PCR results
PCR analysis of both the healthy and affected samples of zoea were negative for seventeen known shrimp pathogens, including DHPV, MrBdv, SHIV, WSSV, PvNV, CMNV, IMNV, YHV, GAV, TSV, MrNV, XSV, Spiroplasma, NHPB, EHP, AHPND and Haplosporidia (Table 1). The apparently healthy zoea, however, were positive for IHHNV EVE (2/2), WzSV8/PvSV (2/2), RLB (1/2) and Microsporidia (1/2). By comparison, the LBS affected zoea were positive for IHHNV EVE (1/2), Vibrio spp. (2/2), RLB (2/2) and Microsporidia (1/2). In summary, the only difference between the two sets of samples were the detection of Vibrios in the zoea affected with LBS and WzSV8 in the healthy zoea.
Table 1 about here
Microbiology
The concentration of total bacteria (TSA) and presumptive Vibrios (TCBS and CHROMagarTM Vibrio) are shown in Table 2. The concentration of total bacteria in the affected zoea were almost an order of magnitude higher than that determined for the sample of healthy zoea. Presumptive Vibrios were almost two orders of magnitude higher in the affected zoea than in the healthy zoea. Presumptive Vibrios were found to represent 17% and 6% of the total bacteria population in the unhealthy and healthy zoea, respectively. Green colonies (on TCBS) and purple colonies (on CHROMagarTM Vibrio)represented 0.2% and 82% of the those recovered from the affected zoea, while a reverse picture of 56% and 1% was found from the analysis of the healthy zoea (Table 2).
Table 2 about here
A total of eleven strains were isolated from the LBS affected zoea and three from the healthy zoea (Table 3a). Biochemical profiling of the eleven strains recovered from the affected zoea, identified six as V. alginolyticus, two as V. fluvialis and one as V. vulnificus, one Aeromonas (undetermined) and one Pasteurella (undetermined). Of the three strains isolated from the healthy zoea, one strain was identified as V. alginolyticus, one as V. fluvialis and one as Aeromonas. Interestingly, all 14 strains were positive for Vibrio by PCR, which suggests that 27% of the results returned by the API 20E biochemical profiles were false negatives. Two sequences were used to identify Vibrio bacteria, all except by LBS-4 were positive for both methods. None of the 14 bacteria were positive for V. parahaemolyticus or for ToxR nor for the high virulent protein genes tested (Vhp-1, Vhp-2, Vhp-3) (Table 3a). The strains identified as Aeromonas and V. fluvialis were Pir AB positive (AHPND). The strain H-3 was positive for V. harveyi.
Table 3b shows the API 20E characterization of the Vibrio spp. that were isolated. This table is divided between LBS zoea (i.e., Vibrio spp. isolated from affected zoea), V. alginolyticus and Vibrio spp. (with 6 and 5 strains), against the three species of Vibrio isolated from the healthy zoea.
The availability of fermentation/oxidation of Arabinose was the main difference between Vibrio spp isolated from affected and the other two groups of bacteria, namely vs V. alginolyticus from LBS affected animals and Vibrio spp from healthy zoeas. On the other hand, V. alginolyticus from LBS affected animals differentiate from the other two groups in been unable to fermentation/oxidation of Amygdalin but with the capability of Acetoin production (Table 3b).
None of the 14 strains were ToxR positive or luminescent.
Table 3a and 3b about here
Histopathology
In fresh mounts of normal healthy zoea 2 larvae, algal material was seen in the digestive tract, giving it a brown coloration (Fig. 1a). By comparison, the affected / diseased larvae had an empty digestive tract and the presence of a round-shaped material that appeared as lipid droplets (Fig. 1b), this general clinical sign is the one usually described by shrimp hatchery technicians and biologists for what has been known as “Las Bolitas Syndrome”.
Figure 1 about here
Eight microscopy slides stained under H&E containing approximately 120 larvae of normal larvae at zoea 3 stage and a similar number from each diseased tank were examined. Normal healthy zoea 3 larvae had algae present in their digestive tract. At this stage the digestive tract is still in formation; the lateral lobes that will eventually develop into a fully functional hepatopancreas could also be seen in the tissue sections (Figs. 2a, b) (Abubakr & Jones 1992). An intact peritrophic membrane was also present and the algal material that had been consumed at this stage was seen as small sized brown cells (Fig. 2c). No sloughed cell material was observed in the lumen of the early forming digestive system of the healthy larvae (Fig. 2d).
Figure 2 about here
Approximately 60% of the larvae from the affected tanks with mortality had brown-green, hyaline spheres, the detached material within appeared to a B-cell (Figs. 3a & b). In the intestine, sloughed cells and material was observed (Figs. 3c & d) giving a contrasting difference to the intestines of the healthy larvae (Fig. 2c). The folds that constitute the early, developing hepatopancreas had sloughed cells that could be differentiated by their colour (Fig. 3e). Interestingly, some of these sloughed cells were inside the peritrophic membrane (Fig. 3f). The faeces from these larvae contained the same material that was seen within the intestine (Fig. 4a) and was contained within a peritrophic membrane. In the sections stained with Twort’s stain, the presence of what appeared to be bacterial cells was observed both within and outside the faecal strands (Fig. 4b).
Figure 3 about here
During this study, no bolitas (i.e., spheres) were observed in the histological samples, and none have ever been documented in the literature in the zoeal stages. The preparation of tissue samples for histology requires their processing through several solvents and so it is tempting to suggest that nature of the bolitas is lipidic in nature.
Figure 4 about here