosts worldwide. However, information on the epidemiology of toxoplasmosis and neosporosis in cats from Portugal is limited. Thus, this study aims to evaluate anti-T. gondii and anti-N. caninum seroprevalence in client-owned cats from Portugal and to identify risk factors using a panel of well-characterized sera. A total of 183 domestic cats were sampled and screened for antibodies against T. gondii and N. caninum using commercial ELISA assays, and their owners answered an online questionnaire designed to obtain background information. The overall anti-T. gondii and anti-N. caninum seroprevalences were 13.1% (CI: 8.97-18.77) and 3.8% (CI: 1.87-7.68), respectively. Univariate analysis revealed that living strictly indoors was a significant protection factor (cOR: 0.053; CI: 0.005-0.627), and the presence of a chronic disease a significant risk factor (cOR: 3.106; CI: 1.062-9.082) to T. gondii seroprevalence. When performing multivariate analysis, only chronic disease (aOR: 57.527; CI: 1.7-1976.7) and seropositivity to N. caninum (aOR: 7.929; CI:0.8-82.9) were found to be a significant risk factor to anti-T. gondii antibodies. To the best of our knowledge, this is the first report of N. caninum seropositivity in cats from Portugal.

Generalized Lymphadenopathy as the First Manifestation of Systemic Lupus Erythematosus.

Lymphadenopathy (LAP) is a common but nonspecific feature of many diseases, representing a vast spectrum of etiologies such as infectious or inflammatory diseases, malignancies, and drugs. In systemic lupus erythematosus (SLE), it can be the first manifestation. We present the case of a 20-year-old female with a history of fever, night sweats, anorexia, and asthenia for five months. She also had diffuse generalized LAP. Although malignant etiologies were our major concern, an extensive workup for malignancy and infections was unrevealing. However, an autoimmune workup led to the diagnosis of SLE. This case shows that SLE can present as generalized LAP with constitutional symptoms, and hence it should be considered in the differential diagnosis.

Prevalence of Toxoplasma gondii Antibodies and Risk Factor Investigation in Portuguese Veterinarians: A Matched Case-Control Study.

(1) Background: Toxoplasma gondii is a widespread zoonotic agent that greatly impacts Public Health, being responsible for one of the most important parasitic zoonosis worldwide. T. gondii has a heteroxenous life cycle, with cats being the definitive hosts and all warm-blooded animals, including humans, being intermediate hosts. Veterinary practitioners (VP) may be at a higher risk than the general population for T. gondii infection, as they have direct and daily contact with many animal species. The aim of the present study was to ascertain if VP were more likely to be anti-T. gondii IgG seropositive than the general population, as well as to understand if age, accidents with blood-contaminated sharps (cross-blood contamination), gender, working years, and geographic regions play a role as risk factors for T. gondii infection. For this purpose, a case−control study using archived samples was performed. (2) Methods: A total of 350 veterinary practitioners were tested using a commercial semiquantitative enzyme immunoassay for anti-T. gondii IgG. From the general population, 175 anonymous volunteers (matched with cases by region, age, and gender) were studied for anti-T. gondii IgG. (3) Results: There was no statistical difference found between the presence of anti-T. gondii IgG in practitioners (26%; CI = 21.40−30.60%) and the general population (33.14%; CI = 26.17−40.12%) (p = 0.108). Univariate and multivariate analysis showed that only age (older groups) was found to be associated with a higher prevalence of anti-T. gondii IgG, with significant p values (p < 0.05) for both univariate and multivariate analysis. (4) Conclusions: To the best of our knowledge, this is the first case−control study fully focused on the prevalence of anti-T. gondii IgG in VP in Portugal, showing that there was no significant risk for T. gondii infection in veterinarians exposed daily and repeatedly to different species of animals.

Crimean-Congo hemorrhagic fever virus circulating among sheep of Portugal: a nationwide serosurvey assessment.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a widespread zoonotic pathogen that can cause mild to severe hemorrhagic disease in humans. CCHFV may be transmitted through direct contact with tissue or blood of viremic animals; however, the primary transmission route is through infected tick bites. CCHFV RNA has been detected in ticks feeding on domestic and wild animals in western Spain, suggesting an established circulation of CCHFV in Western Europe. Ruminants have been recognized as important CCHFV reservoirs and have been linked to human cases in endemic regions. Given the emergence of CCHF in neighboring Spain, and a report of two CCHFV seropositive humans in southern Portugal in 1985, we investigated the potential circulation of this virus in the country by performing a nationwide anti-CCHFV IgG serosurvey in sentinel sheep of Portugal. Sera (n = 459) randomly selected from widely distributed farms (n = 20) of Portugal were tested using a commercial double-antigen enzyme-linked immunosorbent assay, yielding an overall seroprevalence of 0.4% (95% confidence interval [CI] 0.04-1.56%). Positive sheep were from the southern region of Portugal (Alentejo region), which raise the seroprevalence of this region to 0.74% (95% CI 0.09-2.66%). This is the first study reporting the presence of CCHFV antibodies in sheep of Portugal, thus suggesting a geographical expansion of CCHFV to this country. It seems likely that CCHFV may exist focally in southern Portugal.

Rickettsia massiliae circulation in sheep and attached Rhipicephalus sanguineus in Central Portugal.

Rickettsiosis is considered an emerging/re-emerging vector-borne disease that causes significant public health threats. Ticks are reservoirs and vectors of Rickettsia having a significant role in the transmission of rickettsiae. In Portugal, little is known about tick-borne Rickettsia species in sheep. Therefore, this study aimed to investigate rickettsiae infection in ticks and their sheep host from 27 farms in four districts of central Portugal, to clarify the role of the sheep host in the circulation of this zoonotic agent. Between March and May 2021, EDTA blood samples (n = 100) of healthy grazing sheep and their ticks (n = 100, one tick per animal) were collected during a herd health program in central Portugal. Obtained ticks were identified as Rhipicephalus sanguineus sensu lato by PCR targeting a partial sequence of 16S rRNA gene followed by sequence analysis. Rhipicephalus sanguineus s.l. and host sheep blood were tested for the presence of Rickettsia spp. by PCR targeting a partial sequence of ompB and ompA genes. From a total of 100 paired R. sanguineus s.l. and host sheep, Rickettsia massiliae was detected in 62 ticks and 35 grazing sheep blood samples, collected in central Portugal, 2021. All 35 positive sheep had attached positive R. sanguineus s.l., with matching nucleotidic sequences. These findings suggest that sheep may develop rickettsiemia and are likely capable of transmitting and amplifying the infection to uninfected ticks maintaining rickettsiae in circulation in the domestic cycle.

Prevalence of Toxoplasma gondii Antibodies in Individuals Occupationally Exposed to Livestock in Portugal.

Toxoplasmosis is a worldwide zoonotic disease with different and complex routes for transmission. Workers occupationally exposed to animals or raw meat and viscera (WOE) may be at more risk than the general population, however conflicting data exist on the risk of developing toxoplasmosis due to this close contact. To add knowledge to this topic, the aim of the present study was to ascertain if WOE were more likely to be anti-T. gondii IgG seropositive than the general population as well as to study risk factors for T. gondii infection such as professional activity, gender, age, years of work and region. For this purpose, a case−control study using archived samples was setup. A total of 114 WOE (including pig slaughterhouse workers, butchers, veterinarians and farmers) and 228 anonymous volunteers (matched with cases by region, age and gender) were studied for anti-T. gondii IgG. A significantly higher anti-T. gondii IgG occurrence (p = 0.0282) was found in WOE when compared with the general population (72.8% [CI = 64.6−81.0%] versus 60.1% [CI = 54.6−65.6%]). Multivariate analysis showed that WOE of more than 50 years of age were more likely to be seropositive for anti-T. gondii IgG (aOR = 16.8; 95% CI 3.6−77.5; p < 0.001) than those aged less than 50 years. To our knowledge, this is the first case−control study on the prevalence of anti-T. gondii IgG in WOE in Portugal, also showing an added risk for T. gondii infection in those exposed to animals or their meat and viscera.

A Questionnaire-Based Survey on the Long-Term Management of Canine Leishmaniosis by Veterinary Practitioners.

Canine Leishmaniosis (CanL) is a chronic and potentially fatal disease. In economically disadvantaged regions, costs associated with long-term patient monitoring may determine that some owners decline veterinary follow-up of their dogs. This online, questionnaire-based survey aimed to assess how Portuguese veterinary practitioners perform long-term patient monitoring and recognize relapses. More than 50% of respondents reported that 50-100% of dog owners declared financial restraints. Hence, in these circumstances, most veterinary practitioners only performed clinical examination and serology. However, when owners did not declare financial restriction, other tests were additionally performed, such as renal and hepatic profiles, hemogram, serum protein electrophoresis and urine protein creatinine ratio. The mean number of exams performed when owners presented financial restraints was significantly lower than the number of exams performed without economic limitations. Most veterinary practitioners prescribed allopurinol ad aeternum or until disease remission and domperidone. CanL relapses were recognized by more than half of respondents "Always", through the reappearance or worsening of clinical signs, whereas about a quarter detected an increase in anti-Leishmania antibody levels and identified abnormalities in the serum protein electrophoresis profile. The relapse rate was higher in the Lisbon Metropolitan Area and north, the most economically favored regions of Portugal. This study confirms that owner financial restraints negatively influence veterinary follow-up and relapse recognition, ultimately compromising clinical decision making and favoring the maintenance of Leishmania infantum infection endemicity.

Occurrence and Distribution of Fasciolosis in a Cohort of Ovine Livestock Restricted to a Mountain Plateau in Central Portugal.

Fasciola hepatica is a parasite that is widespread in Europe, having been reported in ruminants of several European countries and causing an important economic impact. This study ascertained the occurrence and distribution of fasciolosis in Portuguese ovine livestock by assessing F. hepatica IgG antibodies in a cohort of confined sheep from a high-altitude region of central Portugal in a 2-year period. Positive animals were found in most locations and in both years, with 18 of the 92 animals (19.6% [95% confidence interval CI: 12.03-19.15]) and 17 of the same 92 animals (18.5% [95% CI: 11.15-27.93]) showing to be seropositive in the first year and second year, respectively (p = 0.85). Pasture contamination by F. hepatica eggs could be reduced by thorough anthelmintic treatments.

Endoscopic and Histopathological Findings of the Esophagus, Stomach, and Duodenum in Patients with Crohn's Disease from a Reference Center in Bahia, Brazil.

(1) The aim of the present study was to describe the endoscopic and histopathological findings in the esophagus, stomach, and duodenum in patients with Crohn's disease. (2) Methods: This was a cross-sectional study that included patients receiving treatment from the inflammatory bowel disease outpatient clinic. Esophagogastroduodenoscopies with biopsies of the stomach and proximal duodenum were performed. Presence of Helicobacter pylori bacteria was assessed by Giemsa staining. (3) Results: We included 58 patients. Erosive esophagitis was identified in 25 patients (43.1%), gastritis was diagnosed in 32 patients (55.2%) and erosive duodenitis was found in eight (13.8%). The most frequent histopathological finding in the H. pylori-positive group was increased inflammatory activity in the gastric body and antrum, with a predominance of mononuclear and polymorphonuclear cells. In turn, the most frequent finding in the H. pylori-negative group was chronic inflammation with predominance of mononuclear cells. Focally enhanced gastritis was identified in four patients (6.9%), all of whom were negative for H. pylori. Granulomas were not observed. H. pylori infection was present in 19 patients (32.8%). (4) Conclusions: Nonspecific endoscopic and histological findings were frequent in patients with Crohn's disease. Focally enhanced gastritis was uncommon and observed only in H. pylori-negative patients. The time from the diagnosis, patient age, and therapy in use may have influenced the nondetection of epithelioid granuloma.

New transitional fossil from late Jurassic of Chile sheds light on the origin of modern crocodiles.

We describe the basal mesoeucrocodylian Burkesuchus mallingrandensis nov. gen. et sp., from the Upper Jurassic (Tithonian) Toqui Formation of southern Chile. The new taxon constitutes one of the few records of non-pelagic Jurassic crocodyliforms for the entire South American continent. Burkesuchus was found on the same levels that yielded titanosauriform and diplodocoid sauropods and the herbivore theropod Chilesaurus diegosuarezi, thus expanding the taxonomic composition of currently poorly known Jurassic reptilian faunas from Patagonia. Burkesuchus was a small-sized crocodyliform (estimated length 70 cm), with a cranium that is dorsoventrally depressed and transversely wide posteriorly and distinguished by a posteroventrally flexed wing-like squamosal. A well-defined longitudinal groove runs along the lateral edge of the postorbital and squamosal, indicative of a anteroposteriorly extensive upper earlid. Phylogenetic analysis supports Burkesuchus as a basal member of Mesoeucrocodylia. This new discovery expands the meagre record of non-pelagic representatives of this clade for the Jurassic Period, and together with Batrachomimus, from Upper Jurassic beds of Brazil, supports the idea that South America represented a cradle for the evolution of derived crocodyliforms during the Late Jurassic.

Genome-Wide Analysis of Experimentally Evolved Candida auris Reveals Multiple Novel Mechanisms of Multidrug Resistance.

Candida auris is globally recognized as an opportunistic fungal pathogen of high concern, due to its extensive multidrug resistance (MDR). Still, molecular mechanisms of MDR are largely unexplored. This is the first account of genome-wide evolution of MDR in C. auris obtained through serial in vitro exposure to azoles, polyenes, and echinocandins. We show the stepwise accumulation of copy number variations and novel mutations in genes both known and unknown in antifungal drug resistance. Echinocandin resistance was accompanied by a codon deletion in FKS1 hot spot 1 and a substitution in FKS1 "novel" hot spot 3. Mutations in ERG3 and CIS2 further increased the echinocandin MIC. Decreased azole susceptibility was linked to a mutation in transcription factor TAC1b and overexpression of the drug efflux pump Cdr1, a segmental duplication of chromosome 1 containing ERG11, and a whole chromosome 5 duplication, which contains TAC1b The latter was associated with increased expression of ERG11, TAC1b, and CDR2 but not CDR1 The simultaneous emergence of nonsense mutations in ERG3 and ERG11 was shown to decrease amphotericin B susceptibility, accompanied with fluconazole cross-resistance. A mutation in MEC3, a gene mainly known for its role in DNA damage homeostasis, further increased the polyene MIC. Overall, this study shows the alarming potential for and diversity of MDR development in C. auris, even in a clade until now not associated with MDR (clade II), stressing its clinical importance and the urge for future research.IMPORTANCECandida auris is a recently discovered human fungal pathogen and has shown an alarming potential for developing multi- and pan-resistance toward all classes of antifungals most commonly used in the clinic. Currently, C. auris has been globally recognized as a nosocomial pathogen of high concern due to this evolutionary potential. So far, this is the first study in which the stepwise progression of multidrug resistance (MDR) in C. auris is monitored in vitro Multiple novel mutations in known resistance genes and genes previously not or vaguely associated with drug resistance reveal rapid MDR evolution in a C. auris clade II isolate. Additionally, this study shows that in vitro experimental evolution can be a powerful tool to discover new drug resistance mechanisms, although it has its limitations.

A 2-Year Longitudinal Seroepidemiological Evaluation of Toxoplasma gondii Antibodies in a Cohort of Autochthonous Sheep from Central Portugal.

(1) Background: Toxoplasma gondii is an important zoonosis and one of the major causes of abortion in sheep worldwide. (2) Methods: We performed a 2-year longitudinal serological anti-T. gondii IgG screening on a cohort of a spatially confined population of a Portuguese autochthonous sheep breed in central Portugal. (3) Results: From the screening of the 2015 and 2016 sera, an increase of seroprevalence was observed (57.7% (95% CI: 49.9-65.3%) versus 69.1% (95% CI: 61.5-75.9), from 2015 and 2016, respectively) (p = 0.031). (4) Conclusions The present study is the first to provide prospective data on the anti-T. gondii serological status of a sheep cohort in Portugal, showing an increase in the occurrence of T. gondii. There is a need to provide a clearer understanding of T. gondii epidemiology in Portugal, ideally by implementing monitoring programs on sentinel herds, not only due to the high impact of T. gondii on animal health but also for it being a zoonosis.

Hernia interna relacionada con la derivación gástrica en Y de Roux: una complicación que representa un reto clínico y diagnóstico / Roux-en-Y related internal hernia: A complication that represents a clinical and diagnostic challenge

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Occupationally Acquired Q Fever in Shepherds and Sheep Milk Cheesemakers.

Q fever is a zoonosis caused by Coxiella burnetii, and transmission to humans is often associated with contact with ovine and caprine livestock. Those exposed to sheep are particularly at high risk of infection. Recent studies show that Q fever is increasing in sheep farms in Portugal raising alerts on spillover to humans. We detected anti-C. burnetii IgG in shepherds and sheep milk cheesemakers (27 [28.1%] in a total of 96; 95% confidence interval [CI] 19.4-38.2%) and in controls (21 [8.1%] in a total of 260; 95% CI 5.1-12.1%), pointing to an increased risk of C. burnetii infection (P = 0.0001), with an odds ratio for anti-C. burnetii of 4.45 (95% CI 2.4-8.4%; P = 0.0001), in individuals with occupational contact with sheep in Portugal.

Increased risk of hepatitis E virus infection in workers occupationally exposed to sheep.

Hepatitis E virus (HEV) is an enteric RNA virus from the family Hepeviridae with five genotypes (genotypes 1-4 and 7) known to infect humans. HEV infection is known to have a zoonotic swine origin in industrialized countries. The role of pigs and wild boars as major reservoirs for human infection is today well-established; however, the list of new animal reservoirs is ever-expanding as new HEV strains are continuously being found in a broad host range. The recent detection of HEV in sheep stools brings concerns on the possibility of HEV transmission from these animals to humans, particularly in those occupationally exposed. The present work investigated the potential occupational risk of HEV infection in shepherds and sheep milk cheesemakers-workers occupationally exposed to ovine (WOEOs; N = 96)-from a region of the Centre of Portugal ('Serra da Estrela') based on the differences of anti-HEV IgG seroprevalence rates between these professionals and the general population (N = 192). The presence of HEV-specific antibodies in sheep (N = 90) from the same region was also evaluated. The HEV seroprevalence in WOEOs (29.3%) was found to be significantly higher (p = .0198) when compared with population controls (16.1%) which suggests an increased risk for HEV infection in these workers. HEV-specific antibodies were also found in 16.6% of the studied sheep showing that HEV circulates in these animals. Further studies are needed to confirm the zoonotic potential of sheep HEV.

Expanding and understanding the CRISPR toolbox for Bacillus subtilis with MAD7 and dMAD7.

The CRISPR-Cas9 system has become increasingly popular for genome engineering across all fields of biological research, including in the Gram-positive model organism Bacillus subtilis. A major drawback for the commercial use of Cas9 is the IP landscape requiring a license for its use, as well as reach-through royalties on the final product. Recently an alternative CRISPR nuclease, free to use for industrial R&D, MAD7 was released by Inscripta (CO). Here we report the first use of MAD7 for gene editing in B. subtilis, in which editing rates of 93% and 100% were established. Additionally, we engineer the first reported catalytically inactive MAD7 (dMAD7) variant (D877A, E962A, and D1213A) and demonstrate its utility for CRISPR interference (CRISPRi) at up to 71.3% reduction of expression at single and multiplexed target sites within B. subtilis. We also confirm the CRISPR-based editing mode of action in B. subtilis providing evidence that the nuclease-mediated DNA double-strand break acts as a counterselection mechanism after homologous recombination of the donor DNA.