Epidemiologic Survey of Crimean-Congo Hemorrhagic Fever Virus in Suids, Spain.

We conducted a cross-sectional study in wild boar and extensively managed Iberian pig populations in a hotspot area of Crimean-Congo hemorrhagic fever virus (CCHFV) in Spain. We tested for antibodies against CCHFV by using 2 ELISAs in parallel. We assessed the presence of CCHFV RNA by means of reverse transcription quantitative PCR protocol, which detects all genotypes. A total of 113 (21.8%) of 518 suids sampled showed antibodies against CCHFV by ELISA. By species, 106 (39.7%) of 267 wild boars and 7 (2.8%) of 251 Iberian pigs analyzed were seropositive. Of the 231 Iberian pigs and 231 wild boars analyzed, none tested positive for CCHFV RNA. These findings indicate high CCHFV exposure in wild boar populations in endemic areas and confirm the susceptibility of extensively reared pigs to CCHFV, even though they may only play a limited role in the enzootic cycle.

Serosurveillance of Trichinella sp. in wild boar and Iberian domestic suids in Mediterranean ecosystems of southwestern Spain.

AIMS: A cross-sectional study was carried out to assess the seroprevalence and risk factors associated with Trichinella spp. exposure in wild boar and Iberian domestic pigs from Mediterranean ecosystems of southwestern Spain. METHODS AND RESULTS: Serum samples from 1360 wild boar and 439 Iberian domestic pigs were obtained during 2015-2020, from regions where Iberian pigs are raised under extensive conditions, hence sharing habitat with wild boar. Seropositivity was found in 7.4% (100/1360; 95% CI: 6.1-8.9) of the wild boar analysed. In this species, the individual seroprevalence ranged from 3.6% (8/223) (hunting season 2016-2017) to 11.4% (37/326) (2018-2019). A significant higher seropositivity was observed during the hunting season 2018-2019 (p < 0.009: OR = 3.07; 95% CI = 1.32-7.18) and one statistically significant cluster was detected within the studied area, in south central Andalusia [Relative Risk (RR) = 2.9; p = 0.037]. Females showed a significantly higher seroprevalence than males (8.7% vs. 5.8%) (p < 0.001: OR = 1.58; 95% CI = 1.08-2.32). No seropositivity to Trichinella spp. was detected in Iberian domestic pigs (0.0%; 95% CI: 0.0-0.9). CONCLUSIONS: Although wild boar play an important role as a reservoir of Trichinella sp. in the Mediterranean ecosystems of southwestern Spain, our results suggest that the wild boar production system does not seem to pose a risk of Trichinella exposure to domestic pigs, despite sharing habitats in these ecosystems.

Large-scale study on virological and serological prevalence of SARS-CoV-2 in cats and dogs in Spain.

The disease produced by the severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) is currently one of the primary concerns worldwide. Knowing the zoonotic origin of the disease and that several animal species, including dogs and cats, are susceptible to viral infection, it is critical to assess the relevance of pets in this pandemic. Here, we performed a large-scale study on SARS-CoV-2 serological and viral prevalence in cats and dogs in Spain in order to elucidate their role and susceptibility. Samples from animals in contact with COVID-19 positive people and/or compatible symptoms (n = 492), as well as from random animals (n = 1024), were taken. Despite the large number of animals analyzed, only 12 animals (eight dogs and four cats), which represents 0.79% of the total analyzed animals (n = 1516), were positive for viral SARS-CoV-2 RNA detection by reverse transcription quantitative PCR (RT-qPCR) in which viral isolation was possible in four animals. We detected neutralizing antibodies in 34 animals, four of them were also positive for PCR. This study evidences that pets are susceptible to SARS-CoV-2 infection in natural conditions but at a low level, as evidenced by the low percentage of positive animals detected, being infected humans the main source of infection. However, the inclusion of animals in the surveillance of COVID-19 is still recommended.

A survey of shared pathogens at the domestic-wild ruminants' interface in Doñana National Park (Spain).

A cross-sectional study was carried out to evaluate shared pathogens that can be transmitted by close or non-close contact at the domestic-wild ruminants' interface. During summer-autumn 2015, a total of 138 cattle and 203 wild ruminants (red deer, Cervus elaphus, and fallow deer, Dama dama) were sampled in Doñana National Park (DNP, south-western Spain), a Mediterranean ecosystem well known for the interaction network occurring in the ungulate host community. Pestiviruses, bovine respiratory syncytial virus (BRSV; Bovine orthopneumovirus), bovine herpesvirus 1 (BoHV-1; Bovine alphaherpesvirus 1) and Mycobacterium tuberculosis complex (MTC) were assessed using serological, microbiological and molecular techniques. The overall seroprevalence against viruses in cattle was 2.2% for pestiviruses, 11.6% for BRSV and 27.5% for BoHV-1. No virus-specific antibodies were found in wildlife. MTC incidence in cattle was 15.9%, and MTC seroprevalence in wild ruminants was 14.3%. The same Mycobacterium bovis spoligotypes (SB1232, SB1230 and SB1610) were identified in cattle, red deer and fallow deer. The serological results for the selected respiratory viruses suggest epidemiological cycles only in cattle. Surveillance efforts in multi-host epidemiological scenarios are needed to better drive and prioritize control strategies for shared pathogens.

The wildlife-livestock interface on extensive free-ranging pig farms in central Spain during the "montanera" period.

The effective management of shared pathogens between wild ungulates and livestock requires the understanding of the processes of interaction between them. In this work, we studied the interspecific frequency of interaction (ifreq) and its spatiotemporal pattern between wild and domestic ungulates that coexist in free-ranging farms. For this purpose, 6 red deer, 6 wild boar, 8 Iberian pigs and 3 cattle were monitored using GPS devices during the "montanera" period (the period in which Iberian pigs are maintained in extensive conditions to feed on acorn). The ifreq was quantified for two spatiotemporal windows: 30 m - 10 min, for inferring potential direct interactions (short window), and 30 m - 12 days for indirect interactions (large window). Secondly, the variation in the ifreq was modelled with regard to 2 temporal (time of the day and week of the year) and 4 environmental factors (distance to water, distance to vegetation cover, Quercus density and distance to feeding points). The interactions at the short window were scarce (N = 13); however, they were very frequent at the large one (N = 37,429), with the red deer as the species with the greatest involvement in the interactions. Models showed that the time of the day and distance to water were the variables that best predicted the ifreq and they were conditioned by differences in the activity pattern of the targeted species. Food resource availability also predicted the ifreq, especially at the short window and between wild species. The results presented here highlight the role that wild ungulates may play in the transmission of pathogens to extensive livestock in general and pigs in particular and show the epidemiological risk of certain areas, periods of time and management practices (for wildlife and livestock) as well as providing useful information in the prevention of the transmission of shared pathogens.

Vaccination with Alpha-Gal Protects Against Mycobacterial Infection in the Zebrafish Model of Tuberculosis.

The alpha-Gal syndrome (AGS) is associated with tick bites that can induce in humans high levels of IgE antibodies against the carbohydrate Galα1-3Galß1-(3)4GlcNAc-R (α-Gal) present in glycoproteins and glycolipids from tick saliva that mediate primarily delayed anaphylaxis to mammalian meat consumption. It has been proposed that humans evolved by losing the capacity to synthesize α-Gal to increase the protective immune response against pathogens with this modification on their surface. This evolutionary adaptation suggested the possibility of developing vaccines and other interventions to induce the anti-α-Gal IgM/IgG protective response against pathogen infection and multiplication. However, the protective effect of the anti-α-Gal immune response for the control of tuberculosis caused by Mycobacterium spp. has not been explored. To address the possibility of using vaccination with α-Gal for the control of tuberculosis, in this study, we used the zebrafish-Mycobacterium marinum model. The results showed that vaccination with α-Gal protected against mycobacteriosis in the zebrafish model of tuberculosis and provided evidence on the protective mechanisms in response to vaccination with α-Gal. These mechanisms included B-cell maturation, antibody-mediated opsonization of mycobacteria, Fc-receptor (FcR)-mediated phagocytosis, macrophage response, interference with the α-Gal antagonistic effect of the toll-like receptor 2 (TLR2)/nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB)-mediated immune response, and upregulation of pro-inflammatory cytokines. These results provided additional evidence supporting the role of the α-Gal-induced immune response in the control of infections caused by pathogens with this modification on their surface and the possibility of using this approach for the control of multiple infectious diseases.

Hypervitaminosis D has no positive effects on goat tuberculosis and may cause chronic renal lesions.

BACKGROUND: There is evidence for a link between vitamin D deficiency and active tuberculosis (TB). In human beings, several trials have evaluated the role of vitamin D supplementation in TB treatment with conflicting results. However, the role of vitamin D supplementation in animal TB control has received less attention. The authors evaluated the benefit of vitamin D supplementation for preventing mycobacterial infection or reducing TB lesions (TBL) in a controlled trial with goats naturally exposed to Mycobacterium caprae. METHODS: Two groups of goats, a vitamin D-supplemented group and a non-supplemented control group, were housed for 10 months in direct contact with M caprae-infected adult goats. Upon contact with the infected adult goats, all animals were TB-tested every two months. RESULTS: No experimental evidence of a protective effect of vitamin D supplementation based on M caprae culture prevalence, TBL prevalence, median TBL score or the proportion of single versus multiple organs presenting TBL was observed. CONCLUSION: The results indicate that, in the conditions used in this study, vitamin D supplementation in goats does not reduce TB infection risk nor the diffusion and severity of TBL. In addition, vitamin D-supplemented goats presented hyperphosphataemia and renal injury with calcifications suggestive of vitamin D intoxication.

Different lesion distribution in calves orally or intratracheally challenged with Mycobacterium bovis: implications for diagnosis.

Animal tuberculosis (TB) remains a major problem in some countries despite the existence of control programmes focused mainly on cattle. In this species, aerogenous transmission is accepted as the most frequent infection route, affecting mainly the respiratory system. Under the hypothesis that the oral route could be playing a more relevant role in transmission, diagnosis and disease persistence than previously thought, this study was performed to assess the course of TB infection in cattle and its effects on diagnosis depending on the route of entry of Mycobacterium bovis. Two groups of five calves each were either endotracheally (EC) or orally (OC) challenged. Necropsies were carried out 12 weeks after challenge except for three OC calves slaughtered 8 weeks later. All animals reacted to the tuberculin skin test and the entire EC group was positive to the interferon-gamma release assay (IGRA) 2 weeks after challenge and thereafter. The first positive IGRA results for OC calves (3/5) were recorded 4 weeks after challenge. Group comparison revealed significant differences in lesion and positive culture location and scoring. TB-compatible gross lesions and positive cultures were more frequently found in the thorax (p < 0.001) and lung (p < 0.05) of EC animals, whereas OC animals presented lesions (p = 0.23) and positive cultures (p < 0.05) mainly located in the abdomen. These results indicate that the infection route seems to be a determining factor for both the distribution and the time needed for the development of visible lesions. Our study suggests that confirmation of TB infection in some skin reactor animals can be problematic if current post-mortem examination and diagnostics are not improved.

Heat-inactivated Mycobacterium bovis protects zebrafish against mycobacteriosis.

Control of mycobacterial infection constitutes a priority for human and animal health worldwide. However, effective vaccines are needed for the control of human and animal tuberculosis (TB). Adult zebrafish have become a useful model for studying the pathophysiology of mycobacterial infection and for the development of novel interventions for TB control and prevention. Recently, parenteral and oral immunization with the heat-inactivated Mycobacterium bovis vaccine (M. bovis IV) protected wild boar against TB. The objectives of this study were to provide additional support for the role of M. bovis IV in TB control using the zebrafish model and to conduct the first trial with this vaccine for the control of fish mycobacteriosis. The results showed that M. bovis IV protected zebrafish against mycobacteriosis caused by low and high infection doses of Mycobacterium marinum and provided evidence suggesting that the protective mechanism elicited by M. bovis IV in zebrafish as in other species is based on the activation of the innate immune response through the C3 pathway, with a role for the regulatory protein Akr2 in this process. These results encourage the use of M. bovis IV for TB control in different species.

Control of mycobacteriosis in zebrafish (Danio rerio) mucosally vaccinated with heat-inactivated Mycobacterium bovis.

BACKGROUND: Mycobacterial infections greatly affect human and animal health worldwide, and vaccines are effective, sustainable and economic interventions for the prevention and control of these infectious diseases. Recent results support the use of zebrafish as a model for studying the pathophysiology of mycobacterial infection and for the development of novel interventions for tuberculosis (TB) control. Recently, we showed that oral immunization with the heat-inactivated M. bovis vaccine (M. bovis IV) protect wild boar against TB, and suggested that this vaccine may controls mycobacterial infection in other species. METHODS: In this study we evaluated the effect of M. bovis IV on the control of mycobacteriosis in zebrafish mucosally vaccinated by immersion and challenged intraperitoneally with Mycobacterium marinum. RESULTS: The results showed that the M. bovis IV administered by immersion protected zebrafish against mycobacteriosis caused by M. marinum by reduction in mycobacterial infection, the number of mycobacteria per granuloma and the number of granulomas per fish. An IgM antibody response against M. bovis antigens was developed in vaccinated fish. Evidences suggested that the protective mechanism elicited by mucosal vaccination with M. bovis IV in zebrafish was based on the activation of the innate immune response through the C3 pathway. CONCLUSIONS: These results support the use of the M. bovis IV administered by immersion for the control of mycobacteriosis in fish.

Absence of protection from West Nile virus disease and adverse effects in red legged partridges after non-structural NS1 protein administration.

The red-legged partridge (Alectoris rufa) is a competent host for West Nile virus (WNV) replication and highly susceptible to WNV disease. With the aim to assess in this species whether the inoculation of non-structural protein NS1 from WNV elicits a protective immune response against WNV infection, groups of partridges were inoculated with recombinant NS1 (NS1 group) or an unrelated recombinant protein (mock group), and challenged with infectious WNV. A third group received no inoculation prior to challenge (challenge group). The NS1 group failed to elicit detectable antibodies to NS1 while in the mock group a specific antibody response was observed. Moreover, no protection against WNV disease was observed in the NS1 group, but rather, it showed significantly higher viral RNA load and delayed neutralizing antibody response, and suffered a more severe clinical disease, which resulted in higher mortality. This adverse effect has not been observed before and warrants further investigations.

HLA-B, HLA-C and KIR improve the predictive value of IFNL3 for Hepatitis C spontaneous clearance.

IFNL3 is the strongest predictor of spontaneous resolution (SR) of hepatitis C virus (HCV), however, consideration of IFNL3 genotype alone is of limited clinical value for the prediction of SR or chronic HCV infection. The objective of this study was to analyze the impact of HLA-B, HLA-C and KIRs on SR, as well as their additive effects on the predictive value of the IFNL3 genotype. We conducted a retrospective study of HIV patients that included both SR and chronic HCV patients. In our study, 61.6% of patients with IFNL3 CC achieved SR, and 81.5% with non-CC genotypes did not achieve SR. HLA-B*44, HLA-C*12, and KIR3DS1 were identified as predictive factors for SR, with percentages of 77.4%, 85.7% and 86.2%, respectively, for patients who did not experience SR. The presence of at least one of these three markers, defined as a genetically unfavorable profile (GUP), combined with the IFNL3 non-CC genotype showed a value of 100% for non-SR. The absence of the three markers, defined as a genetically favorable profile (GFP), in addition to the IFNL3 CC genotype showed a percentage of 74.1% for SR. The combination of these markers in addition to the IFNL3 genotype improves the predictive value of IFNL3 for SR of acute HCV infection in HIV patients, which would be clinically valuable.

Development and evaluation of an interferon gamma assay for the diagnosis of tuberculosis in red deer experimentally infected with Mycobacterium bovis.

BACKGROUND: Red deer (Cervus elaphus) is regarded as an epidemiologically relevant host for Mycobacterium bovis (M. bovis) and closely related members of the Mycobacterium tuberculosis complex that cause animal tuberculosis (TB). The standard antemortem screening test for the detection of TB in deer is the intradermal tuberculin skin test, but the detection of interferon-gamma (IFNγ) produced by white blood cells exposed to M. bovis antigens can be used as an alternative or supplemental assay in most TB eradication/control programs. This study aims to develop an in-house sandwich ELISA for deer IFNγ, based on the cross-reactivity of the antibodies to both cervid and bovine IFNγ, and to evaluate the potential of this assay to detect M. bovis-infected red deer in response to the in vitro stimulation of whole-blood cells with bovine purified protein derivative (bPPD), p22 protein complex derived from bPPD or using the specific tuberculous mycobacterial proteins ESAT-6/CFP-10, Rv3615c and Rv3020c. The positive control stimulant used in this study was pokeweed mitogen, which resulted in a consistent induction of IFNγ in samples from red deer, thus allowing the interpretation of the assay. RESULTS: The percentage of animals correctly classified by this technique as M. bovis non-infected was 100%. The detection of infected animals as positive was high and ranged widely depending upon the antigen and the cut-off value applied, as well as the time after infection. Our findings indicate that this protocol may serve as a reliable assay for the antemortem diagnosis of TB from the initial stage of M. bovis-infection, and may also be adequately sensitive. CONCLUSIONS: The suggested optimal antigens and cut-off are bPPD, p22 and the combination of ESAT-6/CFP-10 and Rv3020c with a 0.05 Δ optical density, which yielded a up to 100% correct classification of TB positive and negatve red deer under our experimental conditions. This technique will aid in TB testing of farmed and translocated deer. Future studies should evaluate the ability of this IFNγ assay to detect specific responses under field conditions.

The response of red deer to oral administration of heat-inactivated Mycobacterium bovis and challenge with a field strain.

Deer species (family Cervidae) are often part of the Mycobacterium tuberculosis complex maintenance host community, and tuberculosis (TB) control in deer, including vaccination, is consequently an area of ongoing research. However, most research into deer vaccination against TB is focused on using the live bacillus Calmette Guerin (BCG). Oral inactivated vaccines represent an interesting alternative to either oral or parenteral BCG, since neither diagnostic cross-reactions nor vaccine strain survival are likely to occur. In order to describe the red deer response to heat-inactivated M. bovis (IV) as compared to BCG and to unvaccinated controls (n=5/group), we ran an experiment with five month-old vaccinated red deer, which were challenged with a virulent M. bovis strain 70days later and necropsied at 60days post-challenge. A reduction in the IV group infection burden was discovered. There were significant differences between the IV group and the control group (53% lesion reduction) as regards to the TB lesion scores, but not between other pairs. Complement component 3 plasma levels increased after challenge, and there were no differences between groups. The plasma cytokines (IL-1ß, TNFα, IFNγ, IL-10 and IL-12) levels did not change after vaccination, but IL-1ß, TNFα and IL-10 did so following the challenge. The IL-1ß level increased in all the groups while TNFα levels had a distinct response pattern in the IV group and IL-10 had a distinct response pattern in control group. The results showed that oral vaccination with IV reduces the TB lesion score in red deer challenged with a M. bovis field strain without interfering with the in vivo diagnosis of infection in this species.

Evaluation of five serologic assays for bovine tuberculosis surveillance in domestic free-range pigs from southern Spain.

In countries where bovine tuberculosis (bTB) is still prevalent the contact among different animal species in extensive systems contributes to the circulation of Mycobacterium bovis (M. bovis) and other members of the Mycobacterium tuberculosis complex (MTC). Thus, free-range pigs can develop subclinical infections and may contribute to disease spread to bovine and wildlife. Serodiagnosis has been proposed as a screening tool for detecting infected pig herds; however, the value of this method to obtain an accurate diagnosis in this species is still not clear. In this study, sensitivity (Se) and specificity (Sp) estimates of four ELISAs and a lateral flow immunochromatographic antibody assay based on different M. bovis antigens, including MPB70 and MPB83 proteins, were evaluated in naturally infected domestic free-range pigs. For this purpose, submandibular lymph nodes and blood samples from 217 pigs from both TB-infected and historically negative farms were sampled at slaughterhouse and analysed by gross examination, histopathology, bacteriological culture and qPCR. Se and Sp estimates of the 5 evaluated assays ranged from 66.1% to 78% (CI95 from 52.6 to 87.7%) and from 98.9% to 100% (CI95 from 93.8 to 100%), respectively. Results of our study suggest that all the evaluated assays could be used as a first screening tool to conduct bTB surveillance in domestic pigs at population level; however, animals from seropositive herds should later be surveyed by other methods in order to reduce false negative results.

Effects of repeated comparative intradermal tuberculin testing on test results: a longitudinal study in TB-free red deer.

BACKGROUND: Diagnosing tuberculosis (TB) in farmed red deer (Cervus elaphus) is challenging and might require combining cellular and humoral diagnostic tests. Repeated skin-testing with mycobacterial purified protein derivatives (PPDs) might sensitize or desensitize the subjects to both kinds of diagnostic tools. We evaluated the effect of repeated (every 6 months) comparative tuberculin skin testing on skin test and ELISA responsiveness in farmed red deer hinds from a TB-free herd. Eighteen 8-month old hinds were inoculated with bovine and avian PPDs and the mitogen phytohaemagglutinin (PHA), as positive control and concurrently tested by ELISA for antibodies against avian (avian PPD, aPPD and protoplasmatic antigen 3, PPA3) and bovine antigens (bPPD and MPB70). Blood serum was also sampled three weeks after each skin testing round and tested for antibodies against aPPD and bPPD, in order to detect eventual antibody level boosts. Testing took place every six months from winter 2012 until winter 2015. RESULTS: The skin test response to both PPDs peaked during the second and third test round, returning to standard values thereafter. Individual variability was particularly high at the first year and early second year testing rounds (first intradermal test and blood sampling; first winter). The antibody response to avian antigens increased through time, while no such increase was recorded for bovine antigens. The antibody boost three weeks after skin testing was more marked for avian PPD. However, there was no consistent trend in the boosting response through time. CONCLUSION: Repeated comparative skin testing at six month intervals did not cause progressive increments in skin test responsiveness or antibody production. Specifically, we observed no loss of the skin test response to bPPD and also no progressive loss of the boosting effect in the ELISA responses. However, we recorded increases through time in the antibody levels against avian mycobacterial antigens, possibly due to the progressive exposure to MAP or to other cross-reacting environmental mycobacteria. These findings should be taken into account in designing and interpreting TB testing schemes in farmed deer.