Supplemental Description of Myxobolus squamalis (Myxozoa) & Epidemiology of M. squamalis at Two Oregon Hatcheries

Myxobolus squamalis is a Myxozoan parasite of salmonids, which contributes to lowered fitness of hatchery and wild fish in the Pacific Northwest of North America. The only GenBank DNA sequence of M. squamalis is from Oncorhynchus tschawytscha (Chinook), but is not linked to a published morphological description. There is a range of salmonid Myxozoans with similar morphology to M. squamalis and we suspect that some “M. squamalis” observations from different host species might be invalid. We provide a clear re-description of M. squamalis from its type host, O. mykiss (rainbow trout and steelhead). Our results confirm the morphological and morphometric data of the original description, which we supplement with a SSU rRNA gene sequence. Based on this sequence, we developed a PCR assay for specific detection of M. squamalis DNA in environmental water samples. We used the assay to compare parasite levels in the water influent and effluent from two hatcheries that rear different salmon and trout species. Our approach allows for early M. squamalis detection, infection level estimates, and estimates for environmental impacts based upon spore release. Early detection of Myxozoan infections is crucial to hatchery and wild fish management, as there is no documented treatment for infected fish

Expanding the diversity of mycobacteriophages: insights into genome architecture and evolution.

Mycobacteriophages are viruses that infect mycobacterial hosts such as Mycobacterium smegmatis and Mycobacterium tuberculosis. All mycobacteriophages characterized to date are dsDNA tailed phages, and have either siphoviral or myoviral morphotypes. However, their genetic diversity is considerable, and although sixty-two genomes have been sequenced and comparatively analyzed, these likely represent only a small portion of the diversity of the mycobacteriophage population at large. Here we report the isolation, sequencing and comparative genomic analysis of 18 new mycobacteriophages isolated from geographically distinct locations within the United States. Although no clear correlation between location and genome type can be discerned, these genomes expand our knowledge of mycobacteriophage diversity and enhance our understanding of the roles of mobile elements in viral evolution. Expansion of the number of mycobacteriophages grouped within Cluster A provides insights into the basis of immune specificity in these temperate phages, and we also describe a novel example of apparent immunity theft. The isolation and genomic analysis of bacteriophages by freshman college students provides an example of an authentic research experience for novice scientists

Transcriptome Analysis and In Situ Hybridization for FcaGHV1 in Feline Lymphoma.

Lymphoma is one of the most common malignancies in domestic cats. The lymphomagenic potential of Felis catus gammaherpesvirus 1 (FcaGHV1), a common infection in domestic cats, is unknown. In other species, including humans, cellular transformation by gammaherpesviruses is typically mediated by viral genes expressed during latency. We analysed tumour RNA, from diffuse large B-cell lymphomas (DLBCL) appearing in cats coinfected with FcaGHV1 and feline immunodeficiency virus (FIV) (n = 10), by high throughput transcriptome sequencing and reverse transcription PCR. A limited repertoire of FcaGHV transcripts was identified in five tumors, including homologs of oncogenic latency-associated transcripts, latency-associated nuclear antigen (LANA, ORF73) and vFLIP (F7), lytic genes (ORF50, ORF6, ORF59, F10), and an ORF unique to FcaGHV1, F20. In situ hybridization of FIV-associated DLBCLs (n = 9), post-transplant lymphomas (n = 6) and high-grade B and T-cell intestinal lymphomas (n = 8) identified a single case in which FcaGHV1 nucleic acid was detectable. These results demonstrate that FcaGHV1 transcripts can be detected in some FIV-associated lymphomas, but at low copy number, precluding assessment of a potential role for FcaGHV1 in lymphomagenesis. Future investigation of the FcaGHV1 transcriptome in clinical samples might employ viral enrichment and greater sequencing depth to enhance the retrieval of viral reads. Our results suggest prioritization of a subset of intestinal T-cell tumors, large granular lymphocyte lymphoma, for study

Transcriptome Analysis and In Situ Hybridization for FcaGHV1 in Feline Lymphoma

Lymphoma is one of the most common malignancies in domestic cats. The lymphomagenic potential of Felis catus gammaherpesvirus 1 (FcaGHV1), a common infection in domestic cats, is unknown. In other species, including humans, cellular transformation by gammaherpesviruses is typically mediated by viral genes expressed during latency. We analysed tumour RNA, from diffuse large B-cell lymphomas (DLBCL) appearing in cats coinfected with FcaGHV1 and feline immunodeficiency virus (FIV) (n = 10), by high throughput transcriptome sequencing and reverse transcription PCR. A limited repertoire of FcaGHV transcripts was identified in five tumors, including homologs of oncogenic latency-associated transcripts, latency-associated nuclear antigen (LANA, ORF73) and vFLIP (F7), lytic genes (ORF50, ORF6, ORF59, F10), and an ORF unique to FcaGHV1, F20. In situ hybridization of FIV-associated DLBCLs (n = 9), post-transplant lymphomas (n = 6) and high-grade B and T-cell intestinal lymphomas (n = 8) identified a single case in which FcaGHV1 nucleic acid was detectable. These results demonstrate that FcaGHV1 transcripts can be detected in some FIV-associated lymphomas, but at low copy number, precluding assessment of a potential role for FcaGHV1 in lymphomagenesis. Future investigation of the FcaGHV1 transcriptome in clinical samples might employ viral enrichment and greater sequencing depth to enhance the retrieval of viral reads. Our results suggest prioritization of a subset of intestinal T-cell tumors, large granular lymphocyte lymphoma, for study

Effect of the macroalgae Asparagopsis taxiformis on methane production and rumen microbiome assemblage.

BackgroundRecent studies using batch-fermentation suggest that the red macroalgae Asparagopsis taxiformis has the potential to reduce methane (CH4) production from beef cattle by up to ~ 99% when added to Rhodes grass hay; a common feed in the Australian beef industry. These experiments have shown significant reductions in CH4 without compromising other fermentation parameters (i.e. volatile fatty acid production) with A. taxiformis organic matter (OM) inclusion rates of up to 5%. In the study presented here, A. taxiformis was evaluated for its ability to reduce methane production from dairy cattle fed a mixed ration widely utilized in California, the largest milk producing state in the US.ResultsFermentation in a semi-continuous in-vitro rumen system suggests that A. taxiformis can reduce methane production from enteric fermentation in dairy cattle by 95% when added at a 5% OM inclusion rate without any obvious negative impacts on volatile fatty acid production. High-throughput 16S ribosomal RNA (rRNA) gene amplicon sequencing showed that seaweed amendment effects rumen microbiome consistent with the Anna Karenina hypothesis, with increased β-diversity, over time scales of approximately 3 days. The relative abundance of methanogens in the fermentation vessels amended with A. taxiformis decreased significantly compared to control vessels, but this reduction in methanogen abundance was only significant when averaged over the course of the experiment. Alternatively, significant reductions of CH4 in the A. taxiformis amended vessels was measured in the early stages of the experiment. This suggests that A. taxiformis has an immediate effect on the metabolic functionality of rumen methanogens whereas its impact on microbiome assemblage, specifically methanogen abundance, is delayed.ConclusionsThe methane reducing effect of A. taxiformis during rumen fermentation makes this macroalgae a promising candidate as a biotic methane mitigation strategy for dairy cattle. But its effect in-vivo (i.e. in dairy cattle) remains to be investigated in animal trials. Furthermore, to obtain a holistic understanding of the biochemistry responsible for the significant reduction of methane, gene expression profiles of the rumen microbiome and the host animal are warranted

Effect of the macroalgae Asparagopsis taxiformis on methane production and rumen microbiome assemblage.

BackgroundRecent studies using batch-fermentation suggest that the red macroalgae Asparagopsis taxiformis has the potential to reduce methane (CH4) production from beef cattle by up to ~ 99% when added to Rhodes grass hay; a common feed in the Australian beef industry. These experiments have shown significant reductions in CH4 without compromising other fermentation parameters (i.e. volatile fatty acid production) with A. taxiformis organic matter (OM) inclusion rates of up to 5%. In the study presented here, A. taxiformis was evaluated for its ability to reduce methane production from dairy cattle fed a mixed ration widely utilized in California, the largest milk producing state in the US.ResultsFermentation in a semi-continuous in-vitro rumen system suggests that A. taxiformis can reduce methane production from enteric fermentation in dairy cattle by 95% when added at a 5% OM inclusion rate without any obvious negative impacts on volatile fatty acid production. High-throughput 16S ribosomal RNA (rRNA) gene amplicon sequencing showed that seaweed amendment effects rumen microbiome consistent with the Anna Karenina hypothesis, with increased β-diversity, over time scales of approximately 3 days. The relative abundance of methanogens in the fermentation vessels amended with A. taxiformis decreased significantly compared to control vessels, but this reduction in methanogen abundance was only significant when averaged over the course of the experiment. Alternatively, significant reductions of CH4 in the A. taxiformis amended vessels was measured in the early stages of the experiment. This suggests that A. taxiformis has an immediate effect on the metabolic functionality of rumen methanogens whereas its impact on microbiome assemblage, specifically methanogen abundance, is delayed.ConclusionsThe methane reducing effect of A. taxiformis during rumen fermentation makes this macroalgae a promising candidate as a biotic methane mitigation strategy for dairy cattle. But its effect in-vivo (i.e. in dairy cattle) remains to be investigated in animal trials. Furthermore, to obtain a holistic understanding of the biochemistry responsible for the significant reduction of methane, gene expression profiles of the rumen microbiome and the host animal are warranted

Effect of the macroalgae Asparagopsis taxiformis on methane production and rumen microbiome assemblage (vol 1, 3, 2019)

Table S1. Quality filtering and OTU distribution at each incubation time. Table S2. Diversity indices at each incubation time. Figures S1A., S1B, S1C Rarefaction curves of equilibration, control and A. taxiformis amended vessels respectively. Figure S2. Principle Coordinate Analysis plot. Table S3. OTU table. Table S4. Raw sequence barcodes for archived 16S rRNA gene amplicon data. Table S5. Results of AMOVA and HOMOVA statistical tests. (XLSX 3751 kb

Expanding the diversity of mycobacteriophages: insights into genome architecture and evolution.

Mycobacteriophages are viruses that infect mycobacterial hosts such as Mycobacterium smegmatis and Mycobacterium tuberculosis. All mycobacteriophages characterized to date are dsDNA tailed phages, and have either siphoviral or myoviral morphotypes. However, their genetic diversity is considerable, and although sixty-two genomes have been sequenced and comparatively analyzed, these likely represent only a small portion of the diversity of the mycobacteriophage population at large. Here we report the isolation, sequencing and comparative genomic analysis of 18 new mycobacteriophages isolated from geographically distinct locations within the United States. Although no clear correlation between location and genome type can be discerned, these genomes expand our knowledge of mycobacteriophage diversity and enhance our understanding of the roles of mobile elements in viral evolution. Expansion of the number of mycobacteriophages grouped within Cluster A provides insights into the basis of immune specificity in these temperate phages, and we also describe a novel example of apparent immunity theft. The isolation and genomic analysis of bacteriophages by freshman college students provides an example of an authentic research experience for novice scientists