Infectious keratoconjunctivitis in semi-domesticated Eurasian tundra reindeer (Rangifer tarandus tarandus): microbiological study of clinically affected and unaffected animals with special reference to cervid herpesvirus 2.

BACKGROUND: Infectious keratoconjunctivitis (IKC) is one of the most common ocular diseases in ruminants worldwide. In addition to keratitis and conjunctivitis, animals with IKC can develop uveitis, corneal ulcer, and in severe cases, blindness. The bacteria Moraxella spp. has been described as the primary causative agent of infectious bovine keratoconjunctivitis (IBK) in cattle (Bos taurus), while Chlamydia spp. and Mycoplasma conjunctivae are considered the main causative agents of IKC in sheep (Ovis aries). Previous studies indicated cervid herpesvirus 2 (CvHV2) as the primary causative agent of IKC in semi-domesticated reindeer (Rangifer tarandus tarandus). The aim of the study was to investigate the presence and prevalence of potential pathogens for IKC in reindeer, and compare the ocular microbiota of animals with IKC, with apparently healthy animals. RESULTS: Semi-domesticated reindeer (n = 341), with (n = 108) or without (n = 113) ocular clinical signs, or with no information on clinical status (n = 120), were sampled in Norway, Sweden and Finland in 2010-2014. Seroprevalence was 37.4% for alphaherpesvirus (95/254), 3.8% for gammaherpesvirus (8/211) and 7.1% for pestivirus (15/211) (ELISA). PCR analyses of conjunctival swab samples revealed a prevalence of 28.5% for CvHV2 (57/200), 11.9% for Chlamydiaceae (16/135) and 1.0% for M. conjunctivae (2/197). Bacteriological cultivation of 202 conjunctival swab samples revealed bacterial growth from 75.2% of the samples, with Moraxella spp. being isolated from 21.6% (11/51) of the animals with and 5.6% (5/84) without ocular clinical signs. A significant association (p < 0.001) existed between the presence of clinical signs of IKC and CvHV2 DNA in the affected eyes, an association that was not present for other microorganisms. CONCLUSIONS: These results support the hypothesis that CvHV2 is the primary agent of IKC in semi-domesticated reindeer in Fennoscandia, with Moraxella bovoculi being a secondary candidate, since it was isolated in two different outbreaks of IKC. Further studies should be carried out to better understand the infection biology and the pathogenesis of IKC in reindeer.

Comparative genomic analysis identifies potential adaptive variation in Mycoplasma ovipneumoniae.

Mycoplasma ovipneumoniae is associated with respiratory disease in wild and domestic Caprinae globally, with wide variation in disease outcomes within and between host species. To gain insight into phylogenetic structure and mechanisms of pathogenicity for this bacterial species, we compared M. ovipneumoniae genomes for 99 samples from 6 countries (Australia, Bosnia and Herzegovina, Brazil, China, France and USA) and 4 host species (domestic sheep, domestic goats, bighorn sheep and caribou). Core genome sequences of M. ovipneumoniae assemblies from domestic sheep and goats fell into two well-supported phylogenetic clades that are divergent enough to be considered different bacterial species, consistent with each of these two clades having an evolutionary origin in separate host species. Genome assemblies from bighorn sheep and caribou also fell within these two clades, indicating multiple spillover events, most commonly from domestic sheep. Pangenome analysis indicated a high percentage (91.4 %) of accessory genes (i.e. genes found only in a subset of assemblies) compared to core genes (i.e. genes found in all assemblies), potentially indicating a propensity for this pathogen to adapt to within-host conditions. In addition, many genes related to carbon metabolism, which is a virulence factor for Mycoplasmas, showed evidence for homologous recombination, a potential signature of adaptation. The presence or absence of annotated genes was very similar between sheep and goat clades, with only two annotated genes significantly clade-associated. However, three M. ovipneumoniae genome assemblies from asymptomatic caribou in Alaska formed a highly divergent subclade within the sheep clade that lacked 23 annotated genes compared to other assemblies, and many of these genes had functions related to carbon metabolism. Overall, our results suggest that adaptation of M. ovipneumoniae has involved evolution of carbon metabolism pathways and virulence mechanisms related to those pathways. The genes involved in these pathways, along with other genes identified as potentially involved in virulence in this study, are potential targets for future investigation into a possible genomic basis for the high variation observed in disease outcomes within and between wild and domestic host species.

Probiotic dosing of Ruminococcus flavefaciens affects rumen microbiome structure and function in reindeer.

Highly cellulolytic bacterial species such as Ruminococcus flavefaciens are regarded essential for the microbial breakdown of cellulose in the rumen. We have investigated the effect of ruminal dosing of R. flavefaciens strain 8/94-32 during realimentation of starved reindeer (males, n = 3). Microbiome function measured as in situ digestion of cellulose and food pellets (percent DMD; dry matter disappearance) decreased after probiotic dosing. Microbial community analyses (>100,000 16S rDNA gene sequences for 27 samples) demonstrated that ruminal dosing influenced the microbiome structure; reflected by increased phylogenetic distances from background samples (unweighted UniFrac analysis) and reduced species diversity and evenness. Despite the inability to detect strain 8/94-32 post-dosing, the relative abundance of its affiliate family Ruminococcaceae remained consistent throughout the trial, whilst a dominant peak in the genus Prevotella and decline in uncharacterized Bacteroidetes (uBacNR) were observed in treatment samples. No clear relationships were observed between the relative abundance of Ruminococcaceae, Prevotella and uBacNR with cellulose DMD; however, Prevotella (negative) and uBacNR (positive) exhibited relationships with pellet DMD. These unexpected effects of ruminal dosing of a cellulolytic bacterium on digestibility are relevant for other studies on rumen manipulation.

Occurrence of non-tuberculous mycobacteria species in livestock from northern China and first isolation of Mycobacterium caprae.

We investigated the presence of Mycobacterium spp. in livestock in northern China. Of the 163 clinical samples selected for this study, 20 were from throat swabs of dairy cows, and 143 were tissue samples (including lung tissue from one reindeer, hilar lymph node tissue from 55 cows, and liver tissue from 87 sheep). A total of 41 mycobacterial isolates were identified including two isolates of M. caprae and 39 non-tuberculous mycobacteria (NTM) isolates. Multi-locus variable-number tandem repeat analysis (MLVA) profiles of the two M. caprae isolates proved to be unique. This is the first report of M. caprae isolates from livestock in China. This study also confirms previous reports that NTM is common in livestock in northern China.

Gut microbiome biogeography in reindeer supersedes millennia of ecological and evolutionary separation.

Ruminants are dependent on their gut microbiomes for nutrient extraction from plant diets. However, knowledge about the composition, diversity, function, and spatial structure of gut microbiomes, especially in wild ruminants, is limited, largely because analysis has been restricted to faeces or the rumen. In two geographically separated reindeer subspecies, 16S rRNA gene amplicon sequencing revealed strong spatial structuring, and pronounced differences in microbial diversity of at least 33 phyla across the stomach, small intestine, and large intestine (including faeces). The main structural feature was the Bacteroidota to Firmicutes ratio, which declined from the stomach to the large intestine, likely reflecting functional adaptation. Metagenome shotgun sequencing also revealed highly significant structuring in the relative occurrence of carbohydrate-active enzymes (CAZymes). CAZymes were enriched in the rumen relative to the small and large intestines. Interestingly, taxonomic diversity was highest in the large intestine, suggesting an important and understudied role for this organ. Despite the two study populations being separated by an ocean and six millennia of evolutionary history, gut microbiome structuring was remarkably consistent. Our study suggests a strong selection for gut microbiome biogeography along the gastrointestinal tract in reindeer subspecies.

[Molecular-genetic characterization of canine and rangiferine brucella isolates from different regions of Russia].

GOAL: Comparative molecular-genetic characterization of Brucella isolates from dogs and reindeers in Russia by molecular-genetic typing methods. MATERIALS AND METHODS: 19 canine and 2 rangiferine Brucella isolates were studied by molecular typing methods based on PCR for differential species and biovar specific molecular targets and MLVA (multiple locus variable number tandem repeats analysis) using primers to 12 known variable loci. RESULTS: Using PCR for differential molecular targets, canine Brucella isolates were characterized as B. canis and rangiferine isolates as B. suis biovar 4. MLVA revealed 5 identical and 7 variable MLVA loci. Using the dendrogram. all the isolates on the data of 12 loci were classified into the close related cluster. On the other hand, high discrimination power of MLVA with a resulting Hunter and Gaston discriminatory index (HGDI) of 0.9842 was shown to reveal genetic diversity for the isolates of 17 MLVA genotypes. CONCLUSION: B. canis and B. suis isolates from different geographical regions in Russia were genetically close related, thereby confirming known genetic relationship between these species. Related MLVA genotypes of isolates were connected to certain regions of preliminary isolation in Russia. To improve the system ofbrucellosis surveillance in Russia MLVA typing of more canine and rangiferine Brucella isolates having epidemiological danger for humans is required to be studied.

Variations in the 16S-23S rRNA internal transcribed spacer of fibrolytic Butyrivibrio isolates from the reindeer rumen.

Strains of Butyrivibrio are principal cellulytic bacteria in the rumen of the High Arctic Svalbard reindeer ( Rangifer tarandus platyrhynchus ). According to phylogenetic analysis based on 16S rRNA gene sequencing, Butyrivibrio can be divided into three subgroups within the Clostridia class of the phylum Firmicutes, but the current phenotypic and genotypic differentiation within the family Lachnospiraceae is insufficient. This current study describes the sequence diversity of the 16S-23S rRNA intergenic transcribed spacer (ITS) region of Butyrivibrio isolates from reindeer. A total of 17 different ITS sequences with sizes between 449 and 784 nt were obtained. Genes encoding tRNA(Ile) and tRNA(Ala) were identified in four of the sequences. Phylogenetic neighbor-joining trees were constructed based on the ITS sequence and compared with a phylogenetic neighbor-joining tree based on 16S rRNA gene sequences previously obtained for the same isolates. These comparisons indicated a better differentiation between strains in the ITS sequence than the 16S rRNA gene based tree. Through this study, a better means for identifying and tracking fibrolytic and potentially probiotic Butyrivibrio strains in reindeer and other ruminants has been provided.

Metagenomic analysis of the cow, sheep, reindeer and red deer rumen.

The rumen microbiota comprises a community of microorganisms which specialise in the degradation of complex carbohydrates from plant-based feed. These microbes play a highly important role in ruminant nutrition and could also act as sources of industrially useful enzymes. In this study, we performed a metagenomic analysis of samples taken from the ruminal contents of cow (Bos Taurus), sheep (Ovis aries), reindeer (Rangifer tarandus) and red deer (Cervus elaphus). We constructed 391 metagenome-assembled genomes originating from 16 microbial phyla. We compared our genomes to other publically available microbial genomes and found that they contained 279 novel species. We also found significant differences between the microbiota of different ruminant species in terms of the abundance of microbial taxonomies, carbohydrate-active enzyme genes and KEGG orthologs. We present a dataset of rumen-derived genomes which in combination with other publicly-available rumen genomes can be used as a reference dataset in future metagenomic studies.

Microbial degradation of usnic acid in the reindeer rumen.

Reindeer (Rangifer tarandus) eat and utilize lichens as an important source of energy and nutrients in winter. Lichens synthesize and accumulate a wide variety of phenolic secondary compounds, such as usnic acid, as a defense against herbivores and to protect against damage by UV-light in solar radiation. We have examined where and to what extent these phenolic compounds are degraded in the digestive tract of the reindeer, with particular focus on usnic acid. Three male reindeer were given ad libitum access to a control diet containing no usnic acid for three weeks and then fed lichens ad libitum (primarily Cladonia stellaris) containing 9.1 mg/g DM usnic acid for 4 weeks. Usnic acid intake in reindeer on the lichen diet was 91-117 mg/kg BM/day. In spite of this, no trace of usnic acid or conjugates of usnic acid was found either in fresh rumen fluid, urine, or feces. This suggests that usnic acid is rapidly degraded by rumen microbes, and that it consequently is not absorbed by the animal. This apparent ability to detoxify lichen phenolic compounds may gain increased importance with future enhanced UV-B radiation expected to cause increased protective usnic acid/phenol production in lichens.

Factors Contributing to Anthrax Outbreaks in the Circumpolar North.

A 2016 outbreak of anthrax on the Yamal Peninsula in Siberia that led to the culling of more than two hundred thousand reindeer and killed one human, resulted in significant media interests and in the reporting was often linked to thawing permafrost and ultimately climate change. Here, we review the historic context of anthrax outbreaks in the circumpolar North and explore alternative explanations for the anthrax outbreak in Western Siberia. Further, we propose a convergence model where multiple factors likely contributed to the outbreak of anthrax, including an expanded population and discontinued vaccination.

Microbiota in foods from Inuit traditional hunting.

The foods we eat contain microorganisms that we ingest alongside the food. Industrialized food systems offer great advantages from a safety point of view, but have also been accused of depleting the diversity of the human microbiota with negative implications for human health. In contrast, artisanal traditional foods are potential sources of a diverse food microbiota. Traditional foods of the Greenlandic Inuit are comprised of animal-sourced foods prepared in the natural environment and are often consumed raw. These foods, some of which are on the verge of extinction, have not previously been microbiologically characterized. We mapped the microbiota of foods stemming from traditional Inuit land-based hunting activities. The foods included in the current study are dried muskox and caribou meat, caribou rumen and intestinal content as well as larval parasites from caribou hides, all traditional Inuit foods. This study shows that traditional drying methods are efficient for limiting microbial growth through desiccation. The results also show the rumen content of the caribou to be a highly diverse source of microbes with potential for degradation of plants. Finally, a number of parasites were shown to be included in the biodiversity of the assessed traditional foods. Taken together, the results map out a diverse source of ingested microbes and parasites that originate from the natural environment. These results have implications for understanding the nature-sourced traditional Inuit diet, which is in contrast to current day diet recommendations as well as modern industrialized food systems.

Mycoplasma ovipneumoniae Associated with Polymicrobial Pneumonia in a Free-Ranging Yearling Barren Ground Caribou (Rangifer tarandus granti) from Alaska, USA.

Mycoplasma ovipneumoniae has been reported in association with respiratory disease in the wild only in members of the subfamily Caprinae of the family Bovidae. We identified M. ovipneumoniae in a cervid: a free-ranging barren ground caribou (Rangifer tarandus granti) yearling with polymicrobial bronchopneumonia.

A novel clinical syndrome and detection of Anaplasma ovis in Mongolian reindeer (Rangifer tarandus).

The Tsaatan (or Dhuka) peoples of northern-western Mongolia are one of the few remaining reindeer-herding cultural groups in the world. Recently a disease condition that involves sudden death of reindeer and cases involving fever, lethargy, and pale mucous membranes has been reported. Examination of blood smears collected in the 2005 field season resulted in the identification of intra-erythrocytic inclusions resembling Anaplasma spp. in smears from clinically sick animals. Using universal polymerase chain reaction (PCR) primers for the amplification of the 60 kDa chaperonin gene (cpn60, also known as hsp60 or groEL), we detected sequences corresponding to Anaplasma ovis in reindeer blood samples. Species-specific PCR primers for A. ovis were designed and validated and used to screen blood samples from Mongolian reindeer. Screening of 66 blood samples collected in the 2006 field season resulted in the detection of A. ovis in 80% of the samples. Our results indicate a high prevalence of A. ovis in the Tsaatan reindeer herds and an association with clinical disease that is likely to be anaplasmosis. To our knowledge this is the first report of natural A. ovis infection in reindeer.

Blood culture and stimulation conditions for the diagnosis of tuberculosis in cervids by the Cervigam assay.

Mitogen- and antigen-induced interferon-gamma (IFN-gamma) responses of peripheral blood leucocytes from cervids were evaluated by a commercial whole-blood assay. The assay was applied to Mycobacterium bovis-infected white-tailed deer and reindeer, M bovis BCG-vaccinated white-tailed deer and elk, and unvaccinated, uninfected white-tailed deer, fallow deer, elk and reindeer. The responses of the M bovis-infected white-tailed deer to pokeweed mitogen (PWM) varied with time and between individuals. The responses of the M bovis-infected reindeer to PWM and M bovis purified protein derivative (PPD) were positively associated. Samples from tuberculosis-free captive herds in various parts of the USA were also evaluated. Four per cent of fallow deer, 20 per cent of elk, 44 per cent of white-tailed deer, and 91 per cent of reindeer had responses to PWM exceeding 0.25 Delta optical density, that is, PWM stimulation minus no stimulation. The specificity of the responses to M bovis PPD and a Mycobacterium tuberculosis complex-specific antigen rESAT-6:CFP-10, excluding animals not responding to PWM, ranged from 78 per cent to 100 per cent and was dependent upon the species and the positive response cut-off value. The results show that the commercial assay is valid for the detection of TB in reindeer; however, further development of the assay will be required before it is used in surveillance programmes for white-tailed deer, fallow deer, and elk.

Systemic nocardiosis in a reindeer (Rangifer tarandus tarandus).

Systemic infection by Nocardia asteroides was diagnosed in a reindeer (Rangifer tarandus tarandus). At necropsy, the animal had enlarged tracheobronchial lymph nodes, pleural and peritoneal effusions, and numerous, multifocal to coalescing, yellow, firm nodules with inspissated pus in the lung, pleura, omentum, liver, heart, adrenal glands, and left kidney. Microscopically, the nodules consisted of foci of pyogranulomatous inflammation. Microscopic lesions were present in the grossly affected organs as well as spleen and brain. Sections stained with Gram and modified Fite-Faraco histochemical stains had numerous gram-positive, variably acid fast, beaded and branching filamentous organisms in the necrotic centers. N. asteroides was isolated from the lung, bronchial lymph nodes, liver, and left kidney. To the authors' knowledge, this is the first report of systemic N. asteroides infection in a reindeer.

Estimating changes in lichen mat volume through time and related effects on barren ground caribou (Rangifer tarandus groenlandicus) movement.

Lichens form a critical portion of barren ground caribou (Rangifer tarandus groenlandicus) diets, especially during winter months. Here, we assess lichen mat volume across five herd ranges in the Northwest Territories and Nunavut, Canada, using newly developed composite Landsat imagery. The lichen volume estimator (LVE) was adapted for use across 700 000 km2 of barren ground caribou habitat annually from 1984-2012. We subsequently assessed how LVE changed temporally throughout the time series for each pixel using Theil-Sen's slopes, and spatially by assessing whether slope values were centered in local clusters of similar values. Additionally, we assessed how LVE estimates resulted in changes in barren ground caribou movement rates using an extensive telemetry data set from 2006-2011. The Ahiak/Beverly herd had the largest overall increase in LVE (median = 0.033), while the more western herds had the least (median slopes below zero in all cases). LVE slope pixels were arranged in significant clusters across the study area, with the Cape Bathurst, Bathurst, and Bluenose East herds having the most significant clusters of negative slopes (more than 20% of vegetated land in each case). The Ahiak/Beverly and Bluenose West had the most significant positive clusters (16.3% and 18.5% of vegetated land respectively). Barren ground caribou displayed complex reactions to changing lichen conditions depending on season; the majority of detected associations with movement data agreed with current understanding of barren ground caribou foraging behavior (the exception was an increase in movement velocity at high lichen volume estimates in Fall). The temporal assessment of LVE identified areas where shifts in ecological conditions may have resulted in changing lichen mat conditions, while assessing the slope estimates for clustering identified zones beyond the pixel scale where forage conditions may be changing. Lichen volume estimates associated with barren ground caribou movement metrics in an expected manner and, as such, show value for future habitat assessments.

Characteristics of Shiga Toxin-Producing Escherichia coli O157 in Slaughtered Reindeer from Northern Finland.

Fecal samples collected from 470 slaughtered reindeer 6 to 7 months of age were screened by real-time PCR (after enrichment) for Shiga toxin genes (stx) and then for Escherichia coli serogroup O157. Shiga toxin genes were found frequently (>30% of samples), and serogroup O157 was detected in 20% of the stx-positive samples. From these samples, a total of 25 E. coli O157:H- isolates (nonmotile but PCR positive for fliCH7) were obtained. Twenty-four of these E. coli O157:H- isolates did not ferment sorbitol and originated from one geographic area. These 24 isolates belonged to the multilocus sequence type 11, typical for Shiga toxin-producing E. coli (STEC) O157:H7 and O157:H-, and harbored genes stx1a, stx2c, eae, and hlyA; the stx2c subtype has been associated with high virulence. In contrast, one E. coli O157:H- isolate (multilocus sequence type 11) did ferment sorbitol, lacked Shiga toxin genes, but was positive for eae, hlyA, and sfpA. This isolate closely resembled an STEC that has lost its Shiga toxin genes. Additional examination revealed that reindeer can be colonized by various other STEC isolates; 21 non-O157 STEC isolates belonged to four multilocus sequence types, harbored stx1a (8 isolates) or stx2b (13 isolates), and in the stx2b-positive isolates the recently described new allelic variants (subAB2-2 and subAB2-3) for subtilase cytotoxin were identified. Hence, slaughtered semidomesticated Finnish reindeer might constitute a little known reservoir for STEC O157:H7/H- and other serogroups, and the risk of direct or indirect transmission of these pathogens from reindeer to humans and domestic livestock must not be overlooked.

Antigen-specific proliferation and activation of peripheral blood mononuclear cells from Mycobacterium bovis-infected reindeer.

To evaluate antigen-specific proliferative and activation-associated responses from Mycobacterium bovis-infected reindeer, blood mononuclear cells from M. bovis- (n = 10) and non-infected reindeer (n = 4) were stimulated with a recombinant early secretory antigenic target-6 and culture filtrate protein-10 fusion protein (rESAT6:CFP10), M. bovis purified protein derivative, pokeweed mitogen, or medium alone and evaluated by flow cytometry using dye tracker analysis and cell surface marker staining. gammadelta TCR+ and CD8+ cells, but not CD4+ cells, from M. bovis-infected reindeer proliferated in response to specific antigen stimulation. Expression (i.e., mean fluorescence intensity) of CD44 was increased and CD62L decreased on proliferative as compared to non-proliferative fractions in antigen- and mitogen-stimulated cultures. In response rESAT6:CFP10 stimulation, MHC II fluorescence intensity was increased on CD4+, gammadelta TCR+, CD172a+, and IgM+ cells from infected reindeer as compared to that of non-stimulated cells from the same reindeer. Recombinant ESAT6:CFP10 stimulation also induced expansion of a CD172a+, MHC II+ population within mononuclear cell cultures from M. bovis-infected reindeer. Despite a moderate challenge dose and extended duration of incubation, experimental infection of reindeer was generally limited to lymph nodes draining the inoculation site, suggestive of host resistance to progressive disease. Present in vitro findings, therefore, may be predictive of host responses by reindeer that limit progression to disseminated disease.

Geographic pattern of serum antibody prevalence for Brucella spp. in caribou, grizzly bears, and wolves from Alaska, 1975-1998.

Blood samples were collected from 2,635 caribou (Rangifer tarandus), 1,238 grizzly bears (Ursus arctos), and 930 wolves (Canis lupus) from throughout mainland Alaska during 1975-98. Sera were tested for evidence of exposure to Brucella spp. Serum antibody prevalences were highest in the northwestern region of the state. In any specific area, antibody prevalences for caribou and wolves were of a similar magnitude, whereas antibody prevalence for bears in these same areas were two to three times higher.