Crassostrea gigas (pacific oyster) has been characterised by a number of successive outbreaks, resulting in dramatic losses of production in France, which has consequently lead to subsequent decline within the industry. The herpes virus (OsHV-1μvar) has often been associated with the deaths of juvenile oysters, along with members of the Vibrionales. The pathogenicity of OsHV-1μvar, as well as several Vibrio, has been demonstrated by experimental infections; however, our understanding of the complexity of these infections is still limited. This study uses specific-pathogen-free (SPF) oysters, infected in estuarine environments, to study the diversity and dynamics of cultured microbial populations during disease expression.
Wild oyster spat was collected and moved to grow in areas within Northern Brittany between 2012 and 2014. Oysters were exposed to disease during spring 2012, and suffered >50% mortality. In 2014, 60 individuals were transferred to experimental facilities, and treated with an antibiotic (chloramphenicol) for 5 days prior to maturation; then reared in controlled conditions until reaching a mean individual wet mass between 0.5 and 3g. qPCR was used to quantify the herpes virus at different developmental stages.
SPF oysters were maintained in another region, during a disease outbreak. Total mortalities resulting in the field were recorded. For cohabitation experiments, field-exposed oysters (“donors”) were brought back to the lab and cohabited in a tank with SPF oysters (“recipient”) under controlled experimental conditions. Chloramphenicol was added to tanks every 2 days to remove any cultivable microbiota, with the number of dead or alive donors being daily counted.
After being killed, oysters had their digestive glands removed and were ground. The total cultivable bacteria and Vibrio microbiota were quantified (cfu/mg of tissue) using serial dilutions on marine agar and TCBS (a type of agar culture plate used in microbiology laboratories to isolate Vibrio sp) selective media. Additionally, the haemolymph from randomly selected colonies surviving either longer than 9 days or less than 24hr was non-destructively acquired and amplified with PCR using the partial hsp60 gene.
OsVH-1 DNA was quantified from ground tissue using PCR. Bacteria were grown under constant agitation at a set temperature for 24h. Intramuscular injections of (107 cfu) were made into the oysters, and bacterial concentration confirmed by conventional dilution plating on agar.
Results
Both adults and juveniles act as reservoirs of pathogens which are able to induce mortalities of young. Only juveniles were shown to transmit the herpes virus, suggesting that the transmitted diseases between the two are distinct and that virus infection is not a prerequisite for juvenile mortality outbreaks. A not-so-surprising discovery was that farmed oysters seem to present higher transmission risks than wild oysters. Additionally, geographic origin of oysters also influences the consortium of pathogens.
Rapid Vibrio colonisation followed by viral replication occurs before oyster death, and no correlation was determined between Vibrio concentration and viral load in co-infected oysters. Instead, the quantity of viral DNA can be used as a predictor of mortality, with OsHV-1 DNA detected in the haemolymph of oysters which died within the first day found to be significantly higher ( up to 4 log units) than those of oysters which lived up to 9 days. Interestingly, the absence of bacteria meant that even a high load of herpes virus is insufficient to induce full disease expression. Juvenile mortalities can occur in the absence of herpes virus, strongly indicating that the herpes virus is neither essential nor sufficient to induce juvenile death, whilst bacteria are essential for disease. Lastly, this paper highlights that the geographic origin, cultivation method and age are all influential factors in disease expression.
I feel that this paper is an interesting read, as it opens up an almost-settled debate on the origin of pathogenicity in Oysters. Herpes virus’ in many papers has been proven as the etiological agent; however, this paper challenges this and shows that it is, in fact, the presence of bacteria, not the herpes virus, which is most important.
Reviewed paper
Petton, B., Bruto, M., James, A., Labreuche, Y., Alunno-Bruscia, M. and Le Roux, F., 2015. Crassostrea gigas mortality in France: the usual suspect, a herpes virus, may not be the killer in this polymicrobial opportunistic disease. Frontiers in microbiology, 6.
Hi Harriet thanks for your review
ReplyDeleteYou mentioned that the herpes virus is often associated with the death of juvenile oysters has there been any suggestions to explain why juvenile oysters are most susceptible? Did the authors mention if adults also showed symptoms?
I think the findings of the paper are really interesting especially in regards to the role bacteria play in this interaction, it appears that bacteria play an important role which may have not been previously recognized with their presence being needed to induce the full disease expression did the authors provide any reason why this may be the case? Maybe some of the bacteria are acting as opportunists after the herpes virus begins to infect juvenile oysters and its more of a polymicrobial disease like the case of black band disease in coral, which is caused by a number of microbes, I’m not entirely convinced that would be the whole story though because as it later mentions its appears that juvenile mortalities can occur in the absence of herpes virus so as you mentioned the presence of bacteria alone appear to play a big role.
I look forward to hopefully reading some of the future follower ups to this study
Hi Alisha,
ReplyDeleteI think that an increasing number of journals will discover that most infections in our oceans are likely to be polymicrbial;especially bivalves such as oysters that filter huge volumes of water daily. Although the results from the paper show that oysters are co-infected by vibrios and virus', the presence of the herpes virus doesn't seem to influence the infection by vibrios and vice versa.
The quantity of herpes DNA was found to be significantly higher ( up to 4 logs) in the haemolylmph of those that died within 24 hours compared to those living 9 days. Contrastingly, the concentration of Vibrios didn't have any correlation.
To determine what occurs in the absence of Bacteria, antibiotics were administrated ( with no changes to the virus concentrations) and the antibiotic treatment resulted in a 2-4 fold decrease in oyster mortality, demonstrating the role of bacteria.
They do try and answer whether there is a synergistic relationship between the vibrios and the viruses, but they did not observe any correlation, so they couldn't answer this. They simply end this section of their paper with "disease oysters that are co-infected by several putative pathogens should sustain much greater losses. However, the authors of this paper were also involved in Lemire et al, (2014), a paper which was in our lectures, which spoke about the progressive replacement of avirulent strains, with those that are virulent.
As you have touched on already, it is a little confusing to decipher what the key point of this paper is. However, I do think that it highlights an important matter, which is simply that Herpes viruses are not the causative agent of disease.
In regard to why juveniles oysters are more susceptible, I believe it is because they are smaller, and also because of their ontogeny. The term 'Juvenile oysters' in many papers, seems to also refer simply to oysters newly introduced to mariculture. I have however stumbled upon this paper which I think will help resolve your query, as it looks at the distinctions between adult and juvenile immune response (Green et al.,2016), and have attached the reference for Lemire.
Lemire, A., Goudenège, D., Versigny, T., Petton, B., Calteau, A., Labreuche, Y. and Le Roux, F., 2015. Populations, not clones, are the unit of vibrio pathogenesis in naturally infected oysters. The ISME journal, 9(7), pp.1523-1531.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4955271/
I hope this has helped!
Harriet
* Juvenile oysters are generally more vulnerable than adults because many vibrios produce cilliostatic toxins (toxins which interfere with cilia) , and this can interfere during the settlement stages of juvenile oysters.
DeleteHi Harriet,
DeleteThanks for the response and link to the paper, it makes sense that in the larvae stage oysters are more vulnerable because of the toxins released by the pathogens that target settlement as you mentioned, I looked over the paper and it helped to answer my query I found it really interesting how there was a difference in the transcriptional response between the juveniles and the adult oysters with different target genes being expressed I guess this could be explained by the different life stages having different vulnerabilities. This difference in gene expression also resulted in the juveniles being more susceptible to OsHV-1, whereas the adults were more susceptible to Vibrio sp. which I also thought was an interesting finding.
Wow a very interesting paper indeed, this idea that mortality is caused by a consortium of pathogens is becoming more and more prevalent! This whole idea of the herpes viruses allowing opportunistic bacteria to infect the host inspired me to do a little bit of further research. I stumbled across a paper that talks about the physiological changes in Crassostrea gigas that are exposed to the herpesvirus.
ReplyDeleteIn this paper they mention that during the infection they can see that there was a reduction in energetic reserves, protein content and the activity of key enzymes. I was wondering if you think that the combination these factors may influence the immune response of the oyster. Did the authors mention anything about the possible reasons behind the opportunistic attack?
Many thanks,
Stefan
Sorry just realized I forgot to include the reference.
DeleteTamayo, D., Corporeau, C., Petton, B., Quere, C. and Pernet, F. (2014). Physiological changes in Pacific oyster Crassostrea gigas exposed to the herpesvirus OsHV-1μVar. Aquaculture, 432, pp.304-310.
Hey Stefan,
ReplyDeleteThanks for the reply and the paper. It's definitely true that more and more researchers are finding consortiums of microbes and polymicrobial factors to be major players in disease.
The paper you have attached says that the changes which you have listed, did not have an effect on metabolic activity, with healthy oysters and diseased oysters being similar in their metabolic scopes. I think it's really hard to point out possible reasons, simply because there are so many factors poorly understood. The opportunistic behaviour of pathogens however is definitely strongly involved in the disease of oysters, but other than the title, the authors did not refer to the opportunism of the bacteria. I think it is the combination of factors such as temperature, rearing conditions, density of population, and energy reserves as well as many others, which determine the reasons behind opportunistic bacteria. Unfortunately, a paper which characterises and studies each of the potential factors behind polymicrobial diseases, in isolation and then cumulatively, is likely to be a steep challenge; but we might just have to wait for such a paper in order to answer these questions.
Sorry that this isn't an 'answer' as such, but hope it makes sense,
Harriet