Specificity of serological test for detection of tuberculosis in cattle, goats, sheep and pigs under different epidemiological situations.

BACKGROUND: Serum antibody detection has potential as a complementary diagnostic tool in animal tuberculosis (TB) control, particularly in multi-host systems. The objective of the present study was to assess the specificity (Sp) of an enzyme-linked immunosorbent assay (ELISA) based on the new multiprotein complex P22 for the detection of specific antibodies against the Mycobacterium tuberculosis complex (MTC) in the four most relevant domestic animals acting as MTC hosts: cattle, goat, sheep and pig. We used sera from an officially TB-free (OTF) country, Norway, and from a non-OTF one, Spain. The samples included sera from goats that had been vaccinated against M. avium subsp. paratuberculosis (MAP) and sheep from a herd in which Corynebacterium pseudotuberculosis had been isolated. RESULTS: In cattle, the Sp ranged from 92.5 (IC95% 90.7-94) to 99.4% (IC95% 98.3-99.8) depending on the cut-off used and the origin of the samples (Spain or Norway). Sp in cattle (cut-off point 100) was significantly higher (P < 0.05) for Norwegian samples. By contrast, Sp in goats was consistently low at the 100 cut-off [30.9 (CI95%23.4-39.5)-78% (CI95% 68.9-85)]. A higher cut-off of 150 improved Sp in Norwegian goats [97% (CI95% 91.6-99)], but still yielded a poor Sp of 56.1% (CI95% 47.3-64.6) in Spanish goats. In Norway at the 100 cut-off the Sp was 58.3 (CI95% 42.2-72.9) and 90.6% (CI95% 81-95.6) in MAP vaccinated and non-vaccinated goats, respectively, indicating interference due to MAP vaccination. Sp in sheep was between 94.4 (CI95% 91.7-96.3) and 100% (CI95% 96.3-100) depending on the cut-off and country, and no diagnostic interference due to infection with C. pseudotuberculosis was recorded. Sp in pigs was 100%, regardless the cut-off point applied, and no significant differences were observed between pigs from Norway and from Spain. CONCLUSIONS: Due to its excellent Sp in pigs and acceptable Sp in cattle and sheep, this ELISA may constitute a suitable option for TB screening at herd level, particularly in OTF-countries.

Biotic and abiotic factors shape the microbiota of wild-caught populations of the arbovirus vector Culicoides imicola.

Biting midges of the genus Culicoides are known vectors of arboviruses affecting human and animal health. However, little is known about Culicoides imicola microbiota and its influence on this insect's biology. In this study, the impact of biotic and abiotic factors on C. imicola microbiota was characterized using shotgun-metagenomic sequencing of whole-body DNA samples. Wild-caught C. imicola adult nulliparous females were sampled in two locations from Sicily, Italy. The climatic variables of temperature and soil moisture from both localities were recorded together with potential host bloodmeal sources. Shared core microbiome among C. imicola populations included Pseudomonas, Escherichia, Halomonas, Candidatus Zinderia, Propionibacterium, and Schizosaccharomyces. Specific and unique taxa were also found in C. imicola from each location, highlighting similarities and differences in microbiome composition between the two populations. DNA and protein identification showed differences in host preferences between the two populations, with Homo sapiens and Canis lupus familiaris L. being the preferred bloodmeal source in both locations. A principal component analysis showed that the combined effect of host preferences (H. sapiens) and local soil moisture factors shape the microbiome composition of wild-caught populations of C. imicola. These results contribute to characterizing the role of the microbiome in insect adaptation and its utility in predicting geographic expansion of Culicoides species with potential implications for the control of vector-borne diseases.

Molecular characterization and antimicrobial resistance of STEC strains isolated from healthy cattle in 2011 and 2013 in Spain.

Prevention of Shiga toxin-producing Escherichia coli (STEC) foodborne outbreaks is hampered by its complex epidemiology. We assessed the distribution of virulence genes (VGs), main serogroups/serotypes for public health [haemolytic uraemic syndrome (HUS)-related], antimicrobial resistance (AMR) profiles and pulsed-field gel electrophoresis (PFGE) patterns in a collection of STEC isolates obtained from cattle hide (n = 149) and faecal (n = 406) samples collected during a national survey conducted in Spain in 2011 and 2013. Isolates were cultured using McConkey and CT-SMAC agar after enrichment, and confirmed as STEC by PCR. STEC prevalence in hides (15·4%) was higher than in faeces (10·7%) and O157:H7 was more frequent in the former (2·7% vs. 0·99%). Non-O157 HUS-related serogroups were present albeit at low frequencies. The non-O157 isolates were more heterogeneous than O157:H7 in their VG patterns, with 25/64 presenting VGs from both STEC and enterotoxigenic pathotypes (hybrid isolates). Of the STEC isolates, 62·5% were resistant at least to one antimicrobial, and no differences in AMR between O157:H7 and non-O157 were detected. All isolates had different profiles by PFGE and did not form a cluster. Overall, our results demonstrated that STEC in the cattle reservoir is still a matter of concern for human health due to the presence of HUS-related serogroups, the occurrence of certain VGs, AMR and the additional risks that hybrid isolates may pose, and thus warrants further investigation.

Contribution of herd management, biosecurity, and environmental factors to the risk of bovine tuberculosis in a historically low prevalence region.

Eradication of bovine tuberculosis (bTB) in certain historically low-prevalence regions remains elusive. A complete characterisation of the husbandry practices, biosecurity, and environment where farms are located is crucial to implement targeted in-farm risk mitigation protocols. Here, a detailed survey performed in 94 dairy cattle farms located in Navarra, a low-prevalence region of Spain between 2016 and 2020 was carried out. Data on 73 biosecurity, farm-, and environmental-level factors potentially associated with the risk of bTB occurrence were evaluated using an ordinal logistic regression model: farms were classified based on their prevalence index, a score linked to each farm to account for the severity and recurrence of bTB cases: 22.3% of the farms had a score of 1, 21.3% a score of 2, 26.6% a score of ≥ 3, and 29.8% were negative herds. A statistically significant association between a higher prevalence index and the frequency of badger sightings along with the lease of pastures to sheep during Winter was identified. Farms that detected badgers on a monthly to daily basis in the surroundings and those that leased pastures for sheep flocks during Winter were four [odds ratio, 95% CI (4.3; 1.1-17.5)] and three (3.1; 1.0-9.9) times more likely to have the highest prevalence index, respectively (predicted probabilityprevalence index≥3 = 0.7; 95% CI 0.3-0.9). Conversely, farms that used a vehicle to transport animals from holdings to pastures were less likely (0.1; <0.1-0.3) to present higher levels of prevalence index compared with farms that used none (on foot). Results suggested that the combined effect of farm- and environmental-level risk factors identified here may be hampering disease eradication in Navarra, highlighting the need to implement targeted protocols on farms and grazing plots. An increased awareness of monitoring sheep and wildlife in direct or indirect contact with cattle herds in historically low bTB prevalence areas should be raised.

Epidemiological factors conditioning sarcoptic mange spreading in wild boar (Sus scrofa).

Sarcoptic mange is a widely distributed disease, with numerous potential hosts among domestic and wild animals. Nowadays it is considered a neglected re-emergent infection in humans. As a difference with domestic pigs, and even with several clinical cases reported in some European countries, it seems that Eurasian wild boars (Sus scrofa) have a low susceptibility to clinical mange. However, because of a case of confirmed transmission from Spanish ibex (Capra pyrenaica) to wild boar in the province of Tarragona, we planned a large-scale ELISA survey in the neighboring Valencian Community (SE Spain). We compared 419 wild boar sera from different management systems (fenced vs. open game estates), different ages (piglets, juveniles, and adults), with different behaviour (gregarious females of all ages and male piglets vs. solitary juveniles and adult males), from areas with different wild boar densities, different wild ruminant densities and different sarcoptic mange epidemiologic situations. The whole prevalence of antibodies against sarcoptic mange in the tested wild boars was 10.5%. No significant differences were found when comparing fenced and free ranging wild boars, males and females, gregarious vs. solitary individuals or among different ages. However, wild boar density was a relevant factor. In areas with a hunting bag of <1 wild boar/km2, considered as a low density of suids, the seroprevalence was 2.94%, but rose to 11.52% in high density districts, constituting a significant difference (p = 0.037). Low wild boar populations would act as a protective factor (OR 0.233; p = 0.049) against coming into contact with the mite. The wild ruminant densities or their sarcoptic mange status did not show any effect on wild boars seroprevalence against this disease. These results reinforce the suggested host-taxon Sarcoptes scabiei specificity and the independence of host-species foci.

Assessment of virulence factors characteristic of human Escherichia coli pathotypes and antimicrobial resistance in O157:H7 and non-O157:H7 isolates from livestock in Spain.

The distribution of virulence factors (VFs) typical of diarrheagenic Escherichia coli and the antimicrobial resistance (AMR) profiles were assessed in 780 isolates from healthy pigs, broilers, and cattle from Spain. VF distribution was broader than expected, although at low prevalence for most genes, with AMR being linked mainly to host species.

Factors driving the circulation and possible expansion of Crimean-Congo haemorrhagic fever virus in the western Palearctic.

AIMS: To produce a spatial risk map regarding spread of Crimean-Congo haemorrhagic fever virus (CCHFV) in the western Palearctic by linking a process-driven model of the main tick vector, Hyalomma marginatum, to a Next Generation Matrix. METHODS AND RESULTS: Process-driven model was composed of deterministic equations that simulate developmental and mortality rates of different tick stages by using temperature and atmospheric water vapour data. The model used climate data at 10-day intervals at a spatial resolution of 10 min over western Palearctic. The model estimated the basic reproduction number, R0, for CCHFV transmission by H. marginatum ticks and evaluated how changes in temperature and biological parameters may alter the geographical range of CCHFV. In particular, variation in the rate of transovarial transmission of CCHFV in the tick produced the greatest change in CCHFV circulation in the tick population. Parameters affecting the rates of tick bite, non-systemic transmission and efficiency of tick-to-tick transmission had little effect on R0. Temperature changes that affect tick development, survival and activity rates increased the suitable area for CCHFV transmission at higher latitudes in the western Palearctic. CONCLUSIONS: Non-systemic transmission had little impact on virus transmission under all scenarios. In the area studied, increase of temperature has no impact on the routes of transmission of CCHFV. However, climate conditions favouring tick survival, which increase infected adult tick populations, together with large numbers of hosts for adults were predicted as the most likely scenario for the spread of the virus in the studied area. SIGNIFICANCE AND IMPACT OF THE STUDY: The proposed framework is able to capture the dynamics and the relative contribution of the different routes (hosts, ticks) in the transmission and spread of an important pathogen affecting human health. The high contribution of the transovarial transmission route makes the process highly dependent upon suitable hosts for adult ticks, like large domestic and wild ungulates. Climate seems to have a very reduced effect on such spread.

Leishmania infantum in free-ranging hares, Spain, 2004-2010.

Iberian hares (Lepus granatensis) were recently deemed responsible for an outbreak of human leishmaniasis affecting metropolitan Madrid, Spain. However, the reservoir potential of hares in Europe is poorly known. We report a retrospective survey on Leishmania infantum, the causal agent of zoonotic endemic leishmaniasis in the Mediterranean basin, infection status of Iberian, European (Le. europaeus) and Broom (Le. castroviejoi) hares in Spain. Spleen samples from 94 hares were tested by polymerase chain reaction. Sequencing and restriction fragment length polymorphism (RFLP) assays were performed on positive samples and RFLP patterns compared with those of strains reported in the scientific literature. DNA prevalence in hare spleen samples was 43.6% (95% confidence interval: 33.6-53.6). In all six regions studied at least one positive sample was found. RFLP revealed existence of specific hare strains of L. infantum differing from those reported in wild carnivores in Spain. The widespread presence of L. infantum in the most abundant Spanish hare species and the recent evidence of the ability of naturally infected hares to transmit the pathogen to Phlebotomus perniciosus, its main vector in the western Mediterranean, suggest that hares may have an unexpected role in the epidemiology of L. infantum in Spain.

Using integrated wildlife monitoring to prevent future pandemics through one health approach.

In the One Health context, Integrated Wildlife Monitoring (IWM) merges wildlife health monitoring (WHM) and host community monitoring to early detect emerging infections, record changes in disease dynamics, and assess the impact of interventions in complex multi-host and multi-pathogen networks. This study reports the deployment and results obtained from a nationwide IWM pilot test in eleven sites representing the habitat diversity of mainland Spain. In each study site, camera-trap networks and sampling of indicator species for antibody and biomarker analysis were used to generate information. The results allowed identifying differences in biodiversity and host community characteristics among the study sites, with a range of 8 to 19 relevant host species per point. The Eurasian wild boar (Sus scrofa) was the most connected and central species of the host communities, becoming a key target indicator species for IWM. A negative relationship between biodiversity and disease risk was detected, with a lower number and prevalence of circulating pathogens in the sites with more species in the community and larger network size. However, this overall trend was modified by specific host-community and environmental factors, such as the relative index of wild boar - red deer interactions or the proximity to urban habitats, suggesting that human-driven imbalances may favour pathogen circulation. The effort of incorporating wildlife population monitoring into the currently applied WHM programs to achieve effective IWM was also evaluated, allowing to identify population monitoring as the most time-consuming component, which should be improved in the future. This first nationwide application of IWM allowed to detect drivers and hotspots for disease transmission risk among wildlife, domestic animals, and humans, as well as identifying key target indicator species for monitoring. Moreover, anthropogenic effects such as artificially high wildlife densities and urbanisation were identified as risk factors for disease prevalence and interspecific transmission.

Establishing a European network for wildlife health surveillance.

Surveillance of wildlife health in Europe remains informal and reporting wildlife diseases is not yet coordinated among countries. At a meeting in Brussels on 15 October 2009, delegates from 25 countries provided an overview of the current status of wildlife health surveillance in Europe. This showed that every year in Europe over 18,000 wild animals are examined as part of general surveillance programmes and over 50,000 wild animals are examined in the course of targeted surveillance. The participants at the Brussels meeting agreed to set up a European network for wildlife health surveillance. The goals of this network, which was established in February 2010, are to improve procedures for the rapid exchange of information, harmonise procedures for investigation and diagnosis of wildlife diseases, share relevant expertise, and provide training opportunities for wildlife health surveillance.

Assessing the risks of SARS-CoV-2 in wildlife.

The novel coronavirus SARS-CoV-2 likely emerged from a wildlife source with transmission to humans followed by rapid geographic spread throughout the globe and severe impacts on both human health and the global economy. Since the onset of the pandemic, there have been many instances of human-to-animal transmission involving companion, farmed and zoo animals, and limited evidence for spread into free-living wildlife. The establishment of reservoirs of infection in wild animals would create significant challenges to infection control in humans and could pose a threat to the welfare and conservation status of wildlife. We discuss the potential for exposure, onward transmission and persistence of SARS-CoV-2 in an initial selection of wild mammals (bats, canids, felids, mustelids, great apes, rodents and cervids). Dynamic risk assessment and targeted surveillance are important tools for the early detection of infection in wildlife, and here we describe a framework for collating and synthesising emerging information to inform targeted surveillance for SARS-CoV-2 in wildlife. Surveillance efforts should be integrated with information from public and veterinary health initiatives to provide insights into the potential role of wild mammals in the epidemiology of SARS-CoV-2.

Effects of density, climate, and supplementary forage on body mass and pregnancy rates of female red deer in Spain.

The influence of short- and long-term (cohort) effects of climate and density on the life-histories of ungulates in temperate regions may vary with latitude, habitat, and management practices, but the life-histories of ungulates in the Mediterranean region are less well known. This study examined the short- and long-term effects of rainfall and absolute density on hinds in two of the southernmost populations of red deer (Cervus elaphus hispanicus) in Europe. One population received supplementary forage. Unlike more northerly latitudes, where red deer hinds lose body mass in winter as a result of adverse weather, in the Spanish populations, hinds did not lose body mass. Hinds in the population that received supplementary forage were heavier and more likely to become pregnant than were the hinds in the unsupplemented population. The likelihood of pregnancy occurring was strongly influenced by hind body mass; the proportion of yearlings that became pregnant was consequently lower in the unsupplemented population than in the population that received supplementary forage. Cohort effects on hind body mass (negative for density and positive for rainfall at birth) and on the probability of pregnancy (negative for density at birth) were apparent only in the unsupplemented population, which implies that supplemental feeding may partially compensate for negative density-dependent factors during early growth, and that supplemented deer hinds may experience reduced selection pressures. These results reflect the particular seasonal variation in the abundance and quality of food in Mediterranean habitats. The delayed effects of climate and density at birth on adult hind body mass and the prevalence of pregnancy probably affects population dynamics and constitutes a mechanism by which cohort effects affect the population dynamics in Iberian red deer. The management of Iberian red deer populations should take into account cohort effects and supplemental feeding practices, which can buffer density- and climate-dependent effects and reduce natural selection pressures.

Evaluation of a new enzyme-linked immunosorbent assay for the diagnosis of tuberculosis in goat milk.

Caprine tuberculosis (TB) is a zoonosis with sanitary and economic repercussions. Caprine TB control programs are based on a test and cull strategy using the intradermal tuberculin tests and slaughterhouse surveillance. However, this approach is not always feasible and may have a limited sensitivity under specific circumstances. In this study, performance of a new experimental test based on the P22 protein complex (P22 ELISA) was evaluated in two TB-infected herds using milk and serum samples and compared with cell-based diagnostic tests. Samples from a low (n = 62, herd 1) and a high (n = 52, herd 2) TB prevalence herd were selected. Moreover, bulk tank milk samples from both herds were analysed using the P22 ELISA. At the end of the study, a group of animals (n = 21) was euthanized and subjected to post-mortem analysis and bacteriological culture. Significant differences (p < .001) on the qualitative and quantitative (ODs) results were observed between herds using both serum and milk samples in the P22 ELISA. The correlation observed in the quantitative results obtained in serum and milk samples was very strong in animals from flock 2 (rs = 0.91) and moderate in animals from flock 1 (rs = 0.46). Among the slaughtered animals, the P22 ELISA detected a higher proportion of lesion-culture positive animals than cell-based diagnostic tests (61.9 and 66.7% using milk and serum samples, respectively). The P22 ELISA using milk samples demonstrated a similar sensitivity compared with serum samples, suggesting it might be a valuable test for TB control in dairy goats.

Risk factors for African swine fever incursion in Romanian domestic farms during 2019.

African swine fever (ASF) entered Georgia in 2007 and the EU in 2014. In the EU, the virus primarily spread in wild boar (Sus scrofa) in the period from 2014-2018. However, from the summer 2018, numerous domestic pig farms in Romania were affected by ASF. In contrast to the existing knowledge on ASF transmission routes, the understanding of risk factors and the importance of different transmission routes is still limited. In the period from May to September 2019, 655 Romanian pig farms were included in a matched case-control study investigating possible risk factors for ASF incursion in commercial and backyard pig farms. The results showed that close proximity to outbreaks in domestic farms was a risk factor in commercial as well as backyard farms. Furthermore, in backyard farms, herd size, wild boar abundance around the farm, number of domestic outbreaks within 2 km around farms, short distance to wild boar cases and visits of professionals working on farms were statistically significant risk factors. Additionally, growing crops around the farm, which could potentially attract wild boar, and feeding forage from ASF affected areas to the pigs were risk factors for ASF incursion in backyard farms.

What does testosterone do for red deer males?

Testosterone has been proposed to have a dual effect, enhancing sexual traits while depressing parasite resistance in males. Here, we test this hypothesis in red deer, examining males from captive populations during the whole annual cycle and males from natural populations during the breeding season. We first explored the effects of body size, age and sampling date on testosterone to avoid confounding effects. Our results show that in captive populations seasonal changes in testosterone levels were mirrored by changes in testes size, and that during the rut there was a strong correlation between both. In natural populations, males with higher testosterone levels had larger testes, improved sperm quality, smaller burr diameter, stronger antlers, higher haematocrit levels, and increased nematode parasite load. By contrast, no significant relationship was found between testosterone and spleen size or tick parasite load. We conclude that testosterone (i) improves males' reproductive investment and physical stamina, (ii) improves antler strength but reduces burr diameter, and (iii) imposes a cost in terms of depressed parasite resistance.

High prevalence of antibodies against Chlamydiaceae and Chlamydophila abortus in wild ungulates using two "in house" blocking-ELISA tests.

Few data are available on the prevalence and relevance of chlamydiae in wild mammals, and even fewer studies have been conducted to determine the prevalence of Chlamydophila abortus in wildlife hosts, most probably due to the absence of suitable species-specific serological assays for testing sera from wild animals. In light of this, we have developed two in-house blocking-ELISA tests for detection of antibodies against Chlamydiaceae and C. abortus in wild ungulates, and analyzed the relationship between geographical and biological factors and the prevalence of antibodies against Chlamydiaceae and C. abortus in 434 wild ungulates from Spain, including sera from European wild boar, Red deer, Fallow deer, Roe deer, Mouflon, Barbary sheep, Southern chamois, and Iberian ibex. Serology revealed that 41.7+/-4% of the sera were positive for the b-ELISA-LPS (Chlamydiaceae-specific) and 18.9+/-3% for the b-ELISA-rPOMP (C. abortus-specific). Antibodies against Chlamydiaceae lipopolysaccharide (LPS) were detected in sera from all eight ungulate species, the prevalence ranging from 23 to 60%. Iberian ibex was the only wild ungulate not showing seropositivity to the C. abortus specific polymorphic outer membrane protein (POMP). The prevalence of anti-POMP antibodies in the other seven wild ungulate species ranged from 7 to 40%. While significant seroprevalence differences were detected among species and among sampling regions, no effect of age and sex was observed. The high prevalence levels found should be considered with regards to livestock and human health, and warrant further research.

Wolves contribute to disease control in a multi-host system.

We combine model results with field data for a system of wolves (Canis lupus) that prey on wild boar (Sus scrofa), a wildlife reservoir of tuberculosis, to examine how predation may contribute to disease control in multi-host systems. Results show that predation can lead to a marked reduction in the prevalence of infection without leading to a reduction in host population density since mortality due to predation can be compensated by a reduction in disease induced mortality. A key finding therefore is that a population that harbours a virulent infection can be regulated at a similar density by disease at high prevalence or by predation at low prevalence. Predators may therefore provide a key ecosystem service which should be recognised when considering human-carnivore conflicts and the conservation and re-establishment of carnivore populations.

Serum haptoglobin response in red deer naturally infected with tuberculosis.

The analysis of haptoglobin (Hp) serum concentration is a very sensitive, but non-specific, indicator of inflammation or infection. Methods to accurately diagnose infection in vivo in wildlife are usually constrained by low sensitivity due to the effects of stress on individual immune response and the challenging logistics of performing tests in the wild. Firstly, we sought to determine serum Hp concentration in red deer (Cervus elaphus) naturally infected with bovine tuberculosis (TB). Secondly, we assessed the complementary diagnostic value of serum Hp levels in conjunction with the cervical comparative skin test (CCT) performed in a subsample (n = 33). Serum Hp concentrations were significantly higher in TB-infected individuals (based on the presence of macroscopic lesions confirmed by culture) compared to those uninfected. In addition, serum Hp significantly changed with the type of animal handling, with captured and handled animals showing higher levels of Hp than hunted animals. Four out of 6 TB positive individuals that tested negative to the CCT (false negatives) showed Hp levels higher than the 95th percentile of healthy animals. These findings indicate that an acute phase response develops in animals with TB. In this paper, we demonstrate for the first time that an acute phase protein can provide a complementary assessment for specific diagnosis tests in wild species.

A new test to detect antibodies against Mycobacterium tuberculosis complex in red deer serum.

Red deer (Cervus elaphus) farming is a growing economic activity worldwide. However, the capacity of this species to act as reservoir of animal tuberculosis (TB) poses a threat to other wildlife and to livestock. Diagnostic assay accuracy in this species is therefore highly relevant for prevention and control measures. Our aim was to evaluate the diagnostic performance of the protein complex P22, obtained from Mycobacterium bovis derived purified protein derivative (bPPD), as a candidate antigen for the detection of antibodies against Mycobacterium tuberculosis complex (MTC). We assessed the performance of this new antigen in indirect enzyme-linked immunosorbent assays (ELISA) in TB-positive and TB-negative red deer, in comparison with a bPPD-based ELISA. The P22 ELISA achieved a higher specificity (Sp) and similar sensitivity (Se) in comparison with the bPPD ELISA at all the cut-off points considered. The P22 ELISA yielded optimal Sp (99.02%; 95% confidence intervals [CI95%]: 96.5-99.8) and appropriate Se (70.1%; CI95%: 63.6-76) at the selected cut-off point of 100%. These results suggest that P22 can be used as an alternative antigen in the immunodiagnosis of animal TB through the use of an ELISA-type detection of antibodies against MTC in red deer, thus contributing to the diagnosis of animal TB in this species as a measure for further disease prevention and control programs.