Giardia and Cryptosporidium on dairy farms and the role these farms may play in contaminating water sources in Prince Edward Island, Canada.

BACKGROUND: Cattle represent a reservoir for Giardia and Cryptosporidium and may contaminate water sources. OBJECTIVES: To determine the distribution of Cryptosporidium and Giardia on dairy farms and in water bodies near the farms. FARMS AND WATER SOURCES: Twenty dairy farms and 20 wells and 13 surface water samples associated with dairy farms. METHODS: Proportions of samples positive for Cryptosporidium or Giardia were determined by a direct immunofluorescence assay. Fecal and water samples were taken at different times. RESULTS: Thirty-two (95% CI: 29-35%) and 14% (95% CI: 12-17%) of fecal samples, and 100 (95% CI: 96-100) and 55% (95% CI: 32-77%) of herds, were positive for Giardia and Cryptosporidium, respectively. Giardia duodenalis assemblage E was detected in high proportions (90%) of fecal samples. Cryptosporidium bovis predominated (51%) in all cattle. C. andersoni predominated in adult cattle (53%), whereas the predominant species in animals < 2 months and 2-6 months was C. bovis, respectively. Only calves < 2 months of age were positive for C. parvum. In 46% (95% CI: 19-75%) and 85% (95% CI: 55-98%) of surface water, concentrations of Giardia cysts and Cryptosporidium oocysts were higher in downstream, than in upstream, locations of farms, whereas only 1 groundwater sample was positive for Cryptosporidium. CONCLUSIONS: This sample of dairy cattle was predominantly infected with nonzoonotic species and genotypes of Cryptosporidium, Giardia, or both. More studies are needed to determine if the presence of Giardia or Cryptosporidium in surface water was associated with shedding in animals from nearby farms.

Lack of detection of a putative retrovirus associated with haemic neoplasia in the soft shell clam Mya arenaria.

Haemic neoplasia (HN) is a leukemia-like disease that affects at least 20 species of marine bivalves including soft shell clam, Mya arenaria. Since the disease was discovered in 1969, the etiology remains unknown. A retroviral etiology has been suggested based on the detection of reverse transcriptase activity and electron microscopic observation of retroviral-like particles using negative staining. To date, however no virus isolate and no retroviral sequence from HN has been obtained. Moreover, transmission of the disease by cell-free filtrate from affected clams has not been reproduced. In the current study, we reinvestigated the association of HN with a putative retrovirus. Sucrose gradient centrifugation followed by assessment of reverse transcriptase activity, electrophoretic analysis of protein and RNA, and electron microscopic examinations of fractions corresponding to retroviral density were employed. Detection of retroviral pol sequences using degenerate RT-PCR approaches was also attempted. Our results showed visible bands at the expected density of retrovirus in HN-positive and HN-negative clam tissues and both with reverse transcriptase activity. Electron microscopy, RNA analysis, protein analysis, and PCR systems targeting the pol gene of retroviruses did not however provide clear evidence supporting presence of a retrovirus. We point out that the retrovirus etiology of HN of Mya arenaria proposed some 25 years ago should be reconsidered in the absence of a virus isolate or virus sequences.

Reverse transcriptase activity in tissues of the soft shell clam Mya arenaria affected with haemic neoplasia.

Since all retroviruses possess reverse transcriptase (RT) enzyme, reverse transcriptase activity has been the main supportive evidence of retroviral etiology of haemic neoplasia (HN) in soft shell clams, Mya arenaria. The objective of the present study was to search for a putative retrovirus in various tissues of diseased clams following quantification of RT activity (biochemical indicator of retroviral infection). The clams were assessed by flow cytometry (FCM) for diagnosis of HN. RT activity was quantified by TaqMan-product enhanced reverse transcriptase (TM-PERT) assay in four different organs, gonad, gills, digestive gland, and mantle, at various stages of HN. The digestive gland, the organ with the highest RT activity, and haemocytes, the target cell of HN, were assessed by EM for presence of retroviruses. All organs were assessed by histology. The results of this study demonstrated that although all organs of healthy clams have some background RT activity, the activity observed in most of organs of diseased clams was significantly increased (p<0.05). An association was observed between the degree of neoplastic cell infiltration and the level of RT activity. Digestive gland showed the highest and most consistent RT activity in both healthy and diseased clams. No evidence for the existence of a retrovirus like particle was found by positive staining EM. The presence of RT activity without indications of retroviral particles in digestive gland and haemocytes suggests a probable endogenous source of RT.

Reverse transcriptase activity associated with haemic neoplasia in the soft-shell clam Mya arenaria.

Reverse transcriptase (RT) activity has been reported in bivalves affected by haemic neoplasia (HN). Since all retroviruses have RT, detection of RT activity was regarded as evidence for the retroviral etiology of HN. This study investigates the relationship between RT levels and the progress of HN as indicated by percentages of tetraploid cells in soft-shell clams Mya arenaria. The percentages of tetraploid cells were estimated by flow cytometry, and the RT levels were quantified using TaqMan product-enhanced RT (TM-PERT) assay. Results demonstrated that the amount of RT was positively correlated with the percentage of tetraploid cells circulating in clam haemolymph (R2 = 0.974, p < 0.001). Compared to HN-negative clams (<5% tetraploid cells), 2 stages with significantly elevated levels of RT activity were observed: the first stage at approximately 10 to approximately 20% tetraploid cells, and the second at approximately 30 to approximately 80% tetraploid cells (p < 0.01). These data support the well established fact from mammalian models that transformed cells express high levels of non-telomeric RT. The observed increase in RT levels at approximately 30% tetraploidy coincides with previously reported p53 gene expression. Taken together, this could indicate that using RT levels as an indicator of HN, > or = 30% tetraploidy is the stage at which the disease process undergoes a change, and perhaps becomes irreversible.

Anti-lipopolysaccharide factors in the American lobster Homarus americanus: molecular characterization and transcriptional response to Vibrio fluvialis challenge.

Two partial mRNA sequences predicted to encode anti-lipopolysaccharide factors (ALFs) were identified among expressed sequence tags generated from the American lobster Homarus americanus and complete cDNA sequences were obtained from library clones. Comparison of the translated amino acid sequences to those publicly available confirmed similarity to arthropod anti-lipopolysaccharide factors. Both protein sequences, designated ALFHa-1 and ALFHa-2, contained an N-terminal signal peptide and two half-cysteines participating in a disulfide bridge, features conserved in other ALFs. Predicted secondary structures were similar to that described for the ALF from the horseshoe crab Limulus polyphemus. As part of an exploratory study of immunity in H. americanus, lobsters were injected with the bacterium Vibrio fluvialis and gill, hematopoietic, and hepatopancreas tissues were sampled for analysis of gene expression of ALFHa-1 and ALFHa-2 by quantitative PCR. The relative abundance of ALFHa-2 mRNA was not significantly affected by Vibrio injection in any of the three tissues tested. In contrast, ALFHa-1 mRNA levels in gills were increased by the treatment some 17-fold. Our results support a molecularly specific regulation of antimicrobial proteins in response to bacterial infection in H. americanus.

Expression of hepatocyte low-density lipoprotein receptor-related protein is post-transcriptionally regulated by extracellular matrix.

The low-density lipoprotein receptor-related protein (LRP) is a multifunctional member of the low-density lipoprotein receptor family that has been implicated in a variety of physiologic and pathologic processes. However, little is known about LRP regulation at the molecular level, and the factors that might mediate LRP have not yet been characterized. This is particularly true of hepatocytes, an important site of LRP expression. Hepatocyte gene expression is known to be dependent on extracellular matrix composition, although the effect of extracellular matrix on lipoprotein receptor expression has not yet been investigated. Also, the mechanisms by which the extracellular matrix affects hepatocyte gene expression are not well understood. In this study, we show that hepatocyte LRP expression decreases rapidly at the mRNA, protein, and functional levels on collagen type I, but remains high on an Engelbreth-Holm-Swarm sarcoma matrix-preparation, Matrigel. LRP function was assessed with ligand binding studies and a novel cytotoxicity assay, using Pseudomonas exotoxin A. Investigation of the mechanism of LRP down-regulation revealed a two-fold longer LRP mRNA half-life in hepatocytes cultured on Matrigel relative to collagen. Taken together, these studies reveal that LRP expression in primary hepatocytes is dependent on the extracellular matrix, and that matrix-dependent differences in hepatocyte LRP mRNA expression are primarily due to changes in mRNA stability, indicating for the first time that the expression of LRP is subject to post-transcriptional regulation.