Accepted Manuscript Variations of immune parameters in the lined seahorse Hippocampus erectus after infection with enteritis pathogen of Vibrio parahaemolyticus Tingting Lin, Dong Zhang, Xin Liu, Dongxue Xiao PII:
S1050-4648(16)30039-0
DOI:
10.1016/j.fsi.2016.01.039
Reference:
YFSIM 3808
To appear in:
Fish and Shellfish Immunology
Received Date: 18 November 2015 Revised Date:
25 January 2016
Accepted Date: 31 January 2016
Please cite this article as: Lin T, Zhang D, Liu X, Xiao D, Variations of immune parameters in the lined seahorse Hippocampus erectus after infection with enteritis pathogen of Vibrio parahaemolyticus, Fish and Shellfish Immunology (2016), doi: 10.1016/j.fsi.2016.01.039. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Variations of immune parameters in the lined seahorse
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Hippocampus erectus after infection with enteritis pathogen
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of Vibrio parahaemolyticus
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Authors: Tingting Lin, Dong Zhang*, Xin Liu, Dongxue Xiao
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East China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences; Key
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Laboratory of East China Sea & Oceanic Fishery Resources Exploitation and Utilization,
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Ministry of Agriculture, Shanghai 200090, PR China
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*Corresponding author: Dong Zhang
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E-mail address: [email protected]
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Tel./fax: +86-21-65684655.
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Abstract: Enteritis has been increasingly recognized as one of the major obstacles for the
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lined seahorse Hippocampus erectus mass culture success. In the present study, the intestinal
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bacteria strains of the lined seahorses H. erectus suffered from enteritis were isolated, then
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their pathogenicities were confirmed by artificial infection, and one pathogenic bacteria strain
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named DS3 was obtained. The median lethal dose (LD50) of strain DS3 for 10 days was
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determined. The seahorses with different infection levels of uninfected (control), early stage
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of infection (ESI) and late stage of infection (LSI) were respectively sampled at 0, 3, 6 and 9
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days post infection, and 12 immune parameters in the plasma were analyzed. The strain DS3
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identified with a biochemical test combined with a molecular method was Vibrio
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parahaemolyticus, and its LD50 for 10 days was 1.3 × 103 cfu/fish. Six parameters including
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monocytes/leucocytes, leucocytes phagocytic rate, interleukin-2, interferon-α, lysozyme and
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immunoglobulin M exhibited a generally similar variation trend: highest in the control,
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second in the ESI and lowest in the LSI throughout the entire experiment. In view of the
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infection level of V. parahaemolyticus to H. erectus is largely decided by the seahorse's own
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immune capacity, therefore, these immune parameters were high in the non- or slightly
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infected seahorses, and low in the severely infected individuals may be an indicator for
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immune level. These immune parameters may be reliable indicators for the juvenile and
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broodstock quality assessment. Moreover, clarification of the enteritis pathogen also provides
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guidances for targeted medicine choice for the lined seahorse.
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Keywords: Lined seahorse Hippocampus erectus; Enteritis; Vibrio parahaemolyticus;
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Immune indicator; Infection level
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1. Introduction Seahorses, Hippocampus spp., are highly specialized marine fishes. Their unique body
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morphology with horse shaped head and curvaceous trunk, unusual life history traits
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including male pregnancy and strict monogamy in the most species, and high traditional
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Chinese medicine value have made them charismatic icons to biologists, aquarium hobbyists
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and medicinal workers [1–3]. In the past ten years, wild seahorse population has been heavily
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reduced due to over-fishing and habitat destruction. From 2004, all seahorse species have
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been listed on the IUCN Red List and the Appendix II of CITES. Aquaculture of seahorses
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has been proposed as one solution to balance the wild population conservation and market
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demand. Seahorse aquaculture started in 1958 and has expanded greatly since 2000 due to the
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improved breeding and rearing protocols [3–6].
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Although seahorse aquaculture has been greatly progressed, there are a few challenges
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affecting commercial seahorse culture, one of which is serious disease [7]. Common seahorse
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diseases include body surface ulcer, ″hair″ and ″gas bubble″, swim bladder inflation, liver
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haemorrhage and gastrointestinal inflammation. These diseases may result from unqualified
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rearing environments [8], inferior diets [9] and pathogen infections including marine leeches,
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ciliates [10], microsporidians [11], fungi [12] and bacteria [13,14]. To date, except bacteriosis
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which can be treated to a great extent with antibiotics, the remaining diseases still have no
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effective measures to control. Therefore, provision of an optimal rearing environment, a high
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quality diet and a periodical quarantine are increasingly considered as fundamental and
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effective measures to prevent seahorse health problems [3].
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The lined seahorse Hippocampus erectus, an ideal species for commercial culture, has
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been reared in captivity successfully [8,15–17]. Unofficial source indicates that the annual
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cultured dried H. erectus has been more than 2.0 t in China since 2014, and mostly used for
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traditional Chinese medicine. However, captive-rearing H. erectus has suffered from diseases 3
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such as H. japonicus [14], H. erectus enteritis also occurs primarily in the juveniles with the
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body height of 4-6 cm. In practice, seriously diseased seahorses with weak swimming
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capabilities, seldomly hold the holdfasts day and night, and their anal openings are obviously
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white. Anatomic symptoms of the diseased seahorse include liver haemorrhage, intestinal
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tract translucence, ascitic fluid hoarding and hindgut erosion. The mortality is quiet high
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(more than 80%), and the seahorses die in 3-5 days after the clinical symptoms appeared.
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In the present study, the pathogenic bacteria causing enteritis in H. erectus was isolated
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and identified, thereby to provide guidances for targeted medicine choice. Thereafter,
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seahorses H. erectus were artificially infected with the pathogenic bacteria, and the variations
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of immune parameters post infection were analyzed, with the aim of screening out several
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immune indicators, thereby to provide reliable indicators for the juvenile and broodstock
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quality assessment.
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2. Materials and Methods
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2.1. Experimental seahorses
Different batches of the healthy cultured juvenile H. erectus (body height: 5.63 ± 0.34
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cm, wet body weigh: 0.74 ± 0.12 g) were collected from Qionghai Research Center of East
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China Sea Fisheries Research Institute, Hainan, China. The seahorses were acclimatized in
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the large tanks (120 × 60 × 30 cm), each with 150 individuals. All tanks plumbed to a central
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filtration system featuring mechanical, biological filtration, and ultraviolet sterilizer. The
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cultured conditions were salinity of 32.0 ± 1.0 ‰, temperature of 27 ± 0.5 °C, light intensity
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of 2000 ± 300 lx, and a photoperiod of 14 h L : 10 h D, respectively. Plastic plants were
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provided for holdfasts. The seahorses were fed twice a day (08:00 and 15:00) with the sterile
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copepods, and the feces in the tanks were siphoned out 3 h after each feeding.
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2.2. Bacterial isolation The moribund seahorses H. erectus with typical clinical symptoms were sampled from
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an enteritis epidemic area in Dongshan, Fujian, China. After shipping to Qionghai Research
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Center, the diseased seahorses were rinsed with sterile 0.01 M phosphate buffered saline
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(PBS) at pH 7.2, then were dissected in a clean bench. The intestinal tracts were collected and
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cut open, and the inclusions were inoculated in a TCBS agar medium. Single bacterial
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colonies were isolated from visible colonies after 24 h of incubation at 28°C, and were
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transferred to fresh medium. Clear colonies were picked up and transferred again, until pure
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colonies were finally obtained. In this way, 3 strains named DS1, DS2 and DS3 were
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isolated.
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2.3. Pathogenicity confirmation
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Three isolated strains were diluted into three concentration gradients of 107, 105 and 103
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cfu/mL with sterile PBS, respectively. Each strain and each gradient was intraperitoneally
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injected into 20 healthy seahorses, and each seahorse with 20 µl of bacterial dilution. Injected
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seahorses were cultured in the small tanks (50 × 30 × 30 cm) with the same cultured
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conditions as for the acclimatized seahorses, and their incidence and mortality were
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monitored and recorded daily for 10 days. As a result, strain DS3 with a high mortality in
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gradients of 107 and 105 cfu/mL were observed, besides, the moribund and dead individuals in
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DS3 treatments also presented highly similar symptoms of the enteritis (Fig. 1). As for the
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remaining two strains, no clinical signs of enteritis and no death occurred. The bacterial strain
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in the moribund individuals in DS3 treatment was also isolated.
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2.4. Pathogenic bacteria identification
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Strain DS3 isolated from the epidemic area of Dongshan together with the bacterial
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strain isolated from artificially infected seahorses in pathogenicity confirmation experiment
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was identified by gram staining, NaCl tolerance test, API 20E strip (BioMeÂrieux, S.A.
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France), and 16S rRNA gene sequence analysis, respectively. The detailed 16S rRNA
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analysis procedure was referenced the previous report in H. japonicus [14].
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2.5. Median lethal dose determination Strain DS3 was diluted into five concentrations (5 × 106, 5 × 105, 5 × 104, 5 × 103 and 5 ×
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102 cfu/mL) with sterile PBS. The healthy seahorses were divided into six groups, one group
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injected with sterile PBS as the control and the other five groups were injected with different
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bacterial dilutions, respectively. Each group had 20 seahorses and each seahorse injected with
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20 µl of bacterial dilution or PBS. Injected seahorses were cultured in the small tanks (50 ×
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30 × 30 cm) with the same cultured conditions as for the acclimatized seahorses, and
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their incidence and mortality were monitored and recorded daily for 10 days. The median
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lethal dose (LD50) was calculated by the modified Karber's method [18].
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2.6. Variation analysis of immune parameters after bacterial infection
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2.6.1. Bacterial infection
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The healthy seahorses were divided into two groups: the control group injected with
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sterile PBS and the bacterial group injected with LD50 of strain DS3. Each group had 5
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replicates and each replicate with 40 seahorses. Injected seahorses were cultured in the
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medium tanks (60 × 60 × 30 cm) with the same cultured conditions as for the acclimatized
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seahorses for 10 days. Dead individuals were taken out immediately as soon as possible. The
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injected seahorses were sampled at 0, 3, 6 and 9 days post injection at night when the
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seahorses usually stopped swimming and held the holdfasts. For the control group, 8
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seahorses were sampled at each sampling time. While for the bacterial group, 4 seahorses
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held the holdfasts (indicating they were slightly infected and classified as early stage of
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infection (ESI)) and 4 seahorses swam weakly and unable to hold the holdfasts (indicating
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they were severely infected and classified as late stage of infection (LSI)) were collected at
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each sampling time.
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2.6.2. Blood sampling and processing The sampled seahorse was placed into a bucket containing a solution of 0.035% MS-222
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(Sigma-Aldrich, Castle Hill, NSW, Australia) in seawater and anaesthetized for 2 min, then
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1/3 of the tail was cut. The remaining tail of amputated seahorses was immediately inserted
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into a 1.5-mL sterile centrifuge tube containing 0.4 mL of anticoagulant (citric acid 0.48 g,
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sodium citrate 1.32 g, glucose 1.47 g, and distilled water 100 mL) and dipped into the
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anticoagulant. Blood was spontaneously collected from the caudal artery and mixed with
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anticoagulant, after about 2 min, the seahorse was removed. The blood of 4 seahorses from
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each sampling time each replicate were pooled. The mixture of blood and anticoagulant was
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let to stand for 10 min at 4 °C, then was centrifuged at 840 × g for 10 min at 4 °C using a
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centrifuge (Sigma 3K18, Sigma GmbH, Osterode am Harz, Germany) to collect the plasma
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and the cell pellet. The plasma was stored at -80°C until used for immune parameters
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including superoxide dismutase (EC 1.15.1.1, SOD), acid phosphatase (EC 3.1.3.2, ACP),
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lysozyme (EC 3.2.1.17,
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interleukin-2 (IL-2), interferon-α (IFN-α), immunoglobulin M (Ig M) and complement 3 (C3)
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determinations.
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interleukin-1β (IL-1β),
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The cell pellet was used for to prepare a leukocyte suspension. Specifically, the cell
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pellet was suspended in PBS and re-centrifuged at 100 × g for 10 min at 4 °C to collect the
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resulting supernatant. The collected supernatant was laid over a discontinuous Percoll
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gradient containing 3 mL of Percoll at a density of 1.070 g/cm3 overlaid with 3 mL of Percoll
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at a density of 1.020 g/cm3. After discontinuous density gradient centrifugation at 840 × g for
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30 min at 4 °C, the leukocytes that were mainly concentrated at the 1.020−1.070 g/cm3
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interface were obtained [19]. The leucocytes were rinsed three times with PBS to remove the
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Percoll and then were re-suspended in PBS and adjusted to a cellular concentration of
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107cells/mL to determine monocytes/ leukocytes, lymphocytes/leucocytes and leucocytes
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phagocytic rate (LPR).
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2.6.3. Immune parameters assays SOD, ACP, MDA, IL-1β, IL-2, IFN-α, Ig M and C3 activities or concentrations were
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measured with commercial ELISA kits (Nanjing Jiancheng Bioengineering Institute, Nanjing,
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China) for fish plasma or serum following the manufacturer's instructions. LZM activity was
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measured via the modified microplate method employing Micrococcus lysodeikticus
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(Sigma-Aldrich) as the target bacteria [20], and the unit of activity was defined as a decrease
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in absorbance of 0.001 per mg plasma protein per min under the specified conditions of pH
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6.2 and 25 °C. The plasma protein concentration was determined by the coomassie brilliant
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blue method using bovine serum albumin (Sigma-Aldrich) as the standard protein [21], and
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the protein concentration of the experimental sample was calculated by applying the sample's
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A595 nm value to a linear regression equation that was developed based on different diluted
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concentrations of the standard and the corresponding A595 nm values.
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Monocytes/leucocytes, lymphocytes/leucocytes and LPR were tested by flow cytometry.
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One milliliter of leukocyte suspension was applied to a well in a 24-well cell plate, and 1 mL of
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fluorescent microspheres (Polysciences, Inc.) was applied to the well and incubated for 1 h in
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the dark at room temperature. Leukocyte suspension with no microspheres was as negative
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control. After incubation, the leukocyte suspension was washed with PBS to remove the
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unphagocytosed microspheres, then was re-suspended in 1 mL of PBS and analyzed using a
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flow cytometer (FACS-Calibur, Becton Dickinson, Heidelberg, Germany) [22]. Twenty
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thousand cells were analyzed and classified into three cellular areas in dotplots according to
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the parameters of cell size (FSC) and cell granularity (SSC), meanwhile formed a positive
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fluorescent curve in histograms according to the parameter of fluorescence intensity (FL1).
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The percentages of monocytes with high FSCs and low SSCs, and lymphocytes with low
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FSCs and low SSCs [23] among the total leukocytes and LPR were calculated using
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CellQuest Pro Software (Becton Dickinson).
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2.7. Statistical analysis The data were analyzed using Origin statistical software (version 8.0, Origin Lab, USA).
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Prior to the analysis, monocytes/leucocytes, lymphocytes/leucocytes and LPR were arc-sine
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transformed, and the normality of all data from immune assays was evaluated using the
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Shapiro–Wilk's W-test, meanwhile, the homogeneity of variances was assessed as well. The
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difference in immune parameters among the control, ESI and LSI from the same sampling
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time was analyzed using one-way analysis of variance (ANOVA) followed by a Tukey's
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multiple comparison test if a significant difference was obtained.
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3. Results
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3.1. Pathogenic bacteria identification
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Strain DS3 from the epidemic area of Dongshan and the bacterial strain from artificial
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infection showed the same characteristics. The colony was blue-green, roundish with a
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diameter of 1–3 mm, and translucent in TCBS agar medium. Strain DS3 was lactose and
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sucrose negative, NaCl tolerance of 3-7%. The detailed physiological and biochemical
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features were listed in Table 1. By comparison the 16S rRNA gene sequence of strain DS3
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and the known sequences deposited in the NCBI database, a similarity of 98.4% to the
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standard strain of Vibrio parahaemolyticus (ATCC 17802) was noted (Fig. 2). Therefore,
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according to the biochemical and molecular results, strain DS3 is identified as V.
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parahaemolyticus. Moreover, the LD50 by strain DS3 for 10 days was figured out of 1.3 × 103
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cfu/fish (Table 2).
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3.2. Variation analysis of immune parameters after bacterial infection
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After bacterial challenge, ACP and SOD activities in LSI significantly decreased at 3
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days post infection, then gradually rebounded to the initial value. However, ACP and SOD in
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ESI was activated by bacterial challenge, and their activities were markedly higher than
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control at day 3 and 6, respectively (Fig. 3). For MDA and C3, bacterial challenge also had an inducement effect for both ESI and
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LSI, they sharply induced at the early period of experiment, then decreased. The slight
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difference between MDA and C3 was the former reduced to the level close to control, while
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the latter reduced to the level below control at the ending period of experiment (Fig. 3 and 4).
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While for LZM, IL-2, IFN-α, IgM, monocytes/leucocytes and LPR, they exhibited a
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generally similar variation trend. After bacterial challenge, these immune parameters in ESI
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and LSI all acutely decreased and reached the valley value at day 3 or day 6. Afterwards, they
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rose up, but still lower than the control at the end of experiment. Differently, the declining
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extent of parameters in LSI was larger than in ESI (Fig. 3 and 4).
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As for lymphocytes/leucocytes, except day 3 when LSI was significant lower than
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control, no difference was observed during the other experimental period. Similarly, no
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difference in IL-1β was obtained throughout the entire experiment (Fig. 3 and 4).
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4. Discussion
As with many fishes in culture, seahorses do not respond well to being kept at high
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densities and are prone to infection. Seahorse pathogen species are diverse including marine
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leeches, ciliates, microsporidians, fungi, and the most concerned of bacteria, particularly
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vibrios [3,7]. Vibriosis in seahorses is common: V. harveyi for haemorrhages disease in H.
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kuda [13, 24]; V. alginolyticus and V. splendidus for skin white spots and tail-rot disease in H.
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guttulatus and H. hippocampus [25] and V. campbellii for white porphyritic ulcer disease in H.
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japonicus [26]. However, V. parahaemolyticus whose virulence was widely confirmed in the
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numerous mariculture animals [27–30], being as one of the pathogens in seahorse diseases
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has still not been reported.
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causative agent for enteritis disease in H. erectus, which is different from the enteritis
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pathogen of Pseudomonas sp. in H. japonicus [14]. Clarification of V. parahemolyticus as the
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pathogen for H. erectus enteritis provides a guidance for targeted medicine choice, such as
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amikacin [30], ampicillin [31] and piperacillin [32]. Additionally, it could partially explains
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why juvenile H. erectus had a better survival and growth performance in salinity of 12−15‰
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than 32‰ in our previous work [33], because of low salinity resulting in a low bacterial
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reproduction (see the NaCl tolerance in Table 1), thereby leading to a low incidence of
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enteritis.
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The LD50 of V. parahemolyticus to H. erectus for 10 days converted into per gram body
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weight was 1.7 × 103 cfu/g·fish. Similarly, the LD50 of Pseudomonas sp. to H. japonicus for
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10 days was 1.4 × 103 cfu/g·fish [14]. However, this is obviously lower than that in the other
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cultured fishes which suffered from enteritis as well, for example, the LD50s of pathogenic
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bacteria to the carp Ctenopharyngodon idella for 14 days [34], to the tilapia Oreochromis
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niloticus for 10 days [35], and to the flounder Paralichthys dentatus for 4 days [36] were 1.1
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× 105, 7.6 × 104 and 5.0 × 103 cfu/g·fish, respectively. The weak resistance to enteritis
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bacteria may be largely due to seahorses lack gut-associated lymphoid tissue, an important
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immune tissue against pathogens invading via mucosal areas of the gastrointestinal tract [37].
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In addition to weak resistance caused by anomalous immune system, high incidence of
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enteritis in cultured seahorses may be also related to their diets, primarily preying on live
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Artemia and copepods which carry a large number of bacteria in Vibrionaceae [38,39].
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Challenge with LD50, the seahorse death peakly occurred from the 1st day to the 6th day
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(Table 2), suggesting the first 6 days may be key time in infection, particularly in the 3rd day
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at which the highest mortality occurred. That's why the seahorses were sampled on day 3 and
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day 6. Additionally, in order to better compare the variations of immune parameters between
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the infection period (i.e., 3rd and 6th day) and the recovery period, the 9th day (i.e., recovery
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period post infection) was also selected as a sampling time. Although most of the vibriosis can be treated with antibiotics, antibiotics should not be
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overused. Overuse of antibiotics is not only easy to disturb the bacterial makeup, to produce
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drug resistant strains, but also likely to result in drug residues to pollute the seahorse's
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medicinal composition [40]. Therefore, improvement of the juvenile and broodstock quality
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to partially replace the use of antimicrobial agents is imperative in aquaculture [41]. Having
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reliable quality indicators is an essential prerequisite for quality assessment. Currently, the
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quality or stress related indicators in fishes are mainly focused on behavior including
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ventilatory frequency, swimming activity, latency to move from an uncomfortable condition
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[42]; morphology such as skin color, eye darkening and hepatosomatic index [43,44]; and
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physiology like cortisol, glucose and lactates concentration [45]. For immunology, only
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lysozyme was reported. In the present study, 6 parameters including monocytes/leucocytes,
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LPR, LZM, IL-2, IFN-α and Ig M were all highest in the uninfected, second in the slight
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infected and lowest in the severely infected throughout the whole experiment, suggesting
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these 6 parameters are very likely to be immune indicators, and could be used as quality
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indicator for the lined seahorse.
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Some of these 6 parameters having the potential to act as an immunity or stress indicator
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were widely confirmed in other fishes including Syngnathidae species. The Victoria labeo
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Labeo victorianus with high LZM and Ig M had a high survivorship when challenged by
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Aeromonas hydrophila [46]; Hippocampus sp. with a low monocytes/leucocytes had a low
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survivorship when challenged by a noisy sound [44]; and the pipefish Syngnathus typhle
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decreased leucocytic proliferation and antimicrobial activity (e.g. LZM and Ig M) when
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challenged by a low salinity or Vibrio sp. [23,47]. However, in recent years, a few studies
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concluding that some immune parameters increase after pathogen infection also have
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emerged. For example, SOD, LZM, C3 and Ig M increased in the large yellow croaker
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Pseudosciaena crocea challenged with Cryptocaryon irritans [48] and in the Atlantic salmon
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Salmo salar challenged with Aeromonas salmonicida [49]. Except for experimental animal difference, the completely different results mentioned
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above might also be attributable to the diversity in infection level. In view of H. erectus dies
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in the first 6 days upon infection with LD50 of V. parahaemolyticus, therefore, the sampling
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times designed on the 3rd and 6th day post infection in the present study could ensure the
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seahorses sampled at that time were severely infected. It's not surprised that a moribund
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seahorse has a weak immune capacity. While in the above-mentioned two counterexamples:
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P. crocea infected with C. irritans at 24,000 theronts/fish, whose survival rate was 96% at 4th
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day, was sampled on day 1, 2 and 3 post infection [48]; and S. salar challenged with A.
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salmonicida at 3.05× 106 cfu/fish, whose symptom appeared from day 4 post infection and no
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death occurred, was sampled on day 2 and 4 [49]. Hence the infection levels in the two fish
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species from their respective sampling time were generally slight. As reported by many
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studies, a slight infection has an activation effect on the host's immunity.
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5. In conclusion
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In the present study, we isolated and identified out the pathogenic bacteria causing
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enteritis in seahorse H. erectus, and screened out six immune parameters promising as the
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indicators for H. erectus immune capacity on protein and cell levels. In the future study, the
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expression variations of these parameter related genes under different severities of infection
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by qPCR will be carried out to better support the prospect of these parameters as immune
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indicators on molecular level.
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Acknowledgements 13
ACCEPTED MANUSCRIPT This work was supported by the Special Fund for China Foundation for International
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Cooperation (Project # 2011DFA33060), the Special Fund for Technology Development and
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Research of Scientific Research Institute (Project # 2014EG1342) and the Special Research
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Fund for the National Nonprofit Institute of China (Project # 2014T08, East China Sea
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Fisheries Research Institute).
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2015;44:307–15.
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Table 1 Physiological and biochemical features of strain DS3.
Items
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Results − + − + + − − + + + − − −
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Gram staining Polar flagellum 0% NaCl growth 3% NaCl growth 7% NaCl growth 10% NaCl growth Lactose Gelatinase Oxidase Mannitol Inositol Rhamnol Sucrose
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Items
Melibiose Amygdalin Arabinose Arginine double enzyme hydrolysis Lysine decarboxylase Ornithine decarboxylase Citrate utilization Glucose acid production Glucose gas production Urease H2S production Indol production VP test
+, Positive; −, Negative.
458 459 460 461 17
Results − − − − + + − + − − − + −
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Table 2 Mortality of seahorse H. erectus artificial infection with different doses of V. parahaemolyticus Injected number (inds)
1d
2d
3d
4d
5d
6
5 × 10 5 × 105
20 20
20 20
1 1
4 2
5 4
5 3
1 2
5 × 104
20
20
1
2
3
2
1
3
20 20
20 20
0 0
0 0
1 0
2 0
20
20
0
0
0
0
9d
10d
6d
7d
18
Mortality (%)
1 1
0 0
0 0
0 0
0 0
17 13
85 65
1
0
0
0
0
10
55
1 0
1 0
0 0
1 1
0 0
0 0
6 1
30
0
0
0
0
0
0
0
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Total death number (inds)
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Injected volume (µl)
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5 0
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Figure caption Fig. 1 The clinical symptoms of healthy seahorses H. erectus infected with DS3 strain. 1: white pus spot in the anal opening; 2: abdominal swelling and ascitic fluid hoarding; 3: hindgut erosion; 4: intestinal tract
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translucence; 5: swim bladder inflation.
Fig. 2 Phylogenetic tree based on 16S rDNA sequences of strain DS3. The numbers at the nodes indicate the bootstrap values based on neighbour-joining analyses of 1000 sample date sets. Bar, 0.02 substitutions
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per nucleotide position.
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Fig. 3 Variations of ACP, LZM, SOD, MDA, IL-1β and IL-2 in the plasma of the control, ESI and LSI seahorses after V. parahaemolyticus infection. The different upper case letters, i.e., A, B and C indicate significant differences among control, ESI and LSI at a fixed sampling time.
Fig. 4 Variations of C3, IFN-α, IgM, monocytes/leucocytes, lymphocytes/leucocytes and LPR in the
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plasma of the control, ESI and LSI seahorses after V. parahaemolyticus infection. The different upper case
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letters, i.e., A, B and C indicate significant differences among control, ESI and LSI at a fixed sampling
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Figure 3
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Figure 4
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ACCEPTED MANUSCRIPT Highlights Pathogenic bacteria for enteritis in H. erectus was isolated and identified
Pathogenic bacteria was Vibrio parahaemolyticus
Six parameters high in slightly infected seahorses, low in severely infected ones
These parameters may be indicators for immune level
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S1050-4648(16)30039-0
DOI:
10.1016/j.fsi.2016.01.039
Reference:
YFSIM 3808
To appear in:
Fish and Shellfish Immunology
Received Date: 18 November 2015 Revised Date:
25 January 2016
Accepted Date: 31 January 2016
Please cite this article as: Lin T, Zhang D, Liu X, Xiao D, Variations of immune parameters in the lined seahorse Hippocampus erectus after infection with enteritis pathogen of Vibrio parahaemolyticus, Fish and Shellfish Immunology (2016), doi: 10.1016/j.fsi.2016.01.039. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Variations of immune parameters in the lined seahorse
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Hippocampus erectus after infection with enteritis pathogen
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of Vibrio parahaemolyticus
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Authors: Tingting Lin, Dong Zhang*, Xin Liu, Dongxue Xiao
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East China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences; Key
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Laboratory of East China Sea & Oceanic Fishery Resources Exploitation and Utilization,
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Ministry of Agriculture, Shanghai 200090, PR China
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*Corresponding author: Dong Zhang
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E-mail address: [email protected]
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Tel./fax: +86-21-65684655.
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Abstract: Enteritis has been increasingly recognized as one of the major obstacles for the
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lined seahorse Hippocampus erectus mass culture success. In the present study, the intestinal
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bacteria strains of the lined seahorses H. erectus suffered from enteritis were isolated, then
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their pathogenicities were confirmed by artificial infection, and one pathogenic bacteria strain
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named DS3 was obtained. The median lethal dose (LD50) of strain DS3 for 10 days was
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determined. The seahorses with different infection levels of uninfected (control), early stage
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of infection (ESI) and late stage of infection (LSI) were respectively sampled at 0, 3, 6 and 9
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days post infection, and 12 immune parameters in the plasma were analyzed. The strain DS3
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identified with a biochemical test combined with a molecular method was Vibrio
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parahaemolyticus, and its LD50 for 10 days was 1.3 × 103 cfu/fish. Six parameters including
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monocytes/leucocytes, leucocytes phagocytic rate, interleukin-2, interferon-α, lysozyme and
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immunoglobulin M exhibited a generally similar variation trend: highest in the control,
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second in the ESI and lowest in the LSI throughout the entire experiment. In view of the
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infection level of V. parahaemolyticus to H. erectus is largely decided by the seahorse's own
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immune capacity, therefore, these immune parameters were high in the non- or slightly
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infected seahorses, and low in the severely infected individuals may be an indicator for
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immune level. These immune parameters may be reliable indicators for the juvenile and
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broodstock quality assessment. Moreover, clarification of the enteritis pathogen also provides
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guidances for targeted medicine choice for the lined seahorse.
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Keywords: Lined seahorse Hippocampus erectus; Enteritis; Vibrio parahaemolyticus;
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Immune indicator; Infection level
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1. Introduction Seahorses, Hippocampus spp., are highly specialized marine fishes. Their unique body
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morphology with horse shaped head and curvaceous trunk, unusual life history traits
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including male pregnancy and strict monogamy in the most species, and high traditional
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Chinese medicine value have made them charismatic icons to biologists, aquarium hobbyists
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and medicinal workers [1–3]. In the past ten years, wild seahorse population has been heavily
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reduced due to over-fishing and habitat destruction. From 2004, all seahorse species have
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been listed on the IUCN Red List and the Appendix II of CITES. Aquaculture of seahorses
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has been proposed as one solution to balance the wild population conservation and market
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demand. Seahorse aquaculture started in 1958 and has expanded greatly since 2000 due to the
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improved breeding and rearing protocols [3–6].
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Although seahorse aquaculture has been greatly progressed, there are a few challenges
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affecting commercial seahorse culture, one of which is serious disease [7]. Common seahorse
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diseases include body surface ulcer, ″hair″ and ″gas bubble″, swim bladder inflation, liver
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haemorrhage and gastrointestinal inflammation. These diseases may result from unqualified
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rearing environments [8], inferior diets [9] and pathogen infections including marine leeches,
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ciliates [10], microsporidians [11], fungi [12] and bacteria [13,14]. To date, except bacteriosis
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which can be treated to a great extent with antibiotics, the remaining diseases still have no
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effective measures to control. Therefore, provision of an optimal rearing environment, a high
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quality diet and a periodical quarantine are increasingly considered as fundamental and
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effective measures to prevent seahorse health problems [3].
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The lined seahorse Hippocampus erectus, an ideal species for commercial culture, has
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been reared in captivity successfully [8,15–17]. Unofficial source indicates that the annual
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cultured dried H. erectus has been more than 2.0 t in China since 2014, and mostly used for
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traditional Chinese medicine. However, captive-rearing H. erectus has suffered from diseases 3
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such as H. japonicus [14], H. erectus enteritis also occurs primarily in the juveniles with the
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body height of 4-6 cm. In practice, seriously diseased seahorses with weak swimming
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capabilities, seldomly hold the holdfasts day and night, and their anal openings are obviously
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white. Anatomic symptoms of the diseased seahorse include liver haemorrhage, intestinal
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tract translucence, ascitic fluid hoarding and hindgut erosion. The mortality is quiet high
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(more than 80%), and the seahorses die in 3-5 days after the clinical symptoms appeared.
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In the present study, the pathogenic bacteria causing enteritis in H. erectus was isolated
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and identified, thereby to provide guidances for targeted medicine choice. Thereafter,
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seahorses H. erectus were artificially infected with the pathogenic bacteria, and the variations
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of immune parameters post infection were analyzed, with the aim of screening out several
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immune indicators, thereby to provide reliable indicators for the juvenile and broodstock
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quality assessment.
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2. Materials and Methods
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2.1. Experimental seahorses
Different batches of the healthy cultured juvenile H. erectus (body height: 5.63 ± 0.34
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cm, wet body weigh: 0.74 ± 0.12 g) were collected from Qionghai Research Center of East
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China Sea Fisheries Research Institute, Hainan, China. The seahorses were acclimatized in
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the large tanks (120 × 60 × 30 cm), each with 150 individuals. All tanks plumbed to a central
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filtration system featuring mechanical, biological filtration, and ultraviolet sterilizer. The
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cultured conditions were salinity of 32.0 ± 1.0 ‰, temperature of 27 ± 0.5 °C, light intensity
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of 2000 ± 300 lx, and a photoperiod of 14 h L : 10 h D, respectively. Plastic plants were
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provided for holdfasts. The seahorses were fed twice a day (08:00 and 15:00) with the sterile
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copepods, and the feces in the tanks were siphoned out 3 h after each feeding.
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2.2. Bacterial isolation The moribund seahorses H. erectus with typical clinical symptoms were sampled from
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an enteritis epidemic area in Dongshan, Fujian, China. After shipping to Qionghai Research
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Center, the diseased seahorses were rinsed with sterile 0.01 M phosphate buffered saline
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(PBS) at pH 7.2, then were dissected in a clean bench. The intestinal tracts were collected and
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cut open, and the inclusions were inoculated in a TCBS agar medium. Single bacterial
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colonies were isolated from visible colonies after 24 h of incubation at 28°C, and were
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transferred to fresh medium. Clear colonies were picked up and transferred again, until pure
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colonies were finally obtained. In this way, 3 strains named DS1, DS2 and DS3 were
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isolated.
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2.3. Pathogenicity confirmation
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Three isolated strains were diluted into three concentration gradients of 107, 105 and 103
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cfu/mL with sterile PBS, respectively. Each strain and each gradient was intraperitoneally
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injected into 20 healthy seahorses, and each seahorse with 20 µl of bacterial dilution. Injected
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seahorses were cultured in the small tanks (50 × 30 × 30 cm) with the same cultured
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conditions as for the acclimatized seahorses, and their incidence and mortality were
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monitored and recorded daily for 10 days. As a result, strain DS3 with a high mortality in
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gradients of 107 and 105 cfu/mL were observed, besides, the moribund and dead individuals in
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DS3 treatments also presented highly similar symptoms of the enteritis (Fig. 1). As for the
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remaining two strains, no clinical signs of enteritis and no death occurred. The bacterial strain
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in the moribund individuals in DS3 treatment was also isolated.
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2.4. Pathogenic bacteria identification
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Strain DS3 isolated from the epidemic area of Dongshan together with the bacterial
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strain isolated from artificially infected seahorses in pathogenicity confirmation experiment
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was identified by gram staining, NaCl tolerance test, API 20E strip (BioMeÂrieux, S.A.
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France), and 16S rRNA gene sequence analysis, respectively. The detailed 16S rRNA
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analysis procedure was referenced the previous report in H. japonicus [14].
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2.5. Median lethal dose determination Strain DS3 was diluted into five concentrations (5 × 106, 5 × 105, 5 × 104, 5 × 103 and 5 ×
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102 cfu/mL) with sterile PBS. The healthy seahorses were divided into six groups, one group
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injected with sterile PBS as the control and the other five groups were injected with different
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bacterial dilutions, respectively. Each group had 20 seahorses and each seahorse injected with
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20 µl of bacterial dilution or PBS. Injected seahorses were cultured in the small tanks (50 ×
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30 × 30 cm) with the same cultured conditions as for the acclimatized seahorses, and
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their incidence and mortality were monitored and recorded daily for 10 days. The median
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lethal dose (LD50) was calculated by the modified Karber's method [18].
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2.6. Variation analysis of immune parameters after bacterial infection
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2.6.1. Bacterial infection
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The healthy seahorses were divided into two groups: the control group injected with
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sterile PBS and the bacterial group injected with LD50 of strain DS3. Each group had 5
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replicates and each replicate with 40 seahorses. Injected seahorses were cultured in the
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medium tanks (60 × 60 × 30 cm) with the same cultured conditions as for the acclimatized
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seahorses for 10 days. Dead individuals were taken out immediately as soon as possible. The
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injected seahorses were sampled at 0, 3, 6 and 9 days post injection at night when the
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seahorses usually stopped swimming and held the holdfasts. For the control group, 8
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seahorses were sampled at each sampling time. While for the bacterial group, 4 seahorses
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held the holdfasts (indicating they were slightly infected and classified as early stage of
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infection (ESI)) and 4 seahorses swam weakly and unable to hold the holdfasts (indicating
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they were severely infected and classified as late stage of infection (LSI)) were collected at
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each sampling time.
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2.6.2. Blood sampling and processing The sampled seahorse was placed into a bucket containing a solution of 0.035% MS-222
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(Sigma-Aldrich, Castle Hill, NSW, Australia) in seawater and anaesthetized for 2 min, then
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1/3 of the tail was cut. The remaining tail of amputated seahorses was immediately inserted
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into a 1.5-mL sterile centrifuge tube containing 0.4 mL of anticoagulant (citric acid 0.48 g,
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sodium citrate 1.32 g, glucose 1.47 g, and distilled water 100 mL) and dipped into the
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anticoagulant. Blood was spontaneously collected from the caudal artery and mixed with
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anticoagulant, after about 2 min, the seahorse was removed. The blood of 4 seahorses from
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each sampling time each replicate were pooled. The mixture of blood and anticoagulant was
156
let to stand for 10 min at 4 °C, then was centrifuged at 840 × g for 10 min at 4 °C using a
157
centrifuge (Sigma 3K18, Sigma GmbH, Osterode am Harz, Germany) to collect the plasma
158
and the cell pellet. The plasma was stored at -80°C until used for immune parameters
159
including superoxide dismutase (EC 1.15.1.1, SOD), acid phosphatase (EC 3.1.3.2, ACP),
160
lysozyme (EC 3.2.1.17,
161
interleukin-2 (IL-2), interferon-α (IFN-α), immunoglobulin M (Ig M) and complement 3 (C3)
162
determinations.
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interleukin-1β (IL-1β),
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The cell pellet was used for to prepare a leukocyte suspension. Specifically, the cell
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pellet was suspended in PBS and re-centrifuged at 100 × g for 10 min at 4 °C to collect the
165
resulting supernatant. The collected supernatant was laid over a discontinuous Percoll
166
gradient containing 3 mL of Percoll at a density of 1.070 g/cm3 overlaid with 3 mL of Percoll
167
at a density of 1.020 g/cm3. After discontinuous density gradient centrifugation at 840 × g for
168
30 min at 4 °C, the leukocytes that were mainly concentrated at the 1.020−1.070 g/cm3
169
interface were obtained [19]. The leucocytes were rinsed three times with PBS to remove the
170
Percoll and then were re-suspended in PBS and adjusted to a cellular concentration of
171
107cells/mL to determine monocytes/ leukocytes, lymphocytes/leucocytes and leucocytes
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phagocytic rate (LPR).
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2.6.3. Immune parameters assays SOD, ACP, MDA, IL-1β, IL-2, IFN-α, Ig M and C3 activities or concentrations were
175
measured with commercial ELISA kits (Nanjing Jiancheng Bioengineering Institute, Nanjing,
176
China) for fish plasma or serum following the manufacturer's instructions. LZM activity was
177
measured via the modified microplate method employing Micrococcus lysodeikticus
178
(Sigma-Aldrich) as the target bacteria [20], and the unit of activity was defined as a decrease
179
in absorbance of 0.001 per mg plasma protein per min under the specified conditions of pH
180
6.2 and 25 °C. The plasma protein concentration was determined by the coomassie brilliant
181
blue method using bovine serum albumin (Sigma-Aldrich) as the standard protein [21], and
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the protein concentration of the experimental sample was calculated by applying the sample's
183
A595 nm value to a linear regression equation that was developed based on different diluted
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concentrations of the standard and the corresponding A595 nm values.
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Monocytes/leucocytes, lymphocytes/leucocytes and LPR were tested by flow cytometry.
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One milliliter of leukocyte suspension was applied to a well in a 24-well cell plate, and 1 mL of
187
fluorescent microspheres (Polysciences, Inc.) was applied to the well and incubated for 1 h in
188
the dark at room temperature. Leukocyte suspension with no microspheres was as negative
189
control. After incubation, the leukocyte suspension was washed with PBS to remove the
190
unphagocytosed microspheres, then was re-suspended in 1 mL of PBS and analyzed using a
191
flow cytometer (FACS-Calibur, Becton Dickinson, Heidelberg, Germany) [22]. Twenty
192
thousand cells were analyzed and classified into three cellular areas in dotplots according to
193
the parameters of cell size (FSC) and cell granularity (SSC), meanwhile formed a positive
194
fluorescent curve in histograms according to the parameter of fluorescence intensity (FL1).
195
The percentages of monocytes with high FSCs and low SSCs, and lymphocytes with low
196
FSCs and low SSCs [23] among the total leukocytes and LPR were calculated using
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ACCEPTED MANUSCRIPT 197
CellQuest Pro Software (Becton Dickinson).
198
2.7. Statistical analysis The data were analyzed using Origin statistical software (version 8.0, Origin Lab, USA).
200
Prior to the analysis, monocytes/leucocytes, lymphocytes/leucocytes and LPR were arc-sine
201
transformed, and the normality of all data from immune assays was evaluated using the
202
Shapiro–Wilk's W-test, meanwhile, the homogeneity of variances was assessed as well. The
203
difference in immune parameters among the control, ESI and LSI from the same sampling
204
time was analyzed using one-way analysis of variance (ANOVA) followed by a Tukey's
205
multiple comparison test if a significant difference was obtained.
206 207 208
3. Results
209
3.1. Pathogenic bacteria identification
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Strain DS3 from the epidemic area of Dongshan and the bacterial strain from artificial
211
infection showed the same characteristics. The colony was blue-green, roundish with a
212
diameter of 1–3 mm, and translucent in TCBS agar medium. Strain DS3 was lactose and
213
sucrose negative, NaCl tolerance of 3-7%. The detailed physiological and biochemical
214
features were listed in Table 1. By comparison the 16S rRNA gene sequence of strain DS3
215
and the known sequences deposited in the NCBI database, a similarity of 98.4% to the
216
standard strain of Vibrio parahaemolyticus (ATCC 17802) was noted (Fig. 2). Therefore,
217
according to the biochemical and molecular results, strain DS3 is identified as V.
218
parahaemolyticus. Moreover, the LD50 by strain DS3 for 10 days was figured out of 1.3 × 103
219
cfu/fish (Table 2).
220
3.2. Variation analysis of immune parameters after bacterial infection
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After bacterial challenge, ACP and SOD activities in LSI significantly decreased at 3
222
days post infection, then gradually rebounded to the initial value. However, ACP and SOD in
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ESI was activated by bacterial challenge, and their activities were markedly higher than
224
control at day 3 and 6, respectively (Fig. 3). For MDA and C3, bacterial challenge also had an inducement effect for both ESI and
226
LSI, they sharply induced at the early period of experiment, then decreased. The slight
227
difference between MDA and C3 was the former reduced to the level close to control, while
228
the latter reduced to the level below control at the ending period of experiment (Fig. 3 and 4).
229
While for LZM, IL-2, IFN-α, IgM, monocytes/leucocytes and LPR, they exhibited a
230
generally similar variation trend. After bacterial challenge, these immune parameters in ESI
231
and LSI all acutely decreased and reached the valley value at day 3 or day 6. Afterwards, they
232
rose up, but still lower than the control at the end of experiment. Differently, the declining
233
extent of parameters in LSI was larger than in ESI (Fig. 3 and 4).
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As for lymphocytes/leucocytes, except day 3 when LSI was significant lower than
235
control, no difference was observed during the other experimental period. Similarly, no
236
difference in IL-1β was obtained throughout the entire experiment (Fig. 3 and 4).
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4. Discussion
As with many fishes in culture, seahorses do not respond well to being kept at high
240
densities and are prone to infection. Seahorse pathogen species are diverse including marine
241
leeches, ciliates, microsporidians, fungi, and the most concerned of bacteria, particularly
242
vibrios [3,7]. Vibriosis in seahorses is common: V. harveyi for haemorrhages disease in H.
243
kuda [13, 24]; V. alginolyticus and V. splendidus for skin white spots and tail-rot disease in H.
244
guttulatus and H. hippocampus [25] and V. campbellii for white porphyritic ulcer disease in H.
245
japonicus [26]. However, V. parahaemolyticus whose virulence was widely confirmed in the
246
numerous mariculture animals [27–30], being as one of the pathogens in seahorse diseases
247
has still not been reported.
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10
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249
causative agent for enteritis disease in H. erectus, which is different from the enteritis
250
pathogen of Pseudomonas sp. in H. japonicus [14]. Clarification of V. parahemolyticus as the
251
pathogen for H. erectus enteritis provides a guidance for targeted medicine choice, such as
252
amikacin [30], ampicillin [31] and piperacillin [32]. Additionally, it could partially explains
253
why juvenile H. erectus had a better survival and growth performance in salinity of 12−15‰
254
than 32‰ in our previous work [33], because of low salinity resulting in a low bacterial
255
reproduction (see the NaCl tolerance in Table 1), thereby leading to a low incidence of
256
enteritis.
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The LD50 of V. parahemolyticus to H. erectus for 10 days converted into per gram body
258
weight was 1.7 × 103 cfu/g·fish. Similarly, the LD50 of Pseudomonas sp. to H. japonicus for
259
10 days was 1.4 × 103 cfu/g·fish [14]. However, this is obviously lower than that in the other
260
cultured fishes which suffered from enteritis as well, for example, the LD50s of pathogenic
261
bacteria to the carp Ctenopharyngodon idella for 14 days [34], to the tilapia Oreochromis
262
niloticus for 10 days [35], and to the flounder Paralichthys dentatus for 4 days [36] were 1.1
263
× 105, 7.6 × 104 and 5.0 × 103 cfu/g·fish, respectively. The weak resistance to enteritis
264
bacteria may be largely due to seahorses lack gut-associated lymphoid tissue, an important
265
immune tissue against pathogens invading via mucosal areas of the gastrointestinal tract [37].
266
In addition to weak resistance caused by anomalous immune system, high incidence of
267
enteritis in cultured seahorses may be also related to their diets, primarily preying on live
268
Artemia and copepods which carry a large number of bacteria in Vibrionaceae [38,39].
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Challenge with LD50, the seahorse death peakly occurred from the 1st day to the 6th day
270
(Table 2), suggesting the first 6 days may be key time in infection, particularly in the 3rd day
271
at which the highest mortality occurred. That's why the seahorses were sampled on day 3 and
272
day 6. Additionally, in order to better compare the variations of immune parameters between
11
ACCEPTED MANUSCRIPT 273
the infection period (i.e., 3rd and 6th day) and the recovery period, the 9th day (i.e., recovery
274
period post infection) was also selected as a sampling time. Although most of the vibriosis can be treated with antibiotics, antibiotics should not be
276
overused. Overuse of antibiotics is not only easy to disturb the bacterial makeup, to produce
277
drug resistant strains, but also likely to result in drug residues to pollute the seahorse's
278
medicinal composition [40]. Therefore, improvement of the juvenile and broodstock quality
279
to partially replace the use of antimicrobial agents is imperative in aquaculture [41]. Having
280
reliable quality indicators is an essential prerequisite for quality assessment. Currently, the
281
quality or stress related indicators in fishes are mainly focused on behavior including
282
ventilatory frequency, swimming activity, latency to move from an uncomfortable condition
283
[42]; morphology such as skin color, eye darkening and hepatosomatic index [43,44]; and
284
physiology like cortisol, glucose and lactates concentration [45]. For immunology, only
285
lysozyme was reported. In the present study, 6 parameters including monocytes/leucocytes,
286
LPR, LZM, IL-2, IFN-α and Ig M were all highest in the uninfected, second in the slight
287
infected and lowest in the severely infected throughout the whole experiment, suggesting
288
these 6 parameters are very likely to be immune indicators, and could be used as quality
289
indicator for the lined seahorse.
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Some of these 6 parameters having the potential to act as an immunity or stress indicator
291
were widely confirmed in other fishes including Syngnathidae species. The Victoria labeo
292
Labeo victorianus with high LZM and Ig M had a high survivorship when challenged by
293
Aeromonas hydrophila [46]; Hippocampus sp. with a low monocytes/leucocytes had a low
294
survivorship when challenged by a noisy sound [44]; and the pipefish Syngnathus typhle
295
decreased leucocytic proliferation and antimicrobial activity (e.g. LZM and Ig M) when
296
challenged by a low salinity or Vibrio sp. [23,47]. However, in recent years, a few studies
297
concluding that some immune parameters increase after pathogen infection also have
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emerged. For example, SOD, LZM, C3 and Ig M increased in the large yellow croaker
299
Pseudosciaena crocea challenged with Cryptocaryon irritans [48] and in the Atlantic salmon
300
Salmo salar challenged with Aeromonas salmonicida [49]. Except for experimental animal difference, the completely different results mentioned
302
above might also be attributable to the diversity in infection level. In view of H. erectus dies
303
in the first 6 days upon infection with LD50 of V. parahaemolyticus, therefore, the sampling
304
times designed on the 3rd and 6th day post infection in the present study could ensure the
305
seahorses sampled at that time were severely infected. It's not surprised that a moribund
306
seahorse has a weak immune capacity. While in the above-mentioned two counterexamples:
307
P. crocea infected with C. irritans at 24,000 theronts/fish, whose survival rate was 96% at 4th
308
day, was sampled on day 1, 2 and 3 post infection [48]; and S. salar challenged with A.
309
salmonicida at 3.05× 106 cfu/fish, whose symptom appeared from day 4 post infection and no
310
death occurred, was sampled on day 2 and 4 [49]. Hence the infection levels in the two fish
311
species from their respective sampling time were generally slight. As reported by many
312
studies, a slight infection has an activation effect on the host's immunity.
313
5. In conclusion
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In the present study, we isolated and identified out the pathogenic bacteria causing
316
enteritis in seahorse H. erectus, and screened out six immune parameters promising as the
317
indicators for H. erectus immune capacity on protein and cell levels. In the future study, the
318
expression variations of these parameter related genes under different severities of infection
319
by qPCR will be carried out to better support the prospect of these parameters as immune
320
indicators on molecular level.
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321 322
Acknowledgements 13
ACCEPTED MANUSCRIPT This work was supported by the Special Fund for China Foundation for International
324
Cooperation (Project # 2011DFA33060), the Special Fund for Technology Development and
325
Research of Scientific Research Institute (Project # 2014EG1342) and the Special Research
326
Fund for the National Nonprofit Institute of China (Project # 2014T08, East China Sea
327
Fisheries Research Institute).
328 329 330
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Table 1 Physiological and biochemical features of strain DS3.
Items
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Results − + − + + − − + + + − − −
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Gram staining Polar flagellum 0% NaCl growth 3% NaCl growth 7% NaCl growth 10% NaCl growth Lactose Gelatinase Oxidase Mannitol Inositol Rhamnol Sucrose
457
Items
Melibiose Amygdalin Arabinose Arginine double enzyme hydrolysis Lysine decarboxylase Ornithine decarboxylase Citrate utilization Glucose acid production Glucose gas production Urease H2S production Indol production VP test
+, Positive; −, Negative.
458 459 460 461 17
Results − − − − + + − + − − − + −
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Table 2 Mortality of seahorse H. erectus artificial infection with different doses of V. parahaemolyticus Injected number (inds)
1d
2d
3d
4d
5d
6
5 × 10 5 × 105
20 20
20 20
1 1
4 2
5 4
5 3
1 2
5 × 104
20
20
1
2
3
2
1
3
20 20
20 20
0 0
0 0
1 0
2 0
20
20
0
0
0
0
9d
10d
6d
7d
18
Mortality (%)
1 1
0 0
0 0
0 0
0 0
17 13
85 65
1
0
0
0
0
10
55
1 0
1 0
0 0
1 1
0 0
0 0
6 1
30
0
0
0
0
0
0
0
M AN U
5 × 10 0
TE D
2
EP
5 × 10
8d
Total death number (inds)
SC
Death number per day (inds)
Injected volume (µl)
AC C
Concentration (cfu/mL)
5 0
ACCEPTED MANUSCRIPT
Figure caption Fig. 1 The clinical symptoms of healthy seahorses H. erectus infected with DS3 strain. 1: white pus spot in the anal opening; 2: abdominal swelling and ascitic fluid hoarding; 3: hindgut erosion; 4: intestinal tract
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translucence; 5: swim bladder inflation.
Fig. 2 Phylogenetic tree based on 16S rDNA sequences of strain DS3. The numbers at the nodes indicate the bootstrap values based on neighbour-joining analyses of 1000 sample date sets. Bar, 0.02 substitutions
SC
per nucleotide position.
M AN U
Fig. 3 Variations of ACP, LZM, SOD, MDA, IL-1β and IL-2 in the plasma of the control, ESI and LSI seahorses after V. parahaemolyticus infection. The different upper case letters, i.e., A, B and C indicate significant differences among control, ESI and LSI at a fixed sampling time.
Fig. 4 Variations of C3, IFN-α, IgM, monocytes/leucocytes, lymphocytes/leucocytes and LPR in the
TE D
plasma of the control, ESI and LSI seahorses after V. parahaemolyticus infection. The different upper case
AC C
time.
EP
letters, i.e., A, B and C indicate significant differences among control, ESI and LSI at a fixed sampling
19
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
Figure 1
20
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
Figure 2
21
Figure 3
AC C
EP
TE D
M AN U
SC
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ACCEPTED MANUSCRIPT
22
Figure 4
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
23
ACCEPTED MANUSCRIPT Highlights Pathogenic bacteria for enteritis in H. erectus was isolated and identified
Pathogenic bacteria was Vibrio parahaemolyticus
Six parameters high in slightly infected seahorses, low in severely infected ones
These parameters may be indicators for immune level
AC C
EP
TE D
M AN U
SC
RI PT
