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Prevalence of the infectious hypodermal and hematopoietic necrosis virus in shrimp (Penaeus vannamei) broodstock in northwestern Mexico
Preventive Veterinary Medicine 117 (2014) 301–304
Contents lists available at ScienceDirect
Preventive Veterinary Medicine journal homepage: www.elsevier.com/locate/prevetmed
Short communication
Prevalence of the infectious hypodermal and hematopoietic necrosis virus in shrimp (Penaeus vannamei) broodstock in northwestern Mexico Fernando Mendoza-Cano a , Tania Enríquez-Espinoza b , Trinidad Encinas-García a , Arturo Sánchez-Paz a,∗ a
Centro de Investigaciones Biológicas del Noroeste (CIBNOR), Campus Hermosillo, Hermosillo, Sonora C.P. 83106, Mexico Departamento de Investigaciones Científicas y Tecnológicas, Universidad de Sonora, Av. Colosio s/n, entre Sahuaripa y Reforma, Hermosillo, Sonora 83000, Mexico b
a r t i c l e
i n f o
Article history: Received 6 May 2014 Received in revised form 19 August 2014 Accepted 18 September 2014 Keywords: IHHNV Penaeus vannamei Broodstock Prevalence Hatcheries Northwestern Mexico
a b s t r a c t The Penaeus stylirostris densovirus (PstDNV or IHHNV) is the smallest of the known shrimp viruses. It causes severe mortalities in juveniles and sub-adults of the blue shrimp Penaeus stylirostris, while specimens of the white shrimp Penaeus vannamei infected by this virus exhibit reduced growth rates and negative effects on the feed-conversion rate (FCR). To date, no descriptive epidemiological surveys on the prevalence of this virus in shrimp broodstock have been performed. In this study, the prevalence of IHHNV in broodstock of the white shrimp P. vannamei from hatcheries on the northwest of Mexico region was estimated. Prevalence vary across different regions from high (63%) to low (6%) in shrimp broodstock. Several factors, as transport of pathogens by human activities, or the absence or implementation of ineffective biosecurity measures, may explain the observed differences. To the best of our knowledge, the present study is the first to examine the prevalence of IHHNV on broodstock. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Global aquaculture production is growing rapidly, and the estimated shrimp production in the Americas accounts for nearly 20% of the global production, being Brazil, Ecuador, Honduras and Mexico the leading producers in the Western Hemisphere (Lightner, 2011). Although Mexico
∗ Corresponding author at: Centro de Investigaciones Biológicas del Noroeste S. C, Laboratorio de Referencia, Análisis y Diagnóstico en Sanidad Acuícola, Hermosa 101, Col. Los Ángeles, Hermosillo, Sonora C. P. 83106, Mexico. Tel.: +52 662 213 15 93l; fax: +52 6622131593. E-mail addresses: [email protected], [email protected] (A. Sánchez-Paz). http://dx.doi.org/10.1016/j.prevetmed.2014.09.006 0167-5877/© 2014 Elsevier B.V. All rights reserved.
has an extensive coastline of 11,122 km, an estimated 90% of the shrimp farms are concentrated along the east coast of the Gulf of California (CONAPESCA, 2012). The Mexican shrimp seed production relies on approximately 20 commercial hatcheries located in Northwestern Mexico, producing up to 10 billion post-larvae annually, which are mainly distributed in Sonora, Sinaloa, Chiapas, Tabasco and Tamaulipas (Fig. 1) (COSAES, 2013; CESASIN, 2012). During the early 1990s, the production of shrimp post-larvae in Latin America was based almost exclusively on the spawning of wild-caught broodstock, however, the occurrence of outbreaks caused by several viruses in shrimp-farming areas led many shrimp hatcheries to develop protocols for a sustainable supply of healthy
302
F. Mendoza-Cano et al. / Preventive Veterinary Medicine 117 (2014) 301–304
Fig. 1. Map of Mexico showing the locations of the five sampled hatcheries (filled circles). In Baja California Sur (BCS) samples were withdrawn from a single hatchery, the samples from Sonora (Son) and Sinaloa (Sin) were obtained from four different hatcheries. The arrows indicate the trade route of shrimp post-larvae.
domesticated stocks; thus, effective biosecurity and health management are considered the most critical of all biological requirements for successful broodstock rearing (Preston et al., 2007). The Penaeus stylirostris densovirus (PstDNV), a nonenveloped icosahedral virus with an average diameter of 22 nm (Bonami et al., 1990), is the smallest of the known penaeid shrimp viruses (Lightner, 2011). PstDNV is also known as infectious hypodermal and hematopoietic necrosis virus (hereinafter referred to as IHHNV) and member of the family Parvoviridae (Mathews, 1982). Several reports have shown that shrimp populations may experience either horizontal or vertical transmission of IHHNV (Tang and Lightner, 2006; Vega-Heredia et al., 2011). Furthermore, IHHNV-infected shrimp Penaeus vannamei exhibit cuticular malformations of the rostrum and reduced growth rates associated to a chronic disease named “Runt Deformity Syndrome” (RDS), which may be associated to an abnormal glycolysis disorder known as Warburg effect (Galván-Alvarez et al., 2012). In addition, studies in P. vannamei show the negative effect on survival and the feed-conversion rate (FCR) of the IHHNV (Singhapan et al., 2004). At present, since no effective treatments against penaeid shrimp viruses are available (Sánchez-Paz et al., 2012), strict and continuous epidemiological surveillance in broodstock has proven to be an invaluable barrier to avoid the dispersion of the Infectious Hypodermal and Hematopoietic Necrosis disease to shrimp farms. Few epidemiological studies have been conducted on farmed aquatic species (Corsin et al., 2002) and studies to determine the frequency of IHHNV in shrimp farms are scarcer. The aim of this study was to estimate the prevalence of
IHHNV in shrimp P. vannamei broodstock, in northwestern Mexico.
2. Materials and methods 2.1. Sample collection and DNA isolation To estimate the presence of IHHNV in shrimp broodstock, 150 organisms were randomly collected on each of 5 different commercial hatcheries identified by letters A through E (Fig. 1), according to the sample size formula of Cannon and Roe (1982) at a 95% confidence interval. This number corresponds to a population of 10,000 individuals per hatchery for an expected prevalence of 2%. A total of 750 individuals of shrimp broodstock (P. vannamei) were collected. Hemolymph samples (100 L) of each organism were withdrawn aseptically from the ventral sinus of each shrimp using a 1 mL syringe (27 gauge) containing 500 L of anticoagulant solution (450 mM NaCl, 10 mM KCl, 20 mM EDTA-Na2, 10 mM HEPES pH 7.3, 850 mOsmkg−1 ) (Vargas-Albores et al., 1993). Hemolymph samples of each laboratory were mixed in a pool from 3 specimens (n = 250 pools) and fixed in 500 L of 70% ethanol. Surveys were conducted during the winter of 2011–2012 in hatcheries located in Baja California Sur, Sonora and Sinaloa. Samples were randomly taken from apparently healthy organisms, before the animals were transferred to the maturation units. Shrimp genomic DNA was isolated by using the silica extraction kit provided with the commercial kit IQ Real Detection and Prevention System (GeneReach
F. Mendoza-Cano et al. / Preventive Veterinary Medicine 117 (2014) 301–304
303
Table 1 Estimated pooled prevalence of IHHNV in shrimp broodstock samples from five different hatcheries from northwestern Mexico. Hatchery
A B C D E Total
Number of pools tested (out of 3)
Number of pools positive (out of 3)
Estimated prevalence (%)
Confidence interval (%)
Std. error
2.5
97.5
50 50 50 50 50
9 33 14 47 27
6 30 10 63 23
3 21 5 46 15
12 41 17 86 32
0.02 0.05 0.03 0.08 0.04
250
130
24
19
28
0.020
Biotechnology Corp.). Genomic DNA was quantified by using a NanoDrop Lite spectrophotometer. 2.2. Primer design, PCR conditions and molecular detection Based on multiple sequence alignment of IHHNV capsid protein sequences, primers IHHNV-C937F (5 -ATT-CAACAA-GAG-CAA-GCC-CAA-G-3 ) and IHHNV-C937R (5 -CATGGT-GCG-TGA-AGA-TAT-TG-3 ) were designed by using the Primer3 software (http://frodo.wi.mit.edu/ primer3/)(Rozen and Skaletsky, 2000) to amplify a 937 bp a fragment of the IHHNV capsid. The PCR products thus obtained were sequenced on both strands using the Big Dye Terminator Cycle Sequencing Kit v3.1 (Applied Biosystems) in the Oligonucleotide Synthesis and Sequencing facilities of the Instituto de Biotecnología de la UNAM with a 16-capillary automated ABI Prism 3130xl Genetic Analyzer (Applied Biosystems). The resulting sequence was compared to existing sequences in GenBank by BLAST (Altschul et al., 1990). 2.3. Estimation of prevalence A total of 250 hemolymph pooled samples from 750 organisms (50 pools per hatchery) were analyzed in this study. Viral prevalence of each commercial hatchery was estimated by using the online pooled prevalence for fixed pool size and tests with known sensitivity and specificity (method 4, which assumes a fixed pool size and that test sensitivity and specificity are known exactly) of the Epitools epidemiological calculators (http://epitools.ausvet. com.au/content.php?page=PPFreq2) (Sergeant, 2014). 3. Results and discussion The current study was conducted to evaluate the prevalence of IHHNV in broodstock of the white shrimp P. vannamei in the northwest of Mexico. Thus, sequence analysis of the amplicon (937 pb) using BLAST showed 100% homology with a DNA fragment of the IHHNV right ORF sequences deposited in GenBank that encodes the capsid protein. In this study, among overall collected pooled samples, 52.0% (130/250) were diagnosed as positive to IHHNV by PCR. Interestingly, nearly all pools from hatchery D amplified positively the gene encoding the IHHNV capsid (47/50), while hatcheries B, E, C and A, the proportion of positive
pools were 33/50, 27/50, 14/50 and 9/50 respectively. Prevalence screening of pooled samples is complicated since it is impossible to determine if a positive result is due to one or more infected organisms. Thus, several methods, as the Minimum Infection Rate and the Frequentist Method (Andreassen et al., 2012), have been developed. In the current study prevalence for pooled samples from different hatcheries was calculated by using the online pooled prevalence calculator of the Epitools site, which provides a measurement of uncertainty in the confidence interval (CI) associated with the apparent prevalence estimation. The highest prevalence value was 63% (95% CI, 46–86%) found in hatchery D, while in hatcheries B, E, C and A, the estimated prevalence were 30% (95% CI 15–32%), 23% (95% CI, 21–41%), 10% (95% CI, 5–17%) and 6% (95% CI 3–12%), respectively (Table 1). Thus, these data show that estimates of IHHNV prevalence vary across different regions from high (>50%), to moderate (10–50%) and low prevalence (1–10%) in shrimp broodstock. A recent study showed that the prevalence of IHHNV in wild shrimp populations of the blue shrimp Penaeus stylirostris had reached almost 100% across the entire Gulf of California by 2005 (Robles-Sikisaka, 2008). Furthermore, a high prevalence of IHHNV in wild crustaceans as P. stylirostris (42%), P. vannamei (40%), Callinectes arcuatus (26%), and P. californiensis (16.6%) was reported (Macías-Rodríguez et al., 2014); however, such results, that may appear as a high prevalence, were based on a too low sample number, limiting its statistical power, reducing thus the statistically certainty of the estimate provided (Button et al., 2013). Thus, these results may represent an initial attempt to define the status of the prevalence of IHHNV in wild populations and they should be followed up carefully. Several factors, as host susceptibility due to life stage (Lightner et al., 1983; Bell and Lightner, 1987), transport of pathogens by human activities or through alternate vectors and hosts, or the absence or implementation of ineffective biosecurity measures, may explain the observed differences in IHHNV prevalence between different regions in northwestern Mexico. However, further studies, which take these variables into account, will need to be undertaken. Similar results were also observed in a study of the prevalence of IHHNV in samples from 26 rearing ponds of seven shrimp farms in northeastern Brazil. The prevalence of this virus in Brazilian ponds ranged from 9.4 to 81%, but no gross signs of RDS were observed, which may suggest that the non-infectious/non-pathogenic form of the virus prevail in this region (Dos Santos-Braz et al., 2009).
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F. Mendoza-Cano et al. / Preventive Veterinary Medicine 117 (2014) 301–304
Prevalence surveys of IHHNV may be considered a particularly useful disease-prevention tool that may help to reduce its deleterious effects. It has been previously recognized that the use of epidemiological principles and logical and science-based approach to identify and manage risks comprising two of the most important components of an effective biosecurity program (Subasinghe, 2005). Several reports have emphasized that IHHNV represents a significant threat to the shrimp health and may also reduce the growth rate of infected shrimp, and may ultimately decrease farm productivity. Molecular studies have detected the presence of IHHNV in ovaries of shrimp females, which reduced significantly the number of nauplii produced by spawn (102,000, against 99,000 nauplii produced by spawning in IHHNV-infected females) (Motte et al., 2003). Thus, such studies highlight the contribution of epidemiological research of IHHNV prevalence to reduce its impact in shrimp broodstock. In this way, culling (the process of removing organisms of a stock based on specific criteria) may become a primary mean contributing to control the spread of IHHNV in shrimp farming facilities, especially considering the lack of effective treatments against this virus. Even if culling remains as a controversial practice, recent studies have demonstrated its positive effects in reducing disease transmission (Yee et al., 2009; Boadella et al., 2012). This work represents the first epidemiological surveillance of virus IHHNV in shrimp P. vannamei broodstock used in northwestern Mexico. Acknowledgments This study was partially supported by Fundación Produce Sonora. We greatly appreciate the valuable help of Dr. Jorge Hernández-López, Diego Galvan Alvarez and Daniel Coronado Molina, staff members of the Laboratorio de Referencia, Analisis y Diagnostico en Sanidad Acuicola by CIBNOR (Hermosillo). References Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic local alignment search tool. J. Mol. Biol. 215, 403–410. Andreassen, A., Jore, S., Cuber, P., Dudman, S., Tengs, T., Isaksen, K., Hygen, H.O., Viljugrein, H., Anestad, G., Ottesen, P., Vainio, K., 2012. Prevalence of tick borne encephalitis virus in tick nymphs in relation to climatic factors on the southern coast of Norway. Parasites Vectors 5, 177. Bell, T.A., Lightner, D.V., 1987. IHHN disease of Penaeus stylirostris: effects of shrimp size on disease expression. J. Fish Dis. 10, 165–170. Boadella, M., Vicente, J., Ruiz-Fons, F., de la Fuente, J., Gortázar, C., 2012. Effects of culling Eurasian wild boar on the prevalence of Mycobacterium bovis and Aujeszky’s disease virus. Prev. Vet. Med. 107, 214–221. Bonami, J.R., Brehelin, M., Mari, J., Trumper, B., Lightner, D.V., 1990. Purification and characterization of IHHN virus of penaeid shrimps. J. Gen. Virol. 71, 2657–2664. Button, K.S., Ioannidis, J.P.A., Mokrysz, C., Nosek, B.A., Flint, J., Robinson, E.S.J., Munafò, M.R., 2013. Power failure: why small sample size undermines the reliability of neuroscience. Nat. Rev. Neurosci. 14 (5), 365–376. Cannon, R.M., Roe, R.T., 1982. Livestock Disease Surveys – A Field Manual for Veterinarians. Australian Government Publishing Service, Canberra, pp. 35.
CESASIN, 2012. Comité Estatal de Sanidad Acuícola de Sinaloa, http:// www.cesasin.com.mx/ CONAPESCA, 2012. Anuario estadístico de acuacultura y pesca. México. Corsin, F., Phi, T.T., Phuoc, L.H., Tinh, N.T.N., Hao, N.V., Mohan, C.V., Turnbull, J.F., Morgan, K.L., 2002. Problems and solutions with the design and execution of an epidemiological study of white spot disease in black tiger shrimp (Penaeus monodon) in Vietnam. Prev. Vet. Med. 53, 117–132. COSAES, 2013. Comité Estatal del Estado de Sonora. Estadísticas, http://www.cosaes.com/ Dos Santos-Braz, R.F., Rodrigues de Oliveira da Silva, C.P., Garcia-Reis, L., Cunha-Martins, P.C., Pereira de Sales, M., Vasconcelos-Meissner, R., 2009. Prevalence of infectious hypodermal and hematopoietic necrosis virus (IHHNV) in Penaeus vannamei cultured in northeastern Brazil. Aquaculture 288, 143–146. Galván-Alvarez, D., Mendoza-Cano, F., Hernández-López, J., Sánchez-Paz, A., 2012. Experimental evidence of metabolic disturbance in the white shrimp Penaeus vannamei induced by the infectious hypodermal and hematopoietic necrosis virus (IHHNV). J. Invertebr. Pathol. 111, 60–67. Lightner, D.V., 2011. Virus diseases of farmed shrimp in the Western Hemisphere (The Americas): a review. J. Invertebr. Pathol. 106, 110–130. Lightner, D.V., Redman, R.M., Bell, T.A., 1983. Infectious hypodermal and hematopoietic necrosis a newly recognized virus disease of penaeid shrimp. J. Invertebr. Pathol. 42, 62–70. ˜ Macías-Rodríguez, N.A., Manón-Ríos, N., Romero-Romero, J.L., CamachoBeltrán, E., Magallanes-Tapia, M.A., Leyva-López, N.E., HernándezLópez, J., Magallón-Barajas, F.J., Perez-Enriquez, R., Sánchez-González, S., Méndez-Lozano, J., 2014. Prevalence of viral pathogens WSSV and IHHNV in wild organisms at the Pacific Coast of Mexico. J. Invertebr. Pathol. 116, 8–12. Mathews, R.E.F., 1982. Classification and nomenclature of viruses. Intervirology 17, 1–199. Motte, E., Yugcha, E., Luzardo, J., Castro, F., Leclercq, G., Rodriguez, J., Miranda, P., Borja, O., Serrano, J., Terreros, M., 2003. Prevention of IHHNV vertical transmission in the white shrimp Litopenaeus vannamei. Aquaculture 219, 57–70. Preston, N., Coman, G., Cowley, J., 2007. Shrimp breeding: knowledge of biological requirements critical to successful broodstock rearing. Glob. Aquac. Advocate, 32–34, July/August. Robles-Sikisaka, R., (Doctoral dissertation) 2008. Population Structure and Genetic Diversity of Litopenaeus stylirostris and its Viral Pathogen, Infectious Hypodermal Hematopoietic Necrosis Virus (IHHNV). Davis and San Diego State University, University of California. Rozen, S., Skaletsky, H., 2000. Primer3 on the WWW for general users and for biologist programmers. Methods Mol. Biol. 132, 365–386. Sánchez-Paz, A., Sotelo-Mundo, R., Muhlia-Almazán, A., 2012. The challenges of developing a treatment that fully protects shrimp against WSSV infection: a perspective. In: Jenkins, O.P. (Ed.), Advances in Zoology Research, Vol. 1. Nova Publishers, St. Louis, Missouri, USA, pp. 1–30. Sergeant, E.S.G., 2014. Epitools Epidemiological Calculators. AusVet Animal Health Services and Australian Biosecurity Cooperative Research Centre for Emerging Infectious Disease. Singhapan, J., Limsuwan, C., Chuchird, N., 2004. Thai study evaluates IHHNV effects on Pacific white shrimp. Glob. Aquac. Advocate, 90, February. Subasinghe, R.P., 2005. Epidemiological approach to aquatic animal health management: opportunities and challenges for developing countries to increase aquatic production through aquaculture. Prev. Vet. Med. 67, 117–124. Tang, K.F.J., Lightner, D.V., 2006. Infectious hypodermal and hematopoietic necrosis virus (IHHNV)-related sequences in the genome of the black tiger prawn Penaeus monodon from Africa and Australia. Virus Res. 118, 185–191. Vargas-Albores, F., Guzman, M.A., Ochoa, J.L., 1993. An anticoagulant solution for haemolymph collection and prophenoloxidase studies of penaeid shrimp (Penaeus californiensis). Comp. Biochem. Physiol. 106A, 299–303. Vega-Heredia, S., Mendoza-Cano, F., Sánchez-Paz, A., 2011. The infectious hypodermal and haematopoietic necrosis virus: a brief review of what we do and do not know. Transbound. Emerg. Dis. 59, 95–105. Yee, K.S., Carpenter, T.E., Cardona, C.J., 2009. Epidemiology of H5N1 avian influenza. Comp. Immunol. Microbiol. Infect. Dis. 32, 325–340.
Contents lists available at ScienceDirect
Preventive Veterinary Medicine journal homepage: www.elsevier.com/locate/prevetmed
Short communication
Prevalence of the infectious hypodermal and hematopoietic necrosis virus in shrimp (Penaeus vannamei) broodstock in northwestern Mexico Fernando Mendoza-Cano a , Tania Enríquez-Espinoza b , Trinidad Encinas-García a , Arturo Sánchez-Paz a,∗ a
Centro de Investigaciones Biológicas del Noroeste (CIBNOR), Campus Hermosillo, Hermosillo, Sonora C.P. 83106, Mexico Departamento de Investigaciones Científicas y Tecnológicas, Universidad de Sonora, Av. Colosio s/n, entre Sahuaripa y Reforma, Hermosillo, Sonora 83000, Mexico b
a r t i c l e
i n f o
Article history: Received 6 May 2014 Received in revised form 19 August 2014 Accepted 18 September 2014 Keywords: IHHNV Penaeus vannamei Broodstock Prevalence Hatcheries Northwestern Mexico
a b s t r a c t The Penaeus stylirostris densovirus (PstDNV or IHHNV) is the smallest of the known shrimp viruses. It causes severe mortalities in juveniles and sub-adults of the blue shrimp Penaeus stylirostris, while specimens of the white shrimp Penaeus vannamei infected by this virus exhibit reduced growth rates and negative effects on the feed-conversion rate (FCR). To date, no descriptive epidemiological surveys on the prevalence of this virus in shrimp broodstock have been performed. In this study, the prevalence of IHHNV in broodstock of the white shrimp P. vannamei from hatcheries on the northwest of Mexico region was estimated. Prevalence vary across different regions from high (63%) to low (6%) in shrimp broodstock. Several factors, as transport of pathogens by human activities, or the absence or implementation of ineffective biosecurity measures, may explain the observed differences. To the best of our knowledge, the present study is the first to examine the prevalence of IHHNV on broodstock. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Global aquaculture production is growing rapidly, and the estimated shrimp production in the Americas accounts for nearly 20% of the global production, being Brazil, Ecuador, Honduras and Mexico the leading producers in the Western Hemisphere (Lightner, 2011). Although Mexico
∗ Corresponding author at: Centro de Investigaciones Biológicas del Noroeste S. C, Laboratorio de Referencia, Análisis y Diagnóstico en Sanidad Acuícola, Hermosa 101, Col. Los Ángeles, Hermosillo, Sonora C. P. 83106, Mexico. Tel.: +52 662 213 15 93l; fax: +52 6622131593. E-mail addresses: [email protected], [email protected] (A. Sánchez-Paz). http://dx.doi.org/10.1016/j.prevetmed.2014.09.006 0167-5877/© 2014 Elsevier B.V. All rights reserved.
has an extensive coastline of 11,122 km, an estimated 90% of the shrimp farms are concentrated along the east coast of the Gulf of California (CONAPESCA, 2012). The Mexican shrimp seed production relies on approximately 20 commercial hatcheries located in Northwestern Mexico, producing up to 10 billion post-larvae annually, which are mainly distributed in Sonora, Sinaloa, Chiapas, Tabasco and Tamaulipas (Fig. 1) (COSAES, 2013; CESASIN, 2012). During the early 1990s, the production of shrimp post-larvae in Latin America was based almost exclusively on the spawning of wild-caught broodstock, however, the occurrence of outbreaks caused by several viruses in shrimp-farming areas led many shrimp hatcheries to develop protocols for a sustainable supply of healthy
302
F. Mendoza-Cano et al. / Preventive Veterinary Medicine 117 (2014) 301–304
Fig. 1. Map of Mexico showing the locations of the five sampled hatcheries (filled circles). In Baja California Sur (BCS) samples were withdrawn from a single hatchery, the samples from Sonora (Son) and Sinaloa (Sin) were obtained from four different hatcheries. The arrows indicate the trade route of shrimp post-larvae.
domesticated stocks; thus, effective biosecurity and health management are considered the most critical of all biological requirements for successful broodstock rearing (Preston et al., 2007). The Penaeus stylirostris densovirus (PstDNV), a nonenveloped icosahedral virus with an average diameter of 22 nm (Bonami et al., 1990), is the smallest of the known penaeid shrimp viruses (Lightner, 2011). PstDNV is also known as infectious hypodermal and hematopoietic necrosis virus (hereinafter referred to as IHHNV) and member of the family Parvoviridae (Mathews, 1982). Several reports have shown that shrimp populations may experience either horizontal or vertical transmission of IHHNV (Tang and Lightner, 2006; Vega-Heredia et al., 2011). Furthermore, IHHNV-infected shrimp Penaeus vannamei exhibit cuticular malformations of the rostrum and reduced growth rates associated to a chronic disease named “Runt Deformity Syndrome” (RDS), which may be associated to an abnormal glycolysis disorder known as Warburg effect (Galván-Alvarez et al., 2012). In addition, studies in P. vannamei show the negative effect on survival and the feed-conversion rate (FCR) of the IHHNV (Singhapan et al., 2004). At present, since no effective treatments against penaeid shrimp viruses are available (Sánchez-Paz et al., 2012), strict and continuous epidemiological surveillance in broodstock has proven to be an invaluable barrier to avoid the dispersion of the Infectious Hypodermal and Hematopoietic Necrosis disease to shrimp farms. Few epidemiological studies have been conducted on farmed aquatic species (Corsin et al., 2002) and studies to determine the frequency of IHHNV in shrimp farms are scarcer. The aim of this study was to estimate the prevalence of
IHHNV in shrimp P. vannamei broodstock, in northwestern Mexico.
2. Materials and methods 2.1. Sample collection and DNA isolation To estimate the presence of IHHNV in shrimp broodstock, 150 organisms were randomly collected on each of 5 different commercial hatcheries identified by letters A through E (Fig. 1), according to the sample size formula of Cannon and Roe (1982) at a 95% confidence interval. This number corresponds to a population of 10,000 individuals per hatchery for an expected prevalence of 2%. A total of 750 individuals of shrimp broodstock (P. vannamei) were collected. Hemolymph samples (100 L) of each organism were withdrawn aseptically from the ventral sinus of each shrimp using a 1 mL syringe (27 gauge) containing 500 L of anticoagulant solution (450 mM NaCl, 10 mM KCl, 20 mM EDTA-Na2, 10 mM HEPES pH 7.3, 850 mOsmkg−1 ) (Vargas-Albores et al., 1993). Hemolymph samples of each laboratory were mixed in a pool from 3 specimens (n = 250 pools) and fixed in 500 L of 70% ethanol. Surveys were conducted during the winter of 2011–2012 in hatcheries located in Baja California Sur, Sonora and Sinaloa. Samples were randomly taken from apparently healthy organisms, before the animals were transferred to the maturation units. Shrimp genomic DNA was isolated by using the silica extraction kit provided with the commercial kit IQ Real Detection and Prevention System (GeneReach
F. Mendoza-Cano et al. / Preventive Veterinary Medicine 117 (2014) 301–304
303
Table 1 Estimated pooled prevalence of IHHNV in shrimp broodstock samples from five different hatcheries from northwestern Mexico. Hatchery
A B C D E Total
Number of pools tested (out of 3)
Number of pools positive (out of 3)
Estimated prevalence (%)
Confidence interval (%)
Std. error
2.5
97.5
50 50 50 50 50
9 33 14 47 27
6 30 10 63 23
3 21 5 46 15
12 41 17 86 32
0.02 0.05 0.03 0.08 0.04
250
130
24
19
28
0.020
Biotechnology Corp.). Genomic DNA was quantified by using a NanoDrop Lite spectrophotometer. 2.2. Primer design, PCR conditions and molecular detection Based on multiple sequence alignment of IHHNV capsid protein sequences, primers IHHNV-C937F (5 -ATT-CAACAA-GAG-CAA-GCC-CAA-G-3 ) and IHHNV-C937R (5 -CATGGT-GCG-TGA-AGA-TAT-TG-3 ) were designed by using the Primer3 software (http://frodo.wi.mit.edu/ primer3/)(Rozen and Skaletsky, 2000) to amplify a 937 bp a fragment of the IHHNV capsid. The PCR products thus obtained were sequenced on both strands using the Big Dye Terminator Cycle Sequencing Kit v3.1 (Applied Biosystems) in the Oligonucleotide Synthesis and Sequencing facilities of the Instituto de Biotecnología de la UNAM with a 16-capillary automated ABI Prism 3130xl Genetic Analyzer (Applied Biosystems). The resulting sequence was compared to existing sequences in GenBank by BLAST (Altschul et al., 1990). 2.3. Estimation of prevalence A total of 250 hemolymph pooled samples from 750 organisms (50 pools per hatchery) were analyzed in this study. Viral prevalence of each commercial hatchery was estimated by using the online pooled prevalence for fixed pool size and tests with known sensitivity and specificity (method 4, which assumes a fixed pool size and that test sensitivity and specificity are known exactly) of the Epitools epidemiological calculators (http://epitools.ausvet. com.au/content.php?page=PPFreq2) (Sergeant, 2014). 3. Results and discussion The current study was conducted to evaluate the prevalence of IHHNV in broodstock of the white shrimp P. vannamei in the northwest of Mexico. Thus, sequence analysis of the amplicon (937 pb) using BLAST showed 100% homology with a DNA fragment of the IHHNV right ORF sequences deposited in GenBank that encodes the capsid protein. In this study, among overall collected pooled samples, 52.0% (130/250) were diagnosed as positive to IHHNV by PCR. Interestingly, nearly all pools from hatchery D amplified positively the gene encoding the IHHNV capsid (47/50), while hatcheries B, E, C and A, the proportion of positive
pools were 33/50, 27/50, 14/50 and 9/50 respectively. Prevalence screening of pooled samples is complicated since it is impossible to determine if a positive result is due to one or more infected organisms. Thus, several methods, as the Minimum Infection Rate and the Frequentist Method (Andreassen et al., 2012), have been developed. In the current study prevalence for pooled samples from different hatcheries was calculated by using the online pooled prevalence calculator of the Epitools site, which provides a measurement of uncertainty in the confidence interval (CI) associated with the apparent prevalence estimation. The highest prevalence value was 63% (95% CI, 46–86%) found in hatchery D, while in hatcheries B, E, C and A, the estimated prevalence were 30% (95% CI 15–32%), 23% (95% CI, 21–41%), 10% (95% CI, 5–17%) and 6% (95% CI 3–12%), respectively (Table 1). Thus, these data show that estimates of IHHNV prevalence vary across different regions from high (>50%), to moderate (10–50%) and low prevalence (1–10%) in shrimp broodstock. A recent study showed that the prevalence of IHHNV in wild shrimp populations of the blue shrimp Penaeus stylirostris had reached almost 100% across the entire Gulf of California by 2005 (Robles-Sikisaka, 2008). Furthermore, a high prevalence of IHHNV in wild crustaceans as P. stylirostris (42%), P. vannamei (40%), Callinectes arcuatus (26%), and P. californiensis (16.6%) was reported (Macías-Rodríguez et al., 2014); however, such results, that may appear as a high prevalence, were based on a too low sample number, limiting its statistical power, reducing thus the statistically certainty of the estimate provided (Button et al., 2013). Thus, these results may represent an initial attempt to define the status of the prevalence of IHHNV in wild populations and they should be followed up carefully. Several factors, as host susceptibility due to life stage (Lightner et al., 1983; Bell and Lightner, 1987), transport of pathogens by human activities or through alternate vectors and hosts, or the absence or implementation of ineffective biosecurity measures, may explain the observed differences in IHHNV prevalence between different regions in northwestern Mexico. However, further studies, which take these variables into account, will need to be undertaken. Similar results were also observed in a study of the prevalence of IHHNV in samples from 26 rearing ponds of seven shrimp farms in northeastern Brazil. The prevalence of this virus in Brazilian ponds ranged from 9.4 to 81%, but no gross signs of RDS were observed, which may suggest that the non-infectious/non-pathogenic form of the virus prevail in this region (Dos Santos-Braz et al., 2009).
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Prevalence surveys of IHHNV may be considered a particularly useful disease-prevention tool that may help to reduce its deleterious effects. It has been previously recognized that the use of epidemiological principles and logical and science-based approach to identify and manage risks comprising two of the most important components of an effective biosecurity program (Subasinghe, 2005). Several reports have emphasized that IHHNV represents a significant threat to the shrimp health and may also reduce the growth rate of infected shrimp, and may ultimately decrease farm productivity. Molecular studies have detected the presence of IHHNV in ovaries of shrimp females, which reduced significantly the number of nauplii produced by spawn (102,000, against 99,000 nauplii produced by spawning in IHHNV-infected females) (Motte et al., 2003). Thus, such studies highlight the contribution of epidemiological research of IHHNV prevalence to reduce its impact in shrimp broodstock. In this way, culling (the process of removing organisms of a stock based on specific criteria) may become a primary mean contributing to control the spread of IHHNV in shrimp farming facilities, especially considering the lack of effective treatments against this virus. Even if culling remains as a controversial practice, recent studies have demonstrated its positive effects in reducing disease transmission (Yee et al., 2009; Boadella et al., 2012). This work represents the first epidemiological surveillance of virus IHHNV in shrimp P. vannamei broodstock used in northwestern Mexico. Acknowledgments This study was partially supported by Fundación Produce Sonora. We greatly appreciate the valuable help of Dr. Jorge Hernández-López, Diego Galvan Alvarez and Daniel Coronado Molina, staff members of the Laboratorio de Referencia, Analisis y Diagnostico en Sanidad Acuicola by CIBNOR (Hermosillo). References Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic local alignment search tool. J. Mol. Biol. 215, 403–410. Andreassen, A., Jore, S., Cuber, P., Dudman, S., Tengs, T., Isaksen, K., Hygen, H.O., Viljugrein, H., Anestad, G., Ottesen, P., Vainio, K., 2012. Prevalence of tick borne encephalitis virus in tick nymphs in relation to climatic factors on the southern coast of Norway. Parasites Vectors 5, 177. Bell, T.A., Lightner, D.V., 1987. IHHN disease of Penaeus stylirostris: effects of shrimp size on disease expression. J. Fish Dis. 10, 165–170. Boadella, M., Vicente, J., Ruiz-Fons, F., de la Fuente, J., Gortázar, C., 2012. Effects of culling Eurasian wild boar on the prevalence of Mycobacterium bovis and Aujeszky’s disease virus. Prev. Vet. Med. 107, 214–221. Bonami, J.R., Brehelin, M., Mari, J., Trumper, B., Lightner, D.V., 1990. Purification and characterization of IHHN virus of penaeid shrimps. J. Gen. Virol. 71, 2657–2664. Button, K.S., Ioannidis, J.P.A., Mokrysz, C., Nosek, B.A., Flint, J., Robinson, E.S.J., Munafò, M.R., 2013. Power failure: why small sample size undermines the reliability of neuroscience. Nat. Rev. Neurosci. 14 (5), 365–376. Cannon, R.M., Roe, R.T., 1982. Livestock Disease Surveys – A Field Manual for Veterinarians. Australian Government Publishing Service, Canberra, pp. 35.
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