Piper betle Leaf Extract Inhibits Multiple Aquatic Bacterial Pathogens and In Vivo Streptococcus agalactiae Infection in Nile tilapia

Abstract

An in vitro assessment of antimicrobial properties of aqueous and ethanol extracts from solo garlic (Allium sativum), garlic chive (Allium tuberosum) and betel leaves (Piper betle) on six bacterial pathogens in aquaculture, and a challenge of Nile tilapia, Oreochromis niloticus with Streptococcus agalactiae were performed. Generally, minimum inhibitory concentrations (MIC) ranged from 26.63 to 53.25 mg mL-1 for aqueous solo garlic (G) and 14.60 to 29.20 mg mL-1 for garlic chive extracts for all pathogens tested. Ethanol extract of betel leaves (P) exhibited the strongest antibacterial activity (0.15 – 0.60 mg mL- 1). P and G incorporated in feed at high and low doses as multiples of MIC [High; H (10X for PH and 3X for GH) and Low; L (3X for PL and 1X for GL)] were fed to tilapia followed by in vivo challenge against S. agalactiae (1 x 108 CFU mL-1). Ethanol extract of P. betle significantly improved survival (P < 0.05; PH=100%, PL =77%). White blood cells (WBC), lymphocytes and monocytes differed significantly (P < 0.05) among treatments and the highest WBC value (1.175 × 103) was for PH. Use of ethanol extract of Piper betle seems promising for sustainable disease management in aquaculture.

Keywords: herbal, antimicrobial, risk, haematology, survival

Source: Turkish Journal of Fisheries and Aquatic Sciences. DOI: 10.4194/1303-2712-v18_5_03

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Evidence of TiLV infection in tilapia hatcheries from 2012 to 2017 reveals probable global spread of the disease

TiLV-Histopathology HT Dong

Histopathological feature of syncytial hepatitis of tilapia (SHT) observed from the liver of  TiLV-infected tilapia (Photo: HT Dong)

Abstract

Recent outbreaks of tilapia lake virus (TiLV) in farmed tilapia in Thailand were the first indication of spread of the virus to the Southeast Asia region. Here we further investigate TiLV infection of archived and newly collected fish samples obtained from Thai hatcheries from 2012 to 2017. Fertilized eggs, yolk-sac larvae, fries, and fingerlings
were tested for the TiLV using an established semi-nested RT-PCR assay. The results revealed that the majority of the tested samples were TiLV positive, including our earliest preserved samples collected in year 2012. DNA sequence analysis of representative amplified products also confirmed the presence of TiLV. Since the
discovery of TiLV in 2012, over 40 countries worldwide have imported tilapia fry and fingerlings, and some may have been unaware of risk that they might be infected with TiLV. Thus, if they have not already done so, we recommend that countries that have imported tilapia for aquaculture carry out surveillance studies for its presence and also add TiLV to their import quarantine inspection list.

Keywords: Disease transmission, TiLV, Tilapia hatcheries

Source: http://www.sciencedirect.com/science/article/pii/S0044848617306695

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Infectious spleen and kidney necrosis disease (ISKND) outbreaks in farmed barramundi (Lates calcarifer) in Vietnam

Highlights

  • The first report on emergence of ISKD in farmed barramundi from Vietnam.
  • The causative agent was identified as Megalocytivirus ISKNV genotype II.
  • A Megalocytivirus RSIV vaccine conferred only partial protection.
  • Structural analysis revealed significant differences between the two viruses.

Abstract

Emergence of a disease with clinical signs resembling megalocytivirus infection seriously affected large-scale barramundi farms in Vietnam in 2012–2014 with estimated losses reaching $435,810 per year. An oil-based, inactivated vaccine against red sea bream iridovirus (RSIV) was applied in one farm for disease prevention without analysis of the causative agent, and the farmer reported inadequate protection. Here we describe histological and molecular analysis of the diseased fish. PCR targeting the major capsid protein (MCP) of megalocytiviruses yielded an amplicon with high sequence identity to infectious spleen and kidney necrosis virus (ISKNV) genotype II previously reported from other marine fish but not barramundi. Detection of the virus was confirmed by positive in situ hybridization results with fish tissue lesions of the kidney, liver, pancreas, and brain of the PCR-positive samples. Based on the complete sequence of the MCP gene, the isolate showed 95.2% nucleotide sequence identity and 98.7% amino acid sequence identity (6 residue differences) with the MCP of RSIV. Prediction of antigenic determinants for MCP antigens indicated that the 6 residue differences would result in a significant difference in antigenicity of the two proteins. This was confirmed by automated homology modeling in which structure superimpositioning revealed several unique epitopes in the barramundi isolate. This probably accounted for the low efficiency of the RSIV vaccine when tested by the farmer.

Keywords

Barramundi; ISKNV; Lates calcarifer; RSIV; Vaccine

Source: http://www.sciencedirect.com/science/article/pii/S1050464817303777

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Comparative genome analysis of fish pathogen Flavobacterium columnare reveals extensive sequence diversity within the species

How to cite: Pattanapon Kayansamruaj, Ha Thanh Dong, Ikuo Hirono,
Hidehiro Kondo, Saengchan Senapin, Channarong Rodkhum , Comparative genome
analysis of fish pathogen Flavobacterium columnare reveals extensive sequence diversity within the species. Infection, Genetics and Evolution (2017), doi: 10.1016/
j.meegid.2017.06.012

Highlights

  • Comparative genomics among fish pathogenic F. columnare strains was investigated
  • Virulence genes were equally distributed among F. columnare strains.
  • Phylogenetic analyses indicated extensive genetic diversity within the species.
  • 16S–RFLP failed to distinguish between genomovar II isolate groups.
  • Tilapia-originated strains from Thailand would be distinct taxonomic groups.

Fc

Abstract

Flavobacterium columnare is one of the deadliest fish pathogens causing devastating mortality in various freshwater fish species globally. To gain an insight into bacterial genomic contents and structures, comparative genome analyses were performed using the reference and newly sequenced genomes of F. columnare including genomovar I, II and I/II strains isolated from Thailand, Europe and the USA. Bacterial genomes varied in size from 3.09 to 3.39 Mb (2714 to 3101 CDSs). The pan-genome analysis revealed open pan-genome nature of F. columnare strains, which possessed at least 4953 genes and tended to increase progressively with the addition of a new genome. Genomic islands (GIs) present in bacterial genomes were diverse, in which 65% (39 out of 60) of possible GIs were strain-specific. A CRISPR/cas investigation indicated at least two different CRISPR systems with varied spacer profiles. On the other hand, putative virulence genes, including those related to gliding motility, type IX secretion system (T9SS), outer membrane proteins (Omp), were equally distributed among F. columnare strains. The MLSA scheme categorized bacterial strains into nine different sequence types (ST 9–17). Phylogenetic analyses based on either 16S rRNA, MLSA and concatenated SNPs of core genome revealed the diversity of F. columnare strains. DNA homology analysis indicated that the estimated digital DNA-DNA hybridization (dDDH) between strains of genomovar I and II can be as low as 42.6%, while the three uniquely tilapia-originated strains from Thailand (1214, NK01 and 1215) were clearly dissimilar to other F. columnare strains as the dDDH values were only 27.7–30.4%. Collectively, this extensive diversity among bacterial strains suggested that species designation of F. columnare would potentially require re-emendation.

Keywords

Comparative genomics; Flavobacterium Columnare; Genetic diversity; Pan-genome; Taxonomy

Source: http://www.sciencedirect.com/science/article/pii/S1567134817301995

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A natural Vibrio parahaemolyticus ΔpirAVp pirBVp+ mutant kills shrimp but produces neither PirVp toxins nor acute hepatopancreatic necrosis disease lesions

How to cite: Phiwsaiya K, Charoensapsri W, Taengphu S, Dong HT, Sangsuriya P, Nguyen GTT, Pham HQ, Amparyup P, Sritunyalucksana K, Taengchaiyaphum S, Chaivisuthangkura P, Longyant S, Sithigorngul P, Senapin S. 2017. A natural Vibrio parahaemolyticus ΔpirAVp pirBVp+ mutant kills shrimp but produces neither PirVp toxins nor acute hepatopancreatic necrosis disease lesions. Appl Environ Microbiol 83:e00680-17. https://doi.org/10.1128/AEM.00680-17.

Abstract

Acute hepatopancreatic necrosis disease (AHPND) of shrimp is caused by Vibrio parahaemolyticus isolates (VPAHPND isolates) that harbor a pVA plasmid encoding toxins PirAVp and PirBVp. These are released from VPAHPND isolates that colonize the shrimp stomach and produce pathognomonic AHPND lesions (massive sloughing of hepatopancreatic tubule epithelial cells). PCR results indicated that V. parahaemolyticus isolate XN87 lacked pirAVp but carried pirBVp. Unexpectedly, Western blot analysis of proteins from the culture broth of XN87 revealed the absence of both toxins, and the lack of PirBVp was further confirmed by enzyme-linked immunosorbent assay. However, shrimp immersion challenge with XN87 resulted in 47% mortality without AHPND lesions. Instead, lesions consisted of collapsed hepatopancreatic tubule epithelia. In contrast, control shrimp challenged with typical VPAHPND isolate 5HP gave 90% mortality, accompanied by AHPND lesions. Sequence analysis revealed that the pVA plasmid of XN87 contained a mutated pirAVp gene interrupted by the out-of-frame insertion of a transposon gene fragment. The upstream region and the beginning of the original pirAVp gene remained intact, but the insertion caused a 2-base reading frameshift in the remainder of the pirAVp gene sequence and in the downstream pirBVp gene sequence. Reverse transcription-PCR and sequencing of 5HP revealed a bicistronic pirABVp mRNA transcript that was not produced by XN87, explaining the absence of both toxins in its culture broth. However,
the virulence of XN87 revealed that some V. parahaemolyticus isolates carrying mutant pVA plasmids that produce no PirVp toxins can cause mortality in shrimp in
ponds experiencing an outbreak of early mortality syndrome (EMS) but may not
have been previously recognized to be AHPND related because they did not cause
pathognomonic AHPND lesions.

Source: http://aem.asm.org/content/early/2017/05/29/AEM.00680-17.abstract

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Urgent update on possible worldwide spread of tilapia lake virus (TiLV)

Tilapia lake virus disease (TiLVD) (also known as syncytial hepatitis of tilapia-SHT) is a newly emerging viral disease of tilapia caused by tilapia lake virus (TiLV), a novel Orthomyxo-like virus (Ferguson et al. 2014; Eyngor et al. 2014; Bacharach et al. 2016; Del-Pozo et al. 2017; NACA, 2017; OIE, 2017). Occurrence of the disease was officially documented earliest in Ecuador and Israel in 2013 and 2014, respectively (Ferguson et al. 2014; Eyngor et al. 2014). The virus, however, is believed to have been responsible for massive mortalities in farmed tilapia in Israel since 2009 (Eyngor et al. 2014). Infection of TiLV was later reported from Colombia (Kembou Tsofack et al. 2017) and Egypt (Fathi et al. 2017) and most recently from Thailand (Dong et al. 2017a; Surachetpong et al. 2017). Natural disease outbreaks result in variable mortalities ranging from 9.2 to 90%, with tilapia fingerlings and juveniles being more vulnerable than larger fish (Ferguson et al. 2014; Fathi et al. 2017; Dong et al. 2017a; Surachetpong et al. 2017). Unlike other viral diseases of tilapia, TiLV appears to be widely spread and so may be present in many countries where it is not yet recognized.

Recently, we released a warning of TiLV in Thailand, including an improved semi-nested RT-PCR method for rapid detection and we urge those involved in Tilapia culture to test for the virus in their country (Dong et al. 2017b). The Fish Health Platform in Centex, BIOTEC/Mahidol University) has also obtained positive test results for TiLV from other countries in Asia where it has not yet been reported, supporting our appeal for wider testing. Further, the majority of our archived samples collected from previous disease outbreaks in several tilapia hatcheries in Thailand during 2012-2017 have tested positive for TiLV (unpublished data), indicating the presence of TiLV in Thailand even before the virus became known to science in 2013. The origin of the disease is currently unknown, but many countries have been translocating tilapia fry/fingerlings prior to and even after the description of TiLV.  Based on records we could obtain about such translocations, we have prepared a map that contains a list of 5 countries with confirmed reports of TiLV infections (red, Fig.1) and a list of 43 other countries that we believe have imported tilapia that may have been infected with TiLV (blue, Fig. 1). We hope that widespread surveillance for TiLV in the Tilapia industry and in translocated fish will help reduce the impact and spread of this disease. To this end, we repeat our willingness to provide on request a free positive control plasmid for use with the method we have developed to detect TiLV by semi-nested PCR (Dong et al. 2017b).

600 dpi

Fig. 1 Possible worldwide distribution map of TiLV. Countries with confirmatory evidence of TiLV circulation (red colour). Blue colour indicates 43 countries that we believe have imported tilapia that may have been infected with TiLV.

List of countries with confirmed evidence of TiLV: Ecuador, Israel, Colombia, Egypt and Thailand

List of countries we believe at high risk of TiLV: Algeria, Bahrain, Bangladesh, Belgium, Burundi, Canada, China, Congo, ElSalvador, Germany, Guatemala, India, Indonesia, Japan, Jordan, Laos, Malaysia, Mexico, Mozambique, Myanmar, Nepal, Nigeria, Pakistan, Philippines, Romania, Rwanda, Saudi Arabia, Singapore, South Africa, Sri Lanka, Switzerland, Tanzania, Togo, Tunisia, Turkey, Turkmenistan, Uganda, Ukraine, United Arab Emirate, United Kingdom, United States, Vietnam and Zambia.

Recommendations

  • We recommend that the 43 countries we have listed quickly initiate surveillance for TiLV in cultured Tilapia, since the virus may have been introduced via direct or indirect translocation of fry/fingerlings from the 5 countries where it has been reported.
  • Biosecurity should be applied to prevent wider spread of the disease especially by countries with no predictive record of TiLV risk.
  • Since TiLV infects very early developmental stages of tilapia (fertilized eggs, fry, and fingerlings) when fish immune system is not fully developed, the use of vaccines may not be an effective control approach.
  • Research should be promoted for the development of methods to clear TiLV from infected tilapia broodstock and allow production TiLV-free fry/fingerlings.
  • Programs should be promoted to develop Tilapia stocks specific pathogen free (SPF) for TiLV and other pathogens as a potential approach to limit impact of Tilapia diseases globally.
  • Since TiLV infections result in highly variable mortality (9.2-90%), it is urgent that research should be promoted to discover the underlying reasons (e.g., research on the correlation between TiLV virulence and genetic types or other factors).

Acknowledgments

The authors acknowledge fish producers who provided fish samples and information for this study. We would also like to thank Prof. T.W. Flegel for his advice and assistance in preparing this announcement.

References

  1. Bacharach, E., Mishra, N., Briese, T., Zody, M.C., Kembou Tsofack, J.E., Zamostiano, R., Berkowitz, A., Ng, J., Nitido, A., Corvelo, A., Toussaint, N.C., Abel Nielsen, S.C., Hornig, M., Del Pozo, J., Bloom, T., Ferguson, H., Eldar, A., Lipkin, W.I., 2016. Characterization of a novel Orthomyxo-like virus causing cass die-offs of tilapia. MBio. 7, e00431-00416.
  2. Del-Pozo, J., Mishra, N., Kabuusu, R., Cheetham, S., Eldar, A., Bacharach, E., Lipkin, W.I., Ferguson, H.W., 2017. Syncytial Hepatitis of Tilapia (Oreochromis niloticus) is associated with Orthomyxovirus-Like virions in hepatocytes. Vet Pathol. 54, 164-170.
  3. Dong, H.T., Siriroob, S., Meemetta, W., Santimanawong, W., Gangnonngiw, W., Pirarat, N., Khunrae, K., Rattanarojpong, T., Vanichviriyakit, R., Senapin, S., 2017a. Emergence of tilapia lake virus in Thailand and an alternative semi-nested RT-PCR for detection. Aquaculture. 476, 111-118.
  4. Dong HT, Siriroob, S., Meemetta, W., Santimanawong, W., Gangnonngiw, W., Pirarat, N., Khunrae, P., Rattanarojpong, T., Vanichviriyakit, R. and Senapin, S., 2017b. A warning and an improved PCR detection method for tilapia lake virus (TiLV) disease in Thai tilapia farms. https://enaca.org/?id=858
  5. Eyngor, M., Zamostiano, R., Kembou Tsofack, J.E., Berkowitz, A., Bercovier, H., Tinman, S., Lev, M., Hurvitz, A., Galeotti, M., Bacharach, E., Eldar, A., 2014. Identification of a novel RNA virus lethal to tilapia. J Clin Microbiol. 52, 4137-4146.
  6. Fathi, M., Dickson, C., Dickson, M., Leschen, W., Baily, J., Fiona, M., Ulrich, K., Weidmann, M., 2017. Identification of Tilapia Lake Virus in Egypt in Nile tilapia affected by ‘summer mortality’ syndrome. Aquaculture. 473, 430–432.
  7. Ferguson, H.W., Kabuusu, R., Beltran, S., Reyes, E., Lince, J.A., del Pozo, J., 2014. Syncytial hepatitis of farmed tilapia, Oreochromis niloticus (L.): a case report. J Fish Dis. 37, 583-589.
  8. NACA (Network of Aquaculture Centres in Asia-Pacific), 2017. Tilapia Lake Virus (TiLV) – an Emerging Threat to Farmed Tilapia in the Asia-Pacific Region. Disease Advisory. https://enaca.org/?id=864&title=tilapia-lake-virus-disease-advisory
  9. OIE (World Organisation For Animal Health), 2017. Tilapia lake virus (TiLV)-A novel Orthomyxo-like virus. May, 2017. http://www.oie.int/en/international-standard-setting/specialists-commissions-groups/aquatic-animal-commission-reports/disease-information-cards/
  10. Surachetpong, W., Janetanakit, T., Nonthabenjawan, N., Tattiyapong, P., Sirikanchana, K., Amonsin, A., Outbreaks of tilapia lake virus infection, Thailand, 2015-2016. Emerg Infect Dis. https://dx.doi.org/10.3201/eid2306.161278
  11. Kembou Tsofack, J.E., Zamostiano, R., Watted, S., Berkowitz, A., Rosenbluth, E., Mishra, N., Briese, T., Lipkin, W.I., Kabuusu, R.M., Ferguson, H., Del Pozo, J., Eldar, A., Bacharach, E., 2017. Detection of tilapia lake virus in clinical samples by culturing and nested reverse transcription-PCR. J Clin Microbiol. 55, 759-767.

Source: https://enaca.org/?id=870&title=urgent-update-on-possible-worldwide-spread-of-tilapia-lake-virus-tilv

 

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A warning and an improved PCR detection method for tilapia lake virus (TiLV) disease in Thai tilapia farms

H.T. Dong, S. Siriroob, W. Meemetta, W. Santimanawong, W. Gangnonngiw, N. Pirarat, P. Khunrae, T. Rattanarojpong, R. Vanichviriyakit and S. Senapin

Tilapia lake virus (TiLV) is a novel RNA virus resembling Orthomyxovirus. It has been reported as a newly emerging virus that causes syncytial hepatitis of tilapia (SHT) in Israel, Ecuador, Colombia, and Egypt (Ferguson et al. 2014; Eyngor et al. 2014; Bacharach et al. 2016; Del-Pozo et al. 2017, Fathi et al. 2017). The disease caused massive mortality up to 90% in farmed tilapia and is considered to be a potential threat to global tilapia farming (Eyngor et al. 2014).

In Thai tilapia farms, disease outbreaks have recently occurred associated with high cumulative mortalities (20-90%), and we have found that the diseased fish show typical histopathological features of SHT. Infection has been confirmed by transmission electron microscopy (TEM), in situ hybridization and high nucleotide sequence identity to TiLV from Israel (Dong et al. 2017). Our research progress was delayed by use of nested RT-PCR primer sequences previously published by Eyngor et al. (2014) together with detailed RT-PCR protocols later released by Tsofack et al. (2016). Our preliminary detection assays performed using the earlier report (Eyngor et al. 2014) with assumed PCR conditions (not specified in the publication) resulted in amplification of nonspecific fish genes. This probably resulted from the fact that 13-18 out of 20 nucleotides for the primer “Nested ext-2” matched fish genes. We, therefore, modified the nested RT-PCR protocols into a semi-nested RT-PCR by omitting the primer “Nested ext-2” to avoid false positive results.

The semi-nested RT-PCR protocol given below may be used freely for non-commercial applications to detect TiLV. Heavily-infected samples will generate 2 amplicon bands of 415 bp and 250 bp while lightly-infected samples will generate a single 250-bp amplicon band (Fig. 1). Since this is the first report of TiLV in Southeast Asia, the authors urge fish health laboratories in Asia to test for TiLV when abnormal mortality of tilapia occurs. Please contact Centex Shrimp (saengchan ‘at’ biotec.or.th) to obtain positive control plasmids (pGEM-415_bp), which will be provided free for non-commercial organisations, and for a fee to the private sector.

PCR gel

Fig. 1 A sample agarose gel of TiLV detection from fish RNA extracts. Expected band sizes of 415 bp and 250 bp represent amplicons from the first and semi-nested PCR, respectively, with lanes marked ++ for a heavy infection, + for a light infection and – for the negative control. The band marked with an asterisk (*) on the right side of the gel probably arose from cross hybridization of the amplified products. M = DNA marker

Source: https://enaca.org/?id=858

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