Cheryl L. Swenson

Retroviral Transmission by the Transplantation of Connective-tissue Allografts. An Experimental Study.

The transmission of a retrovirus by the transplantation of allografts of connective tissues was studied in a feline model with use of the feline leukemia virus, a retrovirus with a replication cycle and pathological characteristics similar to those of the human immunodeficiency virus. The retrovirus was used to infect four specific-pathogen-free cats that were subsequently used as tissue donors. Fresh allografts of menisci, patellar ligaments, and patellar ligament and bone composites were harvested from infected donors and were transplanted into the knee joints of twelve specific-pathogen-free cats. A fresh cancellous-bone allograft was transplanted into the proximal part of the tibia of four additional specific-pathogen-free cats, which served as positive control animals. Additional grafts from infected donors were harvested and were stored at -80 degrees Celsius for ten weeks. A fresh-frozen graft was then transplanted into the knee of twelve other specific-pathogen-free cats. Samples of plasma were obtained weekly from all twenty-eight cats and were tested with both an enzyme-linked immunosorbent assay to detect the presence of viral antigen and an immunofluorescent antibody assay to determine exposure to the virus. All types of fresh and fresh-frozen connective-tissue allografts from the infected donors resulted in transmission of the retrovirus to the recipient cats. The recipients had evidence of viral antigen or rising antibody titers as early as two weeks after the transplantation. Histological examination of specimens of the allografts revealed normal incorporation of the transplanted tissues, with no sign of rejection of the graft.

Retroviral transmission in bone allotransplantation: The effects of tissue processing

The transmission of a retrovirus through transplantation of processed bone allografts was studied using the feline leukemia virus. The long bones of 4 previously infected donor cats were harvested and assigned to 1 of 3 treatment groups: single freeze/thaw cycle, double freeze/thaw cycle, or double freeze/thaw cycle with water flush to remove bone marrow. Cortical bone grafts and corticocancellous bone grafts from each treatment group were transplanted into individual specific-pathogenfree recipients. Samples of plasma were obtained weekly from all recipients and were tested with an enzyme-linked immunosorbent assay to detect viral antigen. For animals that tested consistently negative for viral antigen, plasma samples also were tested for antiviral antibody to feline leukemia virus measured by live cell immunofluorescence. The results of the antigen and antibody testing revealed that all of the cortical and corticocancellous bone allografts in each of the 3 treatment groups transmitted virus. The ability of the treated bone allografts to transmit a feline retrovirus suggests that routine processing and removal of bone marrow may not inhibit their ability to transmit other retroviruses, such as the human immunodeficiency virus.

Options for the control of bovine leukemia virus in dairy cattle

The subclinical impact of bovine leukemia virus (BLV) on the sustainability of the US dairy industry is only now being fully recognized. Findings of recent longitudinal studies conducted in Michigan dairy herds were consistent with the results of previous studies in showing that within-herd prevalence of BLV-infected cattle was negatively associated with milk production and cow longevity. Risk factors relating to routes of hematogenous transmission such as the use of shared hypodermic needles, shared reproductive examination sleeves, and natural breeding were associated with BLV within-herd prevalence. Few US dairy producers know the prevalence of BLV-infected cattle in their herds or are aware of the insidious economic impact of BLV or the options for BLV control. As an increasing number of countries eradicate BLV from their cattle populations, restrictions on the movement of US cattle and cattle products will likely increase. Veterinarians should be aware of recent developments for screening serum and milk samples for antibodies against BLV and the results of research regarding the economic impact of BLV so they can advise their dairy clients of available alternatives for monitoring and controlling BLV infection.

Lyophilization does not inactivate infectious retrovirus in systemically infected bone and tendon allografts.

Background A review of multiple transplantations of human immunodeficiency virus–infected musculoskeletal allografts found that recipients of lyophilized (freeze-dried) bone or tendon from an infected donor all tested negative for human immunodeficiency virus. The finding that 75% of the recipients of fresh-frozen bone from the same donor contracted human immunodeficiency virus has led to speculation that freeze-drying may render retroviral-infected musculoskeletal allografts noninfectious. Hypothesis Lyophilization does not inactivate retrovirus in systemically infected bone and tendon. Study Design Controlled laboratory study. Methods Tendons and cortical bone segments from cats systemically infected with feline leukemia virus were used in this study. Feline embryonic fibroblast cells were cultured in the presence of fresh-frozen or freeze-dried cortical bone or tendon segments. At each passage, feline leukemia virus p27 antigen was measured in media by enzyme-linked immunosorbent assay, and feline leukemia virus (pro)viral nucleic acids were quantified by real-time quantitative polymerase chain reaction in the DNA extracted from cells. Results Enzyme-linked immunosorbent assay results and quantitative polymerase chain reaction results demonstrated retroviral antigen and proviral DNA in all cultured cell replicates after exposure to fresh-frozen or freeze-dried bones or tendons. Conclusion Freeze-drying (lyophilization) of retroviral-infected cortical bone and tendon does not inactivate retrovirus. Clinical Relevance These results conclusively demonstrate that freeze-drying should not be relied on to inactivate infectious retrovirus in systemically infected musculoskeletal allografts.

Demineralization for inactivation of infectious retrovirus in systemically infected cortical bone: in vitro and in vivo experimental studies.

Background: Clinical and experimental studies have demonstrated viral transmission through the transplantation of fresh-frozen infected bone. While sterilization methods sufficient to inactivate the human immunodeficiency virus (HIV) have been shown to markedly alter osteoconductive and osteoinductive properties of bone allografts, the ability of a process for creating demineralized bone matrix to abrogate transmission of a retrovirus has not been investigated, to our knowledge. We hypothesized that a clinically accepted demineralization procedure would alter the nucleic acids of the feline leukemia virus (FeLV, a retrovirus with a structure and replication cycle similar to those of HIV), inactivating the virus in infected bone and rendering it noninfectious.Methods: Bone infected with FeLV was demineralized with a method employed for creating demineralized bone matrix powder. The effects of demineralization on cellular and (pro)viral nucleic acids were examined with use of gel electrophoresis and quantitative polymerase chain reaction, respectively. To compare the infectivity of the demineralized bone matrix with that of mineralized bone particles in cell cultures and in animals in which they had been implanted, we measured FeLV p27 antigen and (pro)viral nucleic acids as well as antiviral antibodies.Results: Demineralization of FeLV-infected bone appeared to inactivate the virus by degradation and fragmentation of the DNA, rendering it noninfectious in both in vitro and in vivo test systems. In contrast, untreated mineralized FeLV-infected bone contained intact nucleic acids and readily transmitted the virus in both test systems.Conclusions: The demineralization process inactivated infectious retrovirus in infected cortical bone, thereby preventing disease transmission.Clinical Relevance: Rigorous donor screening is currently believed to be the best method for ensuring the safety of allografts, but it is not 100% effective. A process for creating demineralized bone matrix powder reliably inactivated a retrovirus from systemically infected bone while maintaining the osteoinductive properties of the bone. Therefore, use of demineralized bone matrix would provide patients and surgeons with an additional margin of safety while sustaining osteoinductive efficacy.

Prevalence of bovine herpesvirus-4 infection in cats in Central Michigan.

The gammaherpesvirus bovine herpesvirus-4 (BHV-4) has been isolated from a wide variety of animals, including lions and domestic cats. Although BHV-4 antibodies have been detected in normal cats and cats with urinary disorders, the epidemiology and pathogenic role of BHV-4 in cats is unknown. The purpose of this study was to determine the prevalence of BHV-4 antibodies and viral nucleic acid in a population of free-roaming cats. Plasma and peripheral blood leukocyte samples were collected from 52 male and 52 female free-roaming cats impounded at a regional animal control facility in Central Michigan. Plasma concentrations of BHV-4 antibodies were measured with an indirect fluorescent antibody test. Peripheral blood leukocyte DNA was isolated, and a 2-stage polymerase chain reaction with heminested primers delineating a conserved portion of the BHV-4 glycoprotein B gene homologue was used to amplify BHV-4-specific DNA sequences. BHV-4 antibodies were detected in 38 (73%) male and 23 (44%) female cats. Seropositive cats were significantly more likely to be male than female (odds ratio = 3.22; P = .007). Cell-associated viremia was detected in 17 (33%) male and 11 (21%) female cats. Of the 61 seropositive cats, 23 (38%) had a detectable viremia; only 5 (12%) seronegative cats had detectable viremia. Seropositive cats were significantly more likely to be viremic than seronegative cats (OR = 4.30: P = .009). Our results suggest that BHV-4 infection may be more widespread in certain cat populations than previously reported. Furthermore, many cats seropositive for BHV-4 antibodies have a concurrent cell-associated viremia.

Generalized calcinosis cutis associated with disseminated paecilomycosis in a dog.

A 4-year-old spayed female mixed breed dog was referred to the Michigan State University, Veterinary Teaching Hospital (MSU-VTH) with vomiting, lethargy and anorexia of 2 weeks duration. Abdominal radiographs and ultrasonography showed hepatosplenomegaly. Cytological evaluation of ultrasound-guided fine needle aspirates of the liver and spleen revealed fungal organisms and pyogranulomatous inflammation; fungal culture documented Paecilomyces variotii infection. The dog received antifungal therapy and supportive care. Multiple firm plaque-like skin lesions, predominantly involving the inguinal region, developed 18 days after initial presentation and were diagnosed histopathologically as calcinosis cutis. While generalized calcinosis cutis has been reported in three dogs with blastomycosis and one dog with leptospirosis, the association with disseminated Paecilomyces spp. infection is novel.

Clinical features and risk factors for development of urinary tract infections in cats

The clinical and diagnostic features of 155 cats with urinary tract infection (UTI) and 186 controls with negative urine culture/s were characterized retrospectively (signalment, clinical signs, urinalysis, urine culture, concurrent diseases, lower urinary tract diagnostic/therapeutic procedures). Multivariable logistic regression was used to identify risk factors associated with UTI. Cats of all ages were affected by UTI with no sex/breed predisposition. Lower urinary tract signs were absent in 35.5% of cats with UTI. Pyuria and bacteriuria had sensitivities of 52.9% and 72.9%, and specificities of 85.5% and 67.7% for detection of UTI, respectively. Risk factors significantly associated with increased odds of UTI were urinary incontinence [odds ratio (OR) = 10.78, P = 0.0331], transurethral procedures (OR = 8.37, P <0.0001), urogenital surgery (OR = 6.03, P = 0.0385), gastrointestinal disease (OR = 2.62, P = 0.0331), decreased body weight (OR = 0.81, P = 0.0259) and decreased urine specific gravity (OR = 0.78, P = 0.0055). Whilst not independently significant, renal disease and lower urinary tract anatomic abnormalities improved statistical model performance and contributed to UTI.

Evaluation of antiviral activity and toxicity of dextran sulfate in feline leukemia virus-infected cats.

The feline leukemia virus (FeLV) disease model was used to conduct a toxicity and antiretrovirus efficacy trial of dextran sulfate (DS; molecular mass, 7,000 to 8,000 Da). In vitro, FeLV infection of feline lymphoid cells was inhibited by 10 micrograms of DS per ml. DS was administered to cats by continuous intravenous infusion at doses of 600, 120, 24, or 4.8 mg/kg of body weight per day, beginning 24 h before FeLV challenge. Doses of 24 mg/kg/day and more were excessively toxic, causing intestinal lesions and death. Similar changes were observed in unchallenged animals receiving 24 mg/kg/day, indicating that toxicity was DS mediated. The dosage of 4.8 mg/kg/day was subtoxic but did not prevent the induction and persistence of FeLV viremia. The results demonstrate that DS by continuous intravenous infusion is excessively toxic at high doses and ineffective at preventing FeLV infection at a subtoxic dose in the FeLV cat model.

Evaluation of modified Wright-staining of dried urinary sediment as a method for accurate detection of bacteriuria in cats

BACKGROUND Urinary sediment examination and quantitative urinary culture results are frequently discordant. OBJECTIVES The aims of this study were to compare accuracy of light microscopic examination of wet-mounted unstained (wet-unstained) and air-dried modified Wright-stained (dry-stained) sedimented preparations of urine with results of quantitative aerobic bacterial culture for detection and characterization of bacteriuria in cats. In addition, the presence of pyuria detected by urinalysis and potential risk factors were assessed. METHODS A blinded prospective study was conducted on 472 urinary samples collected from 410 cats by cystocentesis. The age and sex of each cat were recorded. Complete urinalyses were performed and included quantification of WBCs. Quantity and morphology of bacteria in each specimen were determined by light microscopic examination of wet-unstained (performed by certified medical technologists) and dry-stained (performed by a veterinary clinical pathologist) sedimented preparations of urine and compared with results of quantitative bacterial cultures. RESULTS Of 472 urinary specimens, 29 were positive for bacteriuria by culture and considered true positives and 443 were considered true negatives. Compared with these results, examination of wet-unstained and dry-stained urines had sensitivities of 75.9% and 82.8%, specificities of 56.7% and 98.7%, and test efficiencies of 57.8% and 97.7%, respectively. Positive likelihood ratios were 1.8 and 63.7 and negative likelihood ratios were 0.42 and 0.17 for wet-unstained and dry-stained examinations, respectively. Compared with 29 culture-positive samples, the wet-unstained method had morphologic concordance and misclassification rates of 37.9% and 62.1%, respectively, whereas the dry-stained method had morphologic concordance and misclassification rates of 65.5% and 34.5%, respectively. Only 34% of samples with bacteriuria had pyuria. Frequency of bacteriuria was not significantly different based on age and sex of the cats, but there was a tendency for increased frequency in female cats and in cats >10 years old. CONCLUSIONS Staining dried urinary sediment with a modified Wright-stain significantly improved sensitivity, specificity, and test efficiency of microscopic detection and classification of bacteriuria compared with the wet-unstained method. Pyuria should not be a criterion for determining the presence or absence of bacteriuria.