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Hematology and Clinical Chemistry Reference Ranges for Laboratory-Bred Natal Multimammate Mice (Mastomys Natalensis)

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Hematology and Clinical Chemistry Reference Ranges for Laboratory-Bred Natal Multimammate Mice (Mastomys Natalensis) viruses Article Hematology and Clinical Chemistry Reference Ranges for Laboratory-Bred Natal Multimammate Mice (Mastomys natalensis) David M. Wozniak 1 , Norman Kirchoff 1, Katharina Hansen-Kant 1, Nafomon Sogoba 2, David Safronetz 3,4 and Joseph Prescott 1,* 1 ZBS5—Biosafety Level-4 Laboratory, Robert Koch-Institute, 13353 Berlin, Germany; [email protected] (D.M.W.); [email protected] (N.K.); [email protected] (K.H.-K.) 2 International Center for Excellence in Research, Malaria Research and Training Center, Faculty of Medicine, Pharmacy and Dentistry, University of Sciences, Techniques and Technologies of Bamako, Bamako 91094, Mali; [email protected] 3 Laboratory of Virology, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840, USA; [email protected] 4 Zoonotic Diseases and Special Pathogens, National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, MB R3E 3M4, Canada * Correspondence: [email protected] Abstract: Laboratory-controlled physiological data for the multimammate rat (Mastomys natalensis) are scarce, despite this species being a known reservoir and vector for zoonotic viruses, including the highly pathogenic Lassa virus, as well as other arenaviruses and many species of bacteria. For this reason, M. natalensis is an important rodent for the study of host-virus interactions within laboratory settings. Herein, we provide basic blood parameters for age- and sex-distributed animals in regards to blood counts, cell phenotypes and serum chemistry of a specific-pathogen-monitored M. natalensis Citation: Wozniak, D.M.; Kirchoff, breeding colony, to facilitate scientific insight into this important and widespread rodent species. N.; Hansen-Kant, K.; Sogoba, N.; Safronetz, D.; Prescott, J. Hematology and Clinical Chemistry Reference Keywords: Mastomys natalensis; multimammate mouse; multimammate rat; blood; reference value; Ranges for Laboratory-Bred Natal normal value; baseline; clinical chemistry; Lassa virus Multimammate Mice (Mastomys natalensis). Viruses 2021, 13, 187. https://doi.org/10.3390/v13020187 1. Introduction Academic Editor: Daniel R. Perez Mastomys natalensis are commensal wild-living rodents of the African continent with Received: 16 December 2020 a large host range [1,2]. Even though M. natalensis are often described as “rats”, phyloge- Accepted: 23 January 2021 netically, they are more closely related to mice (Mus) than Rattus [3]. However, since they Published: 27 January 2021 grow to sizes and weights greater than Mus musculus [4], the confusion with rats as a general descriptor is understandable. Seasonal resource limitations [5,6], their grani- and Publisher’s Note: MDPI stays neutral omnivore sustenance and the high reproduction capabilities of M. natalensis [5] cause large with regard to jurisdictional claims in annual fluctuations of local populations. During the dry season, M. natalensis often live published maps and institutional affil- peri-domestically and are found inside human dwellings [6] consuming food scraps and iations. unsecured food stores and thereby contaminate these with their excretions. Mastomys sp. can harbor a diversity of zoonotic pathogens of serious concern for humans, including Capillaria hepatica, Gongylonema sp., Escherichia coli, Salmonella typhimurium [4,7], and, in the case of the visually indistinguishable sister species, M. coucha, Yersinia pestis infection, Copyright: © 2021 by the authors. which could have been partially responsible for human plague clusters in Africa [8]. Licensee MDPI, Basel, Switzerland. The pathogen of highest concern harbored by M. natalensis, as well as other species of This article is an open access article Muroinae, including Hylomyscus pamfi, Mus baoulei, and Mastomys erythroleucus, is Lassa distributed under the terms and virus (LASV, Lassa mammarenavirus, Arenaviridae)[9–11], which is considered a Risk Group conditions of the Creative Commons 4 (RG-4) pathogen. LASV is the etiological agent of the hemorrhagic virus disease called Attribution (CC BY) license (https:// Lassa fever, which is considered a major public health concern in West African countries creativecommons.org/licenses/by/ 4.0/). due to annual outbreaks and high morbidity and mortality. Viruses 2021, 13, 187. https://doi.org/10.3390/v13020187 https://www.mdpi.com/journal/viruses Viruses 2021, 13, 187 2 of 12 Mastomys species, especially Mastomys coucha, have a long standing history as a model for schistosomiasis [12,13], the plague [14] and papilloma virus research [15]. However, basic blood parameters of M. natalensis from controlled rearing are not available. Addi- tionally, up to this point and only recently, one annotated genome has been published of a laboratory strain of M. coucha [16,17]. Therefore, biomolecular analysis testing of M. natalensis is still based on genetic sequences acquired from M. coucha, which due to the close relationship have at least a chance to similarly work on M. natalensis. To effectively study M. natalensis in laboratory settings, new tools and baselines parameters are currently being developed and established. Herein, we report the normal hematological and clinical chemistry values of healthy pathogen-tested animals of the animal species of M. natalensis from our laboratory-bred colony to further facilitate research on this currently understudied, yet important for human health, rodent species. 2. Materials and Methods 2.1. Animals The Mastomys natalensis colony originated from Mali and was brought to the USA in 2013 and has since bred successfully in captivity. Courtesy of Heinz Feldmann and David Safronetz, an off-shoot colony was established at the Bernhard Nocht-Institute for Tropical Medicine (BNITM) in Hamburg, Germany, in 2017, with another off-shoot colony being established at the Robert Koch-Institute (RKI) in Berlin, descending from the BNITM colony in 2018. The animals were bred at the Animal Husbandry Facility of the Robert Koch-Institute in (RKI) Berlin. Groups of 2–4 animals of the same sex were co-housed in filter-top cages (2000P Tecniplast) with small wood chip bedding and paper nesting material at 22 ◦C, 40–50% relative humidity on standard rodent chow and water ad libitum during a 12-h light cycle. The animals used for the study were between 8 and 53 weeks of age (specifically 8, 12, 17, 24, 27, 51, 52, and 56 weeks). The calculated reference ranges are deduced from not completely sex-matched groups (40.7% male against 59.3% females) before removal of statistical outliers. The specific age stratification can be seen in AppendixA Table A1. According to analyses on sentinel animals, the colony was free of most specific pathogens tested with the exception of Rodentibacter sp. (serological), Helicobacter spp. (PCR) and Chilomastix spp. (microscopic). The full list of tested organisms can be seen in AppendixA Table A2. 2.2. Blood Collection M. natalensis were anesthetized with an isoflurane overdose and exsanguinated via cardiac puncture using BD vacutainer system (K2 EDTA 2.0 mL, SST-II 2.5 mL; 21G or 22G needle adapter). The euthanasia conformed to German animal protection laws and was registered under animal euthanasia report number T0168-19 with the Landesamt für Gesundheit und Soziales in Berlin, Germany. 2.3. Blood Cell Counts Whole blood that was collected in tubes containing EDTA (BD Vacutainer K2EDTA) was agitated on a rotator at 6 rpm until measured with an automatic hematology analyzer (Abaxis HM5c). The hematology analyzer was maintained according to manufacturer instructions and correct performance was verified using commercial quality controls of nor- mal and high-range reference blood (Abaxis VetScan HM5 Hematology Control). The sam- ples were measured using the standard mouse settings, without modifications. In addition to automatic hematological analysis, thin-film blood smears stained with Diff-Quik stain- ing (Labor + Technik: LT-SYS®) were prepared from the identical samples, and used for microscopic visualization and manual counting verification. Micrographs were captured using analysis 5.0 (Olympus) software and panels were composed using paint.net v4.1 (dotPDN LLC). Viruses 2021, 13, 187 3 of 12 2.4. Clinical Serum Chemistry Blood that was collected in serum separation tubes (BD Vacutainer SST II) was left to clot for at least 30 min and subsequently centrifuged at 3000× g for 5 min. The sera were aliquoted and stored at −80 ◦C until measured on an automatic clinical chemistry analyzer (FUJI DRI-CHEM NX500i) with the corresponding colorimetric test assays (FUJI). The sam- ples were assessed for concentrations of creatinine (CRE), blood urea nitrogen (BUN), albumin (ALB), Glutamic pyruvate transaminase/Alanine transaminase (GPT/ALT), glu- tamic oxaloacetic transaminase/Aspartate transaminase (GOT/AST) and Alkaline phos- phatase (ALP). If required, due to volume constraints, the samples were diluted with sterile 0.9% NaCl solution. 2.5. Data Analysis and Statistics Data were analyzed using Graphpad Prism 8.4. Strongly hemolytic sera were removed from analysis prior to identification of statistical outliers. Statistical outliers for each group were identified using Robust Regression and Outlier removal (ROUT method) with a Q- coefficient of 1%. Measurements below the limit of detection in an assay were imputed by using l.po.d. as set value. One animal was removed from the analysis due to a skin condition 2 that appeared inflammatory. Normal reference ranges were established by calculating the 2.5% and 97.5% percentiles
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