key: cord-0295728-nqinvoux
authors: Jenckel, Maria; Hall, Robyn; Strive, Tanja
title: Pathogen profiling of Australian rabbits by metatranscriptomic sequencing
date: 2022-02-16
journal: bioRxiv
DOI: 10.1101/2022.02.15.480619
sha: d8f985e2c5bc4d58ea58c46a328ecad8bc5a1c4b
doc_id: 295728
cord_uid: nqinvoux

Australia is known for its long history of using biocontrol agents, like myxoma virus and rabbit haemorrhagic disease virus (RHDV), to manage wild European rabbit populations. Interestingly, while undertaking RHDV surveillance of rabbits that were found dead we observed that approximately 40% of samples were negative for RHDV. To investigate whether other infectious agents are responsible for killing rabbits in Australia we subjected a subset of these RHDV-negative liver samples to metatranscriptomic sequencing. In addition, we investigated whether the host transcriptome data could provide additional differentiation between likely infectious versus non-infectious causes of death. We identified transcripts from several Clostridia species, Pasteurella multocida, Pseudomonas, and Eimeria stiedae in liver samples of several rabbits that had died suddenly, all of which are known to infect rabbits and are capable of causing fulminant disease. In addition, we identified Hepatitis E virus and Cyniclomyces yeast in some samples, both of which are not usually associated with severe disease. In one third of the sequenced liver samples, no infectious agent could be identified. While metatranscriptomic sequencing cannot provide definitive evidence of causation, additional host transcriptome analysis provided further insights to distinguish between pathogenic microbes and commensals or environmental contaminants. Interestingly, three samples where no pathogen could be identified showed evidence of upregulated host immune responses, while immune response pathways were not upregulated when E. stiedae, Pseudomonas, or yeast were detected. In summary, although no new putative rabbit pathogens were identified, this study provides a robust workflow for future investigations into rabbit mortality events. Importance We have observed that approximately 40% of rabbit liver samples submitted for RHDV testing (from rabbits that had died suddenly without obvious cause) are RHDV-negative. Interestingly, a similar finding was reported in pet rabbits in the United Kingdom. This raises the intriguing question of what else is killing rabbits, both in Australia and internationally? Using a metatranscriptomic sequencing approach, we found that Clostridiaceae, Pasteurella multocida, and Eimeria are frequently detected in cases of sudden rabbit death in Australia. While we did not identify any potential new pathogens that could be explored in the context of wild rabbit management, we have validated an approach to explore future mortality events of lagomorphs that may identify candidate novel biocontrols. Furthermore, our findings reaffirm the recommendation to follow good hygiene practices when handling rabbits, since domestic rabbits harboured several pathogens of potential public health significance, including Escherichia, Pasteurella multocida, and Hepatitis E virus.

sequencing methods (i.e., 'metagenomics' and 'metatranscriptomics'), laboratories can now 77 apply these methods to specific disease syndromes and/or mortality events to detect putative 78 associations with known or emerging pathogens (14) (15) (16) . Finally, in the Australian context, 79 these pathogen discovery approaches may reveal candidate future biocontrol agents, or 80 potential ecological interactions between microbes (either synergistic or antagonistic), that 81 may enhance future rabbit management approaches. 82

There are several known causes of sudden death in rabbits. Non-infectious differential 83 diagnoses include degenerative (heart disease, renal disease), developmental (congenital 84 defects), inflammatory (e.g., pancreatitis), neoplastic, nutritional, traumatic, toxic, physical 85 (e.g., liver lobe torsion, intussusception, aspiration pneumonia, heat stroke), and vascular 86 (pulmonary embolism, haemorrhagic syndromes) pathologies (4, 17) . Examples of known 87 infectious causes of acute fatalities in rabbits include pasteurellosis, staphylococcosis, hepatic 88 coccidiosis, enterotoxaemia/epizootic rabbit enteropathy (ERE), colibacillosis, Tyzzer's 89 disease, pseudotuberculosis, tularaemia, myxomatosis, and rabbit haemorrhagic disease (4, 90 11-13, 18) . But are there potentially overlooked pathogens? In this study, we profiled the 91 metatranscriptome of liver samples collected from RHDV-negative rabbits found dead in 92

Australia to determine what putative pathogens may be killing these rabbits. 93 94 Results 95

To identify microbes associated with sudden death of rabbits in Australia, we conducted 97 metatranscriptomic sequencing on 60 RHDV-negative rabbit liver samples collected from 98 Victoria (VIC; n = 38), Tasmania (TAS; n = 8), New South Wales/Australian Capital Territory 99 (NSW/ACT; n = 11), South Australia (SA; n = 2), and Western Australia (WA; n = 1) between 100 2016 and 2020 (Supplementary table 1). Samples were obtained from a mix of breeds of 101 domestic pet, show, and meat rabbits that ranged from 4.5 weeks to 9 years of age, as well as from two wild rabbits; 23 samples were from does and 33 samples were from bucks (for 103 the remaining 4 samples the sex was not specified). Rabbits were reported to have a wide 104 range of clinical signs prior to death, although many were simply found dead (Supplementary  105   table 1) . Notably, six rabbits from Victoria that died between 2017 and 2018, including three 106 from a single shelter facility, were reported with frank haemabdomen. On further investigation, 107 these six rabbits had no access to anticoagulants, there were no clear dietary associations 108 between the cases, and at least four were housed indoors. This prompted us to look more 109 closely at cases from Victorian rabbits, and haemorrhagic signs prior to death were also 110 reported in a further five cases. 111

The 60 sequencing libraries ranged in size from 6,356,968 to 24,147,560 paired-end 112 reads, of which 8.0-93.6% (x̄ 24.11%) did not map to the phylum Chordata (i.e., the vertebrate 113 host). Reads were assembled into contigs, which were used for taxonomic assignment. The 114 transcripts per million (TPM) method was used to normalise the data and to calculate the 115 relative abundance of taxa, where reads were used in place of transcripts. Taxonomic 116 assignment at the kingdom level revealed three clear groupings of samples-bacteria-117 dominant (n = 9), eukaryota-dominant (n = 1), and unassigned-dominant (n = 50) ( Figure 1A) . 118

For comparison, metatranscriptomic sequencing of 24 known RHDV+HEV-positive liver 119 samples almost always showed an extremely high proportion of viral reads (x̄ 52.7%; Figure  120 1B) (19). Overwhelmingly, most unknown samples grouped as unassigned (i.e., most reads 121 were classified as unassigned). 122

Metatranscriptomic sequencing was conducted on liver samples from rabbits that had died 125 suddenly and that were negative for Rabbit haemorrhagic disease virus (RHDV) (A). Reads 126 were assembled into contigs, classified to the kingdom level, and normalised using the 127 transcript per million method by mapping individual reads to contigs. This revealed three 128 distinct clusters of samples: those with a high proportion of 1) bacterial reads, 2) eukaryotic 129 reads (excluding phylum Chordata), or 3) unassigned reads. For comparison, the same 130 analyses were performed on 24 known RHDV+HEV-positive liver samples. In addition to Eimeria and Cyniclomyces yeast, other eukaryotic reads detected included 155 those of the roundworm Toxocara and the coccidian parasites Isospora and Cyclospora 156 ( Figure 2A ). The latter reads correlated strongly with the presence and abundance of Eimeria 157 reads, suggesting that perhaps conserved coccidia regions were misclassified between these 158 three genera. RHDV reads were detected in most samples ( Figure 2C ), however, this most 159 likely reflects cross-contamination of the flow cell during sequencing, since RHDV-positive and 160 -negative samples were combined in the same sequencing run and the abundance of RHDV 161 reads in positive samples is extremely high ( Figure 1B , Figure 2D ). Indeed, samples from run 162 2, which comprised 24 RHDV-positive samples, revealed a higher level of RHDV reads than 163 samples from run 1, which included two RHDV positive samples (that were not part of this 164 study). Other viruses identified included HEV, Eimeria stiedai RNA virus 1 (in two samples, 165

both of which were also positive for Eimeria), retroviruses (likely reflecting rabbit endogenous 166 retroviruses), and a rabbit picobirnavirus in one sample ( Figure 2C ). As well as the dominant 167 bacterial genera discussed above, other putative bacterial pathogens were detected 168 sporadically, such as Escherichia, Staphylococcus, Corynebacterium, and Bacteroides, but 169 typically at low abundance and/or secondary to other dominant microbes. Furthermore, many 170 likely commensal and/or environmental bacterial genera were identified frequently and 171 typically at low abundances. 172

For multiple samples collected from the same 'outbreak' event, there was not always a 173 strong correlation between the dominant microorganism detected (Supplementary table 1) . 174

For example, C. cuniculi was detected in CBN-2 but CBN-1 was classified as unassigned. 175

Similarly, Clostridiacae were detected in COR-5 but not in COR-2. However, for samples CND-176 1 and -2, Cyniclomyces yeast were detected in both cases. C. cuniculi and HEV were detected 177 in both HTL-10 and HTL-11. Clostridiacae were detected in both MGY-1 and MGY-2, although 178 C. cuniculi specifically was only detected in MGY-2. While no infectious agents were identified 179 for KYB-2 and KYB-7, both samples were classified as unassigned. For Victorian samples with a haemorrhagic presentation, most samples were classified as unassigned, with 181

Clostridiacae and HEV each being identified in two of 11 cases. 182

To verify detections of HEV, Clostridiacae, Pasteurella, and Eimeria, and to confirm the 183 RHDV status of the samples, we conducted confirmatory RT-PCR and RT-qPCR testing 184 While metatranscriptomic analyses can identify the presence of microbial reads and high 233 abundance may be suggestive of fulminant infection, these analyses cannot reliably be used 234 to infer pathogenicity/cause of death at an individual level. Therefore, we interrogated the 235 'residual' host transcriptome for gene ontology (GO) terms in the 'biological process' domain 236

for processes related to immune responses and/or defense mechanisms (hereafter described 237 as 'immune response'). Host transcriptome data from three healthy laboratory rabbits 238 generated in a previous study were used as 'known non-infectious cause of death ' controls 239 (20) . GO terms that we considered as indicative of an immune response, based on a positive 240 enrichment score relative to healthy control laboratory rabbits, are detailed in Supplementary 241 table 2. The 24 known RHDV+HEV-positive liver samples (described above) served as 'known 242 infectious cause of death' samples. Through our long-term Australian lagovirus surveillance program, we were surprised to 257

observe that approximately 40% of rabbit liver samples collected from rabbits that had died 258 suddenly were negative for RHDV (3), with a recent UK study presenting similar findings (4). 259

To identify whether other infectious agents may be responsible for these sudden deaths, 260 particularly in outbreak situations where multiple rabbit deaths were reported, we undertook 261 metatranscriptomic sequencing of 60 RHDV-negative rabbit liver samples. While reads from several putative bacterial and eukaryotic pathogens were identified at high relative 263 abundance, including several Clostridiaceae species, Pasteurella, Eimeria, and 264

Pseudomonas, most liver samples were classified as 'unassigned', where no hit could be 265 identified in the NCBI nucleotide database. This suggests that most cases of sudden death in 266 (RHDV-negative) rabbits may be due to non-infectious causes. Alternatively, liver samples 267 may not have been suitable for diagnosis of these cases, or the pathogen may not have been 268 present at high abundance at the time of sampling. Interestingly, three of these 'unassigned' were not ideal for metatranscriptomic analyses (i.e., samples were not snap-frozen at -80 °C), 279 which may have adversely impacted our findings. Importantly, the RHDV+HEV-positive 280 samples were derived from the same sampling program, making them ideal controls since 281 they were subject to the same limitations, and upregulation of immune responses were still 282 detectable in these samples. For metatranscriptomic analyses to be revealing, the pathogen 283 must be transcriptionally active in the sampled tissue at the time of sampling. Since the liver 284 is generally considered to be a sterile site, this organ is infrequently targeted for exploratory 285 metatranscriptomic analyses (21). However, because of the highly vascular nature of the liver, 286

it could be expected that any systemic infection would also be detected in this tissue. Indeed, 287

it may be easier to differentiate pathogens from the healthy commensal microbiome using liver 288 samples compared to, for example, gastrointestinal tract samples. While metatranscriptomic 289 analyses can only provide evidence of association, not causation, several criteria support an 290 agent being potentially pathogenic. For example, in the context of this study, finding an agent 291 at high abundance, consistently across several similar cases, temporally associated with 292 sudden death, particularly if known to be pathogenic in other species, and with corresponding 293 transcriptomic evidence of upregulation of immune responses, would additionally support a 294 putatively causal relationship. 295

The incorporation of host transcriptome analysis into a metatranscriptomic survey offers 296 a novel and innovative approach to the diagnosis of infectious disease via 297 metatranscriptomics, utilising the host mRNA data that is usually discarded in such analyses. 298

While subject to several limitations in the implementation of this study, such as variable post-299 mortem degradation of samples, we clearly observed positive enrichment of immune 300 responses and defense pathways in our known RHDV+HEV-positive controls, which had been 301 subjected to similarly variable sampling and handling regimes. Notably, most of the 302 unassigned cases generally did not show evidence of upregulation of host immune responses. 303

In summary, whilst the detection of upregulated immune genes is still not proof for causation 304 (such as in the case of HEV), inclusion of these data in combination with other findings such 305 as high abundance of a single dominant microorganism can lend additional support to the 306 hypothesis of infection as a contributing factor to death. 307

While HEV was identified in 18 samples, we do not suspect this to be the primary cause 308 of death in these cases, despite many cases also showing evidence of an immune response 309 in their transcriptome profiles. HEV is present globally in wild and domestic rabbit populations 310 at relatively high seroprevalence (3-60%) (22), yet was only identified for the first time in 311 rabbits in 2009 through a serosurvey of farmed rabbits (23). This suggests that it is not a major 312 cause of morbidity or mortality, at least in healthy animals. Experimental infection studies have 313

shown that while rabbits can develop both acute and chronic hepatitis following HEV infection, 314

infection is often subclinical and sudden death has not been observed, except in pregnant rabbits (24-27). A recent study found a seroprevalence of 9% in healthy shot wild rabbits in 316

Australia (19), providing further support that HEV was likely an incidental finding. 317

We identified several clostridial species in rabbit liver samples in this study, including C. 318 cuniculi, Paeniclostridium sordellii, C. spiroforme, and C. perfringens, first through 319 metatranscriptomic sequencing and subsequently verified by RT-PCR. Toxigenic Clostridium 320 species, particularly C. spiroforme but also infrequently C. perfringens and C. difficile, are 321 known to cause enterotoxaemia in rabbits, a major cause of acute diarrhoea leading to severe 322 dehydration and death in 24-48 hours (12). A similar syndrome, epizootic rabbit enteropathy 323 (ERE), has recently been associated with C. cuniculi overgrowth (28, 29). Both syndromes 324 frequently occur in farmed rabbits at weaning with very high (30-95%) mortality (12, 28). The 325 disease is multifactorial, with stress, dietary changes, or antibiotic use triggering 326 gastrointestinal dysbiosis leading to subsequent proliferation of Clostridium species, 327 sometimes with secondary opportunistic overgrowth of coliforms (12). Coinfections with other 328 pathogens (such as enteropathogenic E. coli, C. piliforme, rotaviruses, and Eimeria) are 329 common, with one study identifying coinfections in 86% of rabbits with enterotoxaemia (29, 330 30). Neither C. spiroforme nor C. cuniculi are typically observed in the microbiome of healthy 331 rabbits (12, 28). Given the major disruption to the gut epithelium in both enterotoxaemia and 332 ERE and the high abundance of Clostridium species during fulminant disease, it would not be 333 surprising to observe bacterial translocation into the bloodstream with subsequent detection 334 in the liver. However, there was evidence of a host immune response in only 40% of samples 335 in this study, although poor sample quality could have adversely affected this analysis. While 336 P. sordelii is not classically associated with enterotoxaemia in rabbits, it has been associated 337 with various enteric and histotoxic infections in a wide variety of species (31). However, its 338 role in disease is controversial, as it is a common environmental bacterium found in soil (31). 339

Another important clostridial pathogen of rabbits is C. piliforme, the causative agent of Tyzzer's 340 disease, characterised by diarrhoea, dehydration, multifocal hepatic necrosis, and death in 1-341 compared to other clostridial species and detections could not be verified by PCR (32). It is 343 possible that the contigs mapping to C. piliforme spanned conserved clostridial genomic 344 regions and were misclassified from other species. The lack of specific RT-PCR detections 345

suggests that none of these rabbits succumbed to Tyzzer's disease. 346

Pasteurella multocida is considered to be the most common bacterial pathogen of 347 laboratory rabbits (12) and indeed, we identified P. multocida in 7 of 60 samples. There are 348 multiple clinical manifestations of pasteurellosis, including rhinitis, pneumonia, genital tract 349 infections, otitis media, and septicaemia. P. multocida is also a common commensal in the 350 rabbit nasopharynx; for example, one study showed that 31% of healthy rabbits were infected 351 asymptomatically (33). Septicaemia typically occurs from haematogenous spread following 352 localised disease and is rapidly fatal. In these cases, P. multocida can be recovered from 353 parenchymal organs (12). Therefore, it is highly probable that our detections of this organism Eimeria are apicomplexan parasites that cause coccidiosis. Eleven Eimeria species 360 infect rabbits, resulting in hepatic coccidiosis (E. stiedae) or intestinal coccidiosis (the 361 remaining 10 species) (13). All rabbit Eimeria species can be carried subclinically, typically by 362 adult animals, which serve as the infection source for young animals. Disease is enhanced by 363 stressors such as overcrowding, poor hygiene, poor nutrition, transportation, and weaning. 364

Hepatic coccidiosis is characterised by severe liver disease, resulting in anorexia, ascites, 365 icterus, and death, particularly in young animals 2-3 months of age (13). Intestinal coccidiosis 366 manifests as diarrhoea, the severity of which depends on the pathogenicity of the infecting 367 species. The presence of E. stiedae was confirmed via RT-PCR with E. stiedae specific 368 primers (34, 35). Of note was the detection of Eimeria stiedae virus RNA 1 in two Eimeria-positive samples, further confirming the presence of Eimeria stiedae in those rabbits. This is 370 a double-stranded RNA virus belonging to the family Totiviridae (36) known to specifically 371 infect E. stiedae (37). All Eimeria infections identified here were in young animals and multiple 372 deaths were reported in each case; diarrhoea was not a feature of these cases. None of the 373 positive samples showed a positive enrichment score for host immune responses on 374 transcriptome analysis, which is perhaps not unexpected given that death is due to secondary 375 liver failure. While Cyclospora and Isospora were also identified in our samples, both of which 376 are also coccidian parasites, these species are not known to infect rabbits and the presence 377 and abundance correlated strongly with detections of Eimeria. Therefore, we suggest that 378 these were probably Eimeria contigs spanning conserved genomic regions that were 379 misclassified as Cyclospora or Isospora. 380

Reads matching Cyniclomyces yeast were detected at high relative abundance in two 381 samples, although the clinical significance of this finding remains unclear. The most well-382 characterised species of this genus is C. guttulatus (formerly Saccharomycopsis guttulata), a 383 normal commensal of the gastrointestinal tract of rabbits and rodents (38). Although it has 384 been detected in association with various clinical presentations, such as oculonasal discharge 385 and systemic abscesses, bloat, enteritis, and coccidiosis, most researchers agree that this is 386 likely to be an opportunistic pathogen or a secondary overgrowth following a prior insult (38). 387

We did not find an association with the presence of Eimeria in this study. Following 388 experimental infections with C. guttulatus, healthy rabbits remain asymptomatic (38, 39). The 389 detection of this yeast in liver samples may suggest translocation post-mortem or 390 contamination during sample collection. The lack of enrichment for immune responses on host 391 transcriptome analysis provides further support that these yeasts were not primary pathogens 392 in these samples. 393

Pseudomonas reads were identified at high abundance in two rabbit liver samples. P. 394 aeruginosa is a common environmental bacterium and is well-known to cause opportunistic, 395 often severe, infections in a range of species, including humans. In rabbits, infections are typically associated with dermatitis but there are also reports of abscessation, septicaemia, 397 pneumonia, and diarrhoea (12). Interestingly, we found that Pseudomonas reads were also 398 abundant in many RHDV+HEV-positive samples, although confirmatory RT-PCR analyses 399 were negative. The widespread distribution of this organism in the environment suggests that 400 these detections were likely environmental contaminants rather than co-infections, particularly is 3 × 10 8 capsid copies per mg of tissue, which equates to 1.2 × 10 8 capsid copies per µl of 437 RNA (42). Indeed, the highest relative abundances of RHDV was observed in samples from 438 sequencing run 2, which also included 24 RHDV+HEV positive samples. Both inter-run and 439 intra-run contamination are known concerns with Illumina platforms. For example, several 440 studies have reported that up to 10% of reads from a sample can be incorrectly assigned when 441 multiplexing, particularly with ExAmp chemistry such as that used for the . 442

For this reason, a non-redundant dual-indexing strategy would have been preferable in 443 hindsight. However, we also cannot rule out low-level cross-contamination during sequencing 444 library preparation or RNA extraction since extraction controls were not sequenced. 445

Finally, while our sample size was relatively small, several notable pathogens were not 446 identified in this study. For example, Salmonella enterica, while uncommon in rabbits, can 447 cause epizootics with high morbidity and mortality and can potentially be transmitted to 448 humans (12). Listeria monocytogenes is also an infrequent cause of sudden death in rabbits 449 but is significant from a public health perspective. Francisella tularensis, the causative agent of the zoonotic disease tularaemia, is endemic in wild rabbits and hares in Eurasia and North NovaSeq6000 instrument (SP300 cycle flow cell) at the Biomolecular Resource Facility (BRF), 502

The John Curtin School of Medical Research, Australian National University. Raw reads were 503 deposited in the NCBI Sequence Read Archive under Biosample accession numbers 504 SAMN24852673 -SAMN24852758, BioProject accession number PRJNA796430. 505 506

Raw data were pre-processed using FastQC (v0.11.08), Trimmomatic (v0.38) (51) and FLASh 508 (v1.2.11) (52), as described previously (19). Cleaned reads were mapped against the rabbit 509 reference genome (GCA_000003625.1 OryCun2.0) using Bowtie2 (v2.2.9) (53) to filter out 510 host reads. The remaining reads were assembled into contigs using Trinity (v2.12.0) (54) and 511 contigs were blasted against the NCBI nt database (BLAST+ v2.12.0; default parameters). 512

Results with a query coverage of less than 50% were discarded and TaxonKit (v0.8.0) (55) 513 was used to assign taxonomic lineages to each remaining BLAST hit. All reads used for 514 assembly were then mapped against the assembled contigs to calculate the coverage per 515 contig and the relative abundance of each taxon in TPM (56) (i.e., the proportion of one million 516 randomly selected reads that match the taxon of interest) using SAMtools (v1.12) (57) and R 517 (v4.1.0) (58). Bacterial phyla with an abundance of less than 100 across all samples were 518 excluded for the bar plot. Heats maps were generated using the R package ampvis2 (v2.7.11) 519 (59). 520 521

Raw reads were processed as described above. Additionally, previous transcriptomic data 523 from three healthy laboratory rabbits generated in another study (20) were used as 'known 524 non-infectious cause of death' controls. Reads were mapped against the rabbit reference 525 genome (GCA_000003625.1 OryCun2.0) using TopHat (v2.1.1) (60). Reads per exon were 526 counted using HTSeq (v0.13.5) (61). Exons were matched to Entrez-IDs and exons without 527 an Entrez-ID were discarded, as no further GO information could be gathered. The DESeq2 528 package (v1.32.0) (62) in R (v4.1.0) (58) was used to calculate the log2fold changes and p-529 values for all genes compared to the 'known non-infectious cause of death' control samples. 530

Genes with an adjusted p-value <0.05 were used for a GO Gene Set Enrichment Analysis in 531 the "biological processes" category using the GOSemSim (v2.18.1) (63) and clusterProfiler 532 (v4.0.5) (64). GO-terms with a p-value <0.05 were considered significant. 533 534

Specific RT-PCRs using the SuperScript III One-Step RT-PCR System with Platinum Taq DNA 536

Polymerase (Invitrogen) were run to verify the presence of Clostridiaceae species (65), 537

Clostridium cuniculi (28), Paeniclostridium sordellii (65), Clostridium perfringens (65), 538

Clostridium spiroforme (66), Clostridium piliforme (32), Pasteurella multocida (67), and 539

Eimeria stiedae (34, 35) . Briefly, each 25 µl reaction contained 12.5 µl of reaction mix (2x), 540 9.5 µl of nuclease-free water, 1 µl of 10 µM primer mix, 1 µl of enzyme mix and 1 µl of total 541 RNA. Eimeria and Pasteurella PCR reactions were run under the same cycling conditions: 45 542 °C for 15 min, 94 °C for 2 min, followed by 35 cycles of 94 °C for 15 sec, 55 °C for 30 sec and 543 68 °C for 90 sec and a final elongation at 68 °C for 120 sec. The Clostridium PCR reactions 544 all used 68 °C for 120 sec for elongation, except for C. spiroforme and C. piliforme where the 545 elongation time was reduced to 30 sec. PCR products were visualized on a 1% agarose gel 546 for a band of appropriate size. The presence of Hepatitis E virus and RHDV were verified via 547 RT-qPCR as previously described (19, 42) . 

Viruses for Landscape-Scale Therapy: Biological Control of 565 Rabbits in Australia

Next Step in the Ongoing Arms Race between Myxoma Virus and Wild Rabbits

Australia is a Novel Disease Phenotype

Intergenotypic Recombination Between the Non-Structural and Structural Genes is a Major 571

Driver of Epidemiological Fitness in Caliciviruses

2020. RHDV2 Epidemic in UK Pet 573

Part 2: PCR Results and Correlation with Vaccination Status

Keystone Species in Southern Europe

Emergence of a New Lagovirus Related to Rabbit Haemorrhagic Disease Virus

Variant Rabbit Hemorrhagic Disease Virus in Young Rabbits

Oryctolagus Cuniculus (Errata Version

The IUCN Red List of Threatened Species, The IUCN Red List of Threatened Species

First Description of Hepatitis E Virus in Australian Rabbits

Zoonoses of Rabbits and Rodents

Chapter 14 -Viral Diseases

The Laboratory Rabbit, Guinea Pig, Hamster, and Other Rodents

Chapter 13 -Bacterial Diseases

The Laboratory Rabbit, Guinea Pig, Hamster, and Other Rodents

Chapter 15 -Parasitic Diseases

The Laboratory Rabbit, Guinea Pig, Hamster, and Other Rodents

Expanding the RNA Virosphere by 601

Clinical Metagenomic Sequencing for Diagnosis of 608 Meningitis and Encephalitis

Assessments Improve Diagnosis and Outcomes in Community-Acquired Pneumonia

The Laboratory Rabbit

Ferrets, Rabbits, and Rodents : Clinical Medicine and 617

Genetic Diversity of Hepatitis E Virus in Wild and Domestic Rabbits in Australia

Innate Immunity of Young Rabbits Mediates Resistance to Rabbit Hemorrhagic Disease 623 Caused by Lagovirus Europaeus GI.1 But Not GI.2. Viruses 10

Comparative Analysis of RNA Virome 625

Composition in Rabbits and Associated Ectoparasites

An Overview: Rabbit Hepatitis E Virus (HEV) and Rabbit 627 Providing an Animal Model for HEV Study

A Novel Genotype of Hepatitis E Virus Prevalent among Farmed Rabbits in China

Experimental 632 Infection of Rabbits with Rabbit and Genotypes 1 and 4 Hepatitis E Viruses

Model for Hepatitis E Virus Infection and Vaccine Evaluation

SPF Rabbits

Infected with Rabbit Hepatitis E virus Isolate Experimentally Showing the Chronicity of 638

Experimental 640 Infection of Pregnant Rabbits with Hepatitis E Virus Demonstrating High Mortality

Epizootic Rabbit Enteropathy: Experimental 646 Transmission and Clinical Characterization

Significance of Clostridium spiroforme 648 in the Enteritis-Complex of Commercial Rabbits

Clostridium) sordellii-Associated Enterocolitis in 7 Horses

Detection by PCR of the Tyzzer's Disease 653

Clostridium piliforme) in Feces

Characterization of Pasteurella multocida Isolates from the 655 Nares of Healthy Rabbits with Pneumonia

Development of Molecular Assays 657 for the Identification of the 11 Eimeria Species of the Domestic Rabbit (Oryctolagus 658 cuniculus)

Simultaneous Identification 660 of Three Highly Pathogenic Eimeria Species in Rabbits Using a Multiplex PCR Diagnostic 661

Assay Based on ITS1-5.8S rRNA-ITS2 Fragments

Evolution Analysis of Eimeria stiedai RNA virus 1, a Novel Member of the Family Totiviridae

Identification of Virus-Like Particles in Eimeria stiedae

An Investigation of 669 the Relationship between Cyniclomyces guttulatus and Rabbit Diarrhoea. Pathogens 10

Whole-Genome Comparison of 671

Endogenous Retrovirus Segregation across Wild and Domestic Host Species Populations

Chapter 23 -Infectious Diseases

The Laboratory Rabbit

Multiplex RT-PCR for Australian Rabbit Haemorrhagic Disease Viruses Uncovers a new 679

Recombinant Virus Variant in Rabbits and Hares

Promises and Pitfalls of Illumina Sequencing for HIV Resistance 681

Characterization and Remediation of Sample Index Swaps by Non-Redundant Dual Indexing 685 on Massively Parallel Sequencing Platforms

Index Switching Causes "spreading-of-signal" among Multiplexed 689

Samples in Illumina HiSeq 4000 DNA Sequencing

Francisella tularensis 692 ssp. holarctica in Ringtail Possums

An Outbreak of Fatal Herpesvirus Infection in Domestic Rabbits in Alaska

Acute Hemorrhagic 697 and Necrotizing Pneumonia, Splenitis, and Dermatitis in a Pet Rabbit Caused by a Novel 698

Leporid herpesvirus-4). The Canadian veterinary journal = La revue veterinaire 699 canadienne

Rabbit Hemorrhagic Disease Virus 2 702 (RHDV2; GI.2) Is Replacing Endemic Strains of RHDV in the Australian Landscape within 18 703 Months of Its Arrival

Trimmomatic: A Flexible Trimmer for Illumina 705

FLASH: Fast Length Adjustment of Short Reads to Improve 707

Fast Gapped-Read Alignment with Bowtie 2

Transcriptome Assembly from RNA-Seq Data Without a Reference Genome

TaxonKit: A Practical and Efficient NCBI Taxonomy Toolkit

Measurement of mRNA Abundance Using RNA-seq data: 718 RPKM Measure is Inconsistent Among Samples

The Sequence Alignment/Map format and 721

2021. R: A Language and Environment for Statistical Computing, R Foundation 723 for Statistical Computing

ampvis2: An R Package to Analyse and 725

Aignment of Transcriptomes in the Presence of Insertions, Deletions and Gene Fusions

HTSeq-A Python Framework to Work with

Moderated Estimation of Fold Change and Dispersion for 732 RNA-Seq Data with DESeq2

GOSemSim: An R package for Measuring 734

Semantic Similarity among GO Terms and Gene Products

2021. clusterProfiler 4.0: A Universal Enrichment Tool for Interpreting Omics Data

Design of Species-Specific Primers to 739

Identify 13 Species of Clostridium Harbored in Human Intestinal Tracts

Development of PCR Protocols for 742

Specific Identification of Clostridium spiroforme and Detection of sas and sbs Genes

Development of a 23S rRNA-based PCR Assay for the 745 Identification of Pasteurella multocida

America and can cause sudden death in these species (12) . Recently, four locally acquired 452 human cases of tularaemia have been reported in Australia, linked to contact with infected 453 possums, however an animal reservoir of F. tularensis has not yet been identified locally (46, 454 47) . While our study focussed mainly on domestic rabbits, we did not detect any Francisella 455 contigs in these samples. Surprisingly, we also did not identify MYXV in this study. Recently, 456 a highly lethal immune collapse syndrome was demonstrated in domestic rabbits infected with 457 MYXV isolates from the 1990s (2). Given the active circulation of MYXV in wild rabbit 458 populations in Australia, we had expected to find MYXV as a cause of death in RHDV-negative 459 domestic rabbits. Leporid herpesvirus 4 is a recently emerged alphaherpesvirus that was 460 isolated from a mass mortality event in Alaska in 2008 and from a single pet rabbit in Canada 461 in 2010 (48, 49). It has not been reported elsewhere and was also not detected in the rabbits 462 analysed in our study. Other viruses known to be associated with sudden death in rabbits 463 include rabbit enteric coronavirus. Finally, no fungal contigs were identified in these samples, 464although rabbits appear to be remarkably resistant to systemic mycoses (17). 465In summary, while sudden death in domestic rabbits in Australia can mostly be attributed 466 to RHDV, our study found that Clostridiaceae, Pasteurella multocida, and Eimeria are also 467 frequently detected in cases of sudden rabbit death. Importantly however, most non-RHDV 468 cases of sudden death in Australian rabbits were not able to be attributed to a known pathogen 469 and no novel putative rabbit pathogens were identified. Furthermore, our findings reaffirm the 470 recommendation to follow good hygiene practices when handling rabbits, since domestic 471 rabbits were found to harbour several pathogens of potential public health significance, 472including Escherichia, Pasteurella multocida, and HEV. While this study did not reveal any 473 potential new pathogens that could be explored in the context of wild rabbit management, we 474 have validated an approach to explore future mortality events of lagomorphs either in Australia 475 or internationally that may identify candidate novel biocontrols. Similarly, we demonstrate that 476 the use of host transcriptome data can lend additional support to diagnosing an infectious 477 cause of death or conversely, suggesting absence of infection. 478 479

Samples were selected from a rabbit tissue bank established for lagovirus surveillance (50). 482No animal ethics approvals are required for sampling rabbits that are found dead in Australia. 483Samples from NSW and ACT were grouped together, since the ACT is a small (~2400 km 2 ) 484 enclave within NSW. Since RHDV is hepatotropic, liver was generally the only sample 485 available. Samples were collected post-mortem (at various times post-death) by pet owners 486 and veterinarians and were stored in an RNA preservative solution at -20 °C. RHDV-negative 487 samples were selected initially (n = 45) based on a detailed clinical history, with a preference 488 for cases where sudden deaths had occurred in multiple rabbits over a short time period (42). 489Because of these selection criteria, most cases were from domestic rabbits. Subsequently, 34 490 known HEV-positive domestic rabbit liver samples (from the same sample collection), 24 of 491 which were also RHDV-positive, were sequenced for another study (19)