Identification of Vancomycin Resistance in Methicillin-resistant Staphylococcus aureus in two macaque species and decolonization and long-term prevention of recolonization in Cynomolgus Macaques (Macaca fascicularis).

Methicillin-resistant Staphylococcus aureus (MRSA) is a S. aureus strain with resistance to beta-lactam antibiotics, making it a global human and veterinary health concern. Specifically, immunosuppressed patients have a remarkably higher risk of clinical MRSA infections with significantly increased rates of prolonged clinical recovery, morbidity, and mortality. The current treatment of choice for MRSA is vancomycin. Importantly, we report the first known vancomycin-resistant S. aureus (VRSA) carriers in a cohort of Mauritian cynomolgus macaques (CM) imported to the Oregon National Primate Research Center (ONPRC), with a MRSA carrier rate of 76.9% (10/13 animals). All MRSA isolates also demonstrated resistance to vancomycin with prevalence of vancomycin-intermediate Staphylococcus aureus (VISA) at 30% (3/10 MRSA-positive CMs) and VRSA at 70% (7/10 MRSA-positive CMs). Additionally, we identified VRSA in a rhesus macaque (RM) housed within the same room as the VRSA-positive CMs and identified a MRSA/VISA carrier rate of 18.8% in RMs (3/16 positive for both MRSA and VISA) in unexposed recently assigned animals directly from the ONPRC RM breeding colony. Considering that the MRSA and VRSA/VISA-positive CMs future study aims included significant immunosuppression, MRSA/VRSA/VISA decolonization treatment and expanded "MRSA-free" practices were employed to maintain this status. We report the first controlled study using in-depth analyses with appropriate diagnostic serial testing to definitively show an MRSA decolonization therapy (90% success rate) and expanded barrier practice techniques to successfully prevent recolonization (100%) of a cohort of CMs MRSA-free (up to 529 days with a total of 4,806 MRSA-free NHP days).

Allogeneic immunity clears latent virus following allogeneic stem cell transplantation in SIV-infected ART-suppressed macaques.

Allogeneic hematopoietic stem cell transplantation (alloHSCT) from donors lacking C-C chemokine receptor 5 (CCR5Δ32/Δ32) can cure HIV, yet mechanisms remain speculative. To define how alloHSCT mediates HIV cure, we performed MHC-matched alloHSCT in SIV+, anti-retroviral therapy (ART)-suppressed Mauritian cynomolgus macaques (MCMs) and demonstrated that allogeneic immunity was the major driver of reservoir clearance, occurring first in peripheral blood, then peripheral lymph nodes, and finally in mesenteric lymph nodes draining the gastrointestinal tract. While allogeneic immunity could extirpate the latent viral reservoir and did so in two alloHSCT-recipient MCMs that remained aviremic >2.5 years after stopping ART, in other cases, it was insufficient without protection of engrafting cells afforded by CCR5-deficiency, as CCR5-tropic virus spread to donor CD4+ T cells despite full ART suppression. These data demonstrate the individual contributions of allogeneic immunity and CCR5 deficiency to HIV cure and support defining targets of alloimmunity for curative strategies independent of HSCT.

Correction: Mitigation of endemic GI-tract pathogen-mediated inflammation through development of multimodal treatment regimen and its impact on SIV acquisition in rhesus macaques.

[This corrects the article DOI: 10.1371/journal.ppat.1009565.].

Antimicrobial prophylaxis does not improve post-surgical outcomes in SIV/SHIV-uninfected or SIV/SHIV-infected macaques (Macaca mulatta and Macaca fascicularis) based on a retrospective analysis.

Surgical antimicrobial prophylaxis is indicated when performing contaminated surgeries, when specific surgical implants are placed, and for prolonged surgical procedures. Unnecessary prophylactic antibiotics are often utilized for macaque surgeries, despite medical and veterinary guidelines. In this study we compared complication rates in macaques receiving peripheral lymph node (PLN) and laparoscopic biopsies, with and without antimicrobial prophylaxis. A majority of animals were SIV or SHIV infected at the time of surgery, so we also compared post-operative complication rates based on infection status. We found no significant difference in PLN biopsy complication rates for animals that received antimicrobial prophylaxis versus those that did not. Animals who underwent laparoscopic procedures and received prophylactic antibiotics had a higher complication rate than those who did not receive them. Complication rates did not differ significantly for SIV/SHIV infected versus uninfected animals for both laparoscopic biopsy procedures and PLN biopsy procedures. SIV/SHIV infected animals that underwent PLN biopsies had no significant difference in complication rates with and without antimicrobial prophylaxis, and SIV/SHIV infected animals receiving prophylactic antibiotics for laparoscopic biopsies had a higher complication rate than those that did not. This study suggests that perioperative prophylactic antibiotics have no role in the management of SIV/SHIV-infected and uninfected macaques undergoing clean, minimally invasive surgeries. Additionally, we recommend eliminating unnecessary antibiotic use in study animals due to their potential confounding impacts on research models and their potential to promote antimicrobial resistance.

Mitigation of endemic GI-tract pathogen-mediated inflammation through development of multimodal treatment regimen and its impact on SIV acquisition in rhesus macaques.

Here, we assessed the efficacy of a short-course multimodal therapy (enrofloxacin, azithromycin, fenbendazole, and paromomycin) to eliminate common macaque endemic pathogens (EPs) and evaluated its impact on gastrointestinal (GI) microbiota, mucosal integrity, and local and systemic inflammation in sixteen clinically healthy macaques. Treatment combined with expanded practices resulted in successful maintenance of rhesus macaques (RM) free of common EPs, with no evidence of overt microbiota diversity loss or dysbiosis and instead resulted in a more defined luminal microbiota across study subjects. Creation of a GI pathogen free (GPF) status resulted in improved colonic mucosal barrier function (histologically, reduced colonic MPO+, and reduced pan-bacterial 16s rRNA in the MLN), reduced local and systemic innate and adaptive inflammation with reduction of colonic Mx1 and pSTAT1, decreased intermediate (CD14+CD16+) and non-classical monocytes (CD14-CD16+), reduced populations of peripheral dendritic cells, Ki-67+ and CD38+ CD4+ T cells, Ki-67+IgG+, and Ki-67+IgD+ B cells indicating lower levels of background inflammation in the distal descending colon, draining mesenteric lymph nodes, and systemically in peripheral blood, spleen, and axillary lymph nodes. A more controlled rate of viral acquisition resulted when untreated and treated macaques were challenged by low dose intrarectal SIVmac239x, with an ~100 fold increase in dose required to infect 50% (AID50) of the animals receiving treatment compared to untreated controls. Reduction in and increased consistency of number of transmitted founder variants resulting from challenge seen in the proof of concept study directly correlated with post-treatment GPF animal's improved barrier function and reduction of key target cell populations (Ki-67+ CD4+T cells) at the site of viral acquisition in the follow up study. These data demonstrate that a therapeutic and operational strategy can successfully eliminate varying background levels of EPs and their associated aberrant immunomodulatory effects within a captive macaque cohort, leading to a more consistent, better defined and reproducible research model.

Terumo spectra optia leukapheresis of cynomolgus macaques for hematopoietic stem cell and T cell collection.

Macaques are physiologically relevant animal models of human immunology and infectious disease that have provided key insights and advanced clinical treatment in transplantation, vaccinology, and HIV/AIDS. However, the small size of macaques is a stumbling block for studies requiring large numbers of cells, such as hematopoietic stem cells (HSCs) for transplantation, antigen-specific lymphocytes for in-depth immunological analysis, and latently-infected CD4+ T-cells for HIV cure studies. Here, we provide a detailed protocol for collection of large numbers of HSCs and T-cells from cynomolgus macaques as small as 3 kg using the Terumo Spectra Optia apheresis system, yielding an average of 5.0 × 109 total nucleated cells from mobilized animals and 1.2 × 109 total nucleated cells from nonmobilized animals per procedure. This report provides sufficient detail to adapt this apheresis technique at other institutions, which will facilitate more efficient and detailed analysis of HSCs and their progeny blood cells.

Non-bronchoscopic Bronchoalveolar Lavage as a Refinement for Safely Obtaining High-quality Samples from Macaques.

Nonbronchoscopic bronchoalveolar lavage (NB-BAL) is a minimally invasive diagnostic and research tool used to sample the cells of lower airways and alveoli without using a bronchoscope. Our study compared NB-BAL and bronchoscopic bronchoalveolar lavage (B-BAL) in terms of costs, cell yields, and the number of post-procedural complications in macaques. We also analyzed procedure times, BAL fluid volume yields, and vital signs in a subset of animals that underwent NB-BAL. Compared with the B-BAL technique, NB-BAL was less expensive to perform, with fewer complications, fewer animals requiring temporary or permanent cessation of BALs, and higher cell yields per mL of recovered saline. The average procedure time for NB-BAL was 6.8 ± 1.6 min, and the average NB-BAL lavage volume yield was 76 ± 9%. We found no significant differences in respiration rate before, during, or after NB-BAL but did find significant differences in heart rate and oxygen saturation (SpO2). This study demonstrates that NB-BAL is a simple, cost-effective, and safe alternative to B-BAL that results in higher cell yields per mL, improved animal welfare, and fewer missed time points, and thus constitutes a refinement over the B-BAL in macaques.

Viral opportunistic infections in Mauritian cynomolgus macaques undergoing allogeneic stem cell transplantation mirror human transplant infectious disease complications.

Allogeneic hematopoietic stem cell transplantation (HSCT) and xenotransplantation are accompanied by viral reactivations and virus-associated complications resulting from immune deficiency. Here, in a Mauritian cynomolgus macaque model of fully MHC-matched allogeneic HSCT, we report reactivations of cynomolgus polyomavirus, lymphocryptovirus, and cytomegalovirus, macaque viruses analogous to HSCT-associated human counterparts BK virus, Epstein-Barr virus, and human cytomegalovirus. Viral replication in recipient macaques resulted in characteristic disease manifestations observed in HSCT patients, such as polyomavirus-associated hemorrhagic cystitis and tubulointerstitial nephritis or lymphocryptovirus-associated post-transplant lymphoproliferative disorder. However, in most cases, the reconstituted immune system, alone or in combination with short-term pharmacological intervention, exerted control over viral replication, suggesting engraftment of functional donor-derived immunity. Indeed, the donor-derived reconstituted immune systems of two long-term engrafted HSCT recipient macaques responded to live attenuated yellow fever 17D vaccine (YFV 17D) indistinguishably from untransplanted controls, mounting 17D-targeted neutralizing antibody responses and clearing YFV 17D within 14 days. Together, these data demonstrate that this macaque model of allogeneic HSCT recapitulates clinical situations of opportunistic viral infections in transplant patients and provides a pre-clinical model to test novel prophylactic and therapeutic modalities.