Abstract
Owing to their size, cost, and availability, the cynomolgus macaque (Macaca fascicularis) has surpassed the rhesus macaque in its use as a non-human primate preclinical model for drug safety studies. There are three major regions where cynomolgus macaques are bred: China, Southeast Asia, and the island of Mauritius. Country of origin of the macaque is important, as disease status and background disease incidence in non-human primates from each of these sites can differ. Once a source of macaque has been decided, careful monitoring of the animal during breeding and by the importing vendor while the animals are in quarantine is important. During vendor quarantine, the animals should be monitored and evaluated for disease, response to tuberculosis testing, retroviral status, and both ecto- and endoparasites. After animals arrive at the test facility, additional quarantine and acclimation are important to ascertain health status further and to reduce stress on the animals, thereby providing a better research model. The type of caging, food, water, and enrichment should be carefully selected to best suit the needs of the study while working within Federal Regulations (i.e., Animal Welfare Act and Good Laboratory Practices). Careful prescreening by performing tests (such as physical, neurologic, and ophthalmologic examinations), complete blood count, biochemical profile, urinanalysis, electrocardiograms, and pulse oximetry is important when selecting the most appropriate animals for the study. After the in-life portion of the study begins, animals that present with clinical signs should be examined and an appropriate treatment course begun while maintaining study objectives. As many commonly used medications have immunomodulatory effects, having an understanding of the mechanism of action of test articles will aid in the appropriate choice of treatment of study animals. A tiered approach to the treatment of these animals is a conservative and usually acceptable approach.
Introduction
Macaques (rhesus and cynomolgus) and other Old and New World primates are widely used in research. Non-human primates can be an excellent animal model for drug safety studies. Similar to all research models, there are important considerations that should be taken care when using non-human primates for drug safety studies. These considerations range from the type and geographical origin of non-human primate used, to the health status, treatment and study history of the animal, to the background incidence of disease in the colony, and how the model compares to humans. As cynomolgus macaques are the most common species of non-human primate used in preclinical research, this review has been focused on this species. The impact of source of origin, a brief overview of the considerations of their use on immunotoxicology and toxicology studies, select complications of common secondary infections seen in immunotoxicology studies, and the treatment and management of those infections have been discussed.
Type, source, and differences
Cynomolgus macaques (Macaca fascicularis) account for the majority of non-human primates used in preclinical research (Drevon-Gaillot et al., Citation2006). Captive-bred cynomolgus monkeys have an average life span of approximately 31 years and when fully grown range from 5 to 8 kg (11–17.6 pounds) in weight and from 43 to 58 cm (18–24 inches) in length. Colonies of cynomolgus monkeys can be found in Southeast Asia, China, and the island of Mauritius. Asian macaques found in Mauritius were brought to the island approximately 400 years ago, most probably during the Dutch occupation in the 16th or 17th century (Kawamoto et al., Citation2008). These monkeys have begun to show some isolation effects when compared to those in Asia (Matsubayashiv et al., Citation1992), including a low major histocompatibility complex (MHC) diversity and genetic homogeneity. In drug safety studies, specifically in immunotoxicological studies, low MHC and genetic homogeneity reduces the potential genetic response variable of the test compound. This result makes animals from the Mauritius colonies excellent models for research (Kawamoto et al., Citation2008).
Country of origin and breeding practices are important in determining the potential background incidence of underlying diseases. Cynomolgus monkeys from the island of Mauritius are considered free from simian immunodeficiency virus (SIV), Macacine herpesvirus 1 (McHV-1) (formally known as Cercopithecine herpesvirus-1 [CHV-1 or BV]), measles, simian retrovirus 1 and 2 (SRV-1 and SRV-2), simian T-cell lymphotropic virus type I and III (STLV-I and STLV-III), herpes simplex virus type 1 (HSV-1), SV5, and SV6 (Matsubayashi et al., Citation1992). Individual colonies within any country of origin may be certified free of specific pathogens if rigorous screening and colony management techniques are employed. Early weaning, culling of suspect and infected animals, as well as appropriate housing and husbandry practices can greatly reduce the incidence of diseases such as McHV-1 in the breeding population (Elmore and Eberle, Citation2008). Although the closed nature and island setting of Mauritius may more easily facilitate disease management, disease status should be verified independently regardless of the origin.
Monkeys from different countries can also vary in hematological and biochemical parameters, histopathological findings, and bacterial and parasitic loads. It has been documented that hematologically monkeys from Vietnam have lower amounts of red blood cells (RBCs) and higher hemoglobin levels, whereas monkeys from Mauritius have lower white blood cell (WBC) concentrations and monkeys from the Philippines had higher eosinophilic granulocytes than the other two countries. In addition, biochemistry values vary with country of origin. Serum calcium is higher in Mauritian monkeys and serum phosphorus is lower. Monkeys from the Philippines have lower creatinine and alanine aminotransferase (ALT) levels, and Vietnamese monkeys have lower alkaline phosphatase, higher aspartate aminotransferase (AST). Intestinal tract lymphoplasmacytic infiltrates differs in grade in monkeys from those three countries as well (Drevon-Gaillot et al., Citation2006). Balantidium coli (B. coli), Entamoeba coli, Trichuris trichiura, and Helicobacter species can be found in the intestinal tracts of cynomolgus monkeys from all origins (Matsubayashi et al., Citation1992; Drevon-Gaillot et al., Citation2006).
Vendor surveillance
After importation into the United States, the importer is required to quarantine all cynomolgus monkeys (Code of Federal Regulations, Title 42). This quarantine is an important time to determine or confirm the serologic status for SIV, STLV-1, SRV, and McHV-1. In addition, testing for tuberculosis (skin tests as well as radiographs of the thorax), performing fecal examinations - including aerobic culture for Salmonella, Shigella, Campylobacter jejuni (C. jejuni), and Escherichia coli (E. coli), and a fecal flotation/wet mount for parasites may be done. Prophylactic treatment for internal and external parasites with medications such as praziquantel, ivermectin, and permethrins can also be performed. During this time, vaccinations for measles and hepatitis A should be considered. These diseases, in addition to producing significant morbidity and mortality when present during a drug safety study, can confound study results.
Facility quarantine and acclimation
Institutions should consider internal quarantine when animals arrive at their facility. This may be accomplished on the property of the main research facility or at another designated facility. Quarantine durations vary but, generally, 8 weeks or longer are common. During this time, multiple tests for tuberculosis may be performed and are typically at 2-week intervals. Physical examinations, complete blood counts (CBC), biochemistry profiles, and fecal screening, as well as confirmatory serologic screening for SIV, STLV-1, SRV, McHV-1, and measles are recommended. Additional vaccinations may also be performed. Macaques imported into certain states (e.g., New York State) should also be tested for filoviruses. An acclimation period often runs concurrent with the quarantine period.
Acclimation is performed to ensure that the animals are familiar with the facility, the husbandry, feeding, and enrichment schedule and to allow physiologic parameters to normalize after the potential stresses of shipment, a new environment, and new social structures. It has been established that stress can alter immune system function including WBC and RBC counts, and the function of WBCs (in both innate and humoral immunity) (Coe and Erickson, Citation1997; Brown et al., Citation2008; Pace et al., Citation2008) and hormone levels (Clarke et al., Citation1996; Blecha, Citation2000). In addition, behavior abnormalities, self-injurious behavior and hair picking (Tiefenbacher et al., Citation2005), and changes in heart rate and liver and kidney functions can be seen with increased stress.
Husbandry
Most non-human primates used in drug safety studies live in stainless steel or stainless steel with plastic cages; the size of these cages can vary depending on the facility and the type of study being performed. In the United States, the Federal Government, through the USDA and in accordance with the Animal Welfare Act (Code of Federal Register. Title 9, Chapter 1, Subchapter A: Animal Welfare; Code of Federal Regulations. Title 42, CFR §71.53. 2003.), regulates minimum cage size requirements.
Animals may be singly-, pair-, or group/gang-housed. In addition, animals that are pair or group housed may be separated during portions of the day to facilitate study activities. For example, animals may be separated before dosing and returned after feeding. This allows for the ability of animals to have socialization time with their cohorts while still collecting post-dose individual animal observations and individual feeding behavior information. It is generally accepted that animals that are pair- or group-housed display less stereotypic behaviors than those who are singly caged. Animals may demonstrate increased species-specific behaviors such as increased social interaction and grooming of the cohort, as well as demonstrate lower heart rates in times of stress (Watson et al., Citation1998). For animals that are pair or group housed, familiarity with their partner can be important. Fighting in cynomolgus monkeys can be sudden, even in familiar or established pairs. As the animals age and they reach sexual maturity (females at approximately 46 months-of-age and males at approximately 42–60 months-of-age) (Fox et al., Citation2002), fighting may be more of a concern. These animals are generally larger in size and can cause substantially more damage as their canine teeth are often fully grown.
Along with pair and group housing, additional enrichment may also reduce the amount of stereotypic behavior that animals demonstrate (Laule et al., Citation2003). Examples of enrichment include novel or supplementary daily fruits or vegetables, foraging mixtures (seeds and dried fruits), chew toys, or toys that require manipulation to receive a small food reward and contact with research staff.
For drug safety studies that are following Good Laboratory Practices (GLP), a certified diet should be fed. There are many commercially-available laboratory diets that would be appropriate. Certification helps ensure the nutritional analysis of the food as well as tests for contaminants such as heavy metals, mycotoxins, chlorinated hydrocarbons, and organophosphates that may impact animal health or confound study results. Enrichment toys and other products specifically designed for non-human primates that may be ingested should also be certified.
Similar to food, drinking water should be tested and treated to reduce contaminants or to standardize its quality. Reverse osmosis treatment is one option to reduce contaminants from drinking water. In addition, testing water for various chemicals, chlorine content, coliforms, pseudomonas, and total bacteria counts is commonly performed. Cage cleaning is typically performed daily and full sanitization of the enclosure every other week; this reduces the amount of fecal, urine, and food waste that the animal is exposed to. In studies where immunosuppression is a concern, adequate cleaning and sanitization are important in minimizing secondary infections. In most facilities, cages are changed at least every 2 weeks to facilitate sanitization. This movement, noise, and new environment can add additional stress to the animals which may further suppress the immune system.
Restraint of non-human primates can be accomplished in many different ways. Chemical restraint is common, with ketamine-HCl being the most commonly-used drug. Animals may be fitted with a plastic or aluminum collar and restrained using a pole and chair system. In addition, hand catching and restraint (manually by the technicians) is also performed at some facilities. There are many benefits and potential complications with each of these methods. Regardless of the method, unlike their human counterparts, the necessity for restraint adds a complexity to the study that is not present in human studies (stress). These husbandry differences are important when correlating results from preclinical studies to their clinical counterparts.
Pre-study examinations and activities
Pre-study or pre-test examinations and procedures are an important step in selecting and acclimating animals for study use. Animals can be trained or acclimated to restraint, dosing, or other procedures that will occur during a study. As new partners may be chosen through the randomization procedure and housing conditions may vary during the course of a study, this time is essential for acclimating the animals to these housing changes. Collection of baseline cage-side data, in addition to physical, ophthalmologic, neurologic, electrocardiographic (ECG) examinations, is often performed during this period. CBC, biochemical profiles, pulse oximetry, and special measurements that are program-specific may also be performed during this time.
Reduction of stress during the dosing period of the study is also essential. Performing study-related activities, such as sham dosing, chairing, or hand catching can allow the animal to acclimate to these procedures before the start of the dosing period. Observations by technical staff pre-test are also important. For immunotoxicological studies, injuries that occur during the pre-study period may be a concern for opportunistic bacterial growth during the study. This may impact the suitability of the animal for use. General behavior of the animals can be assessed and documented. This time also allows the animals to become accustomed to the technical staff that will be handling them throughout the duration of the study.
Physical examination including neurologic and ophthalmologic assessments, ECG, pulse oximetry, CBC, biochemistry profile, and coagulation panels are performed before beginning the study. These assessments allow for baseline data on the animals that will be selected for study, as well as to identify animals that may not be appropriate for use in a drug assessment study. For immunotoxicological studies, it is important to select animals carefully that have no clinical history of confounding disease or conditions or have baseline data that would be inappropriate for the objective of the study. Careful selection of animals at this stage may improve study outcome.
Veterinary concerns on study
Once the animals have been selected for study and the dosing period begins, animals that have abnormal clinical signs should be evaluated by a veterinarian. Assessments that are performed at the time of the examination depend on the presenting clinical signs. CBC, biochemical panel, coagulation panel, aerobic and anaerobic cultures of blood, discharges, rectum or tissues, fecal flotation, clostridium toxin examination of feces may be performed. Information such as the number of animals affected, the groups they comprise, the presenting clinical signs, the timing of the presentation, and other information will help determine the cause of the clinical signs and the appropriate response.
Animals affected can present clinical signs at one time, staggered, or independent of each other. This information may help differentiate clinical signs as drug, vehicle, or procedure related, opportunistic or incidental (i.e., spontaneous disease, injuries). Further, this information, as well as severity of presenting signs, is vital when considering the etiology as well as follow-up care.
With this information and the results of any examinations that are performed, the determination of a possible etiology may be made. Issues with administration such as dosing error, vehicle-related, pharmacologic or toxic drug effects, or immunosuppression with or without secondary infections can be considered directly or indirectly study related. Non-study-related clinical signs may be due to trauma or recurrent disease that was previously identified or typical to a non-human primate model.
Background disease and immunosuppression
Background disease incidence is a part of any toxicological study. In monkeys, common conditions include diarrhea, epistaxis, trauma due to fighting or injuries, and a less common gastric dilatation (bloat). Diseases may be related to the source of the monkey, such as SIV, STLV-1, SRV, and McHV-1. In addition, infestation with Plasmodium spp. and exposures to measles, hepatitis A, tuberculosis, endoparasites, and ectoparasites are also possible.
Diarrhea can be found in uncompromised cynomolgus monkeys. Most diarrheas are self-limiting, idiopathic, and some are recurrent (Holmberg et al., Citation1982). However, chronic enteropathies that are associated with infectious etiologies may have an impact on drug safety studies (Sasseville and Diters, Citation2008). These can cause serious clinical illness during the study resulting in changes in the mucosal lining of the intestine. This may interfere with the interpretation of histopathologic findings. Therefore, testing for these agents when animals present with diarrhea should be performed. Diarrhea incidence in the in-life portion of drug safety studies may be attributed, for example, to the vehicle, test article, and toxicity, stress-induced, bacterial (i.e., C. jejuni, enteropathogenic E. coli, Shigella spp., Salmonella spp.), protozoal (i.e., giardiasis, amoeba), or idiopathic. Information obtained by testing allows for treatment as well as potential to attribute the diarrhea to an etiology other than directly test article related. Balantidium coli is a protozoan that is commonly found in cynomolgus monkeys and is often present in animals that present with diarrhea. It is uncertain if this organism is a primary pathogen (Drevon-Gaillot et al., Citation2006).
Epistaxis or blood from the nasal passages can be attributed to a drug-related effect, the bacteria Moraxella catarrhalis (previously known as Branhamella) (VandeWoude and Luzarraga, Citation1991), low humidity, or trauma. Aerobic culture to isolate and identify M. catarrhalis is possible. However, not all animals that are infected with the bacteria will be symptomatic and this may hinder in the diagnosis of the cause of the epistaxis.
Many, diseases such as those caused by SIV, SRV, STLV-1, McHV-1, and plasmodium, are often asymptomatic but can confound study results. These diseases can also lead to further immunosuppression. Incidental issues such as alopecia, dermatitis, and trauma are usually self-limiting. In animals that are immunocompromised due to underlying disease or due to a test article effect, these conditions can be a source of secondary infections.
Treatment of study animals
Animals on drug safety studies, especially those receiving immunomodulating compounds, may present with clinical signs that require treatment. These include, but are not limited to, anorexia, diarrhea, skin, blood or tissue infections, injuries, epistaxis, and neurologic signs. Many medications that are commonly used to treat these conditions have their own side effect profile that could affect the study outcome or make interpretation of study results difficult. A tiered approach to treatment may mitigate some of these effects. In addition, knowledge of the effects and side effects of treatments being considered, and their interaction with the test article, is essential to ensure that study objectives can be met.
After assessment of the animal, a tiered treatment plan may be considered. This depends on the condition of the animal, the clinical signs that the animal presented with, and the duration of those clinical signs. A tiered system could begin with conservative monitoring if deemed appropriate. This monitoring time allows for a determination if resolution during continued dosing is possible. After monitoring, treatments that are designated as “supportive care” should be implemented. This can be extremely important in animals that present with anorexia, dehydration, or vomiting and diarrhea. “Supportive care” can consist of supplemental fluids (i.e., oral, subcutaneous, or intravenous), supplemental food (e.g., fruits, vegetables, or specialty diets), and oxygen and heat supplementation. These treatments provide support for organ function and may stabilize the animal’s condition. If this level of care is ineffective, directed treatments may be required. Directed treatments can consist of anti-microbials, anti-fungals, pain medications, anti-diarrheals, topical medications, or any medication required to treat the condition or disease.
Secondary infections can be a common sequela to immunosuppressive study agents. Antimicrobials may be necessary for some animals that present with infections. In those animals, bacterial cultures and microbial sensitivities should be carried out before treatment. To aid in the selection of the most appropriate antibiotic, antimicrobial susceptibility should be performed on any isolate that is cultured. Common antibiotics are penicillin, amoxicillin and amoxicillin with clavulanic acid, doxycycline and tetracycline, cefazolin and other cephalosporins, metronidazole, and enrofloxacin. These antibiotics have been shown to have many immunomodulatory effects. Toxicities, such as suppression of antibody responses (i.e., doxycycline) (Woo et al., Citation1999), potentiating phagocytosis by WBC (i.e., certain cephalosporins, fluoroquinolones) (Gemmell, Citation1993; Hamilton-Miller, Citation2001), inhibition of transcription factors (i.e., telithromycin) (Leiva et al., Citation2008), reductions in tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-8 production (i.e., tetracycline, doxycycline, fluoroquinolones) (Woo et al., Citation1999; Kolios et al., Citation2006; Lahat et al., Citation2007), modulation of interferon (IFN)-γ levels (i.e., β-lactams) (Brooks et al., Citation2005), modulation of the glutamate transporter GLT1 (i.e., β-lactams) (Rothstein et al., Citation2005), reduction of T-cell activation by modulation of cellular antigen-presentation and impairment of antigen-specific T-cell migration into the CNS rather than a modulation of central glutamate homeostasis” (i.e., β-lactams) (Melzer et al., Citation2008), inhibition of matrix metalloproteases (i.e., doxycycline) (Errami et al., Citation2008), and inhibition of macrophage phagocytic activity (i.e., metronidazole) (Fararjeh et al., Citation2008) are examples of the published effects of some antibiotics (Van Vlem et al., Citation1996).
In addition to secondary infections, non-human primates can be prone to injury. Some injuries are severe enough to require anesthesia. Surgical stress and anesthesia can cause immune system effects, such as dysfunction of natural killer cells, lymphocytes, anti-inflammatory effects, and direct suppressive effects on cellular and humoral immunity (Kurosawa and Kato, Citation2008). Injuries have the potential for secondary infections and preemptive treatment with antibiotics may be necessary in these cases.
Animals with injuries or infections, in addition to those requiring surgery, may also present with pain, and analgesics medications may be required for humane reasons. Selection of an appropriate anesthetic is crucial to minimize the impact on immunotoxicological studies. In these situations, opioids, non-steroidal anti-inflammatory drugs (NSAIDs), and local anesthetics are most commonly used. All three classes of drugs have direct effects on the immune system. Opioids, such as morphine and buprenorphine, have immunosuppressive effects (Sacerdote, Citation2008), such as the reductions of TNF-α levels observed following the administration of an opioid. In addition, decreases in: mitogen-stimulated proliferation of B-cells and T-cells; natural killer cell activity; IFNγ and IL-2 production; and, antibody responses and macrophage phagocytic capacity have been reported with opioid use (Piersma et al., Citation1999; Carrigan et al., Citation2004).
Topical medications have been shown to absorb transdermally or are consumed orally by the monkey, and can affect the animal in a similar way as their parenteral or enteral counterparts (Otto et al., Citation2009). In cases where topical medications are possible, such as topical antibiotics for superficial wounds, these may provide a better alternative than their oral or parenteral counterparts. Care should be used when selecting topical formulations and the amounts that are applied.
If these directed treatments are unsuccessful or not possible due to study design or compound-related concerns, a temporary suspension of dosing should be considered. Animals that present with secondary infections or drug-related clinical signs and those that are not dosed for a short period may be able to resume dosing and complete the study. This information is important in determining the clinical trail monitoring information and strategy.
The welfare of the animals is vital. Animals should be monitored during clinical disease (secondary disease, infections, or drug-related disease) for indications that euthanasia would be required. Prospectively-planned humane euthanasia may facilitate maximal collection of critical terminal data including toxicokinetic, clinical, and anatomical pathology samples. In cases where compounds are known to cause primary disease or predispose to secondary disease, an appropriate number of animals should be selected to ensure that the power of the study is not compromised due to these effects if animals must be removed before completion of the study.
Although many medications have direct effects on the immune system, treatment should be considered even for immunotoxicological studies. A good working relationship between the attending veterinarian and the study director will help to determine the most appropriate method of addressing clinical signs in the animals during the in-life phase of the study.
Conclusion
Non-human primates are used for drug safety studies. Knowledge of the species, origin of the monkey, background incidence of disease in the country of origin, and the background incidence of disease in the facility’s colony help to differentiate test article-related effects, from secondary infections related to immunodeficiencies, or incidental disease. A complete examination and appropriate testing performed when animals display clinical signs of disease while in drug safety studies allows for earlier decisions about treatment. Monitoring, treatment, a temporary suspension of dosing, or humane euthanasia are options. Welfare of the individual animal should always guide the appropriate method of treatment for the animals on the study.
Acknowledgments
This work was supported by ILSI Health and Environmental Sciences Institute and Bristol-Myers Squibb.
Declaration of interest
The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
References
- Blecha, F. 2000. The biology of animal stress. In: The Biology of Animal Stress: Basic Principles and Implications for Animal Welfare (Moberg, G. P., and Mench, J. A., eds.), New York: CABI Publishing, pp. 111–121.
- Brown, A. S., Levine, J. D., and Green, P. G. 2008. Sexual dimorphism in the effect of sound stress on neutrophil function. J. Neuroimmunol. doi:10.1016/j.jneuroim.2008.08.005.
- Brooks, B. M., Hart, C. A., Coleman, J. W. 2005. Differential effects of beta-lactams on human IFN-gamma activity. J Antimicrob Chemother. 56(6):1122–5.
- Carrigan, K. A., Saurer, T. B., Ijames, S. G., and Lysle, D. T. 2004. Buprenorphine produces naltrexone reversible alterations of immune status. Int. Immunopharmacol. 4:419–428.
- Clarke, M. R., Harrison, R. M., and Didier, E. S. 1996. Behavioral, immunological, and hormonal responses associated with social change in rhesus monkeys (Macaca mulatta). Am. J. Prim. 39:223–233.
- Coe, C. L., and Erickson, C. M. 1997. Stress decreases lymphocyte cytolytic activity in the young monkey even after blockade of steroid and opiate hormone receptors. Dev. Psychobiol. 30:1–10.
- Drevon-Gaillot, E., Perron-Lepage, M. F., Clément, C., and Burnett, R. 2006. A review of background findings in cynomolgus monkeys (Macaca fascicularis) from three different geographical origins. Exp. Toxicol. Pathol. 58:77–88.
- Elmore, D., and Eberle, R. 2008. Monkey B virus (cercopithecine herpesvirus 1). Comp. Med. 58:11–22.
- Errami, M., Galindo, C. L., Tassa, A. T., Dimaio, J. M., Hill, J. A., and Garner, H. R. 2008. Doxycycline attenuates isoproterenol- and transverse aortic banding-induced cardiac hypertrophy in mice. J. Pharmacol. Exp. Ther. 324:1196–1203.
- Fararjeh, M., Mohammad, M. K., Bustanji, Y., Alkhatib, H., and Abdalla, S. 2008. Evaluation of immunosuppression induced by metronidazole in Balb/c mice and human peripheral blood lymphocytes. Int. Immunopharmacol. 8:341–350.
- Fox, J. G., Anderson, L. A., Loew, F. M., and Quimby, F. W., (Eds) 2002. Laboratory Animal Medicine, 2nd Edition. New York: Academic Press.
- Gemmell, C. G. 1993. Antibiotics and neutrophil function—potential immunomodulating activities. J. Antimicrob. Chemother. 31(Suppl. B):23–33.
- Hamilton-Miller, J. M. 2001. Immunopharmacology of antibiotics: Direct and indirect immunomodulation of defense mechanisms. J. Chemother. 13:107–111.
- Holmberg, C. A., Leininger, R., Wheeldon, E., Slater, D., Henrickson, R., and Anderson, J. 1982. Clinicopathological studies of gastrointestinal disease in macaques. Vet. Pathol. Suppl. 7:163–170.
- Kawamoto, Y., Kawamoto, S., Matsubayashi, K., Nozawa, K., Watanabe, T., Stanley, M. A., and Perwitasari-Farajallah, D. 2008. Genetic diversity of long-tail macaques (Macaca fascicularis) on the island of Mauritius: An assessment of nuclear and mitochondrial DNA polymorphisms. J. Med. Primatol. 37:45–54.
- Kolios, G., Manousou, P., Bourikas, L., Notas, G., Tsagarakis, N., Mouzas, I., and Kouroumalis, E. 2006. Ciprofloxacin inhibits cytokine-induced nitric oxide production in human colonic epithelium. Eur. J. Clin. Invest. 36:720–729.
- Kurosawa, S., and Kato, M. 2008. Anesthetics, immune cells, and immune responses. J. Anesth. 22:263–277.
- Lahat, G., Halperin, D., Barazovsky, E., Shalit, I., Rabau, M., Klausner, J., and Fabian, I. 2007. Immunomodulatory effects of ciprofloxacin in TNBS-induced colitis in mice. Inflamm. Bowel Dis. 13:557–565.
- Laule, G. E., Bloomsmith, M. A., and Schapiro, S. J. 2003. The use of positive reinforcement training techniques to enhance the care, management, and welfare of primates in the laboratory. J. Appl. Anim. Welfare Sci. 6:163–173.
- Leiva, M., Ruiz-Bravo, A., Moreno, E., and Jiménez-Valera, M. 2008. Telithromycin inhibits the production of pro-inflammatory mediators and the activation of NF-κB in in vitro-stimulated murine cells. FEMS Immunol. Med. Microbiol. 53:343–350.
- Matsubayashi, K., Gotoh, S., Kawamoto, Y., Watanabe, T., Nozawa, K., Takasaka, M., Narita, T., Griffiths, O., and Stanley, M. A. 1992. Clinical examinations on crab-eating macaques in Mauritius. Primates 33:281–288.
- Melzer, N., Meuth, S. G., Torres-Salazar, D., Bittner, S., Zozulya, A. L., Weidenfeller, C., Kotsiari, A., Stangel, M., Fahlke, C., and Wiendl, H. 2008. A beta-lactam antibiotic dampens excitotoxic inflammatory CNS damage in a mouse model of multiple sclerosis. PLoS ONE 3:e3149.
- Otto, A., du Plessis, J., and Wiechers, J. W. 2009. Formulation effects of topical emulsions on transdermal and dermal delivery. Int. J. Cosmet. Sci. 31:1–19.
- Pace, T. W., Negi, L. T., Adame, D. D., Cole, S. P., Sivilli, T. I., Brown, T. D., Issa, M. J., and Raison, C. L. 2008. Effect of compassion meditation on neuroendocrine, innate immune and behavioral response to psychosocial stress. J. Psyneuen. doi:10.1016/j.psyneuen.2008.08.011.
- Piersma, F. E., Daemen, M. A., Bogaard, A. E., and Buurman, W. A. 1999. Interference of pain control employing opioids in in vivo immunological experiments. Lab. Anim. 33:328–333.
- Rothstein, J. D., Patel, S., Regan, M. R., Haenggeli, C., Huang, Y. H., Bergles, D. E., Jin, L., Dykes Hoberg, M., Vidensky, S., Chung, D. S., Toan, S. V., Bruijn, L. I., Su, Z. Z., Gupta, P., and Fisher, P. B. 2005. Beta-lactam antibiotics offer neuroprotection by increasing glutamate transporter expression. Nature 433:73–77.
- Sacerdote, P. 2008. Opioid-induced immunosuppression. Curr. Opin. Support Palliative Care 2:14–18.
- Sasseville, V. G., and Diters, R. W. 2008. Impact of infections and normal flora in non-human primates on drug development. ILAR J. 49:179–190.
- Tiefenbacher, S., Novak, M. A., Lutz, C. K., and Meyer, J. S. 2005. The physiology and neurochemistry of self-injurious behavior: A non-human primate model. Frontiers Biosci. 10:1–11.
- VandeWoude, S. J., and Luzarraga, M. B. 1991. The role of Branhamella catarrhalis in the “bloody-nose syndrome” of cynomolgus macaques. Lab. Anim. Sci. 41:401–406.
- Van Vlem, B., Vanholder, R., De Paepe, P., Vogelaers, D., and Ringoir, S. 1996. Immunomodulating effects of antibiotics: Literature review. Infection 24:275–291.
- Watson, S. L., Shively, C. A., Kaplan, J. R., and Line, S. W. 1998. Effects of chronic social separation on cardiovascular disease risk factors in female cynomolgus monkeys. Atherosclerosis 137:259–266.
- Woo, P. C., Tsoi, H. W., Wong, L. P., Leung, H. C., and Yuen, K. Y. 1999. Antibiotics modulate vaccine-induced humoral immune response. Clin. Diagn. Lab. Immunol. 6:832–837.