Characterization of a single dose methylprednisolone acetate immune suppression model using Cryptosporidium muris and Cryptosporidium parvum

An immunosuppressive dose of methylprednisolone acetate (MPA) was compared with a non-immunosuppressive dose using Cryptosporidium oocyst production as an indicator of immunosuppression. To be classified as immunosuppressive, the dose had to satisfy five criteria. First, the dose had to abrogate normal immune defenses allowing the propagation of an organism to which the host is normally resistant, i.e. Cryptosporidium parvum in adult mice. Second, the dose had to decrease overall circulating CD4 T-lymphocyte numbers by greater than 80%. Third, the immunosuppressive dose had to prolong the infection beyond the normal infection length, and fourth, increase the severity of an active infection. Lastly, after complete recovery from a C. muris infection, immunosuppression must suppress the naturally acquired post infection immunity and allow reinfection. In mice immunosuppression with 600 mgMPA/kg lasted approximately 14 days and satisfied all five criteria. Fecal oocyst production could be perpetuated by dosing at 10-day intervals. A 200 mgMPA/kg dose transiently lowered CD4 counts by over 80%, but failed to override the naturally acquired post infection immunity or allow infection with C. parvum. The immunosuppressed blood profile consisted of an immediate sharp rise of mature segmented neutrophils combined with a severe decrease in circulating T-lymphocyte numbers. The rise and fall of neutrophils proved to be a good indicator of the severity and duration of immunosuppression. The thymus and spleen likewise contracted and then expanded in accordance with the steroid effect. The metabolism of MPA resulted in the eventual recovery of immune function signified by the cessation of C. parvum oocyst production. The recovery blood profile was associated with circulating CD8 counts near control levels, continuing 80% depression of CD4 counts and a dropping total neutrophil count. This study shows that the 600 mg/kg MPA dose is a good model for immunosuppression, which satisfies all five criteria for immunosuppression with low morbidity and low mortality.     

Ice record of delta13C for atmospheric CH4 across the Younger Dryas-Preboreal transition

We report atmospheric methane carbon isotope ratios (delta13CH4) from the Western Greenland ice margin spanning the Younger Dryas-to-Preboreal (YD-PB) transition. Over the recorded approximately 800 years, delta13CH4 was around -46 per mil (per thousand); that is, approximately 1 per thousand higher than in the modern atmosphere and approximately 5.5 per thousand higher than would be expected from budgets without 13C-rich anthropogenic emissions. This requires higher natural 13C-rich emissions or stronger sink fractionation than conventionally assumed. Constant delta13CH4 during the rise in methane concentration at the YD-PB transition is consistent with additional emissions from tropical wetlands, or aerobic plant CH4 production, or with a multisource scenario. A marine clathrate source is unlikely.     

Chinese Box turtle (Cuora flavomarginata) with lymphoid leukemia characterized by immunohistochemical and cytochemical phenotyping

Lymphoid leukemia of T‐cell origin was diagnosed in a male Chinese Box turtle, Cuora flavomarginata, of approximately 25 years of age. The turtle presented with a history of anorexia, open‐mouth breathing, and lethargy for one week. The CBC findings included a mildly increased PCV, and severe leukocytosis due to high numbers of atypical cells interpreted to be blasts. The blasts were medium‐sized cells with round to pleomorphic nuclei, slightly clumped chromatin, indistinct nucleoli, and scant moderate‐to‐dark blue cytoplasm with occasional red‐to‐purple cytoplasmic granulation. Cytochemical and immunohistochemical staining indicated that the neoplastic cells were positive for CD3 and α‐naphthyl butyrate esterase (ANBE), leading to the diagnosis of T‐cell lymphoid leukemia. Histology of tissues collected at necropsy showed multifocal infiltrations of neoplastic round cells in the liver, spleen, kidneys, testicles, pancreas, thyroid, duodenum, bone marrow, epicardium, and myocardium. Transmission electron microscopy failed to identify viral particles within the neoplastic cells. This article describes the hematologic, histologic, and ultrastructural abnormalities associated with lymphoid leukemia in this turtle, and advanced diagnostic methods used for phenotyping the T‐cell origin.     

LHRH Fusion Protein Immunization Alters Testicular Development, Ultrasonographic and Histological Appearance of Ram Testis

This study was designed to evaluate the effectiveness of a luteinizing hormone releasing hormone (LHRH) fusion protein immunization on reproductive traits in ram lambs including the changes in histology and ultrasonographic appearance of testis. Thirty native ram lambs at 19 weeks of age were divided into control (C, n = 10), immunization (I, n = 10) and castration (E, n = 10) groups. Animals in immunization group were immunized against LHRH using ovalbumin‐LHRH‐7 (OL) protein generated by recombinant DNA technology as a primary and a booster injection at 19 and 23 weeks of age respectively. Animals were bled via jugular venepuncture at 2‐week intervals to determine LHRH antibody and testosterone concentrations. Bi‐weekly ultrasonographic examination of the testes was performed to determine the changes in ultrasonographic appearance as the age increased. Biopsied testicular tissues taken at 19, 29 and 41 weeks age were also evaluated. At 41 weeks of age, animals were slaughtered. Semen and epididymis were evaluated for the presence of sperm cells. Testicular development and sperm production were suppressed in the immunized animals. Semineferous tubule diameters decreased, basal membrane of the tubule was thickened and hyalinized in immunized ram lambs. While testes of control animals gained their normal ultrasonographic appearance as the age increased, immunized animals had uniform hypoechogenic testicular structure as observed at 19 weeks of age until slaughter. Simultaneous histological and ultrasonographic evaluations indicated that the changes in testicular histology could partly be monitored via ultrasonographic imaging. Nevertheless, it is difficult to claim that ultrasonographic image reflects the exact changes in such instances. In conclusion, these results indicate that recombinant OL fusion protein is effective in immunocastration in ram lambs and has a potential to be used as an alternative to physical castration. Further research studies should be conducted to help assess reproductive status of testes from ultrasound images.     

Identification of Sperm-Head Morphometric Subpopulations in Iberian Red Deer Epididymal Sperm Samples

Computer-assisted sperm morphometry analysis (CASMA) was used in this study to identify sperm morphometric subpopulations in Iberian red deer epididymal sperm samples. Epididymal sperm samples were collected from 37 mature stags and were divided. One portion was diluted in a Tris–citrate–egg yolk medium. A microscope slide was prepared from single extended sperm samples prior to freezing. The remainder of each sample was frozen in nitrogen vapours using a conventional protocol. After thawing, sperm smears were prepared as described for extended samples. All slides were air-dried and stained with Hemacolor^®. The sperm-head dimensions for a minimum of 145 sperm-heads were analyzed from each sample by means of the Sperm-Class Analyser^®, and the mean measurements recorded. Each sperm-head was measured for four primary sperm-head parameters, and five parameters of head shape. All sperm morphometric parameters evaluated were placed in a statistical database and a multivariate cluster analysis was performed. The clustering analyses, based on 10 867 individual spermatozoa, revealed the existence of three subpopulations (SP₁, SP₂, SP₃) of spermatozoa with different morphometric characteristics (p  <  0.001). The proportion of spermatozoa present in any of the three subpopulations remained constant (p  >  0.05) through the cryopreservation process. Pre-freeze and post-thaw sperm quality was in vitro evaluated by microscopic assessments of individual sperm motility and of plasma membrane and acrosome integrities. In conclusion, our results show that applying the CASMA techniques and multivariate cluster analyses, it was possible to determine that three subtle subpopulations of spermatozoa with different morphometric characteristics coexist in red deer semen.     

Anticoagulant‐dependent in vitro hemagglutination in a cat

Abstract: A 17‐year‐old domestic shorthaired cat was presented to the Cornell University Hospital for Animals for recheck of hyperthyroidism previously treated with radioiodine. Marked agglutination was noted in a blood sample collected into EDTA for a CBC; no other clinical or hematologic evidence of hemolysis was observed and none developed despite persistent agglutination in additional EDTA samples collected over a 2‐month period. Blood drawn into heparin and sodium citrate tubes did not have grossly or microscopically detectable agglutination, unless EDTA was added. Plasma from the cat induced agglutination of washed RBCs from a control cat in the presence of EDTA but not in the presence of heparin. Flow cytometric analysis of samples created by mixing plasma from the patient with washed RBCs from a control cat showed immunoglobulin coating of the control RBCs, predominantly by IgM. These findings suggested an anticoagulant‐dependent antibody‐mediated mechanism for the agglutination. EDTA‐dependent hemagglutination has not been reported previously in cats, although rare cases have been described in humans. The phenomenon needs to be recognized as an in vitro occurrence in order to prevent erroneous diagnosis of immune‐mediated hemolytic anemia.     

Alteration of soil labile organic carbon by invasive earthworms (Pontoscolex corethrurus) in tropical rubber plantations

The invasive earthworm (Pontoscolex corethrurus) is commonly found in rubber plantations of Xishuangbanna, southwestern China. To understand the long-term impact of this invasive earthworm on soil labile organic carbon (LOC), we examined changes in LOC that was protected and unprotected by soil aggregates during a worm-exclusion experiment in rubber plantations of Xishuangbanna. We found that the presence of this invasive earthworm for 20 months increased LOC (up to 35%) protected by aggregates in surface soil layer (0–5 cm). In contrast, the presence of this earthworm increased LOC unprotected by the aggregates and the turnover rate of LOC protected by the aggregates in subsurface soil layer (5–15 cm). Soil total LOC did not differ between the control and worm-exclusion treatments. These findings suggest that the invasion of P. corethrurus can redistribute LOC along soil vertical profiles with accumulation of protected LOC on surface soil layer and unprotected LOC on subsurface soil layer. Earthworms' redistribution of LOC between the protected and unprotected forms and along soil profile may affect long-term soil carbon cycling.     

On the structural and species diversity effects of bark beetle disturbance in forests during initial and advanced early-seral stages at different scales

Following disturbances, early-seral stages of forests provide a variety of structures. Whether this variety is a short-term phenomenon or influences forest succession for several decades or even longer is not known. We tested the hypotheses that after spruce dieback caused by bark beetles, a high spatial heterogeneity of stand structures will persist within stands and among stands even in advanced early-seral stages and that species taxonomical and functional diversity measures will reflect this heterogeneity. We used a chronosequence of unmanaged forests in the Berchtesgaden National Park (Germany) consisting of mature undisturbed spruce stands (control), stands belonging to an initial early-seral stage (~3 years after disturbance) and stands in an advanced early-seral stage (~20 years after disturbance). We analysed diversity and heterogeneity of these forest stands including stand structure, species density, species composition and functional–phylogenetic diversity of vascular plants, wood-inhabiting fungi and saproxylic beetles within plots, among plots of the same successional stage and among stages. Stands of the advanced early-seral stage were characterized by a high spatial heterogeneity of structural attributes, such as crown cover, regeneration density and spatial distribution of trees. Among-plot taxonomic beta diversity was highest in the advanced early-seral stage for beetles, but lowest for fungi, while beta diversity of plants among plots remained unchanged during succession. The mosaic of successional stages initiated by bark beetles increased the gamma diversity of the study area, especially for fungi and beetles. Our findings support the hypothesis that structural heterogeneity continues for at least two decades at stand and landscape scales and that species turnover among successional stages is a major mechanism for gamma diversity in forests after bark beetle disturbance.