Gene discovery and expression profiling in porcine Peyer's patch

Peyer's patches of the intestinal mucosa are essential for host defense and immune regulation in the enteric system. To better understand molecular mechanisms of Peyer's patch function, we have screened for differentially expressed genes specific to Peyer's patch. cDNA libraries were created from normal Peyer's patch, immune stimulated Peyer's patch, and pooled cDNA subtracted with fibroblast RNA. From the subtracted library, 3687 expressed sequence tags (ESTs), representing 2414 unique nucleotide sequences, were isolated, identified by BLAST searches against public databases, and spotted onto a microarray for gene expression profiling. Approximately 30% of these ESTs BLAST to genes of unknown function and 20% have no known homology in the public databases (novel genes). Of the novel genes, 70% are expressed in normal immune tissues by microarray analysis, suggesting that at least 371 of the unidentified EST sequences from the subtracted library are novel porcine genes and can now be further characterized to determine their function in the porcine Peyer's patch. We surmise that the products of these genes participate in biochemical and cellular functions related to the unique immunological and gastroenterological functions of the small intestine. The BLAST and gene ontology information for each of the subtracted library EST sequences, the normal and immune stimulated libraries, and the microarray are all valuable resources that will facilitate further examination of the biological function of porcine Peyer's patch tissue.     

Mitochondrial damage as an early event of monensin-induced cell injury in cultured fibroblasts L929

The present study was designed to identify, submicroscopically, the primary organelle or target structure for monensin in cultured murine fibroblasts L929. In addition, the effect of the drug on cell size and surface membranes of the cells were analysed; cellular proliferation, collagen secretion, and necrosis and apoptosis were re-evaluated. At the lowest concentration of monensin the foremost ultrastructural alteration occurred in the mitochondria, characterized by increased matrix density with disorganized and less distinct crystae. Incubation with monensin at higher concentrations resulted in severe mitochondrial damage and marked dilatation of the Golgi apparatus and rough endoplasmic reticulum cisternae. Fibroblasts exposed to higher concentrations of monensin were enlarged with decreased number of filopodia and hollows in the surface membrane. Moreover, monensin inhibited the cell proliferation, increased immunohistochemical positiveness for collagen type I in a dose-dependent manner, and, at high concentrations, caused cell necrosis whereas apoptosis was not induced. Taken together, these results show that monensin induces early mitochondrial damage, possibly causing an energy deficit that led to inhibition of fibroblasts proliferation and accumulation of collagen causing dilatation of Golgi apparatus and rough endoplasmic reticulum. Moreover, the mitochondrial damage would also explain the monensin-induced necrosis.     

Evaluation of the internal vertebral venous plexus, vertebral canal, dural sac, and vertebral body via nonselective computed tomographic venography in the cervical vertebral column in healthy dogs

OBJECTIVE: To evaluate nonselective computed tomographic (CT) venography for evaluating the cervical internal vertebral venous plexus (IVVP), define the diameter and area dimensions of the IVVP, and determine the relationship between dimensions of the cervical IVVP and other vertebral components in medium-sized dogs. Animals-6 healthy dogs that weighed 18 to 27 kg. Procedure-Helical CT scans were performed from C1 to C7 before and after IV injection of contrast medium (480 mg of iodine/kg) and a continuous infusion (240 mg of iodine/kg). Image data were transferred to a CT workstation, and measurements were performed on displayed transverse images. Diameter and area measurements of the vertebral canal, dural sac, IVVP, and vertebral body were obtained at C3 to C7. RESULTS: Opacification of vertebral venous structures was achieved in all dogs with no adverse reactions. Sagittal diameters of the IVVP for C3 to C7 ranged from 0.6 to 3.2 mm. Transverse diameters ranged from 2.32 to 5.74 mm. The IVVP area represented 12.4% of the mean vertebral canal transverse area and 30.61% of the mean vertebral epidural space area. Area measurements of the IVVP were significantly correlated with vertebral canal area and dural sac area. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that nonselective CT venography is a safe, sensitive method for performing morphometric assessments of the cervical IVVP in dogs. Findings support the theory that there may be a physiologic or developmental relationship between cervical vertebral canal components.     

Hemorrhagic disease in dogs infected with an unclassified intraendothelial piroplasm in southern Brazil

A hemorrhagic disease affecting dogs in Brazil, referred to popularly as "nambiuvú" (bloody ears) and believed to be transmitted by ticks, has been observed in animals infected with an organism described originally in 1910 as a piroplasm, and known locally as Rangelia vitalli. In this series of 10 cases, the disease was characterized by anaemia, jaundice, fever, spleno- and lymphadenomegaly, hemorrhage in the gastrointestinal tract, and persistent bleeding from the nose, oral cavity and tips, margins and outer surface of the pinnae. The ixodid ticks Rhipicephalus sanguineus and Amblyomma aureolatum infested affected dogs from suburban and rural areas, respectively. Laboratory findings included regenerative anaemia, spherocytosis, icteric plasma and bilirubinuria. Those intracellular organisms were found in bone marrow smears but not in blood smears. Microscopically, zoites were seen within the cytoplasm of blood capillary endothelial cells. Parasitized and non-parasitized endothelial cells were positive immunohistochemically for von Willebrand factor (vWF). Langhans-type multinucleate giant cells were observed in the lymph nodes and choroid plexus. There was prominent erythrophagocytosis by macrophages in the lymph node sinuses and infiltration of the medullary cords by numerous plasma cells. Ultrastructurally, this organism had an apical complex that included a polar ring and rhoptries but no conoid. This parasite was contained within a parasitophorous vacuole that had a trilaminar membrane with villar protrusions and was situated in the cytoplasm of capillary endothelial cells. This organism tested positive by immunohistochemistry for Babesia microti. This pathogen was also positive by in situ hybridization for B. microti. Tentative clinical diagnosis in these cases was based on the history, clinical picture, haemogram and favorable response to therapy, and confirmed through microscopic examination of smears from the bone marrow or histological sections of multiple tissues, especially lymph nodes where zoites were most frequently found. The disease was reproduced by intravenous inoculation of blood from a naturally infected dog into an experimental dog. The authors demonstrate in this study that this organism is a protozoa of the phylum Apicomplexa, order Piroplasmorida. This piroplasm seems to be different from Babesia since it has an intraendothelial stage. Molecular phylogenetic analysis is necessary to better characterize this parasite and clarify its taxonomic status.     

Relative sensitivity of a herbicide-resistant rutabaga genotype to cyanazine and metribuzin

Growth and photochemical response of triazine-susceptible and -resistant rutabaga ‘Laurentian’ genotypes to cyanazine (chloro-s-triazine) and metribuzin (methylthio-as-triazine) were evaluated. The young seedlings of the susceptible rutabaga were killed when cyanazine or metribuzin were applied either pre- or post-emergence. The triazine-resistant rutabaga, however, displayed a differential response to these herbicides. Metribuzin applied pre-emergence killed the seedlings at 0.4 kg ha^?1, and at 0.2 kg ha^?1 the growth was severely affected. Cyanazine even at higher rates applied pre- or post-emergence failed to inhibit growth in these plants.Chlorophyll fluorescence in leaf sections at the cotyledonary, 2-leaf and 4-leaf stages in the susceptible plants increased by 130 and 172% in response to 10^?5 M cyanazine and metribuzin, respectively, suggesting that Photosystem II reactions in these plants were severely impaired. In resistant plants, there was little or no leaf chlorophyll fluorescence (LCF) increase at this herbicide concentration. However, at 10^?4 M cyanazine and metribuzin, the LCF in resistant plants increased significantly and the increase was greater in response to metribuzin (86%) than to cyanazine (51%). This indicates that with these triazine-resistant genotypes, metribuzin is relatively more potent and at high rates is capable of interfering with photosynthesis.     

Seroepidemiology of neosporosis in dairy cattle in Uruguay

A seroepidemiological survey of Neospora caninum was done in a dairy farm in Uruguay employing indirect fluorescent antibody test (IFAT). Sera from 217 cattle (155 cows, 31 heifers and 31 calves) were tested for N. caninum antibodies in July 2000 and it was found that abortions were significantly associated with the seropositivity of the cows by chi(2)-test (P = 0.025). The cows with an IFAT titre of 1:3200 were at a significantly higher risk of abortion than seronegative cows (P = 0.0013). Neospora organisms were detected in the brains of two aborted foetuses by immunohistochemical examination. The infection of N. caninum was considered postnatal because of the even distribution of seropositive cows (60%), low seroprevalence (20%) in calves and no significant correlation in the serostatus between calves and their dams (P = 0.43). A fitted binomial generalised linear model revealed that there was an increasing risk of abortion with increasing IFAT titre and increasing age between 2 and 6 years.     

Experimental acute canine monocytic ehrlichiosis: clinicopathological and immunopathological findings

Clinical signs, humoral and cellular immune responses, and microscopic and gross tissue alterations resulting from acute experimental Ehrlichia canis infection in dogs were studied. Four dogs were inoculated with E. canis and four were used as uninfected controls. After a 10-14-day incubation period, infected dogs developed pyrexia up to 41 degrees C for 6-8 days. Antibody titers to E. canis antigen were demonstrable in all inoculated dogs at 30 days post-infection. Necropsy of infected animals revealed pale mucous membranes, generalized lymphadenopathy, splenomegaly, edema and ascites. Microcopically, the main lesions were: lymphoreticular hyperplasia in cortical areas of lymph nodes and spleenic white pulp, periportal accumulation of mononuclear cells and centrolobular fatty degeneration of the liver. Kidneys presented with glomerulonephritis characterized by interstitial mononuclear infiltration. Immunophenotyping of lymphocytes from lymph nodes and spleen sections displayed alterations in IgG, IgM, CD3+ and CD8+ cells population in infected dogs.     

Prevalence of anti-Neospora caninum antibodies in cattle and dogs from Western Amazon, Brazil, in association with some possible risk factors

For evaluation of the prevalence of anti-Neospora caninum antibodies and its associated risk factors, serum samples from 2109 cattle (11 beef, 50 dairy and 25 mixed farms) and 174 dogs were examined in the State of Rond?nia, Western Amazon, Brazil. An inquiry was applied in each farm. Sera were examined by the Indirect Fluorescence Antibody Test (IFAT) using cut off dilution of 1:25 for cattle and 1:50 for dogs. Statistical association between the serologic status and several variables were analyzed by linear and logistic regression. The overall herd prevalence of anti-N. caninum antibodies for 86 farms was 72% (61.3-81.2%). Prevalence values were 100, 70 and 64% in beef, dairy and mixed herds, respectively. Herd prevalence in beef herds was significantly different (P<0.05) from dairy and mixed herds. The overall animal prevalence of N. caninum in cattle was 8.8%. Prevalence values by animal were similar in different production types (P>0.05), with values of 9.5, 11.2 and 9.7% for beef, dairy or mixed cattle, respectively. Antibodies were found in 12.6% of the 174 examined dogs. Sixteen (22.8%) out of 70 farms with dogs had at least one dog with anti-N. caninum antibodies. The occurrence of antibodies in cattle was statistically associated with farms having more than 25 cows (OR 9.7, 95% IC 2.9-32.2; P=0.0002). There was no significant association between the presence of the dogs, jungle contact or reproductive variables with the occurrence of antibodies in cattle.     

Is the decline of birds and amphibians in a steppe lake of northern Patagonia a consequence of limnological changes following fish introduction?

• 1. Laguna Blanca, in Laguna Blanca National Park, is a lake in Patagonia which has been designated as a Ramsar site since 1971 because of bird diversity and abundance and importance for nesting, particularly for the black neck swan, Cygnus melanocoryphus. It is also valued for its populations of endemic amphibians, Atelognathus patagonicus and Atelognathus praebasalticus.
• 2. Avian and amphibian populations have decreased dramatically in recent years. Percichthys colhuapiensis, Percichthys trucha (Pisces, Percichthyidae), Salmo trutta and Oncorhynchus mykiss (Pisces, Salmonidae) were introduced into Laguna Blanca in 1965. Since 1986, no Atelognathus frogs have been found. The abundance of swans and coots, which are strongly associated with macrophytes for nesting and feeding, has diminished drastically, whereas piscivorous birds have increased.
• 3. The fishless condition of some neighbouring small lakes with abundant pond weeds, aquatic birds and endemic amphibians, was assessed in order to compare the physical and chemical characteristics and the quantitative composition of the benthos among lakes. Fish presence at Laguna Blanca and its absence at El Burro, Antiñir and Jabón lakes, were confirmed. Compared with previous results, it seems that the abundance of Amphipoda (Hyalella), Copepoda and Cladocera at Laguna Blanca has decreased, while Acari has increased and Notostraca has disappeared. Water transparency has diminished in Laguna Blanca and now is lower than that of fishless lakes.
• 4. P. colhuapiensis were captured only in Laguna Blanca, with the highest captures in the littoral zone. The population shows rapid individual growth in the early years and an absence of fish older than 6 years. The preponderance of benthos and the presence of macrophytes in the gut contents of adult P. colhuapiensis appear to indicate that they are important consumers of these resources.
• 5. This paper concludes that fish introduction in Laguna Blanca led to a complex trophic cascade effect (fish predation on tadpoles, fish competition for avian food, bottom disturbance, zooplankton reduction) producing deleterious effects on the amphibian and bird populations.

Copyright © 2006 John Wiley & Sons, Ltd.     

Sensitivity of Lasiodiplodia theobromae from Brazilian papaya orchards to MBC and DMI fungicides

Stem rot caused by Lasiodiplodia theobromae is an important postharvest disease of papaya in Brazil, responsible for reducing the quality and quantity of fruits. Fungicide use is one of the main disease management measures. However, there are no estimates available of pathogen sensitivity to commonly employed fungicides. Therefore, the EC₅₀ from 120 isolates of L. theobromae from northeastern Brazil, representative of six populations of the pathogen, was estimated in vitro for fungicides of the methyl benzimidazole carbamates—MBC (benomyl and thiabendazole) and demethylation-inhibiting—DMI (imazalil, prochloraz, tebuconazole) groups. Mycelial growth on fungicide-free media and virulence on papaya fruits of the MBC-sensitive and non-sensitive isolates were compared. For MBCs, 8.4% of isolates were non-sensitive to fungicides. For the remaining 91.6%, the mean EC₅₀ ranged from 0.002 to 0.13 μg ml⁻¹ and 0.36 to 1.27 μg ml⁻¹ for benomyl and thiabendazole, respectively. For DMIs, the mean EC₅₀ range for imazalil was 0.001 to 2.27 μg ml⁻¹, 0.04 to 1.75 μg ml⁻¹ for prochloraz, and 0.14 to 4.05 μg ml⁻¹ for tebuconazole. The EC₅₀ values of non-sensitive isolates were significantly (P≤0.05) higher those for the sensitive isolates for each of the DMI fungicides. Differences (P≤0.05) were found in the levels of sensitivity to DMI fungicides among the isolate populations associated with orchards. The populations from two orchards were less sensitive to DMIs. No solid evidence was found for fitness costs relating to MBC non-sensitive isolates because mycelial growth in fungicide-free media and virulence on papaya fruits were similar to those of sensitive isolates.