Beyond tuberculosis: Diversity and implications of non-tuberculous mycobacteria at the wildlife-livestock interface.

Non-tuberculous mycobacteria (NTM) circulate between the environment, animals and humans entailing a double concern: their ability to interfere with tuberculosis diagnosis and their potential to cause infections in their hosts. However, published records on NTM infections in animals are still scarce. The aims of the present study were to describe the diversity of NTM circulating among wild and domestic species from Spain and to analyze their implications as potential pathogenic microorganisms or as sources of interferences in the diagnosis of bovine tuberculosis. Overall, 293 NTM isolates of 277 animals were obtained from tissue samples collected between 2012 and 2019, and analyzed through a multigene approach for mycobacteria identification. Thirty-one species were identified, being Mycobacterium avium subsp. avium (Maa) and M. avium subsp. hominissuis (Mah), but also M. bouchedurhonense, M. nonchromogenicum and M. lentiflavum, the most abundant ones. Maa and M. lentiflavum were isolated in several animals showing tuberculosis-like lesions. Maa, Mah and M. nonchromogenicum were recovered from many cattle that had reacted to the tuberculin skin test. Other NTM were also associated to these phenomena. These four mycobacterial species were geographically associated between wild boar and other hosts. The findings of the present study suggest that a high diversity of NTM circulates among wildlife and livestock. Wild boar and M. avium seem to play a relevant role in this epidemiological scenario.

Is serology a realistic approach for monitoring red deer tuberculosis in the field?

Tuberculosis (TB) is a zoonotic mycobacterial infection with great importance in human health, animal production, and wildlife conservation. Although an ambitious eradication programme in cattle has been implemented for decades, TB-free status has not yet been achieved in most of Spain, where animal TB persists in a multi-host system of domestic and wild hosts, including the red deer (Cervus elaphus). However, information on long time series and trends of TB prevalence in wildlife is scarce. The diagnosis of TB in wild red deer is often based on gross pathology and bacteriological culture confirmation, although recently serological assays have been developed to detect anti- Mycobacterium tuberculosis Complex (MTC) antibodies. Particularly, protein complex P22 has demonstrated to yield good specificity and sensitivity in the serological diagnosis of MTC for red deer, as well as cattle, goats, sheep, pigs, wild boar, and European badger. Thus, the objective of the present study was to compare the performance of the P22-ELISA with TB-compatible lesion detection, as well as to assess the potential application of each technique for determining spatiotemporal trends and risk factors of MTC infection in wild red deer from low and high TB prevalence areas of Spain over the last two decades. We tested 5095 sera from 13 wild populations by indirect ELISA using P22 as antigen. Mean seroprevalence (13.22%, CI95: 12.32-14.18) was compared with the prevalence of macroscopic TB-compatible lesions (6.94%, CI95: 6.18-7.79). The results evidenced a poor agreement between both techniques (K < 0.3), although generalized TB-lesions and anti-P22 antibodies showed a positive association (χ² = 9.054, P = 0.004). Consequently, TB-lesion based prevalence and seroprevalence cannot be considered as equivalent for TB surveillance in red deer. Regarding the spatiotemporal trend of TB in red deer in Spain, we observed a North-South gradient of TB occurrence [North: 1.23% (CI95: 0.77-1.97) of TB-lesions and 12.55% (CI95: 10.91-14.41) of P22-ELISA; Centre: 7.10% (CI95: 6.04-8.33) and 8.74% (CI95: 7.57-10.08); South: 21.04% (CI95:17.81-24.69) and 23.09% (CI95: 19.73-26.84), respectively]. Overall, there was a stability over time, with higher prevalence in adults belonging to densely populated sites. We conclude that the P22-ELISA alone is not sufficiently reliable for TB surveillance in red deer at large spatiotemporal scales. Instead, we recommend combining gross pathology and P22-ELISA.

Serosurveillance of Schmallenberg virus in wild ruminants in Spain.

Schmallenberg disease (SBD) is an emerging vector-borne disease that affects domestic and wild ruminants. A long-term serosurvey was conducted to assess exposure to Schmallenberg virus (SBV) in all the wild ruminant species present in mainland Spain. Between 2010 and 2016, sera from 1,216 animals were tested for antibodies against SBV using a commercial blocking ELISA. The overall prevalence of antibodies was 27.1% (95%CI: 24.7-29.7). Statistically significant differences among species were observed, with significantly higher seropositivity found in fallow deer (Dama dama) (45.6%; 99/217), red deer (Cervus elaphus) (31.6%; 97/307) and mouflon (Ovis aries musimon) (28.0%; 33/118) compared to Barbary sheep (Ammotragus lervia) (22.2%; 8/36), Iberian wild goat (Capra pyrenaica) (19.9%; 49/246), roe deer (Capreolus capreolus) (17.5%; 34/194) and Southern chamois (Rupicapra pyrenaica) (10.2%; 10/98). Seropositive animals were detected in 81.4% (57/70; 95%CI: 70.8-88.8) of the sampled populations. SBV seroprevalence ranged from 18.8% (48/256) in bioregion (BR)2 (north-central, Mediterranean) to 32.3% (31/96) in BR1 (northeastern or Atlantic, Eurosiberian). Anti-SBV antibodies were not found before 2012, when the first outbreak of SBD was reported in Spain. In contrast, seropositivity was detected uninterruptedly during the period 2012-2016 and anti-SBV antibodies were found in yearling animals in each of these years. Our results provide evidence of widespread endemic circulation of SBV among wild ruminant populations in mainland Spain in recent years. Surveillance in these species could be a useful tool for monitoring SBV in Europe, particularly in areas where wild ruminants share habitats with livestock.

Distribution of Pestivirus exposure in wild ruminants in Spain.

A large-scale study was carried out to determine the prevalence of antibodies against Pestivirus species in wild ruminants and describe their spatial variation in mainland Spain. Serum samples of 1,874 wild ruminants from different regions of this country were collected between the years 2000 and 2017. A total of 6.6% (123/1,874) animals showed antibodies against Pestivirus by both blocking ELISA (bELISA) and virus neutralization tests (VNT). The prevalence of antibodies against pestiviruses was different both among species and regions. Seroprevalence by species was 30.0% (75/250) in Southern chamois (Rupicapra pyrenaica), 7.0% (25/357) in fallow deer (Dama dama), 2.5% (10/401) in red deer (Cervus elaphus), 2.4% (8/330) in Iberian wild goat (Capra pyrenaica), 1.1% (4/369) in roe deer (Capreolus capreolus) and 0.8% (1/130) in mouflon (Ovis aries musimon), not detecting seropositivity (0/37) in Barbary sheep (Ammotragus lervia). The results confirm that exposure to pestiviruses was detected throughout mainland Spain, with significantly higher seroprevalence in Northern regions associated with the presence of Southern chamois. This indicates an endemic circulation of pestiviruses in Southern chamois and a limited circulation of these viruses in the remaining wild ruminant species during the last two decades, thus suggesting that non-chamois species are not true Pestivirus reservoirs in Spain. Nonetheless, the high spatial spread of these viruses points out that new epidemic outbreaks in naïve wild ruminant populations or transmission to livestock may occur, evidencing the usefulness of monitoring pestiviruses in wild ruminants, especially at the wildlife-livestock interface.

Tuberculosis outbreak caused by Mycobacterium caprae in a rabbit farm in Spain.

Animal tuberculosis remains a great source of socioeconomic and health concern worldwide. Its main causative agents, Mycobacterium bovis and Mycobacterium caprae, have been isolated from many different domestic and wild animals. Naturally, occurring tuberculosis is extremely rare in rabbits, and implication of M. caprae has never been reported earlier. This study describes a severe tuberculosis outbreak caused by M. caprae in a Spanish farm of rabbits raised for meat for human consumption. The disease was first identified in a cachectic dam, and then it was confirmed in ten does with similar clinical signs. Subsequently, a depopulation operation was ordered for public health, animal welfare and environmental reasons. To broaden knowledge of spontaneous tuberculosis in rabbits, a study focused on pathological, epidemiological and diagnostic aspects was carried out on 51 does and 16 kittens after receiving the necessary authorizations. These animals were subjected to a modified intradermal test. After being euthanized, rabbits were examined for the presence of visible tuberculosis-compatible lesions. Lung, kidney, caecal appendix and sacculus rotundus samples underwent microbiological and anatomopathological analysis. Infection was revealed by at least one of the methods used in 71% of dams and in 44% of kittens. The intradermal test was shown to be a good indicator of infection. Lung was the tissue for which more animals were positive but renal and intestinal tissues were also affected in many cases. Apparently, M. caprae spread mainly through the aerogenous route. Infection was pathologically characterized by the absence of evident fibrous capsules surrounding granulomas. A spoligotype (SB0415) frequently found in this area was considered responsible for the outbreak but the source could not be established. Regardless of the exceptional nature of animal tuberculosis in this host, rabbit industry might not escape from its effects and therefore, current biosafety and surveillance strategies should also consider this disease.

Distribution of infectious endogenous retroviruses in mixed-breed and purebred cats.

Endogenous retroviruses of domestic cats (ERV-DCs) are members of the genus Gammaretrovirus that infect domestic cats (Felis silvestris catus). Uniquely, domestic cats harbor replication-competent proviruses such as ERV-DC10 (ERV-DC18) and ERV-DC14 (xenotropic and nonecotropic viruses, respectively). The purpose of this study was to assess invasion by two distinct infectious ERV-DCs, ERV-DC10 and ERV-DC14, in domestic cats. Of a total sample of 1646 cats, 568 animals (34.5%) were positive for ERV-DC10 (heterozygous: 377; homozygous: 191), 68 animals (4.1%) were positive for ERV-DC14 (heterozygous: 67; homozygous: 1), and 10 animals (0.6%) were positive for both ERV-DC10 and ERV-DC14. ERV-DC10 and ERV-DC14 were detected in domestic cats in Japan as well as in Tanzania, Sri Lanka, Vietnam, South Korea and Spain. Breeding cats, including Singapura, Norwegian Forest and Ragdoll cats, showed high frequencies of ERV-DC10 (60-100%). By contrast, ERV-DC14 was detected at low frequency in breeding cats. Our results suggest that ERV-DC10 is widely distributed while ERV-DC14 is maintained in a minor population of cats. Thus, ERV-DC10 and ERV-DC14 have invaded cat populations independently.

Myxomatosis and Rabbit Haemorrhagic Disease: A 30-Year Study of the Occurrence on Commercial Farms in Spain.

In this retrospective study, we describe the relative occurrence of clinical myxomatosis, and rabbit haemorrhagic disease (RHD), on 1714 commercial farms visited in Spain, between 1988 and 2018. We determined the annual prevalence based on 817 visits to 394 farms affected by myxomatosis. Myxomatosis was more prevalent from August to March, being lowest in June (3%) and highest in September (8.9%). With regard to RHD, we assessed 253 visits to 156 affected farms. We analyzed mean annual and monthly incidence. Two important RHD epidemics occurred; the first in 1988-1989 due to RHDV GI.1 (also known as RHDV), and the second from 2011 to 2013 due to RHDV GI.2 (RHDV2 or RHDVb). These epidemics occurred at times when effective vaccination had not been carried out. Relative monthly incidence in 2011-2018 was higher from April to August (p < 0.001). The results we obtained from 1404 necropsies on 102 farms did not clearly relate serosanguinous nasal discharge in rabbits with disease caused by GI.2 infection. We also assessed vaccination schedules used on 200 doe farms visited from the end of 2014 to 2018; 95.5% vaccinated against myxomatosis and 97.5% against RHD. Both diseases remain prevalent; however, effective vaccination has produced a steady decline in myxomatosis and RHDV GI.1 and GI.2 on-farm detection. The maintenance of high hygienic standards will be needed to continue and improve this control. However, further studies are required to investigate the causes of sustained virus presence and vaccine breaks.

Tracking the Fate of Endogenous Retrovirus Segregation in Wild and Domestic Cats.

Endogenous retroviruses (ERVs) of domestic cats (ERV-DCs) are one of the youngest feline ERV groups in domestic cats (Felis silvestris catus); some members are replication competent (ERV-DC10, ERV-DC18, and ERV-DC14), produce the antiretroviral soluble factor Refrex-1 (ERV-DC7 and ERV-DC16), or can generate recombinant feline leukemia virus (FeLV). Here, we investigated ERV-DC in European wildcats (Felis silvestris silvestris) and detected four loci: ERV-DC6, ERV-DC7, ERV-DC14, and ERV-DC16. ERV-DC14 was detected at a high frequency in European wildcats; however, it was replication defective due to a single G → A nucleotide substitution, resulting in an E148K substitution in the ERV-DC14 envelope (Env). This mutation results in a cleavage-defective Env that is not incorporated into viral particles. Introduction of the same mutation into feline and murine infectious gammaretroviruses resulted in a similar Env dysfunction. Interestingly, the same mutation was found in an FeLV isolate from naturally occurring thymic lymphoma and a mouse ERV, suggesting a common mechanism of virus inactivation. Refrex-1 was present in European wildcats; however, ERV-DC16, but not ERV-DC7, was unfixed in European wildcats. Thus, Refrex-1 has had an antiviral role throughout the evolution of the genus Felis, predating cat exposure to feline retroviruses. ERV-DC sequence diversity was present across wild and domestic cats but was locus dependent. In conclusion, ERVs have evolved species-specific phenotypes through the interplay between ERVs and their hosts. The mechanism of viral inactivation may be similar irrespective of the evolutionary history of retroviruses. The tracking of ancestral retroviruses can shed light on their roles in pathogenesis and host-virus evolution.IMPORTANCE Domestic cats (Felis silvestris catus) were domesticated from wildcats approximately 9,000 years ago via close interaction between humans and cats. During cat evolution, various exogenous retroviruses infected different cat lineages and generated numerous ERVs in the host genome, some of which remain replication competent. Here, we detected several ERV-DC loci in Felis silvestris silvestris Notably, a species-specific single nucleotide polymorphism in the ERV-DC14 env gene, which results in a replication-defective product, is highly prevalent in European wildcats, unlike the replication-competent ERV-DC14 that is commonly present in domestic cats. The presence of the same lethal mutation in the env genes of both FeLV and murine ERV provides a common mechanism shared by endogenous and exogenous retroviruses by which ERVs can be inactivated after endogenization. The antiviral role of Refrex-1 predates cat exposure to feline retroviruses. The existence of two ERV-DC14 phenotypes provides a unique model for understanding both ERV fate and cat domestication.

Low sequence diversity of the prion protein gene (PRNP) in wild deer and goat species from Spain.

The first European cases of chronic wasting disease (CWD) in free-ranging reindeer and wild elk were confirmed in Norway in 2016 highlighting the urgent need to understand transmissible spongiform encephalopathies (TSEs) in the context of European deer species and the many individual populations throughout the European continent. The genetics of the prion protein gene (PRNP) are crucial in determining the relative susceptibility to TSEs. To establish PRNP gene sequence diversity for free-ranging ruminants in the Northeast of Spain, the open reading frame was sequenced in over 350 samples from five species: Iberian red deer (Cervus elaphus hispanicus), roe deer (Capreolus capreolus), fallow deer (Dama dama), Iberian wild goat (Capra pyrenaica hispanica) and Pyrenean chamois (Rupicapra p. pyrenaica). Three single nucleotide polymorphisms (SNPs) were found in red deer: a silent mutation at codon 136, and amino acid changes T98A and Q226E. Pyrenean chamois revealed a silent SNP at codon 38 and an allele with a single octapeptide-repeat deletion. No polymorphisms were found in roe deer, fallow deer and Iberian wild goat. This apparently low variability of the PRNP coding region sequences of four major species in Spain resembles previous findings for wild mammals, but implies that larger surveys will be necessary to find novel, low frequency PRNP gene alleles that may be utilized in CWD risk control.

The role of wild ruminants as reservoirs of Besnoitia besnoiti infection in cattle.

Bovine besnoitiosis, a parasitic disease caused by Besnoitia besnoiti, has been reported mainly in beef cattle raised under extensive pastoral systems and is considered to be re-emerging in Western Europe. Horizontal transmission probably occurs either by means of blood sucking arthropods or as a consequence of direct contact between infected and non-infected cattle. However, the role that wild ruminants (e.g., red deer (Cervus elaphus) and roe deer (Capreolus capreolus)) may play in the parasite life cycle as putative reservoirs remains elusive. Thus, we investigated the presence of Besnoitia spp. infection in 2608 wild ruminants located in areas where bovine besnoitiosis is present and identified the Besnoitia species detected. First, a serosurvey was conducted in red deer (n=309), roe deer (n=417), Pyrenean chamois (Rupicapra p. pyrenaica, n=383) and Iberian wild goat (Capra pyrenaica hispanica, n=288) from two areas of Aragon, northeastern Spain, where bovine besnoitiosis is endemic. Second, red deer (n=820), roe deer (n=37), fallow deer (Dama dama, n=166), Iberian wild goat (n=86) and European mouflon (Ovis orientalis musimon, n=102) from southwestern Spain, where new outbreaks have recently been reported, were also sampled. The presence of Besnoitia spp.-specific antibodies was confirmed by western blot in one red deer and one roe deer from the Pyrenees, and Besnoitia spp. DNA was detected by ITS1-PCR in the seropositive red deer. Besnoitia genotyping based on 6 microsatellite (MS) analyses was carried out in red deer samples and compared with B. besnoiti genotypes from 7 in vitro isolates and 3 infected bovines, B. tarandi (1 isolate) and B. bennetti (from tissues of an infected donkey) for Besnoitia spp. assignation. Multilocus MS analysis of B. besnoiti, B. tarandi and B. bennetti showed specific genotypes for each species. A restricted genetic diversity with two genotypes by variation in a unique MS marker was revealed among the 7 B. besnoiti isolates. Incomplete Besnoitia spp. genotype of 3 MS markers from red deer samples entirely matched the B. besnoiti genotypes. Accordingly, this work gives clues for the presence of B. besnoiti infection in red deer from Western Europe. Further molecular genotyping is needed to confirm that red deer may act as an intermediate host of B. besnoiti, although the low prevalences that were found indicate that wild ruminant species do not pose a significant risk of transmitting the infection to cattle.

Effects produced by the feeding of larvae of Ornithodoros aff. puertoricensis (Acari: Argasidae) on laboratory mice.

The clinical picture produced by the feeding of larvae of Ornithodoros aff. puertoricensis on laboratory mice, was studied using different larval infestation protocols that included 30, 40 or 50 larvae per mouse and control uninfested groups. Clinical effects appeared around 72 h of larval feeding, having a first stage characterized by hyperaemia in both nasal and ocular mucosa, followed by respiratory symptoms (96-120 h) and nervous incoordination (120-144 h). No one mouse evidenced paralysis, and nervous symptoms were never observed in animals infested with only 30 larvae. High mortality (commonly up to 70%) was observed in mice with respiratory symptoms, while 100% of animals in the nervous phase died between 168 and 192 h after the beginning of larval feeding. When some infested mice were treated with a solution of Amitraz the larvae were killed and reversion of symptoms was observed. These effects are ascribed to the presence of a toxin in the saliva of the feeding larvae.

Genome of a novel circovirus of starlings, amplified by multiply primed rolling-circle amplification.

The genus Circovirus comprises small non-enveloped viruses with a circular single-stranded DNA genome. By using PCR with degenerate primers, a novel circovirus (starling circovirus, StCV) was detected in spleen samples of wild starlings (Sturnus vulgaris and Sturnus unicolor) found dead during an epidemic outbreak of septicaemic salmonellosis in northeastern Spain. Using a specific PCR, StCV was also detected in apparently healthy birds from the same population. The genome was amplified using multiply primed rolling-circle amplification and cloned. Open reading frames (ORFs) with similarities to the replication-associated protein and the capsid protein of circoviruses as well as an additional ORF encoding a protein of 106 aa were evident from the sequence. Phylogenetic analysis of circovirus genomes revealed the highest degree of similarity (67.1 %) between StCV and canary circovirus. A similar analysis of the evolutionarily conserved cytochrome b gene of the circovirus host species revealed a strict co-evolution of circoviruses with their hosts; however, the circoviruses showed about a threefold higher genetic divergence than their hosts.

Characterization of two novel polyomaviruses of birds by using multiply primed rolling-circle amplification of their genomes.

Polyomaviruses are small nonenveloped particles with a circular double-stranded genome, approximately 5 kbp in size. The mammalian polyomaviruses mainly cause persistent subclinical infections in their natural nonimmunocompromised hosts. In contrast, the polyomaviruses of birds--avian polyomavirus (APV) and goose hemorrhagic polyomavirus (GHPV)--are the primary agents of acute and chronic disease with high mortality rates in young birds. Screening of field samples of diseased birds by consensus PCR revealed the presence of two novel polyomaviruses in the liver of an Eurasian bullfinch (Pyrrhula pyrrhula griseiventris) and in the spleen of a Eurasian jackdaw (Corvus monedula), tentatively designated as finch polyomavirus (FPyV) and crow polyomavirus (CPyV), respectively. The genomes of the viruses were amplified by using multiply primed rolling-circle amplification and cloned. Analysis of the FPyV and CPyV genome sequences revealed a close relationship to APV and GHPV, indicating the existence of a distinct avian group among the polyomaviruses. The main characteristics of this group are (i) involvement in fatal disease, (ii) the existence of an additional open reading frame in the 5' region of the late mRNAs, and (iii) a different manner of DNA binding of the large tumor antigen compared to that of the mammalian polyomaviruses.

Phyllodistomum folium (Trematoda: Gorgoderidae) infecting zebra mussels (Dreissena polymorpha) in the Ebro River, Spain.

Zebra mussels (Dreissena polymorpha) were first found in the Ebro River (Spain) in Ribaroja reservoir, in the summer of 2001. This paper reports a study to detect parasites in this bivalve species. From September 2003 to August 2004, a total of 1380 zebra mussels were collected and dissected or sectioned in paraffin and haematoxylin and eosin staining. We observed the presence of Phyllodistomum folium (Olfers, 1816) in two hosts (prevalence 0.14%). Sporocysts containing metacercariae were located within the gill lamellae. One of the mussels was collected in January and the other one in July. In both cases the shell length was >2 cm. P. folium had not been previously reported in Spain and D. polymorpha is its only known intermediate host. It represents a new invasive species in this river basin, presumably introduced together with the zebra mussels.

A novel pestivirus associated with deaths in Pyrenean chamois (Rupicapra pyrenaica pyrenaica).

During investigations into recent population decreases in Pyrenean chamois (Rupicapra pyrenaica pyrenaica) 21 animals found dead or dying were necropsied. Immunohistochemistry revealed the presence of a pestivirus in organs from two of the 21 chamois. From one of these animals a pestivirus was isolated from the spleen, skin and serum. The virus had better growth in ovine than in bovine cells and was neutralized most effectively by an anti-border disease virus (BDV) reference antiserum. Using panpestivirus and genotype-specific primers selected from 5'-untranslated region (UTR) of the pestivirus genome, BDV RNA was demonstrated by RT-PCR. Comparison of the chamois sequences from 5'-UTR, entire N(pro) and E2 gene coding regions with those of other pestivirus genotypes revealed that this virus did not fall into any of the pestivirus genotypes identified so far. Results of phylogenetic analysis suggested that the chamois pestivirus was closely related to BDV and it was typed as BDV-4 genotype.