A histopathological survey of wild and cultured shrimp from the Gulf of Mexico was carried out to determine the prevalence and severity of infectious diseases and parasites, affecting four species of penaeid shrimp (Litopenaeus setiferus, Farfantepenaeus aztecus, F. duorarum, and L. vannamei). Around 60 shrimp were obtained from each of 10 sampling stations during different months in 1999 and 2000. One station was a shrimp culture farm from Tamaulipas State, and nine were wild stations from Tamaulipas (five), Veracruz (one), and Campeche (three) States. Thirty shrimp from each station were used for histological analysis. The remaining shrimp were frozen and kept at −20 C to carry out PCR techniques to determine the presence of white spot syndrome virus (WSSV). Some in situ hybridization analyses were applied to ascertain cases of WSS viral disease. Fresh analysis was carried out in shrimp from only one station for parasite identification. The histopathological survey performed on 688 shrimp showed no evidence of viral diseases. Two shrimp from one station were histologically diagnosed as having presumptive WSSV. Tissues from the same shrimp were processed for in situ hybridization and another 20 samples from the same station were processed to run PCR techniques. In each case, the results were negative. The same PCR procedure was applied to the rest of the frozen samples and none of them showed the presence of the WSSV. No important bacterial infections were observed in any of the wild or cultured shrimp analyzed. Fresh analysis demonstrated the presence of the cestode Prochristianella hispida and the gregarines Cephalolobus penaeus and Nematopsis penaeus. Histological analysis showed that the abundance of P. hispida varied from 7 to 90%, and G1 to G4 grades of severity were observed, with G1 being the most common. Gregarines were commonly observed in the histological slides, with a prevalence varying from 3 to 56% and G1 severity in most cases. Haplosporidan infection was observed in one sample of F. duorarum, which is the first report of this parasite in this host. Two invasive epibionts, the filamentous bacteria Leucothrix sp. and the ciliate Ascophrys sp., were found in four sampled stations. The study showed that in ten stations sampled from 1999 to 2000 in the Gulf of Mexico, shrimp populations had no serious infectious diseases.

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Counts oí' viable heterotrophic bacteria. C'ihrio-like bacteria and total colilòrms were perlòrmed in sca\+ater supply. l'ood stocks and broodstock pools at tuo aquacultural facilities located in Mexico. '1 hc hiph abundance of these bacteria1 groups indicates risl\ areas that must bc monitored to control potential pathogens. such as I 'ihrio spp. I-iltering systems and food supplies, such as Artenw spp. and í'rcsh or frozen sca tòod. wre found to bc the most important sourccs oïpotcntial pathogcns. /L¿,J, wo,-tk: I'rhrio. marine bacteria. shrimp hatcheries. hatchery watcr quality. RESUMEN I:n dos laboratorios situados cn la República Mrxicana. SC rcali/.aron conteos dc bacterias hcterótrofas viables. bacterias tipo I'ibrio y coliformes totales en muestras de agua del sistema de aprovicionamiento. cn alimentos marinos y en estanques dc reproductores. Las altas concentraciones de estos grupos bacterianos indican la existencia de puntos de riesgo. los cuales deben ser vigilados sistemáticamcnte para poder controlar patógenos potenciales. tales como lïhrio spp. En cstc traba.@, se encontró que los sistemas de filtración 5 el alimento utilizado. tal como Artemio spp. y orgamsmos marinos Frescos o conpclados. son las prmcipales fuentes de patógenos potenciales en los laboratorios dc acuicultura estudiados. /~~r/&w.s C/CIW: I 'lhrco. bacterias marinas. criaderos de camarón. calidad del agua del criadero.

To determine if exposure to a sublethal mixture of metals (Cd, Cu, Fe, Mn, Pb and Zn) increases susceptibility to White spot syndrome virus (WSSV) infection, Litopenaeus vannamei juveniles were fed WSSV-infected shrimp tissues after 21 days of exposure to the metal mixture (WS-MM treatment). Other treatments consisted of shrimp not exposed to metals and fed infected tissues (WS), and shrimp fed healthy tissues and exposed (MM) or not exposed to metals (C). The presence of viral DNA and inclusion bodies was detected at 32 hr postinfection (hpi) in the stomach epithelium of shrimp from the WS treatment, and eight hours later in shrimp from the WS-MM treatment, possibly because of an initial negative effect of metals in viral replication. At 40 hpi, the severity of infection represented by the histopathological index increased in both WS and WS-MM treatments, and values were higher in WS-MM than in WS shrimp at the end of the experiment. From 56 hpi to the end of experiment, total hemocyte counts were lower in both WS-MM and WS treatments, and concentrations were particularly low in WS-MM shrimp. Conversely, phenoloxidase activity was higher in the WS-MM treatment from 32 to 56 hpi, suggesting a possible role of the prophenoloxidase (proPO) system in the antiviral defense against WSSV. The presence of heavy metals at sublethal concentrations may increase shrimp susceptibility to WSSV; this is supported by a decrease in circulating hemocytes, an increase in the humoral response, and the development of a higher number of WSSV inclusion bodies. K E Y W O R D S heavy metals, immune responses, Litopenaeus vannamei, proPO activity, WSSV

White spot disease (WSD) causes high mortality in cultured shrimp throughout the world. Its etiologic agent is the white spot syndrome virus (WSSV). The genomic repeat regions ORF 75, ORF 94, and ORF 125 have been used to classify WSSV isolates in epidemiological studies using PCR with specific primers and sequencing. The present study investigated the variation in nucleotide sequences from 107, 150, and 143 isolates of WSSV collected from Litopenaeus vannamei shrimp ponds with WSD outbreaks in northwestern Mexico during the period 2010−2012, in the genomic repeat regions ORFs 75, 94, and 125, respectively. The haplotypic nomenclature for each isolate was based on the number of repeat units and the position of single nucleotide polymorphisms on each ORF. We report finding 17, 43, and 66 haplotypes of ORFs 75, 94, and 125, respectively. The study found high haplotypic diversity in WSSV using the complete sequences of ORFs 94 and 125 as independent variables, but low haplotypic diversity for ORF 75. Different haplotypes of WSSV were found from region-to-region and year-to-year, though some individual haplotypes were found in different places and in more than one growing cycle. While these results suggest a high rate of mutation of the viral genome at these loci, or perhaps the introduction of new viral strains into the area, they are useful as a tool for epidemiological surveys. Two haplotypes from some of the ORFs in the same shrimp were encountered, suggesting the possibility of multiple infections.