Animals were observed twice-daily for 28 days. Samples include blood, cerebrospinal fluid, feces, urine, bronchoalveolar lavage and mucosal swabs. These samples were collected at pre-exposure and post-exposure days and necropsy. Physical examination and imaging (radiography, PET, computed tomography) occurred 7 days prior to exposure and a week after exposure. Following the euthanization of the animals, samples from the left cranial and caudal lung lobes were processed in the media and analyzed. Right lung lobes contained in a fixative to preserve the samples. 

Animals and Procedure

Pulmonary pathology, a measurement of the severity of ARDs, was scored using random forest tissue segmentation algorithms. These algorithms recognized fibrin, edema and cellular inflammation using HALO software. Tissues samples from each of the right lung lobes were analyzed to determine the percentage of inflamed tissue. The percentage of inflammation was converted to a pathology score based on the following scoring system: fibrin and edema score of 0, 0% to 2%; 1, 2% to 5%; 2, 5% to 15%; 3, 15% to 30%; and 4, >30%; and cellular inflammation score of 0, 0% to 0.5%; 1, 0.5% to 3%; 2, 3% to 6%; 3, 6% to 12%; and 4, >12%.

Histopathologic Scoring

The lung samples, specifically 5 micrometer sections, were incubated for 1 hour with antibodies. These include
- SARS-CoV-2 nucleoprotein, mouse IgG1
- ACE2, rabbit polyclonal
- Iba-1, rabbit polyclonal
- Pancytokeratin, rabbit polyclonal

Secondary antibodies dyed with Alexa Fluor fluorochromes and diluted in NGS. 


Immunohistochemistry

The cytokines used in this experiment 
- Interferon (IFN)-γ
- IL-1β
- IL-2
- IL-4
- IL-6
- IL-8
- IL-10
- IL-12p70
- IL-13
- tumor necrosis factor-α

Cytokine Production in Plasma

Analysis of serum samples included testing for IgG antibodies against SARS-CoV-2 S1/S2 using an enzyme-linked immunosorbent assay kit. Positive samples were indicated as a difference of ≥0.300 between absorbance values of the positive viral antigen and those of the negative control antigen (positive samples had an MFIA score of ≥3.0). 

 Detection of Binding IgG Antibody in Plasma

Statistical softwares such as GraphPad Prism was used to analyze the data. In order to compare viral loads between RMs and AGMs, the Mann-Whitney U-Test was performed. The Pearson correlation test determined a correlational relationship between cytokines and viral loads. 

Statistical Analysis

In this experimental procedure, the virus used was SARS-Cov-2. Viral stock was prepared in Vero E6 cells and analyzed using deep sequencing.

Virus used in the Study of the Application of Green African Monkeys

- Virus
- Animals and Procedure
- Histopathologic Scoring
- Immunohistochemistry
- Cytokine Production in Plasma
- Detection of Binding IgG Antibodies in Plasma
- Statistical Analysis

Tulane University

In this study performed by the Tulane National Primate Research Center, reports of physiological symptoms similar to patients with severe infections of SARS-CoV-2 were confirmed. Two cases of ARDS in African green monkeys (AGMs) and mild infections in 2 AGMs and 4 RMs infected with Covid-19 were observed. All the AGMs had increased levels of plasma IL-6 and three of the four infected AGMs had high levels of circulating cytokines. All of these factors indicate an immune response similar to those in humans and support the use of AGMs as animal models useful for observing the in vivo effects of ARDS. 

Study of the Application of Green African Monkeys

Blair, R. V., Vaccari, M., Doyle-Meyers, L. A., Roy, C. J., Russell-Lodrigue, K., Fahlberg, M., . . . Rappaport, J. (2021). Acute respiratory distress in aged, sars-cov-2–infected african green monkeys but not rhesus macaques. The American Journal of Pathology, 191(2), 274-282. doi:10.1016/j.ajpath.2020.10.016

Acute Respiratory Distress in Aged, SARS-CoV-2–Infected African Green Monkeys but Not Rhesus Macaques

Acute Respiratory Disease, ARDS, can be fatal in certain cases. Severely affected individuals present with a fever, cough, dyspnea and bilateral radiographic opacities (filling of the pulmonary tree with foreign materials). In critically ill patients these pathologies can progress to ARDS. 

Acute Respiratory Disease

Materials and Methods

Four AGMs and four RMs were exposed to SARS-CoV-2 either through aerosolization or via multiple routes. 
Experimental Group 1:
Four animals exposed to SARS-CoV-2 via small particle aerosol

- AGM1
- AGM4
- RM3
- RM4

Four animals exposed to SARS-CoV-2 via multiple route installation

- AGM2
- AGM3
- RM1
- RM2

RNA from the virus was detected in mucosal swabs and samples from the pharynx and nasal cavity of all eight animals. Analysis of rectal swabs showed high levels of viral RNA. 


Results of the Study

Other studies recently published have demonstrated the progression of SARS-CoV-2 in AGMs and RMs. Both animals are capable of modeling mild to moderate disease and can be used to analyze the efficacy of vaccines and other therapeutics. However, these other studies have not demonstrated the clinical manifestation, ARDS, of the Covid-19 infection. Results of this study show that aged AGMs infected with Covid-19 can develop ARDS (signified by cytokine elevations similar to those in humans). The study’s findings are associated with the results from other studies, with AGMs presenting mild clinical disease, pathology and viral shedding. 

Learn Before

Related

Learn After