Luminescent vibrios associated with mortality in pond-cultured shrimp penaeus monodon in the Philippines: Species composition

Heavy mortalities due to luminescent vibriosis have been observed among pond-cultured Penaeus monodon shrimp in the Philippines. The species composition of luminescent Vibrio associated with mortalities was determined. A total of 189 luminescent bacteria isolated from the hepatopancreas of affected shrimps, rearing water and seawater from different shrimp farms in 11 provinces of the Philippines were examined for their morphological, physiological and biochemical characteristics. Results revealed a varied composition of Vibrio species. The most dominant luminescent Vibrio species was V. harveyi (65.5%) followed by V. logei (7%), Photobacterium sp. (6%) and V. orientalis (1%). Some isolates, based on their characteristics, were identified as V. campbellii (16%), V. mediterranei (3%), V. fluvialis (0.5%), V. cholerae (0.5%) and V. splendidus II (0.5%), which are known as non-luminescent Vibrios. V. harveyi is thought to be the major etiological agent associated with the luminescent vibriosis in pond-cultured P. monodon and its pathogenicity was confirmed through intramuscular injection to shrimp. Experimental infection showed that V. campbellii was also pathogenic to P. monodon

Qualitative and quantitative comparison of bacterial flora associated with hatchery-reared and wild-caught shrimp postlarvae

Because of high mortality recorded in pond-reared shrimps due to luminescent vibriosis infection, a study was conducted to determine if postlarvae (PLs) could be major sources of luminescent bacteria (LB). Batches of hatchery-reared (PL12 to 18) and wild-caught Penaeus monodon PLs were examined to determine their bacterial load. Results show that although all PLs have associated Vibrio spp., not all of them harbored detectable levels of LB. Fifty eight percent of wild-caught postlarval batches did not have associated LB compared with only 23-44% of hatchery-reared postlarvae. A significant difference in quantitative LB load was noted between hatchery reared and wild-caught PLs with the former harboring up to 3.0 x 105 cfu LB/postlarva. Wildcaught PLs had only up to 3.5 x 102 cfu LB/postlarva. Antimicrobial sensitivity tests using disc diffusion method show significant resistance to Chloramphenicol and Oxytetracycline among isolates from hatchery-reared PLs (33 and 44%) compared with bacteria from wild-caught PLs (3 and 6%) and near shore seawater (0 and 12%). The differences between the quantitative and qualitative bacterial flora of hatchery-reared and wild-caught PLs may have contributed to the occurrence of luminescent vibriosis in grow-out ponds, which generally make use of hatchery-reared postlarvae

Standardization of PCR techniques as the detection method for white spot syndrome virus (WSSV) infection in shrimp (Penaeus monodon)

Several runs were conducted using the three published PCR protocols and the three DNA extraction methods. All three primer pairs designed by individual authors based on the sequence of a cloned fragment of the white spot syndrome virus (WSSV) genome successfully detected the Philippine WSSV isolates. In addition, a high specificity of the assay was also obtained. CTAB and DNAzol extraction methods consistently gave a better DNA quality using the three PCR protocols. Any of the three PCR protocols can be used in the rapid and sensitive detection of WSSV in shrimp (Penaeus monodon) and crab (Episesarma vesicolor)

Prevalence of economically-important viral diseases in wild shrimp (Penaeus monodon) in the Philippines

Prevalences of white spot syndrome virus (WSSV) and monodon baculovirus (MBV) were determined using polymerase chain reaction (PCR) as the detection method in wild black tiger shrimp (Penaeus monodon) collected from seven sampling sites in the Philippines. These seven sampling sites are the primary sources of spawners and broodstocks for hatchery use. Samples for PCR comprised DNA extracts from gills for WSSV detection and from hepatopancreas for MBV. As a result, WSSV was detected in shrimp from all sites except Bohol during dry season, and were negative from all sites except Palawan during wet season. However, all the positive shrimp showed no white spots in the carapace. MBV was also detected from all sites except Palawan, Negros Occidental and Bohol during dry and wet seasons, respectively. The prevalences of WSSV and MBV showed seasonal and sex variations, i.e. the prevalence in dry season (April-May) was higher than in wet season (August-October), and the prevalence in female shrimps was higher than in males. A very low prevalence of dual infection with WSSV and MBV was also observed. These results suggest that the viruses have already established in the local marine environment and in wild populations of P. monodon, and spawners collected during dry season could serve as the main source of contamination for vertical transmission of the virus when they spawn

Occurrence and pathology of Penaeus monodon baculovirus infection in hatcheries and ponds in the Philippines

Samples from Penaeus monodon hatcheries (2-3 day old larvae or Zoea 1 to 34 day old post larvae or PL34) and ponds (15 to 159 days old in the pond) were histologically examined for the presence of eosinophilic occlusion bodies in hypertrophied nuclei of the hepatopancreas which is indicative of P. monodon baculovirus infection. The earliest stage found infected in the hatcheries was PL3. Infected shrimp from ponds had slow growth rates and generally pale yellow to reddish brown hepatopancreata. The infection was also characterized by the necrosis and degeneration of the hepatopancreatic tubules with secondary bacterial invasion

Prevalence of monodon baculovirus (MBV) in wild shrimp Penaeus monodon in the Philippines

Prevalence of monodon baculovirus (MBV) was determined using polymerase chain reaction (PCR) on deoxyribonucleic acid (DNA) extracts from the hepatopancreas of the wild black tiger shrimp, Penaeus monodon collected from 7 sampling sites in the Philippines. These sites are considered as the primary sources of broodstock and spawners used for hatchery operations. MBV was detected from all sites except Palawan during the dry season and Negros Occidental and Bohol during wet season. The prevalence of MBV showed no seasonal, sex and size variations. These results show that MBV is an established viral infection in wild populations of Penaeus monodon in the Philippines. Broodstock collected from the contaminated sites could serve as a reservoir of the virus which could infect the post-larvae used to stock in grow-out ponds.This work was supported by the Government of Japan Trust Fund to SEAFDEC AQD under study code GOJ-TF 6251-100-60. The authors wish to thank our fish disease experts and Trust Fund Managers, Drs. Y. Inui, K. Nagasawa, K. Okuzawa and H. Ogata for their valuable support and guidance. Thanks are also due to R. Traviña, M. Peniero and V. Suarnaba for their technical assistance and to S. Alisasis, D. Aragon, M. Uy, R. Dieta, R. Abrera, R. Seballos and B. Lim who gave access to their farms and stations for sampling and provided wild shrimp samples

Prevalence of white spot syndrome virus (WSSV) in wild shrimp Penaeus monodon in the Philippines

Prevalence of white spot syndrome virus (WSSV) was determined using polymerase chain reaction (PCR) methodology on DNA extracted from the gills of wild black tiger shrimp Penaeus monodon collected from 7 sampling sites in the Philippines. These 7 sampling sites are the primary sources of spawners and broodstock for hatchery use. During the dry season, WSSV was detected in shrimp from all sites except Bohol, but during the wet season it was not detected in any site except Palawan. None of the WSSV-PCR positive shrimp showed signs of white spots in the cuticle. Prevalence of WSSV showed seasonal variations, i.e. prevalence in dry season (April to May) was higher than in the wet season (August to October). These results suggest that WSSV has already become established in the local marine environment and in wild populations of P. monodon. Thus, broodstock collected during the dry season could serve as the main source of WSSV contamination in shrimp farms due to vertical transmission of the virus in hatcheries.This study was funded by the Government of Japan Trust Fund to SEAFDEC AQD under the study code GOJ-TF 6251-100-60. Thanks are due to Y. Inui, K. Nagasawa and K. Okuzawa (fish disease experts) for their guidance during the study and to M. Peniero, R. Traviña and V. Suarnaba for their technical assistance. We are very grateful to S. Alisasis, D. Aragon, M. Uy, R. Dieta, R. Abrera, R. Seballos and B. Lim, who gave us access to their farms and stations for sampling, and provided wild shrimp samples

Establishment of preventive measures against viral nervous necrosis (VNN) in finfish hatcheries

The establishment of preventive measures against viral nervous necrosis (VNN) at the finfish hatcheries of SEAFDEC Aquaculture Department in Iloilo, Philippines, was instituted primarily to minimize the impact of the viral infection in cultured stocks. Selection of VNN-negative broodstocks was conducted and the detection rate ranged from 25-56%. It was confirmed that all species of broodstocks are already contaminated with the virus with varying percentages. The combination of cell culture and RT-PCR in the detection of the virus proved to be more sensitive than the RT-PCR alone. The trash fish food was suspected as a main source of contamination and tests prove that most of the trash fish species were contaminated with the virus. The effect of chemical treatment was tested and a good result was obtained by using 5 and 7 ppm iodine. Several recommendations were formulated in the prevention and control of VNN in the hatchery phase: VNN-negative broodstocks should be selected by using cell culture and PCR; spawning of the selected broodstocks should be limited to only six times and these fertilized eggs should be used for larval productions; fertilized eggs should be washed and disinfected with iodine; and commercial broodstock feeds should be used instead of trash fish

Koi herpesvirus-associated mortalities in quarantined koi carp in the Philippines

Illegally imported koi carp were confiscated at the Ninoy Aquino International Airport (NAIA), Manila, Philippines by the Fisheries Quarantine and Inspection Service Officers of the Bureau of Fisheries and Aquatic Resources (BFAR). The confiscated fish were turned over to the BFAR Fish Health Laboratory where they were held for observation at a water temperature of 28 degree C. After 5 days, some fish were showing abnormal swimming behavior and some had died. The most prominent disease signs in the freshly dead and moribund fish were body ulcerations and pale gills showing white necrotic patches, consistent with the clinical signs of KHV infection. Gills were dissected and fixed in 95% ethanol. All of the samples tested positive for KHV in a 1-step PCR assay. This paper reports the first case of KHV associated mortalities in illegally important koi carp confiscated by the Fisheries Quarantine and Inspection Service Officers of BFAR. This highlights the importance of the quarantine and inspection service s role in preventing the illegal entry of fish into the country and the introduction of exotic aquatic diseases.We would like to thank the Fisheries Quarantine and Inspection Service of BFAR and the personnel of the Bureau of Customs for transferring the confiscated koi carp. Thanks are also due to the Fish Health Section staff of the BFAR and SEAFDEC AQD for their technical assistance. We are also grateful to SEAFDEC AQD and to our fish disease experts and GOJ-Trust fund Co-managers: Drs. Y. Inui, K. Nagasawa, K. Okuzawa, H. Ogata and T. Azuma for their financial support and guidance