Davidson's Fixative Research Articles

The senile plaque: Morphological differences in APP knock-in mice brains by fixatives.

The morphology of senile plaques depends on the APP knock-in mice brain fixative. Solid forms of senile plaques were detected in APP knock-in mice after formic acid treatment with Davidson's and Bouin's fluid fixative as the brain of AD patients. Aβ42 was deposited as cored plaques and Aβ38 accumulated around Aβ42.

Temporal Sequence of Post-Mortem Autolysis in the Mouse Retina

In order to better differentiate ante-mortem lesions from post-mortem retinal autolysis, the temporal sequence of post-mortem changes was studied in a well-controlled mouse model. Mice were of the same strain, age and sex, and were held at a constant ambient temperature. Eyes were collected at various times up to 72h after death and immersion-fixed in either Davidson's fixative or 10% neutral buffered formalin, paraffin-embedded and sections cut and stained with haematoxylin and eosin. The most prominent, and early, autolytic change was retinal detachment, and subsequent folding, which occurred immediately after death in formalin-fixed eyes, but not until 2h post mortem with Davidson's fixative. Retinal separation was complete at 16h, or almost complete by 2h, in formalin, but in Davidson's fixative, was only partial and segmental, the latter not becoming total until much later. Retinal detachment was attended by progressively more severe disruption and dissolution of photoreceptors and, particularly in Davidson's-fixed retinas, the rod outer segment often showed marked homogenization from 30min to 4h after death. The other major early change was nuclear pyknosis in the inner nuclear layer. Ganglion cells initially had cytoplasmic swelling, followed by shrinkage and basophilia (at 4h with formalin and 16h with Davidson's), with nuclear pyknosis becoming increasingly common over time. While the three retinal neuronal layers eventually became more attenuated and depleted of cells, the thickness of these layers was augmented by severe swelling. These findings show that the post-mortem interval at which histological interpretation of retinal changes becomes potentially compromised is dependent on the duration of this interval and the fixative used.

Prenatal developmental study of the ovaries of the Baladi rabbits (oryctolagus cuniculus)

This study was carried out to find out the histological changes of the ovaries of the Baladi rabbits at different prenatal periods. A hundred Baladi rabbit embryos and fetuses were used in this study. The pregnant rabbits were euthanized, dissected and, subsequently, the embryos and fetuses were collected. The samples were fixed with modified Davidson's fixative and 10% neutral buffered formalin for 24 to 48 hours and then subjected to routine paraffin embedding technique. They sections were stained by hematoxylin-eosin, Crossman’s trichrome and Gomori's reticulin stains. Stained sections were examined by light microscope. The study revealed that the ovarian differentiation with the appearance of its cortex and medulla occurred at the 19th day postconception. The oogonial nests were observed within the ovarian cortex at the 24th day of fetal rabbit life. The primitive tunica albuginea ovarii could be recognized at the 28th day postconception. No primordial follicle occurred throughout the fetal rabbit life.

Optimization of fixative solution for retinal morphology: a comparison with Davidson's fixative and other fixation solutions.

Numerous fixative solutions are available but many are not amenable to the histomorphological preservation of retinae. The investigators specifically focused on retinal histological studies, which rather than 4% formaldehyde (FA), often use Davidson's fixative. However the latter has its limitations. The purpose of this study was to produce a new fixative which maintains retinae closer to the in vivo conditions. Experimental design. Four fixative formulations (4% paraformaldehyde, Davidson's fixative, modified Davidson's fixative and an in-house fixative - TB-Fix) were tested on retinae and the outcomes on histomorphology and immunohistochemical staining for selected antigenic markers was compared. TB-Fix markedly improved morphological detail following hematoxylin and eosin staining, most importantly eliminating the spongiform appearance in the plexiform layer and the swelling of somata (including Müller cells), when compared to FA, Davidson's fixative and its modified version. Retinal samples fixed with TB-Fix or FA showed comparable results in immunohistological staining for neurons and glia in the retina. Importantly, while the whole eye fixed with FA collapsed in shape and induced artificial retinal detachment, the eye fixed with TB-Fix avoided deformation and detachment. Furthermore, we found that TB-Fix also prevented detachment from the culture plate when used to fix HEK293 cells, which are known to detach from the plate easily. It was demonstrated that TB-Fix provides an overall improvement in the preservation of retinal morphology and chemical composition.

The Morphogenesis of the Sexually Indifferent Gonads in the Rabbit

The present study aimed to point out the prenatal developmental stages of the indifferent gonads in rabbit. About 50 embryos and fetuses aged 11 to 18 days postcoitum were used in this study. The embryos of 9 days-old up to 13 days-old fetuses were fixed firstly within the uterus by using modified Davidson's fixative and then were transferred with the caudal halves of the fetuses aged from 14 to 18 days postconception into 10% Neutral buffered formalin for 24 to 48 hours. The specimens were prepared using routine histological work. The morphogenesis of the sexually indifferent gonad of the rabbit was studied by high-resolution light microscopy. The results showed that the gonadal ridges were detected at the 12th day postconception as a cellular thickening covered by coelomic epithelium on either sides of the dorsal mesentery and attached to the developing pronephros. As the development progressed the gonadal ridge increased in size and cellular constituents and became housing blood vessels and germ cells. The current work concluded that the gonads of the rabbit remained indifferent and neither testis nor ovary was observed until the 17th day postconception.

Effect of Aeromonas hydrophila infection with high and normal water temperature on total hemocyte count, total plasma protein and histopathological differences in freshwater crayfish Astacus leptodactylus

In this study, the effect of water temperature on pathogenesis of Aeromonas hydrophila in freshwater Crayfish Astacus leptodactylus were investigated. In our study, The treatments Crayfish have exposed to A hydrophila with concentration of 3×10 6 CFUml -1 in high water temperature (25 ˚ C) and normal water temperature(10-15 ċ ). The untreated control groups in high water temperature (25 ˚ C) and normal water temperature (10-15 ˚ C) were disinfected by oxytetracyclin at concentration of 100 ppm for 24 hours. The haemolymph samples were withdrawn for measuring of THC and TPP. For histopathological studie, the Crayfish samples were fixed in Davidson fixative. The results showed that the value of THC in control group1 in comparing with control group2 and two other treatments at 48h after experiment time were significant (p<0.05). Also the difference of THC value between control group 2 with treatment group2 at 144h after experiment time was significant (p<0.05). The finding of TPP value showed that there was significant difference between control group1 and treatment4 in last time after experiment time (336h) (p<0.05). In infected Crayfish to bacteria with high temperature, red and black spots and also wound were observed. The result of histopathology in heptopancreas, gill, heart and digestive tract showed hemocyte aggregation, necrotized tissue, dead cells within pyknosis and karyorrehexis of nucleus and vaculation of hepatopancreas. In treatment group1 and control group1 no pathological changes of heart were observed. In digestive tract no changes were appeared in treatment1 and two others control group, but aggregation of hemocyte with pyknosis of nuclei was revealed in concentration of 3×10 6 CFUml -1 with high temperature.

A Comparison of Two Methods for Colorimetric in situ Hybridization Using Paraffin‐Embedded Tissue Sections and Digoxigenin‐Labeled Hybridization Probes

Two methods for colorimetric in situ DNA probe hybridization (CISH) assays on paraffin-embedded tissue sections were compared. The heated method used heat (90-100°C) to denature DNA in the sample prior to probe hybridization, while the unheated method used a standard hybridization temperature of 42°C. Both procedures were tested on tissue samples that harbored the mollusk protozoan pathogens Perkinsus marinus, P. chesapeaki, or Haplosporidium nelsoni, the protozoan and bacterial fish pathogens Myxobolus cerebralis (myxosporidean) or Renibacterium salmoninarum (bacterial), or the crab viral pathogen Callinectes sapidus reovirus. Samples were fixed in either formalin or Davidson's fixative and embedded in paraffin for histological examination. The heated method is labor intensive and highly prone to human error, while the unheated method is less labor intensive and can be completed in a shorter period of time. Both methods yielded similar hybridization results. The use of complex and expensive prehybridization buffers did not improve the performances of the tested CISH assays. Prehybridization heat denaturation of DNA in assayed samples increased both assay duration and loss of samples but did not improve hybridization signals.

Report of significant WSSV-resistance in the Pacific white shrimp, Litopenaeus vannamei, from a Panamanian breeding program

Three Litopenaeus vannamei families, from a breeding program in Panama and with possible WSSV resistance, were challenged per os with a reference isolate of White spot syndrome virus originally obtained from China in 1995 (WSSV-CN95). These F8, F9 and F12 generation families were developed from founder stocks a decade ago and were survivors of white spot disease. Juvenile shrimp used for WSSV challenge averaged 1.5g, and they were stocked at 50 to 96 animals per tank into nine 1000L fiberglass tanks containing artificial seawater at 30ppt salinity and 26°C. Three of the 1000L tanks were used as negative control tanks, with one tank for each family. Six 1000L tanks were used for challenging the three families with WSSV, with two replicate tanks for each family. A positive control consisting of 20 “Kona” SPF reference line L. vannamei (average weight 1.5g) was included and challenged with WSSV in a 90L glass aquarium. The Kona stock was fed the same batch of WSSV infected tissue as the three Panamanian families to confirm infectivity and to provide a basis with which to compare final survival. WSSV infected minced frozen shrimp tissue was fed at a rate of 5% of average body weight one time on day 0. All tanks were equipped with air diffusers to provide sufficient aeration and an acclimated crushed oyster shell internal recirculating biological filter. Each tank was covered with a plastic sheet to contain aerosols and minimize water temperature fluctuations. The experimental tanks were checked daily and moribund animals were collected when observed and preserved in Davidson's AFA fixative. Mortalities in the three Panamian families ceased at 17days post challenge. Two survivors from each tank were preserved for histology and five shrimp per tank were individually tested by qPCR to determine their WSSV status and viral load. Survival at termination in the negative control families was 95%, 98% and 100%. Survival in the Kona line WSSV positive control was 0% with all the Kona line shrimp dead by day 6 post infection. At termination on day 17, survival of Panamanian selected families in the WSSV challenged groups was 23%, 57% and 26% for families LP-1, LP-2 and LP-3, respectively. This is the first time in the scientific literature that significant resistance of L. vannamei against WSSV under controlled conditions is reported.

Experimentally pathogenesis of Aeromonas hydrophila in freshwater Crayfish (Astacus leptodactylus) in iran

In this research the affect of A. hydrophila on pathogenesis of Crayfish A. leptodactylus was carried out. This study was designed in three groups as treatments and one group as control in triplicate with 20 Crayfish in each glass aquarium. The treatments have exposed to A. hydrophila. The concentrations of the bacteria in treatments were , and CFUmL-1 respectively. The untreated control group was disinfected by oxytetracyclin at concentration of 100 ppm for 24 hours. The haemolymph samples were withdrawn for measuring of THC and TPC within interval hours (2, 6, 12, 24, 48, 96, 144, 240 and 336). The Crayfish samples were then fixed in Davidson fixative. The results showed that the differences of THC value between treatment 4 (95.33±23.16) and control (1.13±.45) were significant (P < 0.05) as well as control and treatments 3 (35.33±16.16) and 4 (95.33±23.16) during 2 initial hours. In case of THC the significant difference (P < 0.05) was observed between control group (72.33±15.04) and treatment 3 (13.13±5.85) after 48 hours. During 240 hours, also the difference of THC value between control group (80.33±24.94) with treatment 2 (14.67±3.51) was significant (P < 0.05). The finding of TPP value showed that there was no significant difference between control group and treatments in during mentioned times (P < 0.05). The result of histopathology in heaptopancreas and gill sections showed that hemocyte aggregation and necrosis within pyknosis of nucleus. In lower concentrations of bacteria no pathological changes of heart were observed. In digestive tract no changes were appeared in treatments, but in concentration of 3×108 CFU ml-1 a low aggregation of hemocytes was revealed.

A Modified Davidson's Fixative Prevents Artifactual Vacuolation of the Optic Nerve

Conventional Davidson is the fixative of choice for ophthalmic pathology; however, this fixative causes optic nerve vacuolation. This study compared the overall histomorphologic structures of eyes fixed with conventional Davidson and four different types of modified Davidson's fixative (namely, Tests 1, 2, 3 and 4) using alcohol, glacial acetic acid, formalin and formaldehyde in different concentrations and proportions. Test 3 was the only fixative that totally prevented optic nerve vacuolation while the conventional Davidson, Tests 1 and 4 caused mild to moderate artifactual vacuolation of the optic nerve. In addition, the collagen fibers in the corneal stroma were disrupted and separated by large clear spaces with conventional Davidson and Tests 1, 2 and 3 but well preserved with Test 4. The nuclei of the rod cells in the outer nuclear layer of the retina were slightly dented in conventional Davidson, Tests 1 and 4. This study has demonstrated that specific fixatives could minimize some fixation artifacts; however, there are no specific rules concerning the choice of fixative, and the selection should be based on the type of ocular investigation. Tests 3 and 4 provided good cellular preservation and diminished the vacuolar artifacts in the optic nerve. (The J Histotechnol 32(4):155–157, 2009)

An antibody panel for immunohistochemical analysis of the retina in Davidson's-fixed, paraffin-embedded eyes of rats

Unlike most other tissues, the optimal fixative for preserving eye morphology is considered to be Davidson's fixative or modified Davidson's rather than formalin. However, the methodology for antibodies to be used in tissues fixed this way is not normally outlined in current antibody datasheets. Additionally, where eyes have been stored in Davidson's fixative, the efficacy of retrospective analysis of eye morphology by immunohistochemistry is largely unknown. The aim of this study was to compare a panel of six antibodies in both Davidson's-fixed and formalin-fixed pigmented and non-pigmented rat eyes, in order to provide optimal methods for future retinal immunohistochemical evaluation with image analysis. The antibodies evaluated were raised against rhodopsin, synaptophysin, glutamine synthetase, glial fibrillary acidic protein (GFAP), cleaved caspase-3 and phospho-histone H3 (PH3). Overall, the staining quality of these antibodies was found to be optimal in Davidson's compared to formalin-fixed tissues after a time period of up to 4 days in fixative. The methods outlined thus provide a platform for future detailed analysis of retinal pathology in Davidson's-fixed eyes.

Effect of modified Davidson's fixative on examined number of lymph nodes and TNM-stage in colon carcinoma

Aims We evaluated the effect of modified Davidson's fixative (mDF) on the number of lymph nodes examined and staging in patients with colon carcinoma. Methods The results of two different fixation methods used in the pathological preparation of the resection specimens were analyzed. A traditional formalin preparation with manual dissection of all nodes was performed in 117 colon specimens between January 2003 and July 2004. After July 2004, the resected specimens of 125 patients was fixated in mDF. Differences in the retrieval and number of nodes and size of suspected nodal metastases were measured. All lymph nodes were stained with conventional H&E methods. Results The median number of examined nodes increased from 5 (0–17) to 13 (0–35) nodes after the introduction of mDF ( p < 0.001). The type of resection and the T-stage influenced the number of retrieved nodes significantly. The percentage of node-positive cases increased from 30% to 41% ( p = 0.077) with mDF, the median size of the retrieved lymph nodes decreased from 9 mm before to 6 mm after mDF ( p < 0.001) and more micrometastases were found (6% vs. 16%, p = 0.03). Conclusions With mDF technique more lymph nodes were retrieved in the resected colon specimens. Smaller nodes and more micrometastases were found, leading to more node positive patients.

399 POSTER Effect of re-fixation in modified Davidson's Fixative on number of lymph nodes and TNM-stage in colon cancer

399 POSTER Effect of re-fixation in modified Davidson's Fixative on number of lymph nodes and TNM-stage in colon cancer

ADP-Ribosylation Factor-Like 3 Is Involved in Kidney and Photoreceptor Development

ADP-ribosylation factor-like 3 (Arl3) is a member of a small subfamily of G-proteins involved in membrane-associated vesicular and intracellular trafficking processes. Genetic studies in Leishmania have shown that the Arl3 homolog is essential for flagellum biogenesis. Mutations in a related human family member, Arl6, result in Bardet-Biedl syndrome in humans, which is characterized by genital, renal, and retinal abnormalities, obesity, and learning deficits. As part of our large-scale phenotypic screen, mice deficient for the Arl3 gene were generated and analyzed. Arl3 (-/-) mice were born at a sub-Mendelian ratio, were small and sickly, and had markedly swollen abdomens. These mutants failed to thrive, and all died by 3 weeks of age. The (-/-) mice exhibited abnormal development of renal, hepatic, and pancreatic epithelial tubule structures, which is characteristic of the renal-hepatic-pancreatic dysplasia found in autosomal recessive polycystic kidney disease. Absence of Arl3 was associated with abnormal epithelial cell proliferation and cyst formation. Moreover, mice lacking Arl3 exhibited photoreceptor degeneration as early as postnatal day 14. These results are the first to implicate Arl3 in a ciliary disease affecting the kidney, biliary tract, pancreas, and retina.

300 IMMUNO-EXPRESSION OF POTENTIAL FACTORS REGULATING SERTOLI CELL PROLIFERATION IN THE PREPUBERTAL BOAR

Today's pork production is very dependent on reproductive efficiency. Any improvements in the production capability (e.g., number of sperm produced) of the animals involved would be invaluable. Many researchers have examined methods to improve oocyte production, but have not focused on the concentration of sperm produced from a single boar used for artificial insemination (AI). The benefit of using AI is that a greater number of females can be bred to a single boar; thus the total amount of sperm per ejaculation is the main factor in the efficiency of AI. An increase in the number of Sertoli cells leads to an increase in testis size and the number of sperm produced because there are a finite number of germ cells that can be supported by the Sertoli cell during spermatogenesis. Therefore, by examining the factors that control the growth and differentiation of Sertoli cells, the amount of sperm per ejaculation in an individual boar can be increased. The purpose of this research is to begin a systematic analysis of cell cycle regulators expressed in the Sertoli cell during testicular development in the pig. By using pigs of different ages, we will establish a baseline of what regulatory factors are present at different time points in the developing Sertoli cell. Identification of certain factors could lead to an increase in sperm production by enabling the growth of Sertoli cells or inhibition of the natural reduction of these cells. Immunohistochemistry (IHC) analysis was performed on testis tissues from different males (n = 2) at various ages (3, 7, 14, 25, and 50 days) prior to puberty. Tissues were fixed in modified Davidson's fixative, embedded in paraffin, and sectioned; slides were processed for IHC. After antigen retrieval, endogenous hydrogen peroxidase was blocked with 0.6% H2O2 and sections were incubated overnight at 4°C with the appropriate antibody. The following antibodies were used to examine the factors controlling Sertoli cell proliferation: GATA-4 (transcription factor specific for developing and adult pig Sertoli cells), Ki67 (a nuclear protein present in all phases of the cell cycle except G0), cyclin-dependent kinase inhibitor p27 (Kip1), and steroid receptors AR (androgen receptor) and LH2 (estrogen receptor). The slides were then incubated with an appropriate secondary antibody, visualized with DAB chromagen, and counterstained with Mayer's hematoxylin. Immunostaining using p27 (Kip1) revealed no positive staining in any of the days tested, as there is cell division during all of these time points. Protein expression for Ki67 stained mildly after Day 25, suggesting that Sertoli cells became more active at this stage of development. The AR weakly stained and GATA-1 stained intensely at all time points. The data for LH2 were inconclusive and the procedure needs to be performed again. These experiments only give a glimpse of the regulatory factors involved in Sertoli cell proliferation in the developing boar testis. Further studies are being planned to explore other potential factors involved.

Evaluation of testicular toxicology: a synopsis and discussion of the recommendations proposed by the Society of Toxicologic Pathology.

Detection of chemically induced effects on male fertility and on testicular spermatogenesis in particular, has become of increasing concern. More stringent regulatory guidelines, introduced by ICH, EPA and OECD (Table 1) have raised the awareness of toxicologists and pathologists for the need to conduct sensitive and careful evaluation of the male reproductive tract for potential toxic effects of administered compounds. With it has come confusion and in many cases, inappropriate procedures, often based on misunderstanding of what is required and on inadequate understanding of spermatogenesis. This article summarizes and discusses the main recommendations recently proposed by the Society of Toxicologic Pathology on recommended approaches for the evaluation of testicular and epididymal toxicity [Lanning LL, Creasy DM, Chapin RE, Mann PC, Barlow NJ, Regan KS, Goodman DG. Toxicologic Pathology 30:518-531, 2002]. The major recommendations are: Use sexually mature animals to evaluate effects on spermatogenesis. Sample left and right testes and epididymides and record organ weights. Use modified Davidson's fixative to fix testes from all species from studies of 13 wks duration and less. Examine transverse sections of the testes (including part of the rete), and longitudinal sections of the epididymides. Embed tissues in paraffin wax. For rodent studies up to 28 days, examine periodic acid-Schiff's-hematoxylin stained sections. For all other studies examine hematoxylin and eosin stained sections. Microscopic evaluation of the testis should be a qualitative evaluation carried out with an awareness of the spermatogenic cycle. Quantitative procedures are inappropriate for screening studies. Nomenclature and grading of findings for spermatogenic disturbances will vary on a case by case basis.

Molecular detection methods developed for a systemic rickettsia-like bacterium (RLB) in Penaeus monodon (Decapoda: Crustacea).

Molecular detection methods were developed to aid in the diagnosis of a rickettsia-like bacterium (RLB) which caused severe mortalities of farm-raised Penaeus monodon in Madagascar. Using primers derived from the 16S rRNA gene of bacteria, a PCR assay was optimized to amplify this region of the genome of the RLB, using extracted DNA from infected P. monodon tissue as the template. The resulting amplified PCR product was sequenced and 2 novel primers were selected from the variable region of the gene. These primers amplified a 532 bp fragment of DNA originating from the rickettsia-infected samples. The PCR assay was optimized and tested on DNA extracted from specific pathogen-free (SPF) P. vannamei tissue and several other strains of bacteria. The PCR assay with the rickettsia-specific primers was specific for this RLB and did not amplify the other DNA samples tested. The 532 bp PCR-amplified fragment was labeled with digoxigenin (DIG) for in situ hybridization assays. This probe was tested on SPF, RLB and bacteria-infected shrimp specimens preserved in Davidson's fixative. The probe was specific for both natural and experimental rickettsial infections. Hybridization with this probe required a stringent temperature of 65 degrees C, otherwise cross-reactivity was observed with other types of bacteria.

A new RNA-friendly fixative for the preservation of penaeid shrimp samples for virological detection using cDNA genomic probes

In situ hybridization analysis of shrimp histological sections, utilizing Taura syndrome virus (TSV) specific cDNA probes, is the most sensitive diagnostic technique presently available for the detection of this penaeid shrimp viral disease. However, false negative genomic probe results are obtained frequently from samples of Pacific white shrimp, Penaeus vannamei, that have been preserved with Davidson's AFA (acetic acid, formaldehyde, alcohol) fixative and that, otherwise, demonstrate pathognomonic TSV lesions by routine histology. This problem was linked to prolonged storage of shrimp samples in Davidson's fixative, which is highly acidic (pH ∼ 3.5–4). Degradation of TSV genomic RNA was hypothesized to be due to either fixative-induced acid hydrolysis and/or acidophilic endogenous ribonuclease activity. Routine H and E histology and in situ hybridization analyses were conducted on equal numbers of TSV infected P. vannamei juveniles that were preserved for four different time periods (2, 6, 10 and 14 days) with either Davidson's fixative or a new, near neutral (pH ∼ 6.0–7.0), RNA-friendly fixative (R-F) that was developed by the authors. In situ hybridization assays were conducted with and without RNase precautions and all of the samples tested contained moderate to severe TSV lesions by routine histology. Davidson's preserved samples produced weak TSV probe signals after 2 days fixation, but did not react with the probes in those samples that were stored for > 6 days in the fixative. In contrast, TSV was detectable by gene probe in all of the time treatment samples preserved with the new R-F fixative. Equivalent in situ hybridization results were obtained when the same samples were analyzed in the absence of RNase-free conditions. These findings suggest that TSV RNA is degraded when samples are stored in an acidic fixative, such as Davidson's, for more than 2 days and that this problem can be prevented through preservation of shrimp samples with R-F fixative. The efficacy of this new fixative is demonstrated and the results show that RNase-free conditions are not necessary for conducting TSV in situ hybridization analyses.

Communications: Comparison of Traditional and Molecular Detection Methods forBaculovirus penaeiInfections in LarvalPenaeus vannamei

Abstract Penaeid shrimp Penaeus vannamei were experimentally infected at a mysis II stage with the penaeid shrimp virus Baculovirus penaei (BP). The larvae were sampled at 0, 8, 12, 18, 24, 48, and 72 h postinfection and were examined for evidence of infection by three different methods: wet mounts, histology, and nucleic acid probes. Wet-mount squashes of the hepatopancreas were prepared and examined for BP tetrahedral occlusion bodies (TOBs) or larvae were fixed in Davidson's fixative and processed for routine histology, then either stained with hematoxylin and eosin (H&E) or reacted with nonradioactive BP gene probes by in situ hybridization. Sections stained with H&E or reacted with labeled gene probes were examined for signs of BP infection, such as cell cytopathology, the presence of TOBs, or the presence of a purple precipitate (which indicates BP-specific nucleic acid in the tissue). Occlusion bodies were initially observed in the wet-mount squash of the hepatopancreas of one shrimp at 18 h postin...

Lymphoidal parvovirus-like particles in Australian penaeid prawns

Prawns in northern Queensland exhib~ted mult~nucleated g ~ a n t cell formation in hypertrophied lymphoid organs. Mostly farmed Penaeus monodon, P, merguiensls and P esculentus were involved The transformation involved mild nuclear hypertrophy, marglnated chromatin and basophilic da rken~ng of increased cytoplasm In a multinucleate giant cell. Areas of transformation were d~scre te , often bounded by elongated f~brocytes and sometimes fully encapsulated Basophilic inclusions occurred con~monly and eos~nophlhc lntranuclear inclus~ons rarely. These inclus~ons talned for DNA with acndlne orange fluorescence. Associated \nth lympho~dal changes was the presence of rounded F l~ke cells with basophilic, pyknolc nucleoli. Under the electron microscope, transformed lympho~dal cells d~splayed virus-hke parlcles of 18 to 20 nm diameter associated with inclusion b o d ~ e s in the nucleus These particles were similar to a parvovlrus and named lymphoidal parvovirus-like (LP\/). Their similarities and relationship to hepatopancreatic parvovirus and infectious hypodermal and haematopoletic necrosis virus are discussed. INTRODUCTION MATERIALS AND METHODS The lymphoid organ (or Oka organ) is a relatively newly discovered organ in penaeid prawns (Oka 1969) and is becoming increasingly important in disease studies. Lightner et al. (1987) described Oka organ hyperplastic spheroids and metastatic foci in ectopic localities in both Penaeus monodon Fabricus and P. penicillatus from Taiwan. Prlmary cell cultures from the lymphoid organ of P. n~onodon were capable of supporting in vitro replication of Monodon baculovirus when inoculated into the cultures (Chen & Kou 1989). Lymphoid t~ssue necrosis and haenlocytic infiltration lead to encapsulation, nodule formation and melanization and are often the earliest changes associated with bacterial infections (Nash 1990). In Australia, the lymphoidal spheroids in Penaeus nlerguiensis de Man have been associated with moribund wild spawners (Owens & Hall-Mendelin 1990). Suggestions were made that there were similarities between tissues showing lymphoid metastasis with the lymphoidal spheroids and tissues attacked by infectious haematopoietic and hypodermal necrosis virus (IHHNV). This paper reports on further investigations of lymphoidal changes in prawns and the probable causahve agent. Banana prawns Penaeus merguiensis were collected by a cast net in the Burdekin Rver , northern Queensland. Tiger prawns (P. monodon and P. esculentus Haswell) were collected from prawn farms in the local area. Prawns were split longitudinally, and fixed in Davidson's fixative. Histological sections were cut and stained using standard techniques (Culling et al. 1985). For electron microscopy, the diced lymphoid organ was fixed in 2 .5 % glutaldehyde/2 O/O paraforn~aldehyde in cacodylate buffer and postfixed in 1 O/O osmium tetroxide. The tissue was dehydrated and mounted in Spurr (Tm) resin. Sections were cut on a LKB Ultratome at 50 nm stained with uranyl acetate/ 70 O/O methanol and lead citrate and viewed at 80 kV on a Joel 2000FX transmission electron microscope