Combined evaluation of commonly used techniques, including PCR, for diagnosis of mouse fur mites.

Our study evaluated and compared the false-negative rates (FNR) of a wide array of fur-mite diagnostic tests, including 2 postmortem tests (pelt exam and sticky paper) and 3 antemortem tests (adhesive tape, fur pluck, and PCR). Past publications examining fur-mite diagnostic techniques primarily used paired comparisons, evaluating tests by their level of agreement with only one other test. However, different combinations or pairs of diagnostics are used in the different studies, making the results of these comparisons difficult to interpret across all available diagnostics. In the current study, mice from a conventionally maintained colony endemic for Myobia musculi were identified as positive based on at least one positive diagnostic test. From this pool of positive animals, the FNR of all tests were quantified. The PCR assay and the pelt exam performed the best, with 0% and 2% FNR respectively, whereas tape, fur-pluck, and sticky-paper tests showed 24%, 26%, and 36% FNR, respectively. Our study shows that for mice in a colony naturally infested with Myobia musculi, PCR testing can be used for reliable antemortem detection, and pelt exam performed by experienced examiners is reliable for postmortem detection.

Long-term follow-up of foamy viral vector-mediated gene therapy for canine leukocyte adhesion deficiency.

The development of leukemia following gammaretroviral vector-mediated gene therapy for X-linked severe combined immunodeficiency disease and chronic granulomatous disease (CGD) has emphasized the need for long-term follow-up in animals treated with hematopoietic stem cell gene therapy. In this study, we report the long-term follow-up (4-7 years) of four dogs with canine leukocyte adhesion deficiency (CLAD) treated with foamy viral (FV) vector-mediated gene therapy. All four CLAD dogs previously received nonmyeloablative conditioning with 200 cGy total body irradiation followed by infusion of autologous, CD34(+) hematopoietic stem cells transduced by a FV vector expressing canine CD18 from an internal Murine Stem Cell Virus (MSCV) promoter. CD18(+) leukocyte levels were >2% following infusion of vector-transduced cells leading to ongoing reversal of the CLAD phenotype for >4 years. There was no clinical development of lymphoid or myeloid leukemia in any of the four dogs and integration site analysis did not reveal insertional oncogenesis. These results showing disease correction/amelioration of disease in CLAD without significant adverse events provide support for the use of a FV vector to treat children with leukocyte adhesion deficiency type 1 (LAD-1) in a human gene therapy clinical trial.

Health Evaluation of Experimental Laboratory Mice.

Good science and good animal care go hand in hand. A sick or distressed animal does not produce the reliable results that a healthy and unstressed animal produces. This unit describes the essentials of assessing mouse health, colony health surveillance, common conditions, and determination of appropriate endpoints. Understanding the health and well-being of the mice used in research enables the investigator to optimize research results and animal care.

Gene therapy for canine leukocyte adhesion deficiency with lentiviral vectors using the murine stem cell virus and human phosphoglycerate kinase promoters.

Children with leukocyte adhesion deficiency type 1 (LAD-1) and dogs with canine LAD (CLAD) develop life-threatening bacterial infections due to mutations in the leukocyte integrin CD18. Here, we compared the human phosphoglycerate kinase (hPGK) promoter to the murine stem cell virus (MSCV) promoter/enhancer in a self-inactivating HIV-1-derived lentiviral vector to treat animals with CLAD. Four CLAD dogs were infused with CD34(+) cells transduced with the hPGK vector, and two CLAD dogs received MSCV vector-transduced CD34(+) cells. Infusions were preceded by a nonmyeloablative dose of 200 cGy total body irradiation. Comparable numbers of transduced cells were infused in each group of animals. Only one of four CLAD animals treated with the hPGK-cCD18 vector had reversal of CLAD, whereas both MSCV-cCD18 vector-treated dogs had reversal of the phenotype. Correction of CLAD depends both upon the percentage of CD18(+) myeloid cells and the level of expression of CD18 on individual myeloid cells. In this regard, the hPGK promoter directed low levels of expression of CD18 on neutrophils compared to the MSCV promoter, likely contributing to the suboptimal clinical outcome with the hPGK vector.

Gene therapy of canine leukocyte adhesion deficiency using lentiviral vectors with human CD11b and CD18 promoters driving canine CD18 expression.

To identify cellular promoters in a self-inactivating (SIN) lentiviral vector that might be beneficial in treating children with leukocyte adhesion deficiency type 1 (LAD-1), we tested lentiviral vectors with human CD11 and CD18 leukocyte integrin proximal promoter elements directing expression of canine CD18 in animals with canine LAD (CLAD). Lentiviral vectors with either the human CD11b (637 bp) proximal promoter or the human CD18 (1,060 bp) proximal promoter resulted in the highest percentages of CD18(+) CLAD CD34(+) cells in vitro. Subsequently, two CLAD dogs were infused with autologous CD34(+) cells transduced with the hCD11b (637 bp)-cCD18 vector, and two CLAD dogs were infused with autologous CD34(+) cells transduced with the hCD18 (1,060 bp)-cCD18 vector. Each dog received a nonmyeloablative dose of 200 cGy total body irradiation (TBI) before the infusion of transduced cells. The two CLAD dogs treated with the hCD18 (1,060 bp)-cCD18 vector, and one of the two dogs treated with the hCD11b (637 bp)-cCD18 vector, had reversal of the CLAD phenotype. These studies using endogenous leukocyte integrin proximal promoters represent an important step in the development of gene therapy for children with LAD-1.

Comparison of carbon dioxide and argon euthanasia: effects on behavior, heart rate, and respiratory lesions in rats.

In this study we compared rat (n = 16) responses to euthanasia with either gradual-fill CO(2) or rapid induction argon gas by evaluating the animals' heart rate via radiotelemetry, behavior, and vocalizations. We also evaluated the histologic effects of the gases. Rats were placed in an open test chamber 24 h before the start of the experiment. During baseline tests, rats were exposed to oxygen to evaluate the effects of the noise and movement of gas entering the chamber; 1 wk later, rats were euthanized by gas displacement with either 10%/min CO(2) or 50%/min argon gas. Rats tended to have higher heart rats and were more active during the baseline test, but these parameters were normal before the euthanasia experiment, suggesting that the rats had acclimated to the equipment. Heart rate, behavior, and ultrasonic vocalizations were recorded for 2 min after gas introduction in both groups. All rats appeared conscious throughout the test interval. The heart rates of rats exposed to argon did not change, whereas those of rats exposed to CO(2) declined significantly. Unlike those exposed to CO(2), rats euthanized with argon gas gasped and demonstrated seizure-like activity. There were no differences in the pulmonary lesions resulting from death by either gas. Our results suggest that argon as a sole euthanasia agent is aversive to rats. CO(2) using a 10%/min displacement may be less aversive than more rapid displacements. Future research investigating methods of euthanasia should allow sufficient time for the rats to acclimate to the test apparatus.

Effective and safe anesthesia for Yorkshire and Yucatan swine with and without cardiovascular injury and intervention.

The goal of this study was to identify an injectable anesthetic protocol that provides sedation sufficient for peripheral vascular catheterization, intubation, and transport while minimizing cardiovascular changes in Yorkshire and Yucatan pigs with and without cardiovascular injury and intervention (CI). Phase 1 examined the safety and efficacy of acepromazine-ketamine, diazepam-ketamine, midazolam-ketamine, and medetomidine-ketamine in 5 healthy Yorkshire pigs. For each drug combination, we obtained multiple measurements of heart rate, blood pressure, respiratory rate, temperature, sedation score, ability to catheterize and intubate, and recovery score. Phase 2 evaluated and refined the dose of the most effective Phase 1 anesthetic combination (midazolam-ketamine) in healthy and CI Yorkshire pigs (n = 53 trials). Phase 3 mirrored Phase 2 but tested midazolam-ketamine in healthy and CI Yucatan pigs (n = 34 trials). Midazolam (0.5 mg/kg)-ketamine (25 to 27 mg/kg) was the most effective anesthetic combination in healthy Yorkshire pigs, but this dose was less effective in healthy Yucatan pigs and CI Yorkshire and Yucatan pigs. Midazolam-ketamine resulted in tachycardia and apnea more frequently in CI pigs than healthy pigs. This combination also caused vomiting in one CI Yucatan pig. Overall, midazolam-ketamine provided safe and effective sedation for catheterization and intubation of both healthy and CI pigs. This study suggests Yucatan pigs may require a higher dose midazolam-ketamine to achieve the same level of sedation as that in Yorkshire pigs. Although anesthetic complication rates were higher in CI pigs, our results indicate that midazolam-ketamine can be safely used for sedation of both pig breeds with and without CI.

Acalculous and clostridial cholecystitis in a pig.

A 21-month-old domestic Hanford pig (Sus scrofa domestica) in a 1-year study for experimental myocardial infarction was euthanized at the end of the study. One week earlier, the animal had symptoms and elevated clinical chemistry results suggestive of hepatobiliary disease, which resolved after medical therapy. At necropsy, the gallbladder was markedly enlarged, discolored, and had a thickened wall. Within the gallbladder, there was abundant friable green-brown material. A culture of the gallbladder luminal material yielded Clostridium perfringens type A. Histopathology of the gallbladder demonstrated multifocal areas of necrosis of varying depths, admixed with an inflammatory infiltrate that was also observed on the serosa and within the associated adipose tissue. Luminal material was composed of cellular debris and bile sludge admixed with numerous bacterial rods. Smooth-muscle hypertrophy of numerous small arterioles with narrowed lumina was observed in the gallbladder. A diagnosis of acalculous cholecystitis presumably because of ischemia of the gallbladder with secondary clostridial infection was made. To the authors' knowledge, this is the first reported case of acalculous cholecystitis with evidence of vascular compromise in a pig, as well as cholecystitis secondarily attributed to Clostridium perfringens type A.

Successful treatment of canine leukocyte adhesion deficiency by foamy virus vectors.

Recent successes in treating genetic immunodeficiencies have demonstrated the therapeutic potential of stem cell gene therapy. However, the use of gammaretroviral vectors in these trials led to insertional activation of nearby oncogenes and leukemias in some study subjects, prompting studies of modified or alternative vector systems. Here we describe the use of foamy virus vectors to treat canine leukocyte adhesion deficiency (CLAD). Four of five dogs with CLAD that received nonmyeloablative conditioning and infusion of autologous, CD34+ hematopoietic stem cells transduced by a foamy virus vector expressing canine CD18 had complete reversal of the CLAD phenotype, which was sustained more than 2 years after infusion. In vitro assays showed correction of the lymphocyte proliferation and neutrophil adhesion defects that characterize CLAD. There were no genotoxic complications, and integration site analysis showed polyclonality of transduced cells and a decreased risk of integration near oncogenes as compared to gammaretroviral vectors. These results represent the first successful use of a foamy virus vector to treat a genetic disease, to our knowledge, and suggest that foamy virus vectors will be effective in treating human hematopoietic diseases.

Correction of the disease phenotype in canine leukocyte adhesion deficiency using ex vivo hematopoietic stem cell gene therapy.

Canine leukocyte adhesion deficiency (CLAD) represents the canine counter-part of the human disease leukocyte adhesion deficiency (LAD). Defects in the leukocyte integrin CD18 adhesion molecule in both CLAD and LAD lead to recurrent, life-threatening bacterial infections. We evaluated ex vivo retroviral-mediated gene therapy in CLAD using 2 nonmyeloablative conditioning regimens--200 cGy total body irradiation (TBI) or 10 mg/kg busulfan--with or without posttransplantation immunosuppression. In 6 of 11 treated CLAD dogs, therapeutic levels of CD18(+) leukocytes were achieved. Conditioning with either TBI or busulfan allowed long-term engraftment, and immunosuppression was not required for efficacy. The percentage of CD18(+) leukocytes in the peripheral blood progressively increased over 6 to 8 months after infusion to levels ranging from 1.26% to 8.37% at 1-year follow-up in the 6 dogs. These levels resulted in reversal or moderation of the severe CLAD phenotype. Linear amplification-mediated polymerase chain reaction assays indicated polyclonality of insertion sites. These results describe ex vivo hematopoietic stem cell gene transfer in a disease-specific, large animal model using 2 clinically applicable conditioning regimens, and they provide support for the use of nonmyeloablative conditioning regimens in preclinical protocols of retroviral-mediated gene transfer for nonmalignant hematopoietic diseases such as LAD.

Reproductive capability in dogs with canine leukocyte adhesion deficiency treated with nonmyeloablative conditioning prior to allogeneic hematopoietic stem cell transplantation.

Nonmyeloablative conditioning regimens are increasingly replacing myeolablative conditioning prior to allogeneic hematopoietic stem cell transplantation (SCT). The recent advent of these conditioning regimens has limited the assessment of the long-term effects of this treatment, including analysis of reproductive function. To address the question of reproductive function after nonmyeloablative transplantation, we analyzed a cohort of young dogs with the genetic disease canine leukocyte adhesion deficiency that were treated with a nonmyeloablative dose of 200 cGy total body irradiation followed by matched-littermate SCT. Five males and 5 females entered puberty; all 5 males and 4 females subsequently sired or delivered litters following transplantation. We demonstrate that fertility is intact and dogs have uncomplicated parturitions following nonmyeloablative conditioning for SCT. These results are encouraging for children and adults of childbearing age who receive similar conditioning regimens prior to allogeneic transplantation.

Nonmyeloablative conditioning with busulfan before matched littermate bone marrow transplantation results in reversal of the disease phenotype in canine leukocyte adhesion deficiency.

Leukocyte adhesion deficiency (LAD)-1, a primary immunodeficiency disease caused by molecular defects in the leukocyte integrin CD18 molecule, is characterized by recurrent, life-threatening bacterial infections. Myeloablative hematopoietic stem cell transplantation is the only curative treatment for LAD-1. Recently, canine LAD (CLAD) has been shown to be a valuable animal model for the preclinical testing of nonmyeloablative transplantation regimens for the treatment of children with LAD-1. To develop new allogeneic transplantation approaches for LAD-1, we assessed a nonmyeloablative conditioning regimen consisting of busulfan as a single agent before matched littermate allogeneic bone marrow transplantation in CLAD. Three CLAD dogs received busulfan 10 mg/kg intravenously before infusion of matched littermate bone marrow, and all dogs received posttransplantation immunosuppression with cyclosporin A and mycophenolate mofetil. Initially, all 3 dogs became mixed chimeras, and levels of donor chimerism sufficient to reverse the CLAD phenotype persisted in 2 animals. The third dog maintained donor microchimerism with an attenuated CLAD phenotype. These 3 dogs have all been followed up for at least 1 year after transplantation. These results indicate that a nonmyeloablative conditioning regimen with chemotherapy alone is capable of generating stable mixed chimerism and reversal of the disease phenotype in CLAD.

Nonmyeloablative hematopoietic stem cell transplantation corrects the disease phenotype in the canine model of leukocyte adhesion deficiency.

OBJECTIVE: The aim of this study was to test a nonmyeloablative hematopoietic stem cell transplant regimen applicable to children with leukocyte adhesion deficiency (LAD) who have a histocompatible sibling donor by using the canine model of LAD, namely canine leukocyte adhesion deficiency or CLAD. METHODS: Thirteen CLAD pups received a hematopoietic stem cell transplant from a dog leukocyte antigen (DLA)-matched littermate donor after pretransplant nonmyeloablative conditioning with 200 cGy total-body irradiation and posttransplant immunosuppression with cyclosporine and mycophenolate mofetil. Donor chimerism following transplant was assessed by flow cytometry for the presence of donor CD18 peripheral blood leukocytes and leukocyte subsets. RESULTS: Eleven of the 13 transplanted animals achieved stable mixed donor chimerism and reversal of the severe CLAD phenotype without graft-vs-host disease. The level of donor chimerism ranged from 3.9 to 95.5% at 1 year following transplant. There was one early death 3 weeks after transplant from thrombocytopenia and hemorrhage, and one dog with donor microchimerism (0.5% CD18+ donor leukocytes) who had attenuation of the CLAD phenotype. CONCLUSION: These results demonstrate that a nonmyeloablative transplant regimen from a DLA-matched littermate donor leads to mixed chimerism and reversal of the severe disease phenotype in dogs with CLAD, and provides support for the use of this approach in children with LAD who possess a histocompatible sibling donor.

Plasma evaluation for ivermectin in llamas (Lama glama) after standard subcutaneous dosing.

Plasma levels of the parasiticide ivermectin were studied by high-performance liquid chromatography in five llamas (Lama glama) after single 200 microg/kg s.c. injections. Ivermectin levels were undetectable in plasma samples drawn up to 4 wk after injection, suggesting that the dosage used was insufficient to reach therapeutic concentrations in this species.

Very low levels of donor CD18+ neutrophils following allogeneic hematopoietic stem cell transplantation reverse the disease phenotype in canine leukocyte adhesion deficiency.

Children with the severe phenotype of the genetic immunodeficiency disease leukocyte adhesion deficiency or LAD experience life-threatening bacterial infections because of molecular defects in the leukocyte integrin CD18 molecule and the resultant failure to express the CD11/CD18 adhesion molecules on the leukocyte surface. Hematopoietic stem cell transplantation remains the only definitive therapy for LAD; however, the degree of donor chimerism and particularly the number of CD18(+) donor-derived neutrophils required to reverse the disease phenotype are not known. We performed nonmyeloablative hematopoietic stem cell transplantations from healthy matched littermates in 9 dogs with the canine form of LAD known as CLAD and demonstrate that in the 3 dogs with the lowest level of donor chimerism, less than 500 CD18(+) donor-derived neutrophils/microL in the peripheral blood of the CLAD recipients resulted in reversal of the CLAD disease phenotype. These results demonstrate the value of a disease-specific, large-animal model for identifying the lowest therapeutic level required for successful cellular and gene therapy.