Growth enhancement and protective potential of feed-based outer membrane proteins against vibriosis in Macrobrachium rosenbergii

The use of antibiotics to curtail vibriosis, which is a major infectious disease, plaguing shrimp and prawn is rather becoming less effective and the need for a better alternative is expedient. The outer membrane proteins (OMPs) of V. alginolyticus were extracted, mixed with powdered commercial feed and fed to the prawns to evaluate its effect on growth performance and protective potential. Sixty prawns were divided into groups A, B and C of 10 prawns each, with two replicates in six (150 L) glass aquaria. Groups A, B and C were fed with OMPs mixed diet, with OMPs-Freund’s incomplete adjuvant mixed diet and OMPs or adjuvant free diet (control diet) respectively. All the prawns were weighed weekly, and haemolymph was collected to determine the total haemocyte count (THC) and phenoloxidase (PO) activity. At the end of the feeding trial, prawns were intramuscularly challenged with 50 μL of 107 CFU V. alginolyticus. The treated groups were significantly higher in growth performance and THC than the control group, but no significant difference between the groups in terms of PO activity and mortality rate. The study, however, submitted that oral administration of OMPs with or without adjuvant is a good growth promoter and has the potential for protection against vibriosis in giant freshwater prawn (Macrobrachium rosenbergii)

The effect of heat stress on the oxidative status of red hybrid tilapia (Oreochromis sp.) infected with Streptococcus Agalactiae

A commercial red hybrid tilapia was experimented with S. agalactiae infection under influences of heat stress which indicated by plasma malondialdehyde (MDA) and erythrocyte superoxide dismutase (SOD) as biomarkers of stress. To achieve these objectives, 110 red hybrid tilapia in good health were divided into five groups of 22 fish each. Group A was challenged with 2.3 109 CFU of S. agalactiae and exposed to heat stress at 33 ± 0.5C on day 1. Group B was challenged on day 1 as in Group A but heat stress was introduced on day 7 post challenge (pc). Group C was exposed to heat stress on day 1 and challenged on day 7 pc while groups D and E served as a positive and negative controls respectively. Blood samples were collected at days 0, 3, 7, 10 and 14 for MDA and SOD analysis. Groups A and B recorded high mortality following exposure to heat stress and bacteria inoculation, with group A reaching 100% mortality at day 7 post inoculation. Overall, Groups A, B, C and Group D showed pattern of increase in MDA level as early as day 3 and decrease pattern for SOD activity. Group E did not show any significant difference in MDA level throughout the study period. Clinical signs such as erratic swimming, exopthalmia, skin haemorrhage and cloudy eye were predominantly observed in group A 24 h post inoculation. Based on the findings of this study, it was concluded that heat stress plays crucial role in the pathogenesis of S. agalactiae, via alteration of the oxidant defence system

Screening and evaluation of local bacteria isolated from shellfish as potential probiotics against pathogenic Vibrios

The present study was carried out to isolate, screen and evaluate potential candidates of local bacteria isolated from tiger shrimp Penaeus monodon and slipper cupped oysters Crassostrea iredalei as probiotics in shellfish aquaculture. A total of 144 of bacteria were successfully isolated from the intestine and stomach of 20 tails of healthy adult tiger shrimp P. monodon, while 136 were successfully isolated from the digestive tract, gills and inner shells of 10 healthy adult C. iredalei. The number of potential isolates was narrowed down to two from tiger shrimp, and one from slipper cupped oyster after in vitro screening assays. The three isolates, labeled as G11, I24 and S66, were identified as Virgibacillus sp., Bacillus sp. and Exiquobacterium sp., respectively, using 16S rDNA gene analysis. The antagonistic ability of the isolates towards Vibrio alginolyticus and Vibrio harveyi were conducted in stagnant and liquid modes via spot lawn and broth co-culture assay, respectively. In these assays, all the potential probionts were inhibitory to both pathogenic vibrios. In the in-vivo assay, Artemia was used as host and treated with different concentrations of potential probionts (10⁴, 10⁶ and 10⁸ CFU ml⁻¹), and challenged with V. alginolyticus ana V. harveyiat 10⁵ CFU ml⁻¹ Respectively, Artemia treated with probiont Gl 1 at all concentrations and challenged with V. alginolyticus had increased survival (70-80 %), which was significantly higher as compared with group with only the pathogen (20 %). Meanwhile, probiont I24 increased the survival of Artemia by 70 % at a concentration of 10⁸ CFU ml⁻¹ after being challenged with V. alginolyticus and Artemia treated with 10⁶ CFU ml⁻¹ of probiont S66 had increased survival of 90% after being challenged with V. harveyi. Thus, the three isolates might have potential applications as probiotics in shellfish aquaculture against vibriosis

Localisation of antigens in the gut post-challenge with Streptococcus iniae in vaccinated and non-vaccinated red hybrid tilapia (Oreochromis sp.)

This study was conducted to determine the localisation of antigen in the gut of vaccinated and non-vaccinated red hybrid tilapia post-challenge via immunohistochemistry and scanning electron microscopy. All groups (1, 2, 3, and 4) were vaccinated orally with feed-based killed whole-cells of Streptococcus iniae (FKV). Group 1 was fed with the FKV for 3 days consecutively only on day 0; group 2 was fed with the FKV for 3 days consecutively on day 0 and booster on day 14; group 3 was fed with the FKV for 3 days consecutively on day 0, followed by a booster on day 14 and 21; and group 4 was fed with the FKV for 5 days consecutively on day 0, followed by a booster on day 14 and 21. The remaining groups, group 5 (negative control group) and group 6 (positive control group), only fed with a commercial feed. Five fish from each group were sacrificed on a weekly basis for the entire 6 weeks for a collection of fresh small intestinal samples including post-challenge starting from 4 to 72 h. Immunohistochemical staining was conducted using the avidin-biotin-peroxidase complex method. Scanning electron microscopy was also used to view the hindgut samples from each group. Positive control group recorded significantly the highest (p < 0.05) antigen positivity, followed by groups 1 and 2, meanwhile, lowest positivity in groups 3 and 4 at 12 h, 24 h, and 72 h, respectively. Immunohistochemistry and scanning electron microscopy showed the presence of antigens in red hybrid tilapia hindgut, protruding in goblet cells, gut-associated lymphoid tissues, and villi dome epithelium in different hours of post-challenge. Significantly, boosted group 4 with a low number of antigens in 72 h showed the stimulation and irregular projection of M-cells with variable morphology and antigen distribution pattern

Vibrio parahaemolyticus and Vibrio harveyi causing Acute Hepatopancreatic Necrosis Disease (AHPND) in Penaeus vannamei (Boone, 1931) isolated from Malaysian shrimp ponds

Acute hepatopancreatic necrosis disease (AHPND) first emerged as a new shrimp disease in 2009 that heavily affected shrimp industry leading to global economic losses. The etiological agent was previously identified as Vibrio parahaemolyticus that carries a plasmid containing toxins (PirA and PirB). However, recent researches revealed that V. parahaemolyticus is not the only bacterial species capable of causing AHPND, thus this study screened on bacterial strains with AHPND toxins from Penaeus vannamei shrimps in Malaysia. Out of the 86 isolated total strains, 12 AHPND positive strains were arbitrarily selected and were evaluated in in vivo assay using Artemia franciscana as a model organism. All the 12 AHPND positive strains with PirA and PirB genes demonstrated significant mortalities (P < 0.05) of A. franciscana compared to the negative control. The 12 AHPND positive strains were identified using molecular methods of 16S rRNA, RctB and RpoD region amplifications belonged to the Harveyi Glade and were closely related to V. parahaemolyticus and Vibrio harveyi. Further test showed that the yellow colony V. harveyi strain BpShHep24 was found to be more virulent than the green colony V. parahaemolyticus strain BpShHep31 in shrimp P. vannamei challenge test. Histological examination of shrimp hepatopancreas challenged with yellow colony V. harveyi strain BpShHep24 showed massive sloughing of hepatopancreas tubules of epithelial cells into the lumen, haemocyte infiltrations, proximal-to-distal lesion of hepatopancreas and collapsed tubule epithelia within 24 h

First report of Pantoea stewartii subsp. indologenes causing leaf blight on rice in Malaysia

In November and December of 2017, leaf samples from rice (Oryza sativa) exhibiting leaf blight symptoms were collected from commercial fields in Kedah and Selangor, Malaysia, with about 80% disease incidence. Symptoms were water-soaked lesions from the upper part of the leaf blade, which later turned into brown stripes (Mondal et al. 2011), as typically caused by Xanthomonas oryzae pv. oryzae (Niño-Liu et al. 2006). Twenty strains of bacteria were isolated from the diseased samples from both fields, and eight isolates (MF1, MF2, MF3, MF4, MF5, MF7, MF8, and MF9) suspected to be Pantoea species were subjected to further characterization. Bacterial colonies were isolated from surface-sterilized infected leaf tissue using 75% ethanol and 1% sodium hypochlorite, rinsed three times in sterilized water, macerated in sterilized water, placed on King’s B agar medium, and incubated for 48 h at 28°C. Single colonies were transferred and maintained on nutrient broth medium. All isolated bacteria were gram-negative facultative anaerobes, motile, positive for catalase and gelatin tests, hydrolyzing starch, incapable of producing hydrosulfuric acid, indole positive, and able to produce acetoin. Colonies were round, smooth with irregular edges, and produced yellow pigment on King’s B agar medium. Polymerase chain reaction using specific primers targeting the gyrb gene of Pantoea genome was performed, and the ∼600-bp amplicons were sequenced (Kini et al. 2017). BLASTn analysis revealed all strains had 99% similarity to the Pantoea stewartii reference strain (ARC646) in the GenBank database (accession no. KX342016). These sequences were later deposited in GenBank (accession nos. MH300616 to MH300621 and MH673052 to MH673054). A phylogenetic tree constructed based on the concatenated nucleotide sequences of the gyrb gene showed that all strains were 99% similar to P. stewartii reference strains (accession nos. KX342015, KF554590, KT7295191, and KX342016). The strains were tested with subspecies-specific primers of P. stewartii subsp. stewartii and P. stewartii subsp. indologenes targeting housekeeping gene gaIE that encodes UDP-glucose 4-epimerase to confirm the subspecies (Gehring et al. 2014). Positive bands were produced by all strains from gaIE indologenes subspecies, each producing a ∼267-bp amplicon. All strains were identical to the reference strain of P. stewartii subsp. indologenes (NCPPB 1845) in GenBank database (accession no. HG792431). To test pathogenicity, 10 ml of 108 CFU/ml bacterial suspension of each strain was inoculated into 4-week-old rice seedlings of MR269 variety using the leaf clipping method (Kauffman et al. 1973), and they were kept in the greenhouse with temperature ranging from 26 to 35°C. All eight strains of P. stewartii subsp. indologenes produced symptoms similar to those originally found in the rice fields, including yellowish necrotic water-soaked lesions, which later turned to typical blight at 2 weeks postinoculation. Strains were reisolated from diseased leaves using the same method mentioned above and confirmed as P. stewartii subsp. indologenes by sequence analysis of the gaIE gene, thus fulfilling Koch’s postulates. To the best of our knowledge, this is the first report of leaf blight disease caused by P. stewartii subsp. indologenes on rice in Malaysia

First report of Pantoea ananatis causing leaf blight disease of rice in Peninsular Malaysia

The genus Pantoea contains pathogenic bacteria causing destructive diseases on rice (Oryzae sativa L.) because they reduce the quality, hence affecting the yield of rice production. Rice infected by Pantoea species can exhibit severe diseases such as leaf blight, stem necrosis, grain discoloration, and damaged germplasm of rice seeds (Mondal et al. 2011). In September 2017 and April 2018, bacterial leaf blight disease of rice (MR269 and CL varieties) was observed in rice fields located in the states of Selangor and Kedah, Malaysia, with 80% disease incidence. Disease symptoms observed were water-soaked stripes with yellowing color, which later turned into brown stripes on the upper part of leaves (Kini et al. 2017). The causal agent was isolated from symptomatic leaf pieces after surface sterilization using 75% ethanol for 30 s and rinsing thrice in sterilized water containing 1% NaClO. Samples were macerated in sterilized water, streaked on semiselective King’s B agar medium, and incubated for 48 h at 28°C. Ten representative isolates (PA1, PA3, PA5, PA6, PA7, PA8, PA9, PA10, PA11, and PA12) were isolated from the diseased samples. Bacterial colonies were yellow pigmented, raised, irregular border, and translucent with smooth margin, resembling the characteristics of Pantoea ananatis (Mondal et al. 2011). All isolates were subjected to biochemical tests, revealing gram-negative facultative anaerobe, motile, positive for catalase and gelatin tests, hydrolyzing starch, not producing hydrosulfuric acid, indole positive, and capable of producing acetoin. The DNA of all isolates was extracted using a Geneaid DNA Isolation Kit (Geneaid Biotech, Taiwan). Polymerase chain reaction (PCR) amplification based on the gyrb gene fragment developed from 26 genomes of Pantoea species was performed on all isolates, each producing a ∼600-bp amplicon (Kini et al. 2017). The amplicons were sequenced, and the sequences were submitted to GenBank database (accession nos. MH698458, MH698460, MH698462, MH698463, MH698464, MH698465, MH698466, MH698467, MH698468, and MH698469). A BLASTn search revealed 100% nucleotide identity of all isolates to P. ananatis ARC60 reference strain (accession no. KX385187). The phylogenetic tree constructed from all isolates based on the gyrb gene sequences indicated 99% similarity to P. ananatis reference strains (accession nos. KX385187, KX342014, and KF554589). PCR amplification with P. ananatis-specific gene, PANA_1080, which encodes for a hypothetical protein of the pathogen, produced a ∼900-bp amplicon each (Asselin et al. 2016), and a BLASTn search showed 96% identity to the P. ananatis LMG 20103 reference strain (accession no. CP001875). The nucleotide sequences of all isolates were later deposited to GenBank database (accession nos. MK348547 to MK348556). To test pathogenicity, 10 ml of 108 CFU/ml bacterial suspension of each isolate was inoculated into 35-day-old rice seedlings of MR269 and CL varieties by using a leaf-clipping method, and they were kept in the greenhouse with temperature ranging from 26 to 35°C, performed in triplicate (Ke et al. 2017). Control rice seedlings were inoculated with sterilized water. All isolates of P. ananatis produced symptoms within 2 weeks postinoculation. Symptoms appeared similar to those of natural infections, including yellowish necrotic water-soaked lesion. Control rice seedlings remained asymptomatic. Bacteria were reisolated from symptomatic leaves and further identified as P. ananatis by PANA_1080 gene sequencing, fulfilling the Koch’s postulates. To the best of our knowledge, this is the first report of P. ananatis causing leaf blight disease of rice in Malaysia. With the aid of molecular-based approaches, a better understanding on the taxonomy of P. ananatis will help to improve and develop effective disease control strategies against this wide-spreading bacterial pathogen of rice in Malaysia