Development and validation of a SYBR green real-time PCR for the quantification of porcine circovirus type 2 in serum, buffy coat, feces, and multiple tissues

The emergence of multiple genotypes of PCV2, as demonstrated by phylogenetic analysis of whole genome or capsid sequences, makes it necessary to have quantitative diagnostic assays that perform equally well on all strains. The objectives of this study were to develop and validate a novel real-time polymerase chain reaction (PCR) assay targeting the highly conserved rep gene (ORF1) and investigate the effects of diagnostic specimen choice on its performance. The assay was tested in naturally infected conventional pigs, experimentally infected gnotobiotic pigs, and plasmid-spiked negative serum, lung tissue, and feces and found to have a linear detection range of 2.2x10(3) to 2.2x10(10) copies of PCV2 per mL. The assay successfully detected and quantified PCV2 DNA in serum, buffy coat, feces, and multiple lymphoid (bronchial, mesenteric, and superficial inguinal lymph nodes; thymus; tonsil; ileal Peyer's patches; and spleen), and non-lymphoid (myocardium; lung; kidney; liver; and gluteal muscle) tissues from naturally infected pigs. Across all tissues and sera of naturally infected pigs, the mean PCV2 concentration was 3.0logs higher in wasting versus non-wasting pigs. PCV2 concentration measured by tissue culture and immunohistochemical staining in homogenized liver samples of experimentally infected gnotobiotic pigs were compared to the concentrations estimated by quantitative PCR. Similar trends were noted with increasing PCV2 concentration detected in subclinically infected to severely PMWS-affected pigs across all assays. Our diagnostic assay was developed with a conserved target sequence, and performed efficiently in quantification of PCV2 in a variety of tissues from naturally and experimentally infected pigs.     

Cardiac lymphoma and pericardial effusion in dogs: 12 cases (1994-2004)

OBJECTIVE: To determine clinical characteristics and clinicopathologic findings, including results of pericardial fluid analysis, and determine the outcome associated with pericardial effusion caused by cardiac lymphoma in dogs. DESIGN: Retrospective case series. ANIMALS: 12 dogs. PROCEDURE: Medical records of affected dogs were reviewed for echocardiographic findings, radiographic findings, results of pericardial fluid analysis, clinicopathologic findings, treatment protocols, and outcomes. RESULTS: Pericardial effusion was detected by echocardiography in all 12 dogs, and lymphoma was detected by cytologic examination of the effusion (11/12 dogs) or histologic examination of pericardium (3/12). Large-breed dogs were overrepresented; median weight was 40.5 kg (89.1 lb). Most hematologic and biochemical changes were mild and non-specific. Survival time for dogs treated with combination chemotherapeutic agents was 157 days and for dogs that did not receive chemotherapy survival time was 22 days. This difference was not significant, but several dogs had long-term survival. CONCLUSIONS AND CLINICAL RELEVANCE: Cardiac lymphoma is an uncommon cause of pericardial effusion, and results suggest that cardiac lymphoma does not always warrant the poor prognosis of other stage V, substage b lymphomas.     

Direct demonstration of an adaptive constraint

The role of constraint in adaptive evolution is an open question. Directed evolution of an engineered beta-isopropylmalate dehydrogenase (IMDH), with coenzyme specificity switched from nicotinamide adenine dinucleotide (NAD) to nicotinamide adenine dinucleotide phosphate (NADP), always produces mutants with lower affinities for NADP. This result is the correlated response to selection for relief from inhibition by NADPH (the reduced form of NADP) expected of an adaptive landscape subject to three enzymatic constraints: an upper limit to the rate of maximum turnover (kcat), a correlation in NADP and NADPH affinities, and a trade-off between NAD and NADP usage. Two additional constraints, high intracellular NADPH abundance and the cost of compensatory protein synthesis, have ensured the conserved use of NAD by IMDH throughout evolution. Our results show that selective mechanisms and evolutionary constraints are to be understood in terms of underlying adaptive landscapes.     

Prognostic factors for survival of dogs with inguinal and perineal mast cell tumors treated surgically with or without adjunctive treatment: 68 cases (1994-2002)

OBJECTIVE: To determine the prognostic factors for survival and tumor recurrence in dogs with cutaneous mast cell tumors (MCTs) in the perineal and inguinal regions treated surgically with or without adjunctive radiation therapy, chemotherapy, or both. DESIGN: Retrospective study. ANIMALS: 68 dogs. PROCEDURE: Medical records of dogs with histologically confirmed MCTs in the perineal region, inguinal region, or both treated surgically with or without adjunctive radiation therapy, chemotherapy, or both were reviewed. RESULTS: Mean tumor-free interval was 1,635 days (median not reached), and 1- and 2-year tumor-free rates were 79% and 71%, respectively. Median survival time was 1,111 days (mean, 1,223 days), and 1- and 2-year survival rates were 79% and 61%, respectively. Factors that negatively influenced survival time were age at diagnosis, tumor recurrence, and treatment with lomustine. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that dogs with MCTs in the inguinal and perineal regions, if appropriately treated, may have survival times and tumor-free intervals similar to dogs with MCTs in other locations.     

Checking that replication breakdown is not terminal

Two new studies help to clarify the relationship between checkpoint proteins, recombination, and replication fork integrity.     

Streptozocin for treatment of pancreatic islet cell tumors in dogs: 17 cases (1989-1999)

OBJECTIVE: To determine toxic effects of streptozocin given in combination with a diuresis protocol in dogs and establish whether streptozocin is efficacious in treatment of pancreatic islet cell tumors in dogs. DESIGN: Retrospective study. ANIMALS: 17 dogs. PROCEDURE: Medical records were reviewed to obtain information regarding signalment, tumor stage and staging tests performed, number of streptozocin treatments, adverse effects, results of biochemical and hematologic monitoring during streptozocin treatment, tumor dimensions, duration of normoglycemia, and date of death, when applicable. Dogs were compared with a historical control group of 15 dogs treated surgically and medically. RESULTS: 58 treatments were administered to the 17 dogs. Only 1 dog developed azotemia. Serum alanine aminotransferase activity increased in some dogs but decreased when treatment was discontinued. Hematologic toxicoses were rare. Vomiting during administration was uncommon but occasionally severe. Two dogs developed diabetes mellitus after receiving 5 doses. Median duration of normoglycemia for 14 dogs with stage-II or -III insulinoma treated with streptozocin was 163 days (95% confidence interval, 16 to 309 days), which was not significantly different from that for the control dogs (90 days; 95% confidence interval, 0 to 426 days). Two dogs had rapid resolution of paraneoplastic peripheral neuropathy, and 2 others had measurable reductions in tumor size. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that streptozocin can be administered safely to dogs at a dosage of 500 mg/m2, IV, every 3 weeks when combined with a protocol for induction of diuresis and may be efficacious in the treatment of dogs with metastatic pancreatic islet cell tumors.     

Feline Lymphoma (145 Cases): Proliferation Indices, Cluster of Differentiation 3 Immunoreactivity, and Their Association with Prognosis in 90 Cats

Paraffin-embedded, formalin-fixed tissue samples from 145 cats with lymphoma were analyzed for cluster of differentiation 3 (CD3, a surface antigen) immunoreactivity, argyrophilic nucleolar organizer region (AgNOR) frequency, and proliferating cell nuclear antigen labeling index (PCNA-LI). This information along with signalment, anatomic site, and feline leukemia virus (FeLV) antigen status was used to determine the potential of these indicators to predict response to therapy, remission, and survival times, and to characterize cats with lymphoma in the era of general availability of FeLV testing and vaccination. Alimentary lymphoma, primarily occurring in older, FeLV-negative cats, was the most common site of involvement. Although the majority of tumors from FeLV-positive cats were CD3-immunoreactive, only one half of CD3-immunoreactive tumors occurred in FeLV-positive cats. Median remission duration and survival times were 126 days and 143 days, respectively, for all cats. Measures of tumor cell proliferation (AgNOR frequency and PCNA-LI) and CD3-immunoreactivity were not predictive of outcome. When all prognostic factors were accounted for by multivariate analysis, response to therapy, FeLV status, and clinical substage were predictive of outcome. FeLV-negative cats that achieved a complete response following induction therapy were likely to have durable (ie, > 6-month) responses, particularly when doxorubicin was included in the chemotherapy protocol. However, FeLV-positive cats had significantly shorter remission and survival times with available chemotherapeutic protocols.     

Proinsecticides effective against insecticide-resistant peach-potato aphid (Myzus persicae (Sulzer))

A range of potential proinsecticides was synthesised and tested against insecticide-susceptible and -resistant clones of Myzus persicae (Sulzer). They were all esters of compounds known to be toxic or pharmacologically active, and were designed to have increased lipophilicity and to be subject to more rapid activation by hydrolysis in resistant than in susceptible aphids due to the increased amount of esterase present in the resistant clones. The most potent toxins were esters of monofluoroacetic acid. When applied topically, the toxicity of these esters to M. persicae was directly proportional to the esterase content of the aphids. Such compounds would not be suitable as commercial insecticides, but the results serve to illustrate the potential benefits of exploiting a resistance mechanism against one class of compounds to render another class more toxic, i.e. to design compounds that show negative cross-resistance. © 1998 SCI     

Environmental and lifestyle risk factors for oral squamous cell carcinoma in domestic cats

Oral squamous cell carcinoma (SCC) is a common malignancy in cats, but little currently is known about its etiology. We examined the relationship between risk of oral SCC and factors such as environmental tobacco smoke, flea control products, and diet in 36 domestic cats with histologically confirmed oral SCC and 112 renal disease control cats presented to a large veterinary referral hospital between 1994 and 2000. Questionnaires were mailed to owners of all study and control cats to assess demographic characteristics, lifestyle factors, and level of chemical exposures 2 years before diagnosis. Multivariate relative risks (RR) were used to estimate the relationships between the various factors and the risk of oral SCC. Flea control product use and diet were significantly associated with risk of oral SCC. Cats that wore a flea collar had 5 times the risk of oral SCC as nonusers, after adjustment for other factors (RR = 5.3; P = .002). In contrast, use of flea shampoo substantially reduced risk. Compared to cats eating mostly dry food, those with high canned food intake had a 3-fold increase in risk (RR = 3.6; P = .014); canned tuna fish intake was independently associated with risk (RR = 4.7; P = .004). Exposure to household environmental tobacco smoke was associated with a nonsignificant 2-fold increase in risk (P = .11). Results of this study suggest that flea control products, diet, and perhaps environmental tobacco smoke might be associated with risk of oral SCC and indicate that further investigation into these relationships is warranted.