Enteromyxum leei IN MEDITERRANEAN GILTHEAD SEA BREAM FARMS: HOW MUCH IS OUT THERE?

Introduction

Enteromyxum leei (Myxozoa) is an important pathogen of gilthead seabream (GSB) ,  Sparus aurata and other Sparidae fish  cultured in the Mediterranean. In GSB , E. leei  induces  chronic enteritis followed by anorexia, cachexia, and  even death .  It can cause significant growth delay and mortality in marine cage farms as well as land-based mariculture systems. Wa ter temperature and water recirculation are critical risk factors in the contagion and onset of enteromyxosis ( Palenzuela et al., 2006, Sitjà-Bobadilla and Palenzuela 2012). However,  the  relevance  of  other risks factors like farm management practices,  and the epidemiological situation in gilthead sea bream farms, have not been thoroughly determined.  In this study, a risk assessment questionnaire and cross-sectional epidemiological study was designed to get insights in the distribution and incidence of E. leei in more than 50  Mediterranean and Atlantic  GSB farms throughout Western Europe.

Methods

A questionnaire for GSB cage farms  was designed to ascertain information about farm background and self-awareness of risk relative to E. leei infection, as well as details on the facilities, production and farm management routines. In addition, the farms were invited to participate in a cross-sectional targeted epidemiological survey to test the presence of the parasite in their stock. The participant farms were recruited by local expert pathologists and consultants acting as nodes in each geographical region, and the samples and questionnaires were coded and studied blindly.

For those farms participating in the biological sampling,  a protocol was submitted to sample one batch of fish at harvest time. The s ampling was designed to detect prevalence higher than 9. 5% with a 95% confidence . The intestinal rectal ampoules (portion with maximum predictive value for E. leei detection in GSB) were taken from  at least 30 randomly selected fish during routine harvest from one or several cages.  For  the  validation of the survey protocol,  a land-based  farm  which is enzootic for the parasite was enrolled in the study and two different GSB lots were sampled as positive controls .  The samples were fixed in 80-90 º Ethanol and paired  into 15 t ubes, which were shipped  to  the  central diagno sis laboratory at IATS-CSIC.

 The  routine  procedures for diagnosis of E. leei  consist of a SYBR-green qPCR test with primers specific for the parasite against a standard curve containing known numbers of copies of the target gene. Upon reception at the laboratory the samples were individually processed and homogenised. DNA was extracted from an aliquot using a robotic system,  its  quantity  and quality were evaluated, and samples were tested by qPCR at two different dilutions . They were considered positive when the cycle threshold (Ct) was <38.

Results and Discussion

A total of 45 cage farms from Croatia (12), Greece (12), Italy (13), Spain (7) and Malta (1) completed  the questionnaires . Among them, 32%  declared  to have  previous records of the parasite:  11 of them at some point in the last 5 years, 3 in t he most recent production  cycle,  and 4  having infections recurrently.  Among the farms with previous records , the disease was considered not relevant in 7, controllable in 7, and problematic in 4 sites .

 A total of 40 cage farms sent a total of 133 0 fish biological samples which were tested by qPCR.  The results show that the infection persists unnoticed at many farms (Table 1) ,  Famers were most aware of the presence of the infection in Greek waters. On the contrary, Croatian farmers were unanimously unaware of the presence of the parasite although it was detected at most of the tested sites. Prevalence of infection was  generally  lower  in sites  where the infection was not  believed to be  present, suggesting that low level infections, although widespread, do not cause any noticeable problems at many farms.

 All countries sampled had the infection, and the only countries with farms  in which the  infection was not detected were Croatia and Italy. Prevalence was highest on average in Greece (Table 2).

 The statistical analysis  considered prevalence against farm level factors as well as batch level factors.   For most variables no significant effect was observed.  At a farm level , farms removing mortalities more frequently , and those with stronger water exchange have a lower prevalence of infection. The number of hatcheries supplying the farm and the size of fish at harvest showed the strongest positive association with prevalence. Larger farms with higher numbers of cages, cage sizes and annual production tended to have higher prevalence. At the batch level, fish which had experienced highest minimum temperatures and those introduced to the cages at larger sizes had higher prevalence, whereas shorter times on the farm and higher specific growth rates were associated with lower prevalence. 

References

 Palenzuela  O  (2006) Myxozoan infections in Mediterranean mariculture . Parassitologia 48 (1-2).

Sitjà-Bobadilla A, Palenzuela O (2012). Enteromyxum species In: Woo PTK & Buchmann (Eds) Fish Parasites: Pathobiology and Protection, p 163-176. CABI Publishing.