ABSTRACT
Background: White spot syndrome virus is a pathogen of major economic importance to cultured penaeid shrimp industries globally. White spot disease can cause mortalities reaching 100% within 3-10 days of gross signs appearing. WSSV replicates in tissues from mesoderm and ectoderm embryonic origin and characteristically induces cell nuclei hypertrophy and intra nuclear inclusion bodies. WSSV also has an extremely broad host range including marine and freshwater crabs and crayfishes, copepods and other arthropods in addition to shrimp. Water temperature can affect the progress of WSD in crustaceans but there have been conflicting reports of higher temperatures protecting Litopenaeus vannamei shrimp but lower temperatures protecting Marsupenaeus japonicas. Here we have examined how 2 water temperatures affect the progression of WSD in the freshwater crayfish Astacus leptodactylus. Materials, Methods & Results: Freshwater Astacus leptodactylus crayfi sh (20 ± 0.5 g) were obtained from Aras dam, Iran. Crayfish were acclimated for 10 days in an aerated indoor cement tank with flow-through of dechlorinated freshwater with the flow rate set at 0.5 L/s, water temperature 15 ± 1ºC and dissolved oxygen 5.2 ppm. Two groups of 25 crayfish were allocated to tanks being supplied 15 ± 1°C water and 2 groups of 25 crayfish were allocated to tanks being supplied 25 ± 1°C water. Each crayfish was injected intramuscularly into soft tissue at the base of swimming legs with either 50 µL inoculum containing 103.2 lethal units/mL. A negative control group of crayfish was injected with PBS. Mortality amongst groups was monitored for 30 days and WSSV DNA present in haemolymph collected on Days 3, 5 and 10 post-injection was detected by nested-PCR. Morbidity and mortalities amongst crayfish held in lower temperature water were delayed and WSSV DNA was detected by nested-PCR at Day 10 pi compared to being clearly detected at Days 3, 5 and 10 pi amongst crayfish held in higher temperature water. Evidence of eosinophilic intranuclear inclusions detected by histology correlated with when WSSV was first detected by nested-PCR. Discussion: The data indicate that low water temperature retards WSSV replication in A. leptodactylus crayfi sh. Similar temperature-related effects on WSD progression have been reported in other freshwater crayfish species. For example, no mortality occurred amongst WSSV-infected Pacifastacus leniusculus held in either 4 ± 2°C or 12 ± 2°C water, but all crayfish died with WSD symptoms after they were transferred to 22 ± 2°C water. In another study, Procambarus clarkia held in 24 ± 1°C water all died by Day 9 post-challenge with WSSV but amongst crayfish held in 18 ± 1°C water, mortality only started on Day 10 post-challenge and in took until Day 22 before all had died, and amongst crayfish held in 10 ± 1°C water, no deaths occurred up to Day 24 post-challenge when the bioassay was terminated. A. leptodactylus injected with WSSV and maintained in 15 ± 1°C water only became moribund and nested-PCR-positive for WSSV by Day 10 pi in contrast to crayfish held in 25 ± 1°C water that started to die and were PCR-positive on Day 3 pi. Unlike WSD in shrimp, no histological evidence of basophilic intra nuclear inclusions was observed in WSSV-infected crayfish tissues even in the late stages of disease. This might be attributed to the over expression of anti-lipopolysaccharide factor in crayfish and/or the low numbers of apoptotic haemocytes that develop in WSSV-infected crayfish compared to the shrimp. Whether the over-expression of anti-lipopolysaccharide factor in combination with lowered WSSV replication induced by lowered water temperature both contributed to slowing the progress of WSD in Astacus leptodactylus warrants further investigation.