Prevalence of monodon baculovirus in wild shrimp Penaeus monodon in the Phils

Leobert D. de la Peña*, Celia R. Lavilla-Pitogo, Corina Belle R. Villar, Milagros G. Paner and Geimbo C. Capulos

Southeast Asian Fisheries Development Center, Aquaculture Department
Tigbauan 5021, Iloilo, Philippines

Viral diseases are considered as a major problem in shrimp aquaculture industry in the Philippines and have caused severe losses to the shrimp farmers. Lightner and Redman (1998) reported that there are nearly 20 viral pathogens that can cause serious epizootics in farmed shrimp. Among the economically important shrimp viral pathogens is monodon baculovirus (MBV). The importance of MBV in shrimp culture is exemplified by problems related to infection that is usually encountered in hatchery and grow-out operations. MBV should be eliminated in the culture system because its infection can slow the growth, especially in intensive culture systems thereby decreasing productivity (Fegan et al., 1991; Flegel, 2006). MBV was first described in cultured black tiger shrimp in Taiwan and other MBV-like baculoviruses have been described for a number of penaeid species in most parts of the Indo-Pacific region where penaeid shrimp are cultured. MBV is a nuclear polyhedrosis virus of the family Baculoviridae composed of a double-stranded, circular DNA genome of 80 - 100 × 106 Da within a rod-shaped, enveloped particle often found occluded within proteinaceous bodies of polyhedrin proteins (Rohrmann, 1986; Belcher and Young, 1998). Viral replication takes place in cell nuclei where polyhedral occlusion bodies are formed (Lightner and Redman, 1981). MBV infects the hepatopancreatic and midgut epithelial cells. Viral transmission occurs via ingestion of free virus, occlusion bodies and by cannibalism (Fegan et al., 1991). Studies on MBV were mostly conducted in the cultured penaeid shrimp populations. This study resulted from sampling of shrimp to determine the prevalence of MBV in the wild populations. The observation of continued occurrence of MBV in hatcheries is an outcome of infection in broodstock.

Shrimp samples were collected from 7 sites that serve as the primary sources of broodstock and spawners used for hatchery operations. The 7 sites are: Quezon and Palawan for Luzon area; Capiz, Negros Occidental, and Bohol for Visayas; and Surigao del Sur and Misamis Occidental for Mindanao. Portions of the hepatopancreas were individually and aseptically dissected, fixed in 95% ethanol and stored at 4ºC until DNA extraction. Genomic DNA was extracted from the hepatopancreas tissue using DNAzol Reagent (MRC, USA) following the manufacturer’s instruction.

Detection of the virus was done using polymerase chain reaction (PCR) based on the work of Belcher and Young (1998) with modifications. The primary PCR yielded a 533bp amplicon while the nested step generated a 381 bp amplicon, respectively.

Statistical analyses were done using statistical program for the social sciences (SPSS ver. 10.0). A t-test was carried out to determine if there is a significant difference in the prevalence between dry and wet seasons, male and female sexes and between big and small size categories.

A total of 1,427 shrimp were collected, individually weighed and classified into small and big size categories based on the median measurements in their weights. Average body weight (ABW) was 87 g and 116 g in the dry and wet season, respectively. Shrimp collected during the dry season were significantly smaller than that of the wet season (p0.05) in the mean prevalence between seasons.

Table 1. Prevalence of MBV in wild P. monodon in the Philippines

	Season
Sampling site	Dry (%)	Wet (%)
Luzon
Quezon	18/103 (17%)	11/106 (10%)
Palawan	0/102 (0%)	5/103 (5%)
Sub-total	18/205 (9%)	16/209 (8%)
Visayas
Capiz	34/100 (34%)	12/103 (12%)
Negros Occidental	22/101 (22%)	0/101 (0%)
Bohol	2/102 (2%)	0/100 (0%)
Sub-total	58/303 (19%)	12/304 (4%)
Mindanao
Misamis Occidental	33/100 (33%)	25/100 (25%)
Surigao del Sur	31/105 (30%)	8/101 (8%)
Sub-total	64/205 (31%)	33/201 (16%)
Grand Total	140/713 (20%)	61/714 (9%)

Table 2 gives comparison of the prevalence between male and female wild P. monodon during the dry and wet season samplings. Although the prevalence for females (20% and 9%) was observed to be higher than in males (18% and 6%) for both dry and wet season, the difference was not statistically significant (p>0.05).

Table 2. Prevalence of MBV in male and female wild P. monodon in the Philippines.

	Dry season		Wet season
Sampling site	Male (%)	Female (%)	Male (%)	Female (%)
Luzon
Quezon	7/40 (18%)	11/63 (17%)	2/23 (9%)	9/83 (11%)
Palawan	0/44 (0%)	0/58 (0%)	1/39 (3%)	4/64 (6%)
Visayas
Capiz	15/50 (30%)	19/50 (38%)	1/31 (3%)	11/72 (15%)
Negros Occidental	13/50 (26%)	9/51 (18%)	0/16 (0%)	0/85 (0%)
Bohol	1/48 (2%)	1/54 (2%)	0/29 (0%)	0/71 (0%)
Mindanao
Misamis Occidental	8/19 (42%)	25/81 (31%)	2/8 (25%)	23/92 (25%)
Surigao del Sur	5/14 (36%)	26/91 (29%)	5/29 (17%)	3/72 (4%)
Total	49/265 (18%)	91/448 (20%)	11/175 (6%)	50/539 (9%)

The prevalence on small and big size shrimp was examined in order to determine if there is a significant size effect on the prevalence and this is shown in Table 3. Although the prevalence in both seasons was relatively higher in larger shrimp (23% and 10%) than in smaller ones (18% and 7%), the results showed no significant difference (p>0.05).

Table 3. Prevalence of MBV in small (12-102 g) and big (103-266 g) wild P. monodon in the Philippines.

	Dry season		Wet season
Sampling site	Small (%)	Big (%)	Small (%)	Big (%)
Luzon
Quezon	11/62 (18%)	7/41 (17%)	3/27 (11%)	8/79 (10%)
Palawan	0/97 (0%)	0/5 (0%)	4/90 (4%)	1/13 (8%)
Visayas
Capiz	13/48 (27%)	21/52 (40%)	1/26 (4%)	11/77 (14%)
Negros Occidental	21/85 (25%)	1/16 (6%)	0/16 (0%)	0/85 (0%)
Bohol	1/71 (1%)	1/31 (3%)	0/46 (0%)	0/54 (0%)
Mindanao
Misamis Occidental	13/34 (38%)	20/66 (30%)	3/12 (25%)	22/88 (25%)
Surigao del Sur	27/77 (35%)	4/28 (14%)	7/47 (15%)	1/54 (2%)
Total	86/474 (18%)	54/239 (23%)	18/264 (7%)	43/450 (10%)

This study confirms the presence of MBV in wild P. monodon broodstock and spawners populations in the Philippines. These broodstock and spawners could serve as carrier for the vertical and horizontal transmissions of the virus in hatcheries. MBV is a common infectious agent in wild shrimp and is considered as endemic in cultured P. monodon in the Philippines (Baticados et al., 1991).

In all samples that were tested, positive results were obtained only after the nested step PCR. This suggests a low viral load in the samples and consequently, in wild populations of P. monodon which may serve as carriers for the vertical and horizontal transmissions of the virus in the hatchery phase. Infection may be expressed when these carriers are exposed to stress such as spawning. Interestingly, our results show no seasonal, sex and size variations in MBV prevalence.

Our results show that MBV is an established viral infection in wild populations of P. monodon in the Philippines. Thus, broodstock collected from the contaminated sites could serve as a reservoir of the virus, which bring infection into the hatchery and could infect the post-larvae that are used to stock in grow-out ponds. Based on our findings, we suggest that the collection of the brooders should be done in areas that are not yet contaminated by the virus such as the provinces of Negros Occidental and Bohol during the wet season and Palawan during the dry season. Alternatively, disinfection of newly spawned eggs should be practiced to prevent horizontal and vertical transmissions after spawning.

Acknowledgements

This work was supported by the Government of Japan Trust Fund to SEAFDEC AQD. 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 stations for sampling and provided wild shrimp samples.

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