Individual subject meta-analysis of parameters for Cryptosporidium parvum shedding and diarrhoea in animal experimental models.

Cryptosporidium is a zoonotic protozoan parasite with public health importance worldwide. The objectives of this study were to (1) conduct a meta-analysis of published literature for oocyst shedding and diarrhoea outcomes, and (2) develop recommendations for standardization of experimental dose-response studies. Results showed that for the outcome of oocyst shedding in faeces, the covariates 'experimental species', 'immunosuppression', 'oocyst dose' and 'oocyst dose' × 'age' were all significant (P≤0.05). This study suggests that exposing mice, piglets, or ruminants, and using immunosuppressed experimental hosts, is more likely to result in oocyst shedding. For the outcome of diarrhoea in experimentally infected animal species, the key covariates 'experimental species', 'age' and 'immunosuppression' were significant (P≤0.2). Therefore, based on the results of this meta-analysis, these variables should be carefully reported and considered when designing experimental dose-response studies. Additionally, detection of possible publication bias highlights the need to publish additional studies that convey statistically non-significant as well as significant results in the future.

Endotoxin: stimulation of bone resorption in tissue culture.

Bacterial endotoxins can stimulate the release of previously incorporated calcium-45 and tritiated proline from fetal rat bone in tissue culture. Endotoxin from Bacteroides melaninogenicus, an organism regularly found in the gingival crevice of man, produces a response similar to parathyroid hormone and is effective at doses as low as 0.1 microgram per milliliter. This response is inhibited by serum and dependent upon the presence of albumin. Endotoxins may play a role in the bone loss characteristic of human periodontal disease.

Reducing microbial contamination in storm runoff from high use areas on California coastal dairies.

High use areas are a fundamental part of California coastal dairies and grazing livestock ranches as feeding areas, nurseries, and sick pens. High stocking densities and daily use in these areas lead to soil surfaces devoid of vegetation and covered in manure, with high potential for manure transport during winter rains to receiving waters regulated for shellfish harvesting and recreation. We characterized the association between California's Mediterranean climate and a series of existing and proposed management practices on fecal coliform bacteria (FCB) transport from high use areas on dairies and ranches. Results from 351 storm runoff samples collected below 35 high-use areas indicate that removal of cattle during winter, locating high use areas on level ground, application of straw and seeding, and vegetative buffer strip implementation were significantly associated with FCB concentration and load reductions. These results complement our findings for reductions of specific pathogens in runoff from these areas. These findings have practical significance because they document surface water quality benefits that the studied management practices provide in application on working farms and ranches. This direction is critical and timely for on-farm management efforts seeking to reduce microbial pollution in runoff and comply with indicator bacteria water quality criteria.

Type X Toxoplasma gondii in a wild mussel and terrestrial carnivores from coastal California: new linkages between terrestrial mammals, runoff and toxoplasmosis of sea otters.

Sea otters in California are commonly infected with Toxoplasma gondii. A unique Type X strain is responsible for 72% of otter infections, but its prevalence in terrestrial animals and marine invertebrates inhabiting the same area was unknown. Between 2000 and 2005, 45 terrestrial carnivores (lions, bobcats, domestic cats and foxes) and 1396 invertebrates (mussels, clams and worms) were screened for T. gondii using PCR and DNA sequencing to determine the phylogeographic distribution of T. gondii archetypal I, II, III and Type X genotypes. Marine bivalves have been shown to concentrate T. gondii oocysts in the laboratory, but a comprehensive survey of wild invertebrates has not been reported. A California mussel from an estuary draining into Monterey Bay was confirmed positive for Type X T. gondii by multilocus PCR and DNA sequencing at the B1 and SAG1 loci. This mussel was collected from nearshore marine waters just after the first significant rainfall event in the fall of 2002. Of 45 carnivores tested at the B1, SAG1, and GRA6 typing loci, 15 had PCR-confirmed T. gondii infection; 11 possessed alleles consistent with infection by archetypal Type I, II or III strains and 4 possessed alleles consistent with Type X T. gondii infection. No non-canonical alleles were identified. The four T. gondii strains with Type X alleles were identified from two mountain lions, a bobcat and a fox residing in coastal watersheds adjacent to sea otter habitat near Monterey Bay and Estero Bay. Confirmation of Type X T. gondii in coastal-dwelling felids, canids, a marine bivalve and nearshore-dwelling sea otters supports the hypotheses that feline faecal contamination is flowing from land to sea through surface runoff, and that otters can be infected with T. gondii via consumption of filter-feeding marine invertebrates.

Novel and canine genotypes of Giardia duodenalis in harbor seals ( Phoca vitulina richardsi).

Feces of harbor seals (Phoca vitulina richardsi) and hybrid glaucous-winged/western gulls (Larus glaucescens / occidentalis) from Washington State's inland marine waters were examined for Giardia and Cryptosporidium spp. to determine if genotypes carried by these wildlife species were the same genotypes that commonly infect humans and domestic animals. Using immunomagnetic separation followed by direct fluorescent antibody detection, Giardia spp. cysts were detected in 42% of seal fecal samples (41/97). Giardia-positive samples came from 90% of the sites (9/10) and the prevalence of positive seal fecal samples differed significantly among study sites. Fecal samples collected from seal haulout sites with over 400 animals were 4.7 times more likely to have Giardia spp. cysts than samples collected at smaller haulout sites. In gulls, a single Giardia sp. cyst was detected in 4% of fecal samples (3/78). Cryptosporidium spp. oocysts were not detected in any of the seals or gulls tested. Sequence analysis of a 398 bp segment of G. duodenalis DNA at the glutamate dehydrogenase locus suggested that 11 isolates originating from seals throughout the region were a novel genotype and 3 isolates obtained from a single site in south Puget Sound were the G. duodenalis canine genotype D. Real-time TaqMan PCR amplification and subsequent sequencing of a 52 bp small subunit ribosomal DNA region from novel harbor seal genotype isolates showed sequence homology to canine genotypes C and D. Sequence analysis of the 52 bp small subunit ribosomal DNA products from the 3 canine genotype isolates from seals produced mixed sequences at could not be evaluated.

Farm factors associated with reducing Cryptosporidium loading in storm runoff from dairies.

A systems approach was used to evaluate environmental loading of Cryptosporidium oocysts on five coastal dairies in California. One aspect of the study was to determine Cryptosporidium oocyst concentrations and loads for 350 storm runoff samples from dairy high use areas collected over two storm seasons. Selected farm factors and beneficial management practices (BMPs) associated with reducing the Cryptosporidium load in storm runoff were assessed. Using immunomagnetic separation (IMS) with direct fluorescent antibody (DFA) analysis, Cryptosporidium oocysts were detected on four of the five farms and in 21% of storm runoff samples overall. Oocysts were detected in 59% of runoff samples collected near cattle less than 2 mo old, while 10% of runoff samples collected near cattle over 6 mo old were positive. Factors associated with environmental loading of Cryptosporidium oocysts included cattle age class, 24 h precipitation, and cumulative seasonal precipitation, but not percent slope, lot acreage, cattle stocking number, or cattle density. Vegetated buffer strips and straw mulch application significantly reduced the protozoal concentrations and loads in storm runoff, while cattle exclusion and removal of manure did not. The study findings suggest that BMPs such as vegetated buffer strips and straw mulch application, especially when placed near calf areas, will reduce environmental loading of fecal protozoa and improve stormwater quality. These findings are assisting working dairies in their efforts to improve farm and ecosystem health along the California coast.

Cryptosporidium and Giardia in marine-foraging river otters (Lontra canadensis) from the Puget Sound Georgia Basin ecosystem.

Species of Cryptosporidium and Giardia can infect humans and wildlife and have the potential to be transmitted between these 2 groups; yet, very little is known about these protozoans in marine wildlife. Feces of river otters (Lontra canadensis), a common marine wildlife species in the Puget Sound Georgia Basin, were examined for species of Cryptosporidium and Giardia to determine their role in the epidemiology of these pathogens. Using ZnSO4 flotation and immunomagnetic separation, followed by direct immunofluorescent antibody detection (IMS/DFA), we identified Cryptosporidium sp. oocysts in 9 fecal samples from 6 locations and Giardia sp. cysts in 11 fecal samples from 7 locations. The putative risk factors of proximate human population and degree of anthropogenic shoreline modification were not associated with the detection of Cryptosporidium or Giardia spp. in river otter feces. Amplification of DNA from the IMS/DFA slide scrapings was successful for 1 sample containing > 500 Cryptosporidium sp. oocysts. Sequences from the Cryptosporidium 18S rRNA and the COWP loci were most similar to the ferret Cryptosporidium sp. genotype. River otters could serve as reservoirs for Cryptosporidium and Giardia species in marine ecosystems. More work is needed to better understand the zoonotic potential of the genotypes they carry as well as their implications for river otter health.

RECODE: a database of frameshifting, bypassing and codon redefinition utilized for gene expression.

The RECODE database is a compilation of 'programmed' translational recoding events taken from the scientific literature and personal communications. The database deals with programmed ribosomal frameshifting, codon redefinition and translational bypass occurring in a variety of organisms. The entries for each event include the sequences of the corresponding genes, their encoded proteins for both the normal and alternate decoding, the types of the recoding events involved, trans-factors and cis-elements that influence recoding. The database is freely available at http://recode.genetics. utah.edu/.

Barley yellow dwarf viruses.

Barley yellow dwarf viruses represent one of the most economically important and ubiquitous groups of plant viruses. This review focuses primarily on four research areas in which progress has been most rapid. These include (a) evidence supporting reclassification of BYDVs into two genera; (b) elucidation of gene function and novel mechanisms controlling gene expression; (c) initial forays into understanding the complex interactions between BYDV virions and their aphid vectors; and (d) replication of a BYDV satellite RNA. Economic losses, symptomatology, and means of control of BYD are also discussed.

Minus-strand initiation by brome mosaic virus replicase within the 3' tRNA-like structure of native and modified RNA templates.

An RNA-dependent RNA polymerase (replicase) extract from brome mosaic virus-infected barley leaves has been shown to initiate synthesis of (-) sense RNA from (+) sense virion RNA. Initiation occurred de novo, as demonstrated by the incorporation of [gamma-32P]GTP into the product. Sequencing using cordycepin triphosphate to terminate (-) strands during their synthesis by the replicase generated sequence ladders that confirmed that copying was accurate, and that initiation occurred very close to the 3' end. The precise site of initiation was further defined by testing the replicase template activity after stepwise removal of 3'-terminal nucleotides. Whereas removal of the terminal A did not decrease template activity, removal of the next nucleotide (C-2) did. Thus, initiation almost certainly occurs opposite the penultimate 3'-nucleotide (C-2) in vitro. The structure of the double-stranded replicative form of RNA isolated from brome mosaic virus-infected leaves was consistent with such a mechanism occurring in vivo, in that it lacked the 3'-terminal A found on virion RNAs. The specific site of (-) strand initiation and normal template activity were retained for RNAs with as many as 15 to 30 A residues added to the 3' end. However, only limited oligonucleotide 3' extensions can be present on active templates. In order to assess the 5' extent of sequences required for an active template, a 134-nucleotide-long fragment of brome mosaic virus RNA, corresponding to the tRNA-like structure, was generated. This RNA had high template activity, but a shorter 3' (85-nucleotide) fragment was inactive. RNAs with various heterologous sequences 5' to position 134 also showed high template activity. Thus, the 3'-terminal tRNA-like structure common to all four brome mosaic virus virion RNAs contains all of the signals required for initiation of replication, and sequences 5' to it do not play a role in template selection.

Satellite cereal yellow dwarf virus-RPV (satRPV) RNA requires a douXble hammerhead for self-cleavage and an alternative structure for replication.

The 110 nt hammerhead ribozyme in the satellite RNA of cereal yellow dwarf virus-RPV (satRPV RNA) folds into an alternative conformation that inhibits self-cleavage. This alternative structure comprises a pseudoknot with base-pairing between loop (L1) and a single-stranded bulge (L2a), which are located in hammerhead stems I and II, respectively. Mutations that disrupt this base-pairing, or otherwise cause the ribozyme to more closely resemble a canonical hammerhead, greatly increase self-cleavage. In a more natural multimeric sequence context containing the full-length satRPV RNA and two copies of the hammerhead, wild-type RNA cleaves much more efficiently than in the 110 nt context. Mutations in the upstream hammerhead, including a knock-out in the catalytic core, affect cleavage at the downstream cleavage site, indicating that multimers of satRPV RNA cleave via a double hammerhead. The double hammerhead includes base-pairing between two copies of the L1 sequence which extends stem I. Disruption of L1-L1 base-pairing slows cleavage of the multimer. L1-L2a base-pairing is required for efficient replication of satRPV RNA in oat protoplasts. Mutations that affect self-cleavage of the multimer do not correlate with replication efficiency, indicating that the ability to self-cleave is not a primary determinant of replication. We present a replication model in which multimeric satRPV RNA folds into alternative conformations that cannot form in the monomer. One potential metastable intermediate conformation involves L1-L2a base-pairing that may facilitate formation of the double hammerhead. However, we conclude that L1-L2a also performs some other essential function in the satRPV RNA replication cycle, because the L1-L2a base-pairing is more important than efficient self-cleavage for replication.

A sequence required for -1 ribosomal frameshifting located four kilobases downstream of the frameshift site.

Programmed ribosomal frameshifting allows one mRNA to encode regulate expression of, multiple open reading frames (ORFs). The polymerase encoded by ORF 2 of Barley yellow dwarf virus (BYDV) is expressed via minus one (-1) frameshifting from the overlapping ORF 1. Previously, this appeared to be mediated by a 116 nt RNA sequence that contains canonical -1 frameshift signals including a shifty heptanucleotide followed by a highly structured region. However, unlike known -1 frameshift signals, the reporter system required the zero frame stop codon and did not require a consensus shifty site for expression of the -1 ORF. In contrast, full-length viral RNA required a functional shifty site for frameshifting in wheat germ extract, while the stop codon was not required. Increasing translation initiation efficiency by addition of a 5' cap on the naturally uncapped viral RNA, decreased the frameshift rate. Unlike any other known RNA, a region four kilobases downstream of the frameshift site was required for frameshifting. This included an essential 55 base tract followed by a 179 base tract that contributed to full frameshifting. The effects of most mutations on frameshifting correlated with the ability of viral RNA to replicate in oat protoplasts, indicating that the wheat germ extract accurately reflected control of BYDV RNA translation in the infected cell. However, the overall frameshift rate appeared to be higher in infected cells, based on immunodetection of viral proteins. These findings show that use of short recoding sequences out of context in reporter constructs may overlook distant signals. Most importantly, the remarkably long-distance interaction reported here suggests the presence of a novel structure that can facilitate ribosomal frameshifting.

New genotypes and factors associated with Cryptosporidium detection in mussels (Mytilus spp.) along the California coast.

A 3 year study was conducted to evaluate mussels as bioindicators of faecal contamination in coastal ecosystems of California. Haemolymph samples from 4680 mussels (Mytilus spp.) were tested for Cryptosporidium genotypes using PCR amplification and DNA sequence analysis. Our hypotheses were that mussels collected from sites near livestock runoff or human sewage outflow would be more likely to contain the faecal pathogen Cryptosporidium than mussels collected distant to these sites, and that the prevalence would be greatest during the wet season when runoff into the nearshore marine environment was highest. To test these hypotheses, 156 batches of sentinel mussels were collected quarterly at nearshore marine sites considered at higher risk for exposure to livestock runoff, higher risk for exposure to human sewage, or lower risk for exposure to both faecal sources. Cryptosporidium genotypes detected in Haemolymph samples from individual mussels included Cryptosporidium parvum, Cryptosporidium felis, Cryptosporidium andersoni, and two novel Cryptosporidium spp. Factors significantly associated with detection of Cryptosporidium spp. in mussel batches were exposure to freshwater outflow and mussel collection within a week following a precipitation event. Detection of Cryptosporidium spp. was not associated with higher or lower risk status for exposure to livestock faeces or human sewage sources. This study showed that mussels can be used to monitor water quality in California and suggests that humans and animals ingesting faecal-contaminated water and shellfish may be exposed to both host-specific and anthropozoonotic Cryptosporidium genotypes of public health significance.

Translational frameshifting by barley yellow dwarf virus RNA (PAV serotype) in Escherichia coli and in eukaryotic cell-free extracts.

The open reading frame (39K ORF) at the 5' end of the genome of barley yellow dwarf virus, PAV serotype (BYDV-PAV), overlaps with a 60K ORF by 13 nucleotides. Several approaches were used to show that the 60K ORF (putative polymerase gene) is translated by a low-frequency frameshift event in which some ribosomes shift into the 60K ORF rather than terminate at the 39K ORF stop codon. A sequence encompassing this region of overlap induced minus one (-1) translational frameshifting in heterologous and native contexts. In Escherichia coli, with the alpha subunit of lacZ used as a reporter gene, the rate of frameshifting caused by the BYDV-PAV sequence was approximately 3%. Amino acid sequencing of the transframe protein confirmed that ribosomes slip into the -1 frame in the overlapping region which includes a consensus shifty heptanucleotide: GGGUUUU. In a wheat germ translation system, BYDV-PAV genomic RNA from virions frameshifted about twice as efficiently as full-length transcripts from a cDNA clone. Frameshifting in rabbit reticulocyte lysates was much lower for either template. The identity of the 99-kDa wheat germ translation product was verified as the transframe protein by immunoprecipitation with antibody specific for the 60K ORF. These results support our previous observations of frameshifting in protoplasts and illustrate a subtle molecular control mechanism between this pathogen and its host cells.

Pericentric inversion of chromosome 16 in a large kindred: spectrum of morbidity and mortality in offspring.

Constitutional pericentric inversions of chromosome 16 are rare in the general population. We report here a large kindred who carry an inv(16)(p13q22) rearrangement. In general, individuals with the inv(16) are in good health but prone to reproductive loss. Two different types of recombinant offspring were identified in this family and analyzed at the molecular level using probes from the alpha-globin and polycystic kidney disease loci. Both were associated with serious major malformations.

Dysgenesis of testicular and streak gonads in the syndrome of mixed gonadal dysgenesis: perspective derived from a clinicopathologic analysis of twenty-one cases.

The clinical and pathologic aspects of 21 cases of mixed gonadal dysgenesis (MGD) were studied. The gonads in 15 patients consisted of a macroscopic testis and a streak gonad; six patients had variants, including two with bilateral testes and four with bilateral streak gonads or tumors. Functionally, the gonads were incompetent. Testes 1) failed to completely inhibit müllerian development, 2) failed to support full differentiation of mesonephric duct structures, 3) failed to adequately masculinize development of the external genitalia, or 4) often failed to mediate their own descent, resulting in asymmetry of the internal and external genitalia. None of the streak gonads mediated normal female adolescent development or fertility. Microscopic examination revealed that every gonad, regardless of its gross appearance, was morphologically abnormal. Although gonads with seminiferous tubules usually developed to a moderately advanced state, macroscopically resembling testes, the hilar zone remained architecturally disorganized; the cortex invariably lacked more than a rudimentary tunica albuginea or exhibited partial ovarian differentiation, sometimes even with a rare primordial follicle. Over time, the seminiferous tubules atrophied and hyalinized. Gonads that grossly resembled streak gonads were observed microscopically to be composed of a stroma resembling that of normal ovarian cortex. In patients more than several years of age, the entire complement of germ cells in streak gonads disappeared. It is suggested that patients with MGD be raised as females. Early removal of gonads will prevent the development of gonadoblastoma and dysgerminoma. If the uterus is retained and the patient is subsequently given exogenous estrogen, care should be taken to detect early any signs of the development of endometrial carcinoma or its precursor, to which these patients may be prone.

Enamel matrix proteins in normal and abnormal amelogenesis.

Histochemical techniques have been used to study three stages of enamel maturation in normal (fetal calf, newborn human, adult human) and abnormal amelogenesis (odontodysplasia, fluorosis, a compound-complex odontome, and an invaginated odontome). The amount of Type I matrix secreted decreases as amelogenesis progresses. Pockets of Type II matrix may be left at the dentino-enamel junction after maturation of enamel to Type III. Fluorosed enamel contains pockets which may extend the width of the enamel. All stages of enamel maturation may be seen in the compound-complex odontoma.

A microbiological study of normal flora of macropod dental plaque.

The percentage bacterial composition of dental plaques from 12 macropods was determined. The major group of organisms was gram-positive filamentous and pleomorphic rods whose median representation was 92.2%. These were mainly Actinomyces viscosus (median value 62.5%) and Bacterionema matruchotii (median value 23.2%). Streptococci were poorly represented (median value 4.2%) as were anaerobic gram-negative organisms. Aerobic gram-negative bacteria were identified as neisseriae and Moraxella sp. The macropod plaque flora was compared with that of human beings and rodents and the differences discussed in relation to dietary differences and the early externalization of the marsupial young.

Pharmacotherapy specialty certification examination. IV. 1992 results and process modifications, including recertification. The 1992 Specialty Council on Pharmacotherapy, Board of Pharmaceutical Specialties.

Certification of pharmacotherapy specialists is proceeding smoothly. Modifications to the examination process, which include reapportioning domains, offering the examination at several sites, and establishing the recertification process, have occurred. The guidelines for petitioners and structure of specialization continue to receive the attention and interest of prospective candidates, pharmacy organizations, and the BPS. To date, 674 specialists have been certified in the approved specialties: 175 nuclear pharmacists, 236 nutrition pharmacists, and 263 pharmacotherapy specialists.

Atypical fibromuscular hyperplasia. Report of two cases.

Two cases of atypical fibromuscular hyperplasia of the internal carotid artery (ICA) are reported. These cases are unique because the changes involve the intima of the vessels without involvement of medial structures as seen in the more common form of fibromuscular hyperplasia. Case 1 is only the fourth report of a symptomatic fibromuscular stenosis at the origin of the ICA. Case 2 is the first report of fibromuscular hyperplasia involving the common carotid artery. Changes similar to those in Case 2 (an elongated area of tubular fibromuscular stenosis) have been reported in the ICA. These atypical or intimal forms of fibromuscular hyperplasia appear to be more common in males and blacks, and may be more often unilateral as well.