A case of equine multicentric lymphoma: Clinical, microscopical, and molecular findings.

Background: Although relatively uncommon, lymphoma is the most prevalent haematopoietic neoplasia in horses, and multicentric lymphoma remains the most common presentation of the disease. The pathogenesis of equine lymphoma is still poorly understood and the diagnosis is usually confirmed at an advanced stage of the disease, compromising the prognosis. This study investigated the clinical, pathological, and molecular features of a case of equine multicentric lymphoma. Case Description: An apparently healthy 5-year-old crossbreed mare hospitalized at the Centre of Animal Reproduction of Vairão, Portugal, suddenly presented clinical signs of supraorbital oedema and mandibular lymph node enlargement, developing fever, facial oedema, and generalized lymphadenopathy. The mare ended up dying twenty-four days after the first clinical signs due to multisystem organ failure. Haematological and biochemical analyses, necropsy, and microscopic and molecular evaluation of affected tissues were performed. At necropsy, the main findings were multiple multinodular lesions, distributed along the serous surface of oropharynx, trachea, pericardium, gastrointestinal tract, and mesentery. Microscopically, these consisted of solid proliferations of neoplastic round cells that exhibited immunopositivity for CD3 (T cells). Based on these findings, a medium-grade multicentric T-cell lymphoma was diagnosed. Conclusion: There is still very little research regarding the molecular characterization of lymphoma in horses. As an entity itself is quite heterogeneous, it is important to describe the interspecies particularities to understand its development and behaviour.

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.

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.

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.

Serological Cross-Reactivity in Zoonotic Flaviviral Infections of Medical Importance.

Flaviviruses are enveloped RNA viruses from the family Flaviviridae that comprise many important human pathogenic arboviruses such as Yellow Fever, Dengue, and Zika viruses. Because they belong to the same genus, these viruses show sequence and structural homology among them, which results in serological cross-reactivity. Upon infection, the immune system produces both species-specific and cross-reactive antibodies, and depending on the virus, in a successive flavivirus infection, cross-reactive antibodies either enhance protection or exacerbate the disease-the latter usually due to antibody-dependent enhancement. These antigenic relationships between different flaviviruses that lead to serological cross-reactivity make them difficult to be identified through serological methods, especially when it comes to successive flavivirus infections. We present here an overview of the main structural, epidemiological, and immunological aspects of flaviviruses, highlighting the role of neutralizing antibodies in fighting viral infections and in the "original antigenic sin" problem. Finally, we draw attention to the importance of developing a rapid serological diagnostic test for flaviviruses with high sensitivity and specificity, especially when considering that cross-reactive immunity can influence the outcome of these infections.

Draft Genome Sequence of a Leptospira kirschneri Serovar Mozdok Type 2 Strain Isolated from a Horse in Portugal.

Leptospira kirschneri is an agent causing leptospirosis in animals and humans. We report the draft genome sequence of Leptospira kirschneri serovar Mozdok type 2 strain Horse 112, comprising 485 contigs and having a genome size of 4,301,784 bp. This genome will facilitate studying important mechanisms for clinical outcomes.

Viral, host and environmental factors that favor anthropozoonotic spillover of coronaviruses: An opinionated review, focusing on SARS-CoV, MERS-CoV and SARS-CoV-2.

Environmental factors play a key role in the zoonotic transmission of emerging pathogenic viruses as mankind is constantly disturbing wildlife's ecosystems usually by cutting down forests to build human settlements or by catching wild animals for food, which deprives the viruses of their natural hosts and gives them opportunity to infect humans. In December 2019, a new coronavirus emerged from bats and was named SARS-CoV-2 by the International Committee for Taxonomy of Viruses, and the disease it causes named COVID-19 by the World Health Organization. Disease outbreaks such as SARS in 2002-2003, MERS in 2012 and the current COVID-19 pandemic are the result of higher mutation rates of coronaviruses and their unique capacity for genetic recombination, resulting in adaptations that make them more suitable to cross the species barriers and infect other species. This ability for host switching and interspecies infection is often attributed to the great diversity of these viruses, which is a result of viral and host factors such as the low fidelity of their RNA-dependent RNA polymerase, the high frequency of their homologous RNA recombination, and the adaptation of the S protein to bind host receptors like the angiotensin converting enzyme 2 (ACE2) in the case of SARS-CoV and SARS-CoV-2, and dipeptidyl peptidase 4 (DDP4) in MERS-CoV. This review presents an overview of the zoonotic transmission of SARS, MERS and COVID-19, focusing on the viral, host and environmental factors that favor the spillover of these viruses into humans, as well as the biological and ecological factors that make bats the perfect animal reservoir of infection for these viruses.

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.

Hepatitis E virus genotype 3 in echinoderms: First report of sea urchin (Paracentrotus lividus) contamination.

Hepatitis E virus (HEV) deriving from manure application runoffs and faecal waste spill over of swine and human origin bypass wastewater treatment plants and contaminate coastal waters. Shellfish bioaccumulate enteric viruses such as HEV from fecally contaminated coastal waters and under current European Regulations, shellfish sanitary status surveillance is mandatory but only by means of bacterial faecal indicators. The sea urchins are under the same regulations and their vulnerability to fecal contamination has been pointed out. Since they are consumed raw and with no steps to control/reduce hazards, sea urchin contamination with enteric viruses can represent a food safety risk. Hence, the aim of the present study was to screen sea urchin gonads destined for human consumption for the presence of HEV. HEV was detected and quantified in gonads of sea urchins collected in north Portugal by a reverse transcription-quantitative PCR (RT-qPCR) assay targeting the ORF3 region, followed by genotyping by a nested RT-PCR targeting the ORF2 region. Sequencing and phylogenetic analysis clustered the HEV sequence within genotype 3, subgenotype e. This the first study reporting HEV contamination of sea urchins. We hypothesize that like shellfish, sea urchins can also be a food vehicle for HEV transmission to humans.

Mumps antibodies in the cord blood: Association with maternal recall of mumps. Possible consequences for vaccination strategies.

The present study aims to contribute to the evaluation of the serological impact of vaccination against mumps in Portugal, measuring anti-mumps IgG (MuIgG) levels in cord sera and the corresponding proportions of seropositive newborns, and their association with potential predictive variables. The data from this study came from 198 umbilical cord sera. Detailed vaccination records were available for all mothers. MuIgG were measured in the sera, using a commercial immunoassay. The geometric mean concentration (GMC) of MuIgG was 31.7 RU/ml. Seropositive/immune sera (concentration ≥16 RU/ml) were 75.3%. While 49 mothers were "unsure" about ever having had mumps, 46 said they had had the disease and 103 said they had not had it. Eighty eight women did not receive a single dose of MMR while the other received 1 or 2 doses, with different combinations of vaccine strains. This study found that recalling mumps was predictive of higher MuIgG GMC and seropositivity. Maternal age and vaccination status were not associated with GMC or seropositivity. Nevertheless, in the small subset of newborns from vaccinated mothers not recalling mumps, receiving two doses was predictive of higher GMC than just receiving one. Maternal recall of mumps is highly predictive of seropositivity while not recalling the disease results in numerous false-negatives. This is consistent with other studies and with the fact that infection with mumps virus can result in a wide range of clinical manifestations. We agree on the need for further research to support a recommendation of a three (or more)-dose MMR strategy but we also believe that evidence is fast accumulating in favour of a higher dose strategy. The issue of waning immunity due to vaccines when vaccination succeeds in controlling (and nationally eliminating) target diseases like measles and mumps must be urgently taken into account.

Short communication: detection and molecular characterization of hepatitis E virus in domestic animals of São Tomé and Príncipe.

As in most of the African continent, the status of hepatitis E virus (HEV) infection in domestic animals in São Tomé and Príncipe, an archipelago off the western equatorial coast of Central Africa, is also completely unknown. In the present study, we investigated the presence of HEV among domestic animals in São Tomé and Príncipe. A total of 93 stool samples from different animal species (goat, cow, pig, chicken, duck, and monkey) were tested for HEV RNA using two real-time RT-PCR assays, followed by a nested RT-PCR assay for sequencing and phylogenetic analysis. A total of six samples (1 cow stool and 5 pig stools) were found to be positive for HEV RNA of which one pig stool was positive by broad spectrum nested RT-PCR. Phylogenetic analysis showed that the retrieved sequence clustered within HEV subgenotype 3f, similar to zoonotic strains of European countries and posing interesting questions on past introduction of European HEV into São Tomé and Príncipe archipelago. This is the first report describing the presence and molecular characterization of HEV in São Tomé and Príncipe.

Prospective Serosurvey of Coxiella burnetii Antibodies in Selected Sheep of Portugal.

Q fever is a zoonotic disease caused by Coxiella burnetii that is highly prevalent across the world. In this study, a prospective serosurvey was performed to study C. burnetii circulation in a population of sheep in the central region of Portugal. Blood from a representative sample of 168 animals was drawn in both 2015 and 2016, and sera were tested for IgG anti-C. burnetii by EIA. In 2015, 7.7% (13/168) animals tested positive for IgG anti-C. burnetii, while in 2016, 17.3% (29/168) tested positive, showing a statistically significant (P = 0.008) increase in anti-C. burnetii seroprevalence. Results support the notion that Q fever is emerging in central Portugal.

The use of Escherichia coli total antigens as a complementary approach to address seropositivity to Leishmania antigens in canine leishmaniosis.

Canine leishmaniosis (CanL) is a major veterinary concern and a public health issue. Serological data are essential for disease management. Several antigens used in serological assays have specificity related problems preventing relevant seropositivity values establishment. Herein we report significant seropositivity level disparity in a study cohort with 384 dogs from eight countries, for antigens traditionally used in CanL - soluble promastigote Leishmania antigens (SPLA) and K39 recombinant protein (rK39): 43·8 and 2·9% for SPLA and rK39, respectively. To better understand the reasons for this disparity, CanL-associated serological response was characterized using, for complement serological evaluation, a ubiquitous antigen - soluble Escherichia coli antigens (SECAs). Using cohorts of CanL dogs and dogs without clinical evidences of CanL from non-endemic regions of Portugal, the serological response of CanL animals followed specific trend of seropositivity rK39 > SPLA > SECA absent in non-diseased animals. Using receiver operating characteristic curve analysis, these characteristic trends were converted in ratios, SPLA/SECA, rK39/SECA and rK39/SPLA, that presented high predictive for discriminating the CanL cohort that was potentiated when applied in a scoring system involving positivity to four out of five predictors (rK39, SPLA, SPLA/SECA, rK39/SECA and rK39/SPLA). In fact, this approach discriminated CanL with similar sensitivity/specificity as reference antigens, diminishing seropositivity in European cohort to 1·8%. Ultimately, non-related antigens like SECA and seropositivity ratios between antigens enable different perspectives into serological data focusing on the search of characteristic serological signatures and not simple absolute serology values contributing to comprehensive serological status characterization.

Seroprevalence of Hepatitis E Virus Antibodies in Portuguese Children.

BACKGROUND: Hepatitis E virus (HEV) has become a growing public health concern in industrialized countries. Most of the HEV seroprevalence studies have focused on adult populations, and reports regarding HEV seroepidemiology among children are scarce in these countries. The aims of this work were to perform a nationwide seroprevalence study on HEV infection in Portuguese children and to compare the HEV seropositivity in this 2015 children cohort with results in sera performed 20 years earlier. METHODS: Sera (N = 352) from children collected in 2015 from all regions of Portugal were screened for anti-HEV IgG and IgM using the commercial enzyme-linked immunosorbent assay recomWell HEV IgG/IgM (2015 version; Mikrogen, Neuried, Germany), and positive samples were retested by immunodot assay recomLine HEV IgG/IgM (2015 version; Mikrogen, Neuried, Germany). For the comparative assessment of HEV seropositivity of the 2 children cohorts, children's sera (N = 71) archived since 1995 were screened for anti-HEV IgG and results were compared with that of the 2015 cohort, matched by sex, age and region. RESULTS: Anti-HEV antibodies were detected in 4 children giving an overall HEV seroprevalence in the 2015 cohort of 1.1%. A healthy 10-15-year-old female was found positive for anti-HEV IgM indicating a current or recent HEV infection. No statistically significant difference was observed in HEV seroprevalence regarding gender, age group and region of residence. Comparison of the HEV seropositivity of the 2 children cohorts showed a statistical significant decrease with time (P = 0.024). CONCLUSIONS: This is the first national study of HEV seroprevalence in Portuguese children and the first to demonstrate a decrease of anti-HEV antibodies in this age group over time.

Reassortment among picobirnaviruses found in wolves.

We conducted a viral metagenomics study in diarrheic free-ranging wolves in Portugal, revealing for the first time the presence of reassortant picobirnaviruses. These viruses shared identical capsid segments together with diverse RNA-dependent RNA polymerase segments. Even though causality between these picobirnaviruses and diarrhea could not be established, the study nonetheless confirms for the first time that wolves are a potential reservoir for picobirnaviruses, which might play a role as enteric pathogens.

Measles antibodies in cord blood in Portugal: Possible consequences for the recommended age of vaccination.

The optimum age to give the first dose of measles vaccine must balance the risks of disease and vaccine failure. Both are influenced by the levels of transplacentally acquired maternal antibodies. This study was conducted in the Obstetric service of Portuguese hospital, in 2012-2013. Mothers were recruited after informed consent. Measles IgG was measured in 206 cord sera, using a commercial immunoassay. Geometric mean concentrations (and 95% CI) were 1849mIU/ml (1196-2857) and 790mIU/ml (618-1008) in cord sera of newborns from unvaccinated and vaccinated mothers respectively. Maternal age and vaccination status were both associated with the concentration in cord sera, but maternal age was the major predictor. The likely explanation is the same already mentioned in other studies: as a vaccination program progresses, vaccination coverage increases as measles incidence decreases. That results newborns from younger vaccinated mothers having less measles antibodies while the older mothers are more likely to have been infected with the wild virus. As the proportion of vaccinated mothers increase, developed countries tend to anticipate the recommended age of the first dose to 12 months of age. Models using hypothetical measles antibody decay rates in infancy were explored. Anticipating the first dose of MMR1 in Portugal to the age of 12 months might have not been the best decision but results were not conclusive, and arguments supporting or not the anticipation were discussed.

Toxoplasma gondii Infection in Hunted Wild Boars (Sus scrofa): Heart Meat Juice as an Alternative Sample to Serum for the Detection of Antibodies.

Toxoplasmosis is a global zoonosis caused by the protozoan parasite Toxoplasma gondii. Detection of antibodies to T. gondii in serum samples from hunted animals may represent a key step for public health protection. It is also important to assess the circulation of this parasite in wild boar population. However, in hunted animals, collection of blood is not feasible and meat juice may represent an alternative sample. The purpose of the present study was to evaluate heart meat juice of hunted wild boars as an alternative sample for post-mortem detection of antibodies to T. gondii by modified agglutination test (MAT). The agreement beyond chance between results from meat juice assessed with Cohen's kappa coefficient revealed that the 1:20 meat juice dilution provided the highest agreement. McNemars's test further revealed 1:10 as the most suitable meat juice dilution, as the proportion of positive paired samples (serum and meat juice from the same animal) did not differ at this dilution. All together, these results suggest a reasonable accuracy of heart meat juice to detect antibodies to T. gondii by MAT and support it as an alternative sample in post-mortem analysis in hunted wild boars.

Persistence of measles antibodies, following changes in the recommended age for the second dose of MMR-vaccine in Portugal.

In populations vaccinated with two doses of combined measles-mumps-rubella vaccine (MMR), the serum levels of antibodies against measles depend on the vaccination schedule, time elapsed from the last dose and the area-specific epidemiological situation. Variables measuring "schedule" are age at first and second doses of MMR and intervals derived from that. Changes in vaccination schedules have been made in Portugal. The specific objectives of this study were to measure the association between those potential determinants and the concentration of measles-specific IgG antibodies, after the second dose of MMR. Convenience samples of three Portuguese birth cohorts were selected for this study (41, 66 and 60 born, respectively, in 2001-2003, 1990-1993 and 1994-1995). Geometric mean concentrations (GMC) for measles IgG were, respectively, 934, 251 and 144mIU/ml; p<0.001). Anti-measles-IgG serum concentration decreased with time since last vaccination (waning immunity) and was not influenced by any other component of vaccination schedule, namely age at vaccination with the second dose of MMR. Waning levels of measles antibodies have been observed elsewhere but not as fast as it was observed in Portuguese birth cohorts in this study. Changes in the vaccination schedules might have to be considered in the future.

Unraveling Sarcocystis miescheriana and Sarcocystis suihominis infections in wild boar.

Sarcocystis species are worldwide spread cyst-forming protozoa that can infect wild boar but little is known about the prevalence of these parasites. In this study we assessed the prevalence of Sarcocystis spp. infections in wild boars from northeastern Portugal, for which novel PCR testing assays targeting Sarcocystis genus, S. miescheriana and S. suihominis were implemented, and risk factors potentially associated with these infections were evaluated. Samples from muscle tissue, namely diaphragm (n=102), oesophagus (n=96) and heart (n=101), were collected from a total of 103 wild boar hunted between October 2011 and February 2012. Diaphragm muscle was used for the PCR detection of Sarcocystis nucleic acids since a higher proportion of samples showed the presence of cysts during histological examination. PCR assay targeting Sarcocystis genus yielded a 73.8% infection rate, which indicate a high level of exposure to these protozoan parasites among wild boars. These samples showed to be positive with the S. miescheriana-specific PCR assay and no sample was positive with the S. suihominis-specific assay, suggesting that a single species infecting wild boar is circulating in Portugal. These results were confirmed by the partial sequencing of the 18S rRNA gene amplified from selected samples from different geographic regions. Adults, young adults and female wild boars were found to be more likely infected. Hunters have an important role in the life cycle of S. miescheriana since potentially infected viscera and carcasses can be left behind promoting the protozoan dissemination to the scavenging final hosts. If hunting dogs bite and ingest infected meat they can perpetuate the life cycle of Sarcocystis spp. spreading oocysts or sporocysts in the environment.