Diagnosis and treatment of chronic T-lymphocytic leukemia in a spotted hyena (Crocuta crocuta).

Physical examination of an asymptomatic 20-yr-old intact female spotted hyena (Crocuta crocuta) revealed a midabdominal mass. A complete blood count (CBC) revealed peripheral lymphocytosis. Abdominal ultrasonography and laparoscopy confirmed severe splenomegaly. Cytologic examination of a bone-marrow core and histologic examination of spleen and liver biopsy samples revealed neoplastic small lymphocytes. Immunohistochemical staining of liver and spleen samples with the use of leukocyte-specific monoclonal antibodies showed that the neoplastic lymphocytes were immunoreactive to T-lymphocyte CD3 receptor and immunonegative to B-lymphocyte CD79a receptor. The morphology and distribution of neoplastic T-lymphocytes within the spleen, liver, peripheral blood, and bone marrow was most consistent with chronic T-lymphocytic leukemia. Treatment with chlorambucil and prednisone effectively decreased the lymphocyte count, but was associated with thrombocytopenia, which resolved after chlorambucil treatment was temporarily discontinued. Chemotherapy was resumed with a single dose of L-asparaginase, followed by a lower dosage of chlorambucil and continued prednisone. Two years after initial diagnosis, the hyena developed a hemoabdomen and was euthanized. Neoplastic T-lymphocytes were present in spleen, liver, visceral and peripheral lymph nodes, lungs, heart, kidney, adrenal glands, mesentery, intestines, pancreas, brain, and bone marrow.     

Chromatin immunoprecipitation: optimization, quantitative analysis and data normalization

Background

Chromatin remodeling, histone modifications and other chromatin-related processes play a crucial role in gene regulation. A very useful technique to study these processes is chromatin immunoprecipitation (ChIP). ChIP is widely used for a few model systems, including Arabidopsis, but establishment of the technique for other organisms is still remarkably challenging. Furthermore, quantitative analysis of the precipitated material and normalization of the data is often underestimated, negatively affecting data quality.

Results

We developed a robust ChIP protocol, using maize (Zea mays) as a model system, and present a general strategy to systematically optimize this protocol for any type of tissue. We propose endogenous controls for active and for repressed chromatin, and discuss various other controls that are essential for successful ChIP experiments. We experienced that the use of quantitative PCR (QPCR) is crucial for obtaining high quality ChIP data and we explain why. The method of data normalization has a major impact on the quality of ChIP analyses. Therefore, we analyzed different normalization strategies, resulting in a thorough discussion of the advantages and drawbacks of the various approaches.

Conclusion

Here we provide a robust ChIP protocol and strategy to optimize the protocol for any type of tissue; we argue that quantitative real-time PCR (QPCR) is the best method to analyze the precipitates, and present comprehensive insights into data normalization.     

Evidence of an acute phase response in dogs naturally infected with Babesia canis

The erythrocyte sedimentation rate (ESR), white blood cell count (WBC), haematocrit (HCT) and platelet number (PLT) were quantified and compared with the acute phase proteins (APPs) in dogs naturally infected with Babesia canis and healthy dogs. Both groups were treated with imidocarb dipropionate on the day of admission and both groups were monitored for all parameters on the admission day and on the first, second, third, fourth and seventh days in order to determine the presence of an acute phase reaction, to assess the diagnostic value of these markers in uncomplicated canine babesiosis and to evaluate the use of APPs in treatment monitoring. It was demonstrated that an acute phase response occurs in dogs naturally infected with Babesia canis, with significant increases in the concentration of major acute phase proteins. The serum concentration of C-reactive protein (CRP), serum amyloid A (SAA) and the erythrocyte sedimentation rate (ESR) decreased daily after treatment and approached reference range values by the eighth day. PLT and haematocrit (HCT) increased daily after treatment and approached reference range values by the fourth day. WBC and haptoglobin increased after treatment and then decreased from the third and fourth days, respectively, to the eighth day. The diagnostic sensitivity of CRP, SAA and PLT was significantly higher compared to haptoglobin, ESR, HCT and the WBC count. CRP and SAA were of clinical use in monitoring the response to antibabesial treatment.     

Monitoring and efficacy of selected insecticides for European corn borer (<Emphasis Type="Italic">Ostrinia nubilalis</Emphasis> Hubn., Lepidoptera: Crambidae) control

The European corn borer (ECB), Ostrinia nubilalis (H?bner) (Lepidoptera: Crambidae), is the major arthropod pest of corn in Croatia. However, chemical control is carried out only in maize for seed production, and in sweet corn. A 3-year investigation was carried out in corn fields in northwest Croatia to establish the most attractive pheromone lure for ECB monitoring, the optimal timing of insecticide applications, and the efficacy of selected insecticides against ECB. During each of the 3 years, the pheromone lures E, Z and E/Z («Isagro») were evaluated in the field from May to July. Insecticides were sprayed 14–20 days after the maximal capture in 2002 and 2003. In 2004, three different trials were set up: one trial with one early treatment (when corn was in the R49 development stage according to the extended BBCH scale); one with one late treatment (when corn was in the R65 development stage according to the BBCH scale); and one with two treatments in both stages. In the trials studying Bacillus thuringiensis var. kurstaki (B.t.k.), insect growth regulators (IGR), spinosad and classical chemical insecticides were applied. Pheromone lure E was the most attractive for monitoring ECB flights throughout 2 years of investigation. Insecticide efficacy depended on application timing. The insecticides most suitable for integrated pest management (IPM) programs, including spinosad, B.t.k. and IGR, resulted in high (spinosad) to moderate (B.t.k.) efficacy. The efficacy of IGRs indicates the need for earlier application. The efficacy of chemical insecticides depended on the year and the insecticide. The organophosphate (OP) insecticides and pyrethroids gave moderate to good results; imidacloprid did not provide significant efficacy. One application of all insecticides based on proper (early) timing resulted in the same efficacy as two applications of the same insecticides.     

How to protect a diverse,poorly known,inaccessible fauna: identification and protection of source and sink habitats in the epikarst

• 1. Aquatic subterranean species are often geographically and numerically scarce. Many of these species are denizens of epikarst, the uppermost zone of karst with semi‐isolated solutional openings and channels, and are only known from drip pools in caves where they accumulate as a result of animals dripping out of the epikarst.
• 2. The question of whether these pool communities adequately reflected the epikarst community was addressed by directly collecting animals from drips in a continuous collecting device.
• 3. The study area was six caves in Slovenia, where a total of 35 drips and associated pools were sampled for copepods for a period of approximately one year. A total of 37 copepod species were found, 25 of them stygobionts and 16 epikarst specialists.
• 4. Overall, the frequency of stygobionts was 1.5 times higher in drips compared with pools and the frequency of epikarst specialists was three times higher in drips compared with pools, and the frequency of immature individuals was higher in drips compared with pools, with the exception of one artificially enlarged pool in ?kocjanske jame. The cause of this difference is probably increased juvenile mortality in pools and reduced reproduction, indicating that pools are not source populations.
• 5. The results of this research suggest that epikarst per se, not just the sampling sites (including pools) in caves, needs to be the focus of conservation planning. Copyright © 2010 John Wiley & Sons, Ltd.

     

Clinical, Serologic, and Parasitologic Follow-Up after Long-Term Allopurinol Therapy of Dogs Naturally Infected with Leishmania infantum

Canine leishmaniasis usually is treated with antimony compounds, but frequent relapses, adverse effects, high costs, and development of resistance to long-term antimonial therapy emphasize the importance of searching for alternative antileishmanial drugs. Allopurinol was used at a dosage of 10 mg/kg/day PO to treat 10 dogs naturally infected with Leishmania infantum for a period of 2-24 months. Nine dogs recovered within 2-6 months of chemotherapy, and no relapses were observed during the treatment of up to 20 months. However, 3 of 4 dogs relapsed after treatment was discontinued. These dogs again recovered clinically when therapy was resumed. Parasite-specific immunoglobulin concentrations (IgG2) were high in all dogs before therapy and remained high even in clinically cured dogs during or after therapy. On the other hand, specific IgG1 reactions, which have been shown to be detectable in symptomatic animals, persisted in 7 dogs for long periods after clinical recovery. Three of these dogs relapsed within 2-4 weeks after interrupting therapy. However, 1 dog with no detectable specific IgG1 reaction at the end of therapy did not relapse in the following 4 months. Parasites could be detected in 8 of 9 dogs after clinical improvement by in vitro cultivation or polymerase chain reaction (PCR) testing of lymph node aspirates. In 4 of these dogs, parasites also were detected in blood samples by PCR. Hence, these clinically cured dogs must be regarded as reservoirs of Leishmania and allopurinol cannot be recommended in endemic areas.     

Status quo of adoption of precision agriculture enabling technologies in Swiss plant production

Precision Agriculture - This paper presents the state of application of Precision Agricultural enabling Technology (PAT) in Swiss farms as an example for small-scale, highly mechanised Central...     

Physiological and molecular characterization of Fe acquisition by tomato plants from natural Fe complexes

The aim of this work is to evaluate the capability of tomato plants to use different Fe sources, such as Fe citrate, Fe phytosiderophores, and Fe complexed by a water-extractable humic substances (Fe-WEHS) also in relation to physiological and molecular adaptations induced by these complexes at the root level. Tomato plants acquired higher amounts of Fe from Fe-WEHS than from the other two sources and this phenomenon occurred only when the treatment lasted 24 h. The higher acquisition of Fe from Fe-WEHS than other sources depended on a reductive mechanism and on rhizosphere acidification and appeared to be due neither to a higher apoplastic loading nor to a higher resistance of WEHS to microbial degradation. Supply of the different Fe complexes to deficient plants induced a transient upregulation of Fe(III)-chelate reductase (LeFRO1) and Fe transporter genes, LeIRT1 and LeIRT2. In Fe-WEHS-fed plants, where a quicker and higher upregulation of these genes was evident, a coordination in the expression of LeFRO1, LeIRT1, and LeIRT2 genes occurred already after 1 h treatment when the amount of Fe acquired by the plants from the three sources was similar. Iron from Fe-WEHS could be efficiently acquired in a mixture of natural Fe complexes possibly occurring in the rhizosphere. This phenomenon is due to an altered expression of Fe uptake-related genes and to the root capacity to create favorable conditions for the micronutrient uptake into the rhizosphere.     

Pathology in Continuous Infusion Studies in Rodents and Non-Rodents and ITO (Infusion Technology Organisation)-Recommended Protocol for Tissue Sampling and Terminology for Procedure-Related Lesions

Many variables may affect the outcome of continuous infusion studies. The results largely depend on the experience of the laboratory performing these studies, the technical equipment used, the choice of blood vessels and hence the surgical technique as well the quality of pathological evaluation. The latter is of major interest due to the fact that the pathologist is not involved until necropsy in most cases, i.e. not dealing with the complicated surgical or in-life procedures of this study type. The technique of tissue sampling during necropsy and the histology processing procedures may influence the tissues presented for evaluation, hence the pathologist may be a source of misinterpretation. Therefore, ITO proposes a tissue sampling procedure and a standard nomenclature for pathological lesions for all sites and tissues in contact with the port-access and/or catheter system.