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The murine bacterial pathogen Chlamydia muridarum (Cm) has been used to study human Chlamydia infections in various mouse models. CD4+ T-cells, natural killer cells, and interferon-gamma (IFN-γ)-mediated immunity are important to control experimentally induced Cm infections. Despite its experimental use, natural infection by Cm has not been documented in laboratory mice since the 1940s. In 2022, the authors reported the discovery of natural Cm infections in numerous academic institutional laboratory mouse colonies around the globe. To evaluate the impact of Cm infection in severely immunocompromised mice, 19 NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice were cohoused with Cm shedding, naturally infected immunocompetent mice and/or their soiled bedding for 4 weeks and subsequently euthanized. Clinical disease, characterized by lethargy, dyspnea, and weight loss, was observed in 11/19 NSG mice, and 16/18 NSG mice had neutrophilia. All mice exhibited multifocal to coalescing histiocytic and neutrophilic bronchointerstitial pneumonia (17/19) or bronchiolitis (2/19) with intraepithelial chlamydial inclusions (CIs). Immunofluorescence showed CIs were often associated with bronchiolar epithelium. CIs were frequently detected by immunohistochemistry in tracheal and bronchiolar epithelium (19/19), as well as throughout the small and large intestinal epithelium without lesions (19/19). In a subset of cases, Cm colonized the surface epithelium in the nasopharynx (16/19), nasal cavity (7/19), and middle ear canal (5/19). Endometritis and salpingitis with intraepithelial CI were identified in a single mouse. These findings demonstrate that Cm infection acquired through direct contact or soiled bedding causes significant pulmonary pathology and widespread intestinal colonization in NSG mice.
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Infecciones por Chlamydia , Chlamydia muridarum , Neumonía , Femenino , Animales , Ratones , Humanos , Ratones Endogámicos NOD , Ratones SCID , Infecciones por Chlamydia/veterinaria , Infecciones por Chlamydia/microbiología , Neumonía/veterinaria , Proteínas de Unión al ADN , Proteína Quinasa Activada por ADN , Subunidad gamma Común de Receptores de InterleucinaRESUMEN
Three isolates of a previously reported novel catalase-negative, Gram-stain-positive, coccoid, alpha-haemolytic, Streptococcus species that were associated with meningoencephalitis in naïve weanling mice were further evaluated to confirm their taxonomic status and to determine additional phenotypic and molecular characteristics. Comparative 16S rRNA gene sequence analysis showed nearly identical intra-species sequence similarity (≥99.9â%), and revealed the closest phylogenetically related species, Streptococcus acidominimus and Streptococcuscuniculi, with 97.0 and 97.5â% sequence similarity, respectively. The rpoB, sodA and recN genes were identical for the three isolates and were 87.6, 85.7 and 82.5â% similar to S. acidominimus and 89.7, 86.2 and 80.7â% similar to S. cuniculi, respectively. In silico DNA-DNA hybridization analyses of mouse isolate 12-5202T against S. acidominimus CCUG 27296T and S. cuniculi CCUG 65085T produced estimated values of 26.4 and 25.7â% relatedness, and the calculated average nucleotide identity values were 81.9 and 81.7, respectively. These data confirm the taxonomic status of 12-5202T as a distinct Streptococcus species, and we formally propose the type strain, Streptococcusazizii 12-5202T (=CCUG 69378T=DSM 103678T). The genome of Streptococcus azizii sp. nov. 12-5202T contains 2062 total genes with a size of 2.34 Mbp, and an average G+C content of 42.76 mol%.
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Meningoencefalitis/microbiología , Ratones/microbiología , Filogenia , Streptococcus/clasificación , Animales , Técnicas de Tipificación Bacteriana , Composición de Base , ADN Bacteriano/genética , Femenino , Genes Bacterianos , Masculino , Hibridación de Ácido Nucleico , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Streptococcus/genética , Streptococcus/aislamiento & purificaciónRESUMEN
Chlamydia muridarum (Cm) has reemerged as a prevalent bacterial contaminant of academic research mouse colonies. A study was conducted to assess the effectiveness of husbandry and cage sanitization methods in preventing intercage transmission of Cm. To assess intercage transmission during cage change, a cage housing 2 Cm-free Swiss Webster (Tac:SW; SW) sentinel mice was placed randomly on each of 12 individually ventilated cage racks, housing cages with Cm-shedding mice, located in 1 of 2 animal holding rooms. Husbandry staff blinded to the study cages, changed all cages in the animal holding rooms weekly using microisolator cage technique. PCR testing performed 180 days post-placement confirmed all mice remained negative for Cm. To assess the effectiveness of cage sanitization to eliminate Cm, we investigated transmission of Cm to a naïve Cm-free SW and NOD.Cg- Prkdc scid Il2rg tm1Wjl /SzJ (NSG) mouse co-housed for 7 days (repeated weekly for 4 weeks) in cages assigned to 1 of 3 groups (n=10 pairs of mice/group). Cages that previously housed 2 Cm-shedding BALB/c mice were either washed in a tunnel washer (82.2°C [180°F] final rinse for an average of 16 seconds per run; n=10) with and without post-washing autoclaving (121°C for 20 minutes; n=10), or were untreated (bedding change only; n=10). Pre- and post-sanitization swabs of each cage were assayed for Cm by PCR. All pre-treatment swabs tested positive, while post-treatment swabs from all cages (excluding bedding change) tested negative. All SW and NSG mice, irrespective of group, remained negative for Cm as determined by PCR. These findings suggest that infectious Cm does not persist in untreated cages nor after mechanical washing with and without autoclaving. Collectively, these findings suggest that neither our husbandry protocols nor inadequate cage sanitization methods likely contributed to the observed prevalence of Cm in contemporary research mouse colonies.
RESUMEN
Chlamydia muridarum (Cm) has reemerged as a prevalent bacterial contaminant of academic research mouse colonies. A study was conducted to assess the effectiveness of husbandry and cage sanitization methods in preventing intercage transmission of Cm. To assess intercage transmission during cage change, a cage housing 2 Cm-free Swiss Webster (SW; Tac:SW) sentinel mice was placed randomly on each of 12 individually ventilated cage racks, housing cages with Cm-shedding mice, located in one of 2 animal holding rooms. Husbandry staff blinded to the study cages changed all cages in the animal holding rooms weekly using a microisolation cage technique. PCR testing performed at 180 d postplacement confirmed all mice remained negative for Cm. To assess the effectiveness of cage sanitization to eliminate Cm, we investigated transmission of Cm to a naive Cm-free SW and NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mouse cohoused for 7 d (repeated weekly for 4 wk) in cages assigned to one of 3 groups (n = 10 pairs of mice/group). Cages that previously housed 2 Cm-shedding BALB/c mice were either washed in a tunnel washer (82.2 °C [180 °F] final rinse for an average of 16 s per run; n = 10) with and without postwashing autoclaving (121 °C for 20 min; n = 10), or were untreated (bedding change only; n = 10). Pre- and postsanitization swabs of each cage were assayed for Cm by PCR. All pretreatment swabs tested positive, while posttreatment swabs from all cages (excluding bedding change) tested negative. All SW and NSG mice, irrespective of group, remained negative for Cm as determined by PCR. These findings suggest that infectious Cm does not persist in untreated cages or after mechanical washing with and without autoclaving. Collectively, these findings suggest that neither our husbandry protocols nor inadequate cage sanitization methods likely contributed to the observed prevalence of Cm in contemporary research mouse colonies.
RESUMEN
Chlamydia muridarum (Cm), an intracellular bacterium of historical importance, was recently rediscovered as moderately prevalent in research mouse colonies. Cm was first reported as a causative agent of severe pneumonia in mice about 80 y ago, and while it has been used experimentally to model Chlamydia trachomatis infection of humans, there have been no further reports of clinical disease associated with natural infection. We observed clinical disease and pathology in 2 genetically engi- neered mouse (GEM) strains, Il12rb2 KO and STAT1 KO, with impaired interferon-γ signaling and Th1 CD4+ T cell responses in a colony of various GEM strains known to be colonized with and shedding Cm. Clinical signs included poor condition, hunched posture, and poor fecundity. Histopathology revealed disseminated Cm with lesions in pulmonary, gastrointestinal, and urogenital tissues. The presence of Cm was confirmed using both immunohistochemistry for Cm major outer membrane protein-1 antigen and in situ hybridization using a target probe directed against select regions of Cm strain Nigg. Cm was also found in association with a urothelial papilloma in one mouse. These cases provide additional support for excluding Cm from research mouse colonies.
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Infecciones por Chlamydia , Chlamydia muridarum , Ratones Noqueados , Factor de Transcripción STAT1 , Animales , Infecciones por Chlamydia/patología , Infecciones por Chlamydia/veterinaria , Infecciones por Chlamydia/microbiología , Ratones , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT1/metabolismo , Femenino , Receptores de Interleucina-12/deficiencia , Receptores de Interleucina-12/genética , Masculino , Enfermedades Pulmonares/microbiología , Enfermedades Pulmonares/patología , Enfermedades Pulmonares/veterinariaRESUMEN
Chlamydia muridarum (Cm) has reemerged as a moderately prevalent infectious agent in research mouse colonies. Despite its' experimental use, there are limited studies evaluating Cm's effects on immunocompetent mice following its natural route of infection. A Cm field isolate was administered (orogastric gavage) to 8-week-old female BALB/cJ (C) mice. After confirming shedding (through 95d), these mice were cohoused with naïve C57BL/6J (B6), C, and Swiss (J:ARC[S]) mice (n=28/strain) for 30 days. Cohoused mice (n=3-6 exposed and 1-6 control/strain) were evaluated 7, 14, 21, 63, 120, and 180 days post-cohousing (DPC) via hemograms, serum biochemistry analysis, fecal qPCR, histopathology, and Cm MOMP immunohistochemistry. Immunophenotyping was performed on spleen (B6, C, S; n=6/strain) and intestines (B6; n=6) at 14 and 63 DPC. Serum cytokine concentrations were measured (B6; n=6 exposed and 2 control) at 14 and 63 DPC. All B6 mice were shedding Cm by 3 through 180 DPI. One of 3 C and 1 of 6 S mice began shedding Cm at 3 and 14 DPC, respectively, with the remaining shedding thereafter. Clinical pathology was nonremarkable. Minimal-to-moderate enterotyphlocolitis and gastrointestinal associated lymphoid tissue (GALT) hyperplasia was found in numerous infected mice. Cm antigen was frequently detected in GALT-associated surface intestinal epithelial cells. Splenic immunophenotyping revealed increased monocytes and shifts in T cell population subsets in all strains/timepoints. Gastrointestinal immunophenotyping (B6) revealed sustained increases in total inflammatory cells and elevated cytokine production in innate lymphoid cells and effector T cells (large intestine). Elevated concentrations of pro-inflammatory cytokines were detected in the serum (B6). These results demonstrate that while clinical disease was not appreciated, 3 commonly utilized strains of mice are susceptible to chronic enteric infections with Cm which may alter various immune responses. Considering the widespread use of mice to model GI disease, institutions should consider excluding Cm from their colonies.
RESUMEN
Chlamydia muridarum (Cm) is a moderately prevalent, gram-negative, intracellular bacterium that affects laboratory mice, causing subclinical to severe disease, depending on the host's immune status. The effectiveness of various antibiotic regimens aimed at eradicating Cm in both immunodeficient and immunocompetent laboratory mice was evaluated. NSG mice were cohoused with Cm-shedding BALB/cJ mice for 14 days to simulate natural exposure. Four groups of 8 infected NSG mice were treated for 7 days with either 0.08% sulfamethoxazole and 0.016% trimethoprim (TMS) in water, 0.0625% doxycycline in feed, 0.124%/0.025% TMS in feed, or 0.12% amoxicillin in feed. A control group was provided standard water and feed. The impact of treatment on gastrointestinal microbiota (GM) was performed using next-generation shotgun sequencing on the last day of treatment. TMS and Amoxicillin had negligible effects on GM, while doxycycline had the largest effect. All antibiotic treated NSG mice exhibited clinical disease, including dehydration, hunched posture, >20% weight loss, and dyspnea, leading to euthanasia 21-40 days post-treatment (32.6 ± 4.2 days; mean ± SD). Untreated controls were euthanized 14-33 days post-exposure (23.75 ± 5.9 days). All mice were fecal PCR positive for Cm at euthanasia. Histological evaluation revealed multifocal histiocytic and neutrophilic bronchointerstitial pneumonia and/or bronchiolitis featuring prominent intralesional chlamydial inclusion bodies in all mice. Subsequently, groups of 8 C57BL/6J, BALB/cJ, NOD.SCID, and NSG mice infected with Cm were treated with 0.124%/0.025% TMS in feed for 7 (BALB/cJ and C57BL/6J) or 21 days (NSG and NOD.SCID). All immunocompetent and NOD.SCID mice were negative for Cm by PCR 14 days post-treatment, remained clinically normal and had no evidence of Cm infection at necropsy, all NSG mice remained Cm positive and were euthanized. While these findings highlight the difficulties in eradicating Cm from highly immunodeficient mice, eradication of Cm from immunocompetent or moderately immunocompromised mice with antibiotics is feasible.
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A breeding pair of genetically engineered laboratory mice (Mus musculus) presented in apparent copulatory lock (coital tie). After anesthetizing the animals, gentle traction was used to separate the pair at which point a vaginal prolapse was detected and the penis was covered with black, firm, dry crusts and noted to have a solid pale, tan, firm cylindrical mass adhering to its glans. The vaginal prolapse was reduced and the female was returned to its cage. The male mouse had a severely distended bladder which could not be expressed and was euthanized. Histopathologic examination of the distal two-thirds of the penis revealed diffuse, acute coagulative necrosis. The mass adhered to the distal penis was a homogenous granular eosinophilic material consistent with a copulatory plug. While copulatory plugs and locks have been described in some rodent species, they have not been reported in laboratory mice. While the cause of the adherence of the plug to the penis could not be determined, we hypothesize that its adherence to both the penis and the vagina led to the lock and subsequently to ischemic necrosis of the distal penis.
Asunto(s)
Conducta Sexual Animal , Prolapso Uterino , Femenino , Masculino , Ratones , Animales , Humanos , Copulación , Pene , NecrosisRESUMEN
Mouse kidney parvovirus (MKPV) causes inclusion body nephropathy in severely immunocompromised mice and renal interstitial inflammation in immunocompetent mice. Here we sought to determine the effects of MKPV on pre-clinical murine models that depend on renal function. To assess the effects of MKPV infection on the pharmacokinetics of 2 renally excreted chemotherapeutic agents, methotrexate and lenalidomide, we measured drug concentrations in the blood and urine of MKPV-infected or uninfected immunodeficient NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) and immunocompetent C57BL/6NCrl (B6) female mice. No differences in plasma pharmacokinetics were observed for lenalidomide. However, the AUC of methotrexate was 1.5-fold higher in uninfected NSG mice compared with infected NSG mice, 1.9-fold higher in infected B6 mice compared with uninfected B6 mice, and 4.3-fold higher in uninfected NSG mice compared with uninfected B6 mice. MKPV infection did not significantly affect the renal clearance of either drug. To assess effects of MKPV infection on the adenine diet model of chronic kidney disease, MKPV-infected and uninfected B6 female mice were fed a 0.2% adenine diet, and clinical and histopathologic features of disease were assessed over 8 wk. MKPV infection did not significantly alter urine chemistry results, hemogram findings, or serum concentrations of BUN, creatinine, or symmetric dimethylarginine. However, infection did influence histologic outcomes. As compared with uninfected mice, MKPV-infected mice had more interstitial lymphoplasmacytic infiltrates after 4 and 8 wk of diet consumption and less interstitial fibrosis at week 8. Macrophage infiltrates and renal tubular injury were similar between in infected and uninfected mice. These findings indicate that MKPV infection had minimal effects on the renal excretion of 2 chemotherapeutics and on serum biomarkers of renal function. However, infection significantly influenced two histologic features of the adenine diet model of chronic renal disease. MKPV-free mice are critically important in studies evaluating renal histology as an experimental outcome.
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Mice are commonly infected with Nippostrongylus brasiliensis (Nb) to study their immune responses. However, biosecurity measures have not been established for housing Nb-infected mice and rats. Transmission reportedly does not occur when infected mice are cohoused with naive mice. To test this, we inoculated female NOD. Cg-Prkdcscid Il2rgtm1Wjl /Sz(NSG;n = 12) and C57BL/6J (B6;n = 12) mice with 750 Nb L3 larvae. These mice were then cohoused with naïve NSG ( n = 24) and B6 ( n = 24) mice (1 infected and 2 naïve mice per cage (24 cages) for 28 d in static microisolation cages that were changed every 14 d. We also did several studies to determine the conditions that favor horizontal transmission. First, we assessed in vitro development to the L3 stage of Nb egg-containing fecal pellets maintained under 4 environmental conditions (dry, moist, soiled bedding, and control). Second, we assessed infection of naïve NSG mice ( n = 9) housed in microisolation cages that contained soiled bedding spiked with infective L3 larvae (10,000/cage). Third, we gavaged NSG mice ( n = 3) with Nb eggs to model the potential for infection after coprophagy. We found that naïve NSG (9 of 24) and B6 (10 of 24) mice cohoused with an infected cagemate passed Nb eggs in feces as early as 1 d after cohousing and intermittently thereafter for varying periods. This shedding was presumably the result of coprophagy because adult worms were not detected in the shedding mice at euthanasia. Although eggs developed in vitro into L3 larvae under moist and control environmental conditions, none of the NSG mice housed in cages with L3 -spiked bedding or gavaged with eggs became infected with Nb. These findings indicate that infectious horizontal transmission does not occur when mice are housed with Nb-shedding cage mates in static microisolation cages with a 14-d cage-changing interval. Results from this study can be used to inform biosecurity practices when working with Nb-infected mice.
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Bioaseguramiento , Nippostrongylus , Ratones , Animales , Ratas , Femenino , Ratones Endogámicos NOD , Ratones Endogámicos C57BL , Vivienda para Animales , Ratones SCIDRESUMEN
Corynebacterium bovis (Cb), the cause of hyperkeratotic dermatitis in various immunocompromised mouse strains, significantly impacts research outcomes if infected mice are used. Although Cb has been isolated from a variety of species, including mice, rats, cows, and humans, little is known about the differences in the infectivity and clinical disease that are associated with specific Cb isolates. The infectious dose that colonized 50% of the exposed population (ID50 ) and any associated clinical disease was determined in athymic nude mice (Hsd:Athymic Nude-Foxn1 nu ) inoculated with Cb isolates collected from mice (n = 5), rat (n = 1), cow (n = 1), and humans (n = 2) The same parameters were also determined for 2 of the mouse isolates in 2 furred immunocompromised mouse strains (NSG [NOD. Cg-Prkdcscid Il2rgtm1Wjl /Sz] and NSG-S [NOD. Cg-Prkdcscid Il2rgtm1Wjl Tg(CMV-IL3, CSF2, KITLG)1Eav/MloySzJ]). To determine the ID 50, mice (n= 6/dose; 3 of each sex) were inoculated topically in 10-fold increments ranging from 1 to 10 8 bacteria. Mice were scored daily for 14 days for the severity of clinical signs. On days 7 and 14 after inoculation, buccal and dorsal skin swabs were evaluated by aerobic culture to determine infection status. The mouse isolates yielded lower ID50values (58 to 1000 bacteria) than did the bovine (6460 to 7498 bacteria) and rat (10,000 bacteria) isolates. Human isolates did not colonize mice or cause disease. Mouse isolates produced clinical disease of vary- ing severity in nude mice. Despite significant immunodeficiency, furred NSG and NSG-S mice required a 1000- to 3000-fold higher inoculum for colonization than did athymic nude mice. Once colonized, clinically detectable hyperkeratosis did not develop in the haired strains until 18 to 22 d after inoculation, whereas athymic nude mice that developed clinically detect- able disease showed hyperkeratosis between 6 and 14 d after inoculation. In conclusion, there are significant differences in Cb's ID 50, disease course, and severity of clinical signs between Cb isolates and among immunodeficient mouse strains.
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Infecciones por Corynebacterium , Corynebacterium , Femenino , Ratones , Ratas , Humanos , Animales , Bovinos , Ratones Desnudos , Ratones Endogámicos NOD , Infecciones por Corynebacterium/veterinaria , Infecciones por Corynebacterium/microbiología , Ratones SCIDRESUMEN
Infectious agents have varying susceptibilities to thermal inactivation and/or mechanical removal from cages by the use of heated, pressurized water. In this study, we tested whether 5 specific infectious organisms (Candidatus savagella [segmented filamentous bacterium (SFB)], Helicobacter sp., mouse norovirus (MNV), Tritrichomonas sp., and Entamoeba muris) could survive the cage wash process and still infect naïve mice. These 5 organisms were chosen due to their prevalence in rodent colonies, environmental stability, and/or potential to influence experimental outcomes. Cages that had housed mice shedding all 5 organisms were assigned to 1 of 3 treatment groups: 1) sanitization in a tunnel washer followed by autoclaving (121 °C [250 °F] for 20 min; n = 40 cages); 2) sanitization in a tunnel washer (82 °C [180 °F] for an average of 30 s; n = 40 cages); or 3) control (bedding change only; n = 40 cages). The presence of these agents in the cage was assessed by performing PCR on swabs of the empty soiled cage interior before and after the treatment. In addition, to determine if any residual nucleic acid was infectious, 2 Swiss outbred (J:ARC(S)) female mice were housed for 7 d in cages from each treatment group. The above procedures were then repeated so that every week each pair of J:ARC(S) mice ( n = 10 pairs of mice/treatment group) were housed in another cage that underwent the same treatment; this was done for a total of 4 consecutive, 1-wk-long periods. Swabs collected from soiled cages were PCR-positive for SFB, Helicobacter, MNV, Tritrichomonas, and Entamoeba in 99%, 97%, 39%, 63%, and 73% of the cages tested, respectively. Cages in the tunnel wash group that were PCR-positive for SFB, Helicobacter, Tritrichomonas, and Entamoeba before treatment remained PCR-positive in 8%, 15%, 43%, and 10% of positive cages, respectively. None of the cages from the autoclave group were PCR-positive for any of the agents after treatment. None of the mice housed in cages in either the autoclave or tunnel wash groups became infected with any of the agents. However, 80%, 60%, and 100% of the pairs of mice housed in untreated cages were PCR-positive for SFB, MNV, and Entamoeba, respectively. None of the mice housed in untreated cages were positive for Helicobacter or Tritrichomonas. Our results suggest that nucleic acids from these bacterial and protozoal organisms may remain in cages after mechanical cage washing, but these nucleic acids are not infectious, and autoclaving is not necessary to prevent transmission.
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Vivienda para Animales , Norovirus , Animales , Femenino , Ratones , Reacción en Cadena de la Polimerasa/veterinaria , BacteriasRESUMEN
Rotifers, Brachionus plicatilis, are a valuable first exogenous feed for zebrafish because they can provide continuous nutrition for growing zebrafish larvae when used in a rotifer-zebrafish polyculture. Typically cultured at high salinities (>10 ppt), B. plicatilis are temporarily immobilized when moved to lower salinities (5 ppt) used for polycultures, decreasing their accessibility and attractiveness to the larvae. The nutritional value of rotifers varies based on their diet, typically live algae, which has limited nutritional value and may pose biosecurity risks. After confirming that rotifers consume and can reproduce when fed an irradiated, processed larval fish diet (PD), they were reared at 5 or 15 ppt, and fed various combinations of an algae mix and/or PD. Population densities and percentages of egg-bearing rotifers were quantified daily until the population density plateaued, and then their nutritional value was assessed. Results indicated that rotifers thrived at both salinities. Those fed PD were successfully maintained at >500 rotifers per mL and contained a greater ω-6/ω-3 fatty acid ratio. Our findings indicate that enriching rotifers with PD raised at 5 ppt can potentially eliminate rotifer immobilization in polyculture, while providing a nutritious, attractive diet for zebrafish larvae and decreasing biosecurity risks.
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Perciformes , Rotíferos , Animales , Pez Cebra , Salinidad , Suplementos Dietéticos , LarvaRESUMEN
Embryo surface disinfection in either an iodine or sodium hypochlorite (NaOCl) solution is commonly performed on imported zebrafish embryos to decrease pathogen introduction into a facility. The impact of the consecutive use of these disinfectants and the conductivity of the culture media on embryo survival and development post-disinfection have not been evaluated. Iodine (12.5-25 ppm) is effective at eliminating several Mycobacterium species, whereas NaOCl (50-100 ppm) reduces the number of viable Pseudoloma neurophilia spores. Casper and T5D (tropical 5D wild type) embryos reared in media of differing conductivities (0-10, 100-200, 750-950, and 1500-2000 µS) with and without exposure to NaOCl 100 ppm at 6 h post-fertilization were evaluated for survival, hatching success, and morphological defects at 5 days post-fertilization. Additionally, the consecutive use of iodine (12.5 ppm for 2 min) followed by NaOCl (75 or 100 ppm for 10 min), as well as the inverse, was evaluated. Embryo survival was not impacted by embryo rearing media alone; however, survival significantly decreased when embryos were disinfected with 100 ppm NaOCl in media with a conductivity >750-950 µS. Iodine (12 ppm) and NaOCl (75 ppm) used sequentially resulted in >50% survival, whereas the use of 100 ppm NaOCl resulted in high levels of embryo mortality.
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Desinfectantes , Yodo , Animales , Hipoclorito de Sodio/farmacología , Desinfección/métodos , Pez Cebra , Yodo/farmacología , Desinfectantes/farmacología , Medios de CultivoRESUMEN
Mouse kidney parvovirus (MKPV), the etiology of murine inclusion body nephropathy, has been identified globally in mice used for research, with an estimated prevalence of 10% in academic colonies. In immunodeficient strains, MKPV causes significant morbidity and mortality, and severe renal pathology. In contrast, in immunocompetent mice, the infection is subclinical and causes minimal pathology. We investigated viral infectivity and shedding in inbred C57BL/6NCrl (B6), outbred Crl:CD1(ICR) (CD1), and highly immunocompromised NOD. Cg - Prkdc scid Il2rg tm1Wjl/SzJ (NSG) mice. Four doses, ranging from 1.16 × 10 3 to 1.16 × 10 6 viral copies per microliter, of an MKPV inoculum were administered oronasally to 3 mice per dose per mouse type. All 3 types (B6, CD1, and NSG) had persistent infection with prolonged shedding in urine and feces. Viral copy number in the urine generally increased over time, while shedding in the feces was more variable. Among the 3 populations, CD1 mice developed viral shedding in urine earliest (4 wk after inoculation) and at higher levels (greater than 1 × 10 7 viral copies per microliter). B6 mice become viruric later (7 wk after inoculation), with lesser virus shed (1 × 10 6 viral copies per microliter or less). In CD1 and B6 mice, peak urine shedding occurred at 11 to 14 wk after inoculation, after which levels gradually declined until 35 wk after inoculation (study endpoint). In contrast, NSG mice did not become viruric until 10 wk after inoculation and continued to shed large amounts of virus (greater than 1 × 107 viral copies per microliter) in urine until the study endpoint. Two commercial immunofluorescent serologic assays failed to detect serum antibodies to MKPV nonstructural protein 1 as late as 58 wk after inoculation, whereas immunohistochemistry of infected renal tissue successfully detected anti-MKPV serum antibodies. These results increase our knowledge of the biology of MKPV and have practical application for development of effective screening programs for this pathogen.
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Infecciones por Parvoviridae , Parvovirus , Esparcimiento de Virus , Animales , Ratones , Riñón , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Ratones Endogámicos NOD , Ratones SCID , Infecciones por Parvoviridae/veterinaria , Parvovirus/patogenicidadRESUMEN
Mouse kidney parvovirus (MKPV), a newly identified parvovirus of the genus Chaphamaparvovirus, causes inclusion body nephropathy in severely immunocompromised mice and is prevalent in research mouse colonies. As nonenveloped viruses, mammalian parvoviruses are stable and generally resist thermal inactivation; however, as a novel and highly divergent parvovirus, the thermal stability of MKPV is undefined. This study aimed to evaluate the ability of cage sanitization in a mechanical washer to eliminate MKPV. Cages contaminated by MKPV-infected mice were assigned to 1 of 3 treatment groups: 1) control (bedding change only); 2) sanitization in a tunnel washer (88°C final rinse for 20 s); or 3) sanitization in a tunnel washer followed by autoclave sterilization (121 °C for 20 min). The presence of MKPV on the cage's interior surface was assessed by PCR of cage swab extracts collected before and after cage treatment. After treatment and swabbing, each cage housed 4 MKPV-negative CD1 mice. Each group of naive CD1 mice was assigned to one of the treatment groups and was housed in a cage from this group for two, 1 wk periods. At 12, 17, and 20 wk after the first exposure, renal tissue was collected from 1 test mouse per cage and assessed for MKPV by PCR. MKPV was detected by PCR on the surface of 63% of the pretreatment cages. All cages sanitized in a tunnel washer with or without sterilization were PCR negative after treatment. Seven of 10 mice housed in untreated cages contained a mouse positive for MKPV by 20 wk after exposure. None of the mice housed in cages sanitized in a tunnel washer with or without sterilization tested positive for MKPV at any time point. This study indicates that MKPV contaminated caging can result in MKPV infection of mice, and the use of a tunnel washer at the temperature and duration evaluated was sufficient to remove MKPV nucleic acid and prevent MKPV transmission.
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Infecciones por Parvoviridae , Parvovirus , Animales , Vivienda para Animales , Riñón , Ratones , Infecciones por Parvoviridae/prevención & control , Infecciones por Parvoviridae/veterinaria , EsterilizaciónRESUMEN
Chlamydia muridarum (Cm) was detected in 2 colonies of mice with lymphoplasmacytic pulmonary infiltrates by using PCR and immunohistochemistry. This discovery was unexpected, as Cm infection had not been reported in laboratory mice since the 1940s. A Cm specific PCR assay was developed and testing implemented for the resident colonies of 8 vivaria from 3 academic institutions, 58 incoming mouse shipments from 39 academic institutions, and mice received from 55 commercial breeding colonies (4 vendors). To estimate Cm's global prevalence in research colonies, a database containing 11,387 metagenomic fecal microbiota samples from 120 institutions and a cohort of 900 diagnostic samples from 96 institutions were examined. Results indicate significant prevalence among academic institutions, with Cm detected in 63% of soiled bedding sentinels from 3 institutions; 33% of incoming mouse shipments from 39 academic institutions; 14% of 120 institutions submitting microbiota samples; and 16% of the diagnostic sample cohort. All samples from commercial breeding colonies were negative. In addition, naïve NOD. Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice exposed to Cm-shedding mice and/or their soiled bedding developed clinical disease at 21 to 28 d after exposure. These mice had a moderate-to-severe histiocytic and neutro- philic bronchointerstitial pneumonia, with their respiratory epithelium demonstrating inclusions, chlamydial major outer membrane protein immunostaining, and hybridization with a Cm reference sequence (GenBank accession no. U68436). Cm was isolated from lungs, cecum, and feces of a Cm-infected NSG mouse by using HeLa 229 cells. The considerable prevalence of Cm is likely due to widespread global interinstitutional distribution of unique mouse strains and failure to recognize that some of these mice were from enzootically infected colonies. Given that experimental Cm colonization of mice results in a robust immune response and, on occasion, pathology, natural infection may confound experimental results. Therefore, Cm should be excluded and eradicated from enzootically infected mouse colonies.
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Chlamydia muridarum , Animales , Heces , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Reacción en Cadena de la PolimerasaRESUMEN
Murine astrovirus 2 (MuAstV2) is a novel murine astrovirus recently identified in laboratory and wild mice. MuAstV2 readily transmits between immunocompetent mice yet fails to transmit to highly immunocompromised mouse strains-a unique characteristic when contrasted with other murine viruses including other astroviruses. We characterized the viral shedding kinetics and tissue tropism of MuAstV2 in immunocompetent C57BL/6NCrl mice and evaluated the apparent resistance of highly immunocompromised NOD- Prkdcem26Cd52Il2rgem26Cd22 /NjuCrl mice to MuAstV2 after oral inoculation. Temporal patterns of viral shedding were determined by serially measuring fecal viral RNA. Tissue tropism and viral load were characterized and quantified by using in-situ hybridization (ISH) targeting viral RNA. Cellular tropism was characterized by evaluating fluorescent colocalization of viral ISH with various immunohistochemical markers. We found a rapid increase of fecal viral RNA in B6 mice, which peaked at 5 d after inoculation (dpi) followed by cessation of shedding by 168 dpi. The small intestine had the highest percentage of hybridization (3.09% of tissue area) of all tissues in which hybridization occurred at 5 dpi. The thymus displayed the next highest degree of hybridization (2.3%) at 7 dpi, indicating extraintestinal viral spread. MuAstV2 RNA hybridization was found to colocalize with only 3 of the markers evaluated: CD3 (T cells), Iba1 (macrophages), and cytokeratin (enterocytes). A higher percentage of CD3 cells and Iba1 cells hybridized with MuAstV2 as compared with cytokeratin at 2 dpi (CD3, 59%; Iba1, 46%; cytokeratin, 6%) and 35 dpi (CD3, 14%; Iba1, 55%; cytokeratin, 3%). Neither fecal viral RNA nor viral hybridization was noted in NCG mice at the time points examined. In addition, mice of mixed genetic background were inoculated, and only those with a functioning Il2rg gene shed MuAstV2. Results from this study suggest that infection of, or interaction with, the immune system is required for infection by or replication of MuAstV2.
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Astroviridae , Animales , Biología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , TropismoRESUMEN
One limitation of HSV1-tk reporter positron emission tomography (PET) with nucleoside analogues is the high background radioactivity in the intestine. We hypothesized that endogenous expression of thymidine kinase in bacterial flora could phosphorylate and trap such radiotracers, contributing to the high radioactivity levels in the bowel, and therefore explored different strategies to increase fecal elimination of radiotracer. Intestinal radioactivity was assessed by in vivo microPET imaging and ex vivo tissue sampling following intravenous injection of 18F-FEAU, 124I-FIAU, or 18F-FHBG in a germ-free mouse strain. We also explored the use of an osmotic laxative agent and/or a 100% enzymatically hydrolyzed liquid diet. No significant differences in intestinal radioactivity were observed between germ-free and normal mice. 18F-FHBG-derived intestinal radioactivity levels were higher than those of 18F-FEAU and 124I-FIAU; the intestine to blood ratio was more than 20-fold higher for 18F-FHBG than for 18F-FEAU and 124I-FIAU. The combination of Peptamen and Nulytely lowered intestinal radioactivity levels and increased (2.2-fold) the HSV1-tk transduced xenograft to intestine ratio for 18F-FEAU. Intestinal bacteria in germ-free mice do not contribute to the high intestinal levels of radioactivity following injection of radionucleoside analogues. The combination of Peptamen and Nulytely increased radiotracer elimination by increasing bowel motility without inducing dehydration.
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Herpesvirus Humano 1/enzimología , Intestinos/efectos de la radiación , Laxativos/farmacología , Tomografía de Emisión de Positrones/métodos , Protección Radiológica/métodos , Radiofármacos/farmacocinética , Timidina Quinasa/biosíntesis , Análisis de Varianza , Animales , Arabinofuranosil Uracilo/análogos & derivados , Arabinofuranosil Uracilo/farmacocinética , Electrólitos/farmacocinética , Motilidad Gastrointestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Ratones , Oligopéptidos/farmacocinética , Polietilenglicoles/farmacocinética , Ratas , Timidina Quinasa/análisis , Imagen de Cuerpo EnteroRESUMEN
Embryo surface disinfection is utilized in aquaculture to decrease the risk of pathogen introduction into established colonies. Zebrafish embryos are commonly disinfected with unbuffered sodium hypochlorite at 25-50 ppm for 10 min with or without concurrent treatment with chemicals, including pronase (Pron), sodium thiosulfate, and/or methylene blue; however, the impact of these chemicals on embryo survival and development has not been evaluated. In this study, AB and casper embryos were exposed to disinfection protocols that used Pron, sodium thiosulfate, and/or methylene blue (given alone, in various combinations, or all three combined) with 50 and 100 ppm sodium hypochlorite performed 6 and 24 h postfertilization (HPF). All groups were evaluated for survival, hatching, and malformations at 5 days postfertilization. Maximal survival (69%-97%) and hatching rates (66%-94%) were generally observed with sodium hypochlorite disinfection followed by exposure to both Pron and sodium thiosulfate and maintenance in standard embryo medium without methylene blue. Methylene blue had variable effects on survival and hatching. Higher survival and hatching rates were seen in AB embryos disinfected at 6 HPF and casper embryos disinfected at 24 HPF. Susceptibility to sodium hypochlorite toxicity differed by strain, emphasizing the need to test disinfection protocols on small embryo cohorts.