Non-invasive surveillance of shared pathogens in the Eurasian brown bear (Ursus arctos) human interface.

Multi-host communities are perfect scenarios for the emergence and spread of pathogens, threatening the recovery of endangered, isolated, or inbred populations, such as the brown bear (Ursus arctos) in northwestern Spain. The population recovery in recent years has forced bears to occupy highly anthropized areas, increasing their interaction with human and domestic animals, with potential consequences for global health. During 2022-2023 a survey of parasites, bacteria and viruses shared between wildlife, domestic animals and humans was performed in this population using non-invasive surveillance, i.e., bear fecal samples (n = 73) and sponge-based sampling of trees (n = 42; 14 rubbed trees and 28 control trees). Pathogen detection rates were defined as the percentage of qPCR or culture-positive samples. Generalized linear models were fitted to assess their relationship with environmental variables including dispersion of the human population, and percentage of agricultural and periurban habitats in a 6 km-buffer around each sample. Canine Adenovirus type 1 (45.2%), Giardia spp. (15.1%), Salmonella spp. (12.3%), and extended-spectrum-beta-lactamases (ESBL) Escherichia coli (1.4%) were identified in fecal samples. In contrast, only five sponges from three rubbed and two control trees resulted positive to E. coli (14.3%). The results suggest that several pathogens are common in the Cantabrian brown bear population and that anthropization of the territory modulates their prevalence and richness. The effective design of management programs for bear conservation will require a one-health approach, in which genetic analysis of non-invasive samples can be key tools for the sanitary surveillance at the wildlife-livestock-human interface.

Forensic findings in urban red fox mortality in the metropolitan area of Madrid, 2014-2022.

The expanding presence of red foxes (Vulpes vulpes) in urban and suburban regions could potentially lead to increased instances of human aggression towards this species. We studied 10 deceased red foxes that were submitted by law enforcement agencies in the metropolitan area of Madrid in 2014-2022 because of suspected abuse. Forensic autopsies were performed to establish the cause and manner of death. In 4 of the 10 cases, the cause of death was deemed unnatural, involving blunt-force trauma (n = 2), asphyxia resulting from hanging (n = 1), and firearm injury (n = 1). Among the remaining cases, most had succumbed to natural causes (n = 4), often marked by severe emaciation and a high burden of parasites, primarily Sarcoptes scabiei. In 2 cases, death was undetermined given the poor preservation of the carcass. The growing prevalence of wildlife species in urban areas, particularly red foxes, may require forensic veterinary investigation of deaths potentially related to abuse.

Time, temperature and media: the three keys to improve the recovery of Campylobacter fetus subsp. venerealis from preputial bull samples.

The isolation of Campylobacter fetus subsp. venerealis (Cfv) from clinical samples is the gold standard for confirming cases of bovine genital campylobacteriosis, an important cause of infertility in cattle and a potential public health concern. Furthermore, isolation is also necessary for the development of autologous vaccines, characterization of strains for antimicrobial susceptibility patterns, etc. Nevertheless, the sensitivity of culture methods is usually low, and there is no standardized protocol to maximize the recovery of Cfv from clinical samples. The aim of the current study is to design a protocol for the culture of Cfv from preputial samples by evaluating the combination of different transport, enrichment and culture media considering the impact of certain factors (time between collection and enrichment, temperature, and use of filters). The use of modified Lander's transport medium and storing the sample for 24 h at 21 ± 2 °C led to the highest recovery of Cfv CFUs. In contrast, the storage of the samples during 24-48 h in PBS and Thomann rarely allowed the recovery of Cfv regardless of the temperature. The enrichment medium yielding the best results was Preston (significantly higher recovery than Brucella medium), while Cfv could not be isolated with Bolton. Regarding our diagnostic assay (using Lander as transport medium and Preston as enrichment medium), the best protocol in terms of maximizing Cfv recovery as well as limiting contaminations is to culture the samples in i) solid media Preston or Skirrow, and ii) using 0.65 µm filters and incubating plates at 37 °C in microaerophilic conditions.

Forensic investigations of suspected livestock depredation by vultures: scientific tools for compensation programmes.

Human-wildlife conflicts may have devastating consequences for fauna due to targeting by humans of wildlife populations suspected to have predated livestock. Suspicion of depredation of extensively raised livestock by vultures in Europe has triggered public administration-led forensic investigations intended to distinguish between predation and scavenging in order to compensate farmers for attacks on their livestock. In this study, gross and histological analyses were carried out on suspected cases of domestic animal depredation by griffon vultures (Gyps fulvus) over a 1-year period. Fifty-eight animals were affected (n = 41, suspected depredation), including domestic cattle (75.9%), sheep (22.4%) and a goat (1.7%). All the adults affected were female and most cases of suspected depredation occurred during the peripartum period (56%). Histological investigations distinguished between post-mortem (84% of diagnostic samples) and ante-mortem (16% of diagnostic samples) cases, and gross examinations revealed significant differences (P ≤ 0.002) between ante-mortem, post-mortem and non-diagnostic samples. This study highlights the need to optimize sampling protocols to increase the success of forensic studies. The forensic investigations presented here may be applied to resolve human-wildlife conflicts involving not only vultures but other endangered carnivores and contribute to human-wildlife coexistence in rural areas by protecting the interests of both the livestock sector and endangered species.

Effect of a recent intradermal test on the specificity of P22 ELISA for the diagnosis of caprine tuberculosis.

Caprine tuberculosis (TB) is a zoonotic disease caused by members of the Mycobacterium tuberculosis complex. TB eradication programs in goats are based on the single and comparative intradermal tuberculin tests (SITT and CITT, respectively). Antibody-based diagnostic techniques have emerged as potential diagnostic tools for TB. P22 ELISA has been previously evaluated using samples collected after the intradermal tuberculin tests to maximize the sensitivity, a phenomenon known as booster effect. However, there is no information available on whether the use of this diagnostic strategy could lead to a decrease of its specificity (Sp). The aim of the present study was to elucidate the interference effect of a recent CITT on the Sp of the P22 ELISA in serum and milk samples collected at different times after the CITT from a TB-free herd (n = 113). The number of reactors to P22 ELISA was significantly higher (p < 0.01) on serum samples collected 15 days post-CITT compared to day 0, showing a decrease in Sp from 99.1% (95% CI; 95.2-99.8%) to 88.5% (95% CI; 81.3-93.2%). The number of reactors and the quantitative values of P22 ELISA were significantly higher (p < 0.01) in serum samples compared to milk. No significant (p > 0.05) changes in the Sp of the P22 ELISA were observed throughout the different time samplings using milk No significant (p > 0.05) changes were observed on days 30 and 60 post-CITT. In conclusion, the booster effect strategy may significantly decrease the Sp of P22 ELISA in TB-free herds when serum samples are used but not when milk is tested.

Heat-inactivated mycobacteria activate the toll-like receptor 2 and 4 pathways in the zebrafish model of tuberculosis.

Based on previous evidence demonstrating the efficacy of inactivated mycobacteria for the control of fish mycobacteriosis, we explored the protective efficacy of two inactivated Mycobacterium bovis administered via parenteral and mucosal routes against Mycobacterium marinum infection mimicking natural conditions in the zebrafish model of tuberculosis. Although we did not observe a clear effect of any of the immunostimulants on mycobacterial burden, the results showed a significant increase in TLR2 and TLR4 gene expression levels in fishes parenterally immunized with inactivated Bacillus Calmette-Guérin (BCG). Our findings demonstrated that the TLR2 and the TLR4 signaling pathways are involved in the immune response elicited by inactivated mycobacteria in the zebrafish model of tuberculosis and support the use of inactivated mycobacteria in vaccine formulations for the control of mycobacteriosis.

A retrospective study of SARS-CoV-2 seroprevalence in dogs and cats in the Community of Madrid, Spain.

To date, susceptibility to SARS-CoV-2 infection in domestic animals including cats and dogs has been described. However, it is important to carry out passive surveillance of these animals to be aware of any changes in the outcomes of the disease in these species that may occur. In this study, we have performed a retrospective study in which we analyzed sera (n = 1,640) from random animals: dogs (n = 1,381) and cats (n = 259) belonging to both homes (n = 1,533) and animal protection centers (n = 107) in the Community of Madrid, Spain. Neutralizing antibodies were evaluated between November 2021 and May 2022 using a surrogate ELISA kit to determine the seroprevalence. Based on the results obtained, a few animals (both cats and dogs) presented neutralizing antibodies to SARS-CoV-2 (2.3%), all of them from private owners. However, the seroprevalence in cats (4.6%) resulted to be almost twice as much as in dogs (1.9%) which reinforces that cats' susceptibility to the infection seems higher than in the case of dogs, maybe due to the lower ACE2 expression of the dogs in the respiratory tract. These findings also confirm that the probability of infection is considerably higher in domestic animals in close contact with infected owners, compared to animals living in animal shelters whose contact with humans is markedly lower.

New insights into the pathogenesis and transmission of Brucella pinnipedialis: systemic infection in two bottlenose dolphins (Tursiops truncatus).

IMPORTANCE: Brucella spp. are zoonotic pathogens that can affect both terrestrial and marine mammals. Brucella ceti has been identified in various cetacean species, but only one sequence type (ST27) has been reported in humans. However, it is important to conduct surveillance studies to better understand the impact of marine Brucella species on marine mammals, a typically understudied host group. Here, we describe a systemic infection by two related strains of Brucella pinnipedialis (ST25) in a couple of live-stranded bottlenose dolphins, with more severe lesions in the younger animal. Furthermore, B. pinnipedialis was first detected in milk from a female cetacean that stranded with its offspring. Our study reveals novel insights into the epidemiology and pathological consequences of B. pinnipedialis infections in cetaceans, emphasizing the crucial importance of ongoing surveillance and accurate diagnosis to understand the impact of this pathogen on marine mammal populations.

Genotypic Comparison of Pasteurella multocida from Healthy Animals at Entry to the Feedlots with That and from Bovine Respiratory Disease-Affected Animals during the Fattening Period.

The aim of this study was to investigate the possible genotypic differences between commensal Pasteurella multocida isolates from apparently healthy animals (AHA) at the time of entry to feedlots and those from BRD-affected animals (BRD-AA). A total of 20 batches of beef calves in seven feedlots were followed-up during the fattening period. P. multocida was isolated from 28.1% of AHA and 22.9% of BRD-AA. All isolates belonged to the A: L3 genotype. Most isolates from clinical cases (81.0%) grouped into a PFGE cluster were significantly associated with BRD cases (OR, 24.9; 95% CI, 6.4-96.2). The whole genomes of 14 isolates representative of the pulsotypes most frequently detected in BRD-AA and AHA were sequenced and compared with 53 bovine genomes belonging to the identified ST13, ST79, and ST80 genotypes for a global comparison. No differences were found in the virulence-associated gene content between sequence types (STs) globally or between BRD-AA and AHA isolates in this study. Significantly, ST79 isolates harbored ARGs, conferring resistance to different antimicrobials, including macrolides and tetracyclines, which are commonly used for the treatment of BRD. Two Spanish ST79 isolates carried an ICE highly similar to ICE Tn7407, which was recently detected in Germany, suggesting that ST79 P. multocida isolates in Europe and North America may be associated with different ICEs.

An Outbreak of Aeromonas salmonicida in Juvenile Siberian Sturgeons (Acipenser baerii).

Aeromonas salmonicida is one of the major threats to world aquaculture, causing fish furunculosis and high mortality rates in cultured fish, particularly salmonids. Although Aeromonas spp. is a thoroughly studied pathogen, little is known regarding aeromoniasis in sturgeons. After a mortality outbreak, four juvenile sturgeons (Acipenser baerii) were submitted for autopsy and tissue samples were collected for histopathological and microbiological studies. The external examination revealed size heterogenicity, skin hyperpigmentation and reduced body condition of sturgeons. Within the abdominal cavity, mild hepatomegaly and splenomegaly were observed, as well as generalized organic congestion. Histology revealed severe multifocal haemorrhagic and ulcerative dermatitis, mainly localized in the dorsal and latero-ventral areas of fish. The histological study also showed moderate to severe inflammation of gills and organic lesions compatible with septicaemia. Bacterial isolates were identified as Aeromonas salmonicida subsp. salmonicida using MALDI-TOF MS and PCR. Overall, the lesions first described here are consistent with those previously reported in other cultured fish species and contribute to a better understanding of the pathogenesis of Aeromonas salmonicida subsp. salmonicida in the Siberian sturgeon, aside from providing new diagnostic tools for bacterial diseases impacting the fast-growing industry of caviar.

Will we ever eradicate animal tuberculosis?

Two characteristics of the Mycobacterium tuberculosis complex (MTC) are particularly relevant for tuberculosis (TB) epidemiology and control, namely the ability of this group of pathogens to survive in the environment and thereby facilitate indirect transmission via water or feed, and the capacity to infect multiple host species including human beings, cattle, wildlife, and domestic animals other than cattle. As a consequence, rather than keeping the focus on certain animal species regarded as maintenance hosts, we postulate that it is time to think of complex and dynamic multi-host MTC maintenance communities where several wild and domestic species and the environment contribute to pathogen maintenance. Regarding the global situation of animal TB, many industrialized countries have reached the Officially Tuberculosis Free status. However, infection of cattle with M. bovis still occurs in most countries around the world. In low- and middle-income countries, human and animal TB infection is endemic and bovine TB control programs are often not implemented because standard TB control through testing and culling, movement control and slaughterhouse inspection is too expensive or ethically unacceptable. In facing increasingly complex epidemiological scenarios, modern integrated disease control should rely on three main pillars: (1) a close involvement of farmers including collaborative decision making, (2) expanding the surveillance and control targets to all three host categories, the environment, and their interactions, and (3) setting up new control schemes or upgrading established ones switching from single tool test and cull approaches to integrated ones including farm biosafety and vaccination.

Importance of genomic surveillance of SARS-CoV-2 in cats during reverse zoonosis events: potential viral evolution may occur.

The apparition of new variants of severe acute respiratory syndrome coronavirus 2 and lineages is constantly happening because of the high viral mutation rate. Since numerous reverse zoonosis events have been reported so far, genomic surveillance should be conducted in susceptible species to evaluate potential adaptations that may trigger the apparition of new variants. Here, we evaluate the evolution of the infection in a cat naturally infected in parallel with its owner, performing a comparative phylogenetic analysis. Sequencing analysis showed that both were infected with the Omicron BA.5/BF.1 lineage and revealed the presence of nucleotide substitution in the viral genome recovered from the cat with respect to the viral genome from the human sample. This nucleotide substitution (C11897A) produced the amino acid change Orf1a: Q3878K. Therefore, genomic surveillance in the case of reverse zoonosis events is still necessary in order to control possible adaptations of the virus to other susceptible species. IMPORTANCE Genomic surveillance of pets for severe acute respiratory syndrome coronavirus 2 is important to monitor the emergence of new variants of the virus associated with these animals. Pets can serve as a potential reservoir for the virus, and their close contact with humans increases the risk of transmission. By conducting genomic surveillance in pets, it is possible to detect and track new variants early on, allowing for more effective control measures to be put in place. This can help prevent the spread of these variants to human populations and potentially mitigate the impact of the pandemic. Furthermore, it may also provide insight into the evolution and spread of the virus within the animal population.

Oral immunization with heat-inactivated Mycobacterium bovis reduces local parasite dissemination and hepatic granuloma development in mice infected with Leishmania amazonensis.

Aiming to explore whether oral immunization with heat-inactivated Mycobacterium bovis (HIMB) protects mice against Leishmania infection, 18 female BALB/c mice were randomly assigned to the immunized group, that received oral HIMB, or the control group, and were infected by inoculation of 10,000 Leishmania amazonensis promastigotes in the footpad. Spleen culture was positive in 55.55% of immunized mice and in 100% of control mice (p = 0.082). The number of immunolabeled amastigotes number in the popliteal lymph node was lower in the immunized group (p = 0.009). The immunized group presented fewer mature granulomas in the liver (p = 0.005) and more Lys + macrophages (p = 0.002) and fewer CD3+ T lymphocytes (p < 0.001) per hepatic granuloma. We conclude that immunization with HIMB via the oral route limited local parasite dissemination and hepatic granuloma development in mice challenged with Leishmania amazonensis through stimulation of macrophages, which is compatible with trained immunity.

Bovine tuberculosis in Spain, is it really the final countdown?

Bovine tuberculosis (bTB) is a severe zoonotic disease that has major impacts on both health and the economy, and which has been subjected to specific eradication programmes in many countries for decades. This manuscript highlights the relevance of this disease in the context of the European Union (EU) and summarizes the epidemiological situation and the main tools (e.g. antemortem diagnostic tests, slaughterhouse surveillance, laboratories, comprehensive databases, etc.) used to control and eradicate bTB in the various EU countries with a focus on the situation in Spain. A comprehensive description of the specific bTB epidemiological situation in Spain is provided, together with an assessment of the evolution of different epidemiological indicators throughout the last decades. Moreover, the main features of the Spanish bTB eradication programme and its control tools are described, along with the studies carried out in Spain that have allowed the updating of and improvement to the programme over the years with the aim of eradication, which has been established for 2030.

Immunopathology of early and advanced epididymis lesions caused by Brucella ovis in rams.

Ovine brucellosis is an infectious disease that causes alterations in the reproductive tract in ram and abortion in ewes. Their negative economic impact in ovine production warrants a thorough understanding the interactions between B. ovis and the host. Here, epididymis lesions of rams infected by B. ovis were histopathologically staged into early and advanced. Expression by immunohistochemistry of Brucella antigens, inflammatory cell markers (CD3, CD79αcy) and cytokines (IFN-γ, TNF-α, TGF-ß1) was assessed in both stages. Early lesions were characterized by epithelial changes, interstitial inflammation, and mild fibrosis; whereas advanced lesions displayed caseous granulomas containing numerous macrophages, multinucleated giant cells, lymphocytes, and plasma cells. Expression of Brucella antigens were observed in both stages. The cellular response in B. ovis lesions were predominantly of T-cells (CD3+) whereas low numbers of B-cells and plasma cells (CD79αcy+) were present in both early and advanced lesions. IFN-γ was expressed by lymphocytes in early lesions suggesting that the adaptive immune response against B. ovis is initiated by Th1 cells, this response was also preserved in advanced stages. Expression of TNF-α was observed in neutrophils of epithelial microabscesses and intraepithelial T-cells of early lesions suggesting a promotion of neutrophil phagocytosis triggered by TNF-α. On the other hand, advanced lesions showed a reduction of TNF-α expression which may permit B. ovis persistence in granulomas. Lastly, TGF-ß1 expression (fibroblast, macrophages and less in lymphocytes) were increased with time, suggesting that B. ovis promotes TGF-ß1 secretion promoting chronicity of the lesions.

Antimicrobial Susceptibility and Resistance Mechanisms in Mannheimia haemolytica Isolates from Sheep at Slaughter.

Mannheimia haemolytica is the main pathogen contributing to pneumonic pasteurellosis in sheep. The aim of this study was to investigate the antimicrobial resistance levels in M. haemolytica isolates from the lungs of slaughtered sheep and to examine the genetic resistance mechanisms involved. A total of 256 M. haemolytica isolates, 169 from lungs with pneumonic lesions and 87 from lungs without lesions, were analyzed by the disk diffusion method for 12 antimicrobials, and the whole genome of 14 isolates was sequenced to identify antimicrobial resistance determinants. Levels of phenotypic resistance ranged from <2% for 10 antimicrobials (amoxicillin, amoxicillin-clavulanic, ceftiofur, cefquinome, lincomycin/spectinomycin, gentamicin, erythromycin, florfenicol, enrofloxacin, and doxycycline) to 4.3% for tetracycline and 89.1% for tylosin. Six isolates carried tetH genes and four isolates carried, in addition, the strA and sul2 genes in putative plasmid sequences. No mutations associated with macrolide resistance were identified in 23 rDNA sequences, suggesting that the M. haemolytica phenotypic results for tylosin should be interpreted with care in the absence of well-established epidemiological and clinical breakpoints. The identification of strains phenotypically resistant to tetracycline and of several resistance genes, some of which were present in plasmids, highlights the need for continuous monitoring of susceptibility patterns in Pasteurellaceae isolates from livestock.

First detection of Flavobacterium psychrophilum in juvenile Siberian sturgeons (Acipenser baerii) and description of the pathological findings.

Flavobacterium psychrophilum affects many cultured fish species and is considered one of the most important bacterial pathogens causing substantial economic losses in salmonid aquaculture worldwide. Here, F. psychrophilum was identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and nested PCR as the aetiological agent causing mortality in diseased juvenile Siberian sturgeons (Acipenser baerii) reared on a freshwater fish farm. Diseased sturgeons were lethargic and displayed dark skin pigmentation, increased mucus production and the presence of skin ulcerations and haemorrhages specially on the ventral side and the base of fins. The histological examination of fish revealed proliferative branchitis, ulcerative and necrotizing dermatitis and myositis, lymphoid tissue atrophy, liver and kidney degeneration and thrombosis. To the best of our knowledge, this is the first report describing the infection of Siberian sturgeons by F. psychrophilum. The detection of F. psychrophilum in diseased Siberian sturgeons and the description of the pathological findings observed during the outbreak may contribute to a better understanding of the bacterium pathogenicity and the range of fish species susceptible to infection.

SARS-CoV-2 Seroprevalence Studies in Pets, Spain.

SARS-CoV-2 can infect domestic animals such as cats and dogs. The zoonotic origin of the disease requires surveillance on animals. Seroprevalence studies are useful tools for detecting previous exposure because the short period of virus shedding in animals makes detection of the virus difficult. We report on an extensive serosurvey on pets in Spain that covered 23 months. We included animals with exposure to SARS-CoV-2-infected persons, random animals, and stray animals in the study. We also evaluated epidemiologic variables such as human accumulated incidence and spatial location. We detected neutralizing antibodies in 3.59% of animals and showed a correlation between COVID-19 incidence in humans and positivity to antibody detection in pets. This study shows that more pets were infected with SARS-CoV-2 than in previous reports based on molecular research, and the findings highlight the need to establish preventive measures to avoid reverse zoonosis events.

Genome-wide association reveals host-specific genomic traits in Escherichia coli.

BACKGROUND: Escherichia coli is an opportunistic pathogen which colonizes various host species. However, to what extent genetic lineages of E. coli are adapted or restricted to specific hosts and the genomic determinants of such adaptation or restriction is poorly understood. RESULTS: We randomly sampled E. coli isolates from four countries (Germany, UK, Spain, and Vietnam), obtained from five host species (human, pig, cattle, chicken, and wild boar) over 16 years, from both healthy and diseased hosts, to construct a collection of 1198 whole-genome sequenced E. coli isolates. We identified associations between specific E. coli lineages and the host from which they were isolated. A genome-wide association study (GWAS) identified several E. coli genes that were associated with human, cattle, or chicken hosts, whereas no genes associated with the pig host could be found. In silico characterization of nine contiguous genes (collectively designated as nan-9) associated with the human host indicated that these genes are involved in the metabolism of sialic acids (Sia). In contrast, the previously described sialic acid regulon known as sialoregulon (i.e. nanRATEK-yhcH, nanXY, and nanCMS) was not associated with any host species. In vitro growth experiments with a Δnan-9 E. coli mutant strain, using the sialic acids 5-N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) as sole carbon source, showed impaired growth behaviour compared to the wild-type. CONCLUSIONS: This study provides an extensive analysis of genetic determinants which may contribute to host specificity in E. coli. Our findings should inform risk analysis and epidemiological monitoring of (antimicrobial resistant) E. coli.

A useful tool for the safe diagnosis and control of the two main pandemics of the XXI century: COVID-19 and African Swine Fever disease.

The COVID-19 pandemic and the disease triggered by the African Swine Fever virus are currently two of the main problems regarding public and animal health, respectively. Although vaccination seems to be the ideal tool for controlling these diseases, it has several limitations. Therefore, early detection of the pathogen is critical in order to apply preventive and control measures. Real-time PCR is the main technique used for the detection of both viruses, which requires previous processing of the infectious material. If the potentially infected sample is inactivated at the time of sampling, the diagnosis will be accelerated, impacting positively on the diagnosis and control of the disease. Here, we evaluated the inactivation and preservation properties of a new surfactant liquid for non-invasive and environmental sampling of both viruses. Our results demonstrated that the surfactant liquid effectively inactivates SARS-CoV-2 and African Swine Fever virus in only five minutes, and allows for the preservation of the genetic material for long periods even at high temperatures such as 37°C. Hence, this methodology is a safe and useful tool for recovering SARS-CoV-2 and African Swine Fever virus RNA/DNA from different surfaces and skins, which has significant applied relevance in the surveillance of both diseases.