A pharmacokinetic study of extended-release buprenorphine in cynomolgus monkeys (Macaca fasicularis).

BACKGROUND: A novel buprenorphine (BUP) extended-release formulation (BUP-XR) produced as a lipid-encapsulated, low viscosity BUP suspension for subcutaneous (SC) injection to control pain was evaluated for pharmacokinetics and safety in four adult male cynomolgus monkeys. METHODS: Each animal was given 0.2 mg/kg reformulated BUP-XR SC. Clinical observations were made during the course of the study. Blood samples were obtained from each animal before BUP-XR administration, 6, 24, 48, 72, and 96 h post-BUP-XR injection. Plasma levels of buprenorphine were analyzed using HPLC-MS/MS. The PK values calculated included peak plasma concentration of the BUP analyte, time to peak plasma concentration, plasma half-life, area under the plasma concentration-time curve, clearance, apparent volume of distribution, and elimination rate constant (Cmax , Tmax , T½ , AUC0-t , CL, Vd, and Ke, respectively). RESULTS: Observable adverse clinical signs were not detected. BUP concentration peaked from 6 to 48 h, then declined in a linear fashion. Quantifiable plasma BUP was measured in all monkeys at all time points. Results indicate that a single BUP-XR dose at 0.2 mg/kg can reliably provide plasma levels of BUP reported in the literature to be therapeutically relevant for up to 96 h. CONCLUSIONS: Because of the lack of any clinical observations or adverse effects at the injection site or absence of observable abnormal behaviors, it may be concluded that the use of BUP-XR is safe and efficacious in this species of non-human primate at the dose regimen described in this study for up to 96 h post-administration.

Toward Global Harmonization of Training and Certification of Specialists in Laboratory Animal Veterinary Medicine.

Laboratory animal medicine (LAM) is a corner stone of animal-based research and has been a veterinary specialty for over 60 y. Today 5 Colleges of LAM (American, European, Japanese, Korean, and Indian) that certify specialists (Diplomates) in LAM are members of the International Association of Colleges of LAM (IACLAM). Goals of IACLAM are to support the development of new Colleges of LAM, to harmonize expectations for the knowledge and skills of newly certified LAM Diplomate, and to harmonize the standards (best practices) for training and examination of candidates among the member Colleges. IACLAM recently conducted an in-depth review and comparison of oversight, training, credentialing, and examination standards in the 5 Colleges as part of an initiative to create a framework for harmonization and consistency for these activities across the 5 Colleges. The process has led to an agreement on recommendations for knowledge and skill requirements for a newly certified Diplomate, as described by each College in a detailed role delineation document (RDD). The RDD is based on task analyses of the work responsibilities of laboratory animal veterinary Diplomates. This agreement is an important step toward the goal of global harmonization of LAM Diplomate training. Further efforts are planned for areas such as training, research, publication, and examination. This paper describes the role and content of the RDD and lists the differences and similarities among the RDDs of 5 Colleges of LAM.

Mycobacterium kansasii Isolated from Tuberculinpositive Rhesus Macaques (Macaca mulatta) in the Absence of Disease.

Mycobacterial infections are of primary health concern in NHP colonies in biomedical research. NHP are constantly monitored and screened for Mycobacterium spp. We report 6 Chinese-origin rhesus macaques infected with Mycobacterium kansasii that exhibited positive tuberculin skin tests in the absence of disease. Two of these macaques were being used for research purposes; the remaining 4 macaques were residing at the contract quarantine company. Histopathology and acid-fast staining of fixed tissues from all macaques showed that all were free of disease. Thoracic radiographs were negative for any signs of disease or infection. Samples from bronchial lavage and tissues including lung, spleen, hilar and mesenteric lymph nodes tested negative by PCR assay for Mycobacterium spp. One of the research macaques tested culture-positive for M. kansasii and a poorly characterized M. avium complex organism. One macaque from the contract quarantine facility tested culture positive for M. kansasii. Genomic testing and target gene RNA expression analysis of the 2 M. kansasii isolates were performed to evaluate possible kinship and affected genes that might contribute to susceptibility to mycobacterial infection. Genotyping of the 2 isolates revealed 2 genetically distinct strains (strains 1 and 4). The presence of positive tuberculin skin tests in the absence of disease raises serious concerns regarding diagnostic methods used for infected NHP.

Auditing laboratory rodent biosecurity programs.

A rodent biosecurity program that includes periodic evaluation of procedures used in an institution's vivarium can be used to ensure that best practices are in place to prevent a microbial pathogen outbreak. As a result of an ongoing comprehensive biosecurity review within their North American and European production facilities, the authors developed a novel biosecurity auditing process and worksheet that could be useful in other animal care and use operations. The authors encourage other institutions to consider initiating similar audits of their biosecurity programs to protect the health of their laboratory animals.

Natural and experimental Helicobacter pullorum infection in Brown Norway rats.

Helicobacter pullorum is an enterohepatic Helicobacter species (EHS) that was recently reported as a naturally acquired infection in mice. Faecal samples from 18 out of 20 Brown Norway (BN) rats, housed in the same barrier as the H. pullorum-infected mice, were positive for H. pullorum using species-specific PCR. In addition, we determined whether H. pullorum was able to persistently colonize the gastrointestinal tract and/or biliary tree and elicit tissue inflammation as well as a serum IgG response in BN rats. Six (four male, two female) 6-week-old, H. pullorum-negative BN rats were orally dosed with 4×10(8) c.f.u. of H. pullorum every other day for a total of three doses. At 2 weeks post-infection, all rats were H. pullorum-positive by faecal PCR. Five out of the six BN rats remained H. pullorum-positive for the entire 30 week study. PCR analysis of tissue collected at necropsy confirmed that the colon and caecum were the primary sites of H. pullorum colonization. Rats that were persistently colonized by H. pullorum had a sustained H. pullorum-specific IgG response measured by ELISA. Intestinal or hepatic pathology associated with H. pullorum infection was not noted. To our knowledge, this is the first report documenting that rats can be persistently colonized with an EHS that also infects humans.

Persistent Helicobacter pullorum colonization in C57BL/6NTac mice: a new mouse model for an emerging zoonosis.

Helicobacter pullorum, an enterohepatic Helicobacter species, is associated with gastroenteritis and hepatobiliary disease in humans and chickens. Recently, a novel H. pullorum outbreak in barrier-maintained rats and mice was described. In this study, persistence of infection and serological responses were further evaluated in H. pullorum-infected female C57BL/6NTac and C3H/HeNTac mice obtained from the barrier outbreak. C57BL/6NTac mice (n=36) aged 10-58 weeks were confirmed to be chronically infected with H. pullorum by PCR or culture of caecum, colon and faeces, with no evidence of hepatic infection; two of three C3H/HeNTac mice cleared H. pullorum infection by 26 weeks of age. A quantitative PCR (qPCR) assay based on the cdtB gene specific to H. pullorum demonstrated that colonization was high in the caecum and colon at 10(4)-10(6) c.f.u. equivalents per µg host DNA, and decreased by several logs from 32 to 58 weeks of age. Infected mice were seropositive by ELISA, and H. pullorum-specific IgG levels decreased as colonization was lost over time in selected mice. Consistent with the lack of pathology associated with chronic infection of C57BL/6 mice with other murine enteric helicobacters, C57BL/6NTac and C3H/HeNTac mice infected with H. pullorum did not develop gross or histological lesions of the liver or gastrointestinal tract. The cdtB-based qPCR assay can be used in screening animals, food sources and environmental samples for H. pullorum, as this food-borne pathogen has zoonotic potential. These findings will also allow future studies in murine models to dissect potential pathogenic mechanisms for this emerging pathogen.

A pharmacokinetic study of enrofloxacin and its active metabolite ciprofloxacin after oral and intramuscular dosing of enrofloxacin in rhesus monkeys (Macaca mulatta).

Enrofloxacin is used for treating Shigellosis in non-human primates; however, there are no reports describing its pharmacokinetics in rhesus monkeys. Pharmacokinetic data in intended target species (rhesus) help to determine the proper dose regimen. Blood levels of enrofloxacin and ciprofloxacin (enrofloxacin's active metabolite), were determined after either intramuscular or oral dosing of enrofloxacin for 7 days in a cross-over study. Levels of both antibiotics were determined by solid phase extraction followed by reversed-phase chromatography with tandem mass spectrometry. Results indicate enrofloxacin half-life after intramuscular dosing is estimated to be 2.4 hours. Enrofloxacin given either intramuscular or p.o. rapidly achieves satisfactory therapeutic blood levels of enrofloxacin or ciprofloxacin in rhesus monkeys. Results from these pharmacokinetic study parallel values published for other animal species. Our results show use of enrofloxacin is effective in managing Shigella infections in rhesus monkeys based upon achieving these blood drug levels.

Isolation and characterization of a novel helicobacter species, "Helicobacter macacae," from rhesus monkeys with and without chronic idiopathic colitis.

Chronic idiopathic colitis is a common clinical entity in young captive rhesus monkeys. Eight isolates, cultured from five monkeys in colony 1 with endemic diarrhea and three from colony 2 without diarrhea, were grown under microaerobic conditions on selective agar and were classified by full 16S rRNA sequence, biochemical, and phenotypic analysis as a novel helicobacter, "Helicobacter macacae" (proposed name). All eight strains of H. macacae had 99.5% identical 16S rRNA sequences.

Integrase inhibitors and cellular immunity suppress retroviral replication in rhesus macaques.

We describe the efficacy of L-870812, an inhibitor of HIV-1 and SIV integrase, in rhesus macaques infected with the simian-human immunodeficiency virus (SHIV) 89.6P. When initiated before CD4 cell depletion, L-870812 therapy mediated a sustained suppression of viremia, preserving CD4 levels and permitting the induction of virus-specific cellular immunity. L-870812 was also active in chronic infection; however, the magnitude and durability of the effect varied in conjunction with the pretreatment immune response and viral load. These studies demonstrate integrase inhibitor activity in vivo and suggest that cellular immunity facilitates chemotherapeutic efficacy in retroviral infections.

The implementation of robotic technology in an animal facility: a case study.

The benefits of a robotics system for waste disposal and cage sanitation in an animal care facility include a reduction of ergonomic concerns and an increase in production capability. A modular approach can further provide an ability to relocate the unit to new building construction. There are numerous considerations that can affect the design and operation of a robotic cagewash system. These include facility layout, existing animal husbandry procedures, equipment selection, and employee involvement. The implementation of the complete system in an animal facility requires communication and insight from all members of the animal care staff as well as novel solutions to attain full production and operation. The system itself contains many complex technical and mechanical functions, but a successful installation can only be fully achieved with teamwork and proper training.

The impact of early immune destruction on the kinetics of postacute viral replication in rhesus monkey infected with the simian-human immunodeficiency virus 89.6P.

Set-point viral load is positively correlated with the extent of initial viral replication in pathogenic simian-human immunodeficiency virus (SHIV) infection. To elucidate the mechanisms underlying the correlation, we conducted a systematic investigation in rhesus monkeys infected with the highly pathogenic SHIV 89.6P. This model is widely used in the preclinical evaluation of AIDS vaccine candidates and a thorough understanding of the model's biology is important to the proper interpretation of these evaluations. We found that the levels of peak viremia were positively correlated not only with the levels of set-point viremia but, importantly, with the extent of initial overall immune destruction as indicated by the degree of CD4+ T cell depletion and lymph node germinal center (GC) formation. The extent of initial overall immune destruction was inversely correlated with subsequent development and maintenance of virus-specific cellular and humoral immune responses. Thus, these data suggest that the extent of early immune damage determines the development and durability of virus-specific immunity, thereby playing a critical role in establishing the levels of set-point viral replication in SHIV infection. Vaccines that limit both the initial viral replication and the extent of early immune damage will therefore mediate long-term virus replication control and mitigation of long-term immune destruction in this model of immunodeficiency virus infection.

Increasing supervisory effectiveness in a laboratory animal facility.

Management personnel expressed concern that the allocation of work was not properly aligned to permit maximum efficiency in the supervisor job family. Employees at various levels in the supervisor job family also felt that there needed to be greater definition regarding which tasks needed to be done by which level in the organization. Using data collection and interviews, a team of staff employees and consultants successfully evaluated the facility operation and supervision. The information was analyzed and compared with the existing supervisory job descriptions. An action plan was developed and presented to management personnel to increase efficiency and stratify the responsibility of the existing supervisory levels. Supervisory reassignment was proposed.

The effect of an environmental enrichment device on individually caged rabbits in a safety assessment facility.

The primary enclosure of a laboratory animal's environment should encourage species-typical behavior and enhancement of the animal's well-being, as indicated by the Guide. Enrichment devices have been documented to decrease the incidence of stereotypical behaviors and increase overall activity of rabbits. An 8-week study was performed to evaluate the effect of an environmental enrichment device, stainless-steel rabbit rattles on spring clips, on individually housed rabbits in a Safety Assessment facility. We used 48 New Zealand White rabbits; the devices were placed on cages of 32 study rabbits, and 16 control rabbits had no devices. Food consumption measurements and observations of device manipulations (taken during a predetermined peak interaction 1-h timeframe) were collected 5 days per week. All rabbits were bled for evaluation of hematologic parameters for the stress triad (neutrophilia, lymphopenia, and eosinopenia) and weighed weekly. No significant differences were found between study and control rabbits when body weights, food consumption, and hematologic parameters were analyzed. Our study supports previous findings that interaction with enrichment devices decreases over time, thus indicating the need for frequent rotation of different enrichment devices. In addition, no adverse effects of the analyzed parameters were found, indicating that stainless-steel rabbit rattles on spring clips are suitable devices for safety assessment studies, in which the introduction of new variables is often unacceptable.

Novel training concepts and techniques used to increase safety awareness in the laboratory animal facility.

Training makes an important contribution to maintaining a safe working environment, but trainees may have problems achieving maximum information retention if they are not motivated and interested. The authors describe an innovative safety training program that has been well received by employees and associated with a 62% drop in workplace injuries over a two-year period.

Electronic freedom of information: challenges facing the regulators and the regulated.

FOIA and, more recently, E-FOIA have provided the American public with the means to obtain valuable information from the federal government. The E-FOIA-mandated posting of USDA animal facility inspection reports on the internet, however, has met with opposition from the research community. The authors outline E-FOIA's shortcomings regarding this policy and present potential solutions to the problem.

Antigen recognition by serum antibodies in non-human primates experimentally infected with Mycobacterium tuberculosis.

Tuberculosis is a significant threat to non-human primates and their caretakers. The diagnosis of tuberculosis in living non-human primates is currently based on the tuberculin skin test, which is cumbersome and sometimes inaccurate. Development of an accurate serodiagnostic test requires identification of the key antigens of Mycobacterium tuberculosis involved in antibody production. When sequential serum samples obtained from 17 cynomolgus, rhesus, and African green monkeys up to seven months since experimental infection with M. tuberculosis Erdman were screened for antibody against purified proteins of M. tuberculosis, three highly seroreactive antigens were identified. One protein, ESAT-6, reacted with sera from all infected animals. Two additional proteins, alpha-crystallin and MTSA-10, were recognized by sera from approximately 90% of infected animals. Time course analysis of antibody production indicated that the earliest response was usually to ESAT-6 alone or to ESAT-6 and other antigen(s). These results provide experimental evidence of the potential value of ESAT-6 as an antigen for use in serodiagnosis of tuberculosis in non-human primates.

Clinical and microbiologic characterization of hemorrhagic pneumonia due to extraintestinal pathogenic Escherichia coli in four young dogs.

Over a 21-month period, three Beagle dogs and one mixed-breed dog at our facility developed fatal pneumonia. The four dogs, all purpose bred, came from three vendors and had received the standard canine vaccines prior to shipment. In each instance, the affected dog had been shipped to our facility within the past 10 days. Three cases presented as a peracute clinical syndrome, and all had gross and microscopic findings consistent with hemorrhagic pneumonia. Escherichia coli was isolated from the lungs of all four dogs. Results of testing of lung tissue for canine parainfluenza virus and canine adenovirus were negative. Escherichia coli was also isolated from blood of three of the four dogs. Serotyping of the E. coli isolates indicated that two were serotype 06 and two were 04. Isolates from all four dogs were positive for the virulence factors alpha hemolysin and cytotoxic necrotizing factor 1 and for the adhesin factor class-III papG allele. These traits place the isolates in the class of extraintestinal pathogenic E. coli, which is being increasingly implicated as a cause of extraintestinal infections in animals and humans and may represent a zoonotic risk to humans working with research dogs.

Miniaturization: an overview of biotechnologies for monitoring the physiology and pathophysiology of rodent animal models.

Recent advances in bioengineering technologies have made it possible to collect high-quality reproducible data quantitatively in a wide range of laboratory animal species, including rodents. Several of these technologies are incorporated into a plan called Miniaturization, which aims to design, develop, and maintain rodent animal models to study the pathophysiology and therapy of human diseases. Laser Doppler flowmetry, digital sonomicrometry, bioelectrical impedance, and microdialysis are some of the most widely used methods under the plan because they cause minimal pain and distress, reduce the number of animals used in biomedical research, and allow chronic, nonterminal assessment of physiological parameters in rodents. An overview of each of these technologies and their major applications in rodents used for biomedical research is provided.

Methods in vascular infusion biotechnology in research with rodents.

Infusion of experimental compounds into the vascular system of rodents and the need to collect blood and other biological fluids from small animals comprise an area of emerging importance to biomedical research and drug discovery and development. The advances in the development of transgenic rodents coupled with technical progress in the manufacture and commercial availability of various catheters, swivels, tethers, infusion pumps, and sample collection systems that are described have enabled biomedical scientists to miniaturize vascular infusion and sample collection systems previously used in animal species larger than the rat or mouse. Use of these advanced, miniature vascular infusion systems in rodents is possible only when careful planning of experimental design, expert surgical technique, adequate postoperative care, and fundamental animal welfare considerations are meticulously taken into consideration. Use of these vascular infusion systems in rodents promotes animal welfare and scientific progress through the reduction and refinement of animal models.

Novel Helicobacter species isolated from rhesus monkeys with chronic idiopathic colitis.

Chronic, idiopathic diffuse colitis is a well recognised clinical and pathological entity in captive rhesus monkeys. Six rhesus monkeys were diagnosed with clinically debilitating, chronic diarrhoea. Histologically, colonic tissues were characterised as chronic, moderate to severe colitis and typhlitis, with diffuse mononuclear inflammation of lamina propria, reactive lymphoid hyperplasia and multifocal micro-abscesses. Colonic tissues were cultured for Salmonella spp. and Shigella spp.; all results were negative. Samples were negative for Clostridium difficile A and B toxins, and special stains of colonic tissue for acid-fast bacteria were also negative. The six diarrhoeic monkeys tested gave negative results for serum IgG antibodies to herpes B virus, STLV, SRV and SIV. Colonic tissue from the six diarrhoeic and two clinically normal monkeys with histologically confirmed colitis from the same colony were also subjected to micro-aerobic culture. Micro-aerobic cultures from all eight monkeys incubated at 37 degrees C and 42 degrees C revealed pinpoint or spreading colonies on antibiotic-containing media. Bacteria were identified as gram-negative, oxidase positive and urease negative. Of the nine strains characterised biochemically, two separate biotypes (corresponding to different species by 16S rRNA analysis) were identified. One biotype (type 1), from non-diarrhoeic monkeys and the second biotype (type 2) from diarrhoeic animals with subclinical chronic colonic inflammation, differed by catalase activity, ability to reduce nitrate to nitrite and sensitivity to cephalothin. Complete 16S rRNA analysis of five of the nine strains characterised biochemically indicated that the organisms isolated were two novel Helicobacter spp. By electron microscopy, these novel helicobacters had spiral morphology with bipolar sheathed flagella. This is the first report describing the isolation of novel Helicobacter spp. from inflamed colons of rhesus monkeys. Studies are needed to determine whether these novel Helicobacter spp. play a causal role in the initiation and progression of chronic colitis in macaques. Further microbiological and histological analysis of this chronic idiopathic colitis syndrome in macaques may prove useful in understanding the aetiology and pathogenesis of inflammatory bowel disease in man.