Chromosome-length genome assembly of Teladorsagia circumcincta - a globally important helminth parasite in livestock.

BACKGROUND: Gastrointestinal (GIT) helminthiasis is a global problem that affects livestock health, especially in small ruminants. One of the major helminth parasites of sheep and goats, Teladorsagia circumcincta, infects the abomasum and causes production losses, reductions in weight gain, diarrhoea and, in some cases, death in young animals. Control strategies have relied heavily on the use of anthelmintic medication but, unfortunately, T. circumcincta has developed resistance, as have many helminths. Vaccination offers a sustainable and practical solution, but there is no commercially available vaccine to prevent Teladorsagiosis. The discovery of new strategies for controlling T. circumcincta, such as novel vaccine targets and drug candidates, would be greatly accelerated by the availability of better quality, chromosome-length, genome assembly because it would allow the identification of key genetic determinants of the pathophysiology of infection and host-parasite interaction. The available draft genome assembly of T. circumcincta (GCA_002352805.1) is highly fragmented and thus impedes large-scale investigations of population and functional genomics. RESULTS: We have constructed a high-quality reference genome, with chromosome-length scaffolds, by purging alternative haplotypes from the existing draft genome assembly and scaffolding the result using chromosome conformation, capture-based, in situ Hi-C technique. The improved (Hi-C) assembly resulted in six chromosome-length scaffolds with length ranging from 66.6 Mbp to 49.6 Mbp, 35% fewer sequences and reduction in size. Substantial improvements were also achieved in both the values for N50 (57.1 Mbp) and L50 (5 Mbp). A higher and comparable level of genome and proteome completeness was achieved for Hi-C assembly on BUSCO parameters. The Hi-C assembly had a greater synteny and number of orthologs with a closely related nematode, Haemonchus contortus. CONCLUSION: This improved genomic resource is suitable as a foundation for the identification of potential targets for vaccine and drug development.

Revealing the associated microflora hosted by the globally significant parasite Trichostrongylus colubriformis.

Trichostrongylus colubriformis is a parasitic helminth that primarily infects small ruminants, causing substantial economic losses in the livestock industry. Exploring the microbiome of this helminth might provide insights into the potential influence of its microbial community on the parasite's survival. We characterised the intestinal microbiome of T. colubriformis that had been collected from the duodenum of sheep, and compared the helminth microbiome with the duodenal microbiome of its host, aiming to identify contributions from the helminth's environment. At the same time, we explored the isolation of fastidious organisms from the harvested helminth. Primary alpha and beta diversity analyses of bacterial species revealed statistically significant differences between the parasite and the host, in terms of species richness and ecological composition. 16S rRNA differential abundance analysis showed that Mycoplasmoides and Stenotrophomonas were significantly present in T. colubriformis but not in the duodenal microbiome of the sheep. Furthermore, two bacteria, Aeromonas caviae and Aeromonas hydrophila, were isolated from T. colubriformis. Examinations of the genome highlight differences in genome size and profiles of antimicrobial resistance genes. Our results suggest that T. colubriformis carries a specific bacterial community that could be supporting the helminth's long-term survival in the host's digestive system.

Bacterial communities in the gastrointestinal tract segments of helminth-resistant and helminth-susceptible sheep.

BACKGROUND: Helminth parasitism is a world-wide problem in livestock industries, with major impacts on health, welfare and productivity. The role of the gut microbiota in host-helminth interactions in ruminants has been extensively examined and the present study added to this body of knowledge by assessing the effects of resistance and susceptibility to helminth infection in the gastro-intestinal tract (GIT). Australian Sheep Breeding Values (ASBVs) for faecal egg count (FEC) were used to select the 10 highly helminth-susceptible (High-FEC) and 10 highly helminth-resistant (Low-FEC) sheep. FEC status was confirmed during the experiment. Using samples from the faeces and the lumen of the rumen, abomasum, duodenum, jejunum, ileum, caecum, and colon, DNA was extracted and used for 16 rRNA gene amplicon sequencing. RESULTS: The most frequent genera identified along the GIT were Eubacterium, Oscillibacter, and Ruminococcus. Intersectoral-specialization zones were identified along the GIT, with the duodenum displaying major differences between the High-FEC and Low-FEC animals in values for alpha and beta diversity. After taking all samples into account and adjusting for GIT segment, the High-FEC and Low-FEC sheep differed significantly for four genera Butyrivibrio, Mycoplasma, Lachnoclostridium and Succiniclasticum. In the duodenum, the abundances of Aminipila, Lachnoclostridium and Mogibacterium differed significantly between the High-FEC and Low-FEC sheep. In the ileum, on the other hand, the genus Mycoplasma was significantly depleted in the Low-FEC group. CONCLUSIONS: The gastro-intestinal microbial profile varies widely between helminth-resistant and helminth-susceptible sheep. Each GIT section appears to support a particular bacterial composition leading to inter-sectoral differences among the various microbial communities. The microbial populations were most rich and diverse in the duodenum of helminth-resistant sheep, comprising bacterial genera that generally ferment carbohydrates. This observation suggests that helminth-resistant sheep can reorganize the duodenal microbiome taxa which may restrict the development of parasites.

Confirmation of botulism diagnosis in Australian bird samples by ELISA and RT rtPCR.

We developed a sandwich ELISA that detects Clostridium botulinum C and D toxins and reverse-transcription real-time PCRs (RT-rtPCRs) that detect botulinum C and D toxin genes, respectively, to replace the mouse bioassay. The toxin genes were closely associated with the toxin molecules and used as surrogates for the presence of toxin. Samples (638) from 103 clinical cases of birds (302) with suspected botulinum toxicity came from wild birds and poultry (9 cases). Samples tested included blood serum, other body fluids, various tissues, gut contents, maggots, water, and sediment. Botulism was diagnosed in 34 cases (all of which had positive samples in the ELISA, the C toxin gene RT-rtPCR, or both assays). Botulism was suspected in 16 cases (each of which had 1 positive sample either in the ELISA or the C toxin gene RT-rtPCR). In the remaining 53 cases, no samples were positive, but botulism could not be excluded in 32 of these cases, whereas there was no indication of botulism or another diagnosis in 21 cases. The D toxin gene was not detected in any of the clinical samples. No C or D toxin genes were detected in 71 pooled cloacal swabs from 213 healthy migratory birds. The use of an ELISA that detects botulinum C and D toxins in combination with a RT-rtPCR for the botulinum C toxin gene can help confirm the diagnosis of botulism in birds.

Assessing the risk of residues of the toxin indospicine in bovine muscle and liver from north-west Australia.

Indospicine is a natural toxin occurring only in Indigofera plant species, including the Australian native species I. linnaei. These perennial legumes are resistant to drought and palatable to grazing livestock including cattle. Indospicine accumulates in the tissues (including muscle) of animals grazing Indigofera and these residues persist for several months after exposure. Dogs are particularly sensitive to indospicine with reports in past decades of hepatotoxicosis and mortalities in dogs after dietary exposure to indospicine-contaminated horse and camel meat. The risk for human consumption is not known, and the current study was undertaken to assess indospicine levels in cattle going to slaughter from divergent regions of Western Australia, and to predict the likelihood of significant residues being present. Muscle and corresponding liver samples from 776 cattle originating from the Kimberley and Pilbara Regions in the tropical north of the state, where I. linnaei is prevalent, and 640 cattle from the South West and South Coast Regions in the temperate south west of the state, where the plant is not known to occur, were collected at abattoirs over four seasons in 2015-2017. Indospicine levels were measured by LC-MS/MS and ranged from below detection to 3.63 mg/kg. No indospicine residues were detected in any of the animals originating from the South West and South Coast Regions. Prevalence of indospicine residues in cattle from the Kimberley Region was as high as 33% in spring and 91% in autumn, with positive animals being present in most consignments and on most properties. The average prevalence of indospicine residues from the Kimberley and Pilbara Regions throughout the survey period was 63%. @Risk best fit probability distributions showed ninety-fifth percentile (P95) indospicine concentrations of 0.54 mg/kg for muscle and 0.77 mg/kg for liver in cattle originating from the Kimberley and Pilbara Regions during the survey period. When considered with average Australian meat consumption data, the estimated consumer exposure from this P95 muscle was 0.32 µg indospicine/kg bw/day, which compared favourably with our calculated provisional tolerable daily intake (PTDI) of 1.3 µg indospicine/kg bw/day. However canine exposure is of potential concern, with active working dog exposure calculated to exceed this PTDI by a factor of 25, based on a P95 indospicine concentration of 0.54 mg/kg in muscle.

Diagnostic significance of Neospora caninum DNA detected by PCR in cattle serum.

A nested PCR that successfully detected Neospora caninum DNA in serum of cattle was used for investigation of selected abortion cases and in a study of healthy pregnant cows at an abattoir. N. caninum DNA was not detected in serum from antibody positive dams that aborted due to N. caninum, but was present in serum of some antibody negative dams that aborted due to other causes. N. caninum DNA was also found in the serum of about half of the animals that aborted of undetermined cause, but was not detected in cow sera from two beef cattle herds in Western Australia with no recent history of abortion. In the abattoir study of 79 dams and their foetuses N. caninum DNA was found in serum of 3 dams and in material from 11 foetuses. The majority of the cows and all foetuses were antibody negative. Our findings suggest that there is no obvious relationship between the presence or absence of N. caninum DNA in serum and the presence of antibodies to N. caninum in dams, the presence of N. caninum DNA in foetuses or abortion due to N. caninum. This is the first report of the detection of N. caninum DNA in serum of cattle rather than the white blood cell fraction. It indicates the presence of free tachyzoites and/or parasite DNA in circulation. The results suggest that persistent infection in the absence of antibodies is a possible outcome of N. caninum infection. Infection of foetuses in the absence of antibodies supports the possibility of persistent infection due to immunotolerance to an early in utero infection. It is therefore important to test for N. caninum DNA as well as antibodies for the detection of exposed and/or infected animals. However, the presence or absence of N. caninum antibodies or DNA did not support nor exclude N. caninum as the cause of abortion. Additional criteria are required for a positive diagnosis of abortion caused by N. caninum.

Interactions between gastrointestinal nematode parasites and diarrhoea in sheep: pathogenesis and control.

Diarrhoea is a major impediment to profitable sheep production in many countries as it predisposes animals to blowfly strike and contaminates wool and meat carcasses. While it is accepted that nematode parasites are a major cause of diarrhoea in grazing animals, less is known about what facets of the host-parasite relationship lead to diarrhoea and what the most appropriate control strategies are. In this review, the relationship between gastrointestinal nematode infection and diarrhoea is discussed and it is concluded that in many cases, particularly in immunologically mature sheep, diarrhoea is not due to parasite infection per se but rather due to immunopathological processes. Mechanisms that lead to faecal softening in immune sheep are considered, and the question addressed as to whether anthelmintic treatment and selective breeding of naturally parasite-resistant sheep will effectively reduce the occurrence of diarrhoea.