Understanding the origin, distribution, and biology of different cell populations in chimeric mice is critical for interpreting the pathological changes developed in these models. To this aim, the methodological work presented here illustrates the validation and application of a collection of labeling techniques to differentiate between specific mouse and human tissue/cell components in formalin-fixed paraffin-embedded samples from chimeric mice, especially those bearing human tumor and immune cells. First, broad approaches to identify cells of human origin using ubiquitous immunohistochemical targets such as HLA-A, Ku80, and human mitochondrial 60 kDa protein (hMito) were established using specimens from humanized mice and a human tissue microarray including both normal and neoplastic samples. Due to its crisp membranous immunoreactivity, HLA-A was the most useful marker for visual human cell identification; however, Ku80 and hMito may be suitable options when HLA-A is not expressed in the cells of interest. Importantly, using one or more of these markers provides a broad range of coverage for the vast majority of human-derived cells in chimeric mice. Second, tailored immunohistochemical or in situ hybridization methodologies to distinguish specific human or mouse cell subsets are presented, focusing on immune/inflammatory cells and human chimeric antigen receptor (CAR) T-cells. These diverse approaches are accompanied by descriptions of case examples highlighting practical diagnostic and experimental applications in the context of various humanized mouse models. While not comprehensive, this work represents a valuable starting reference for pathologists and investigators working with humanized mouse models and seeking to add spatial resolution to the complex landscape of chimeric tissues.

Mouse kidney parvovirus (MKPV) causes inclusion body nephropathy, resulting in clinical signs and mortality in immunodeficient mice and subclinical infection in immunocompetent mice. While late-stage renal lesions and viral replication have been characterized, a comprehensive multisystemic investigation of MKPV infection from the initial to the late stages of infection has not been conducted. Our goal was to investigate lesions and viral replication in all major organs at multiple stages of MKPV infection in immunocompetent C57BL/6NCrl (B6) and Crl: CD1(ICR) (CD1) mice and immunodeficient NOD. Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. Following experimental oronasal inoculation with MKPV, mice were evaluated at 15 time points from 1.5 to 112 days post-inoculation (DPI) by histology and in situ hybridization for MKPV RNA on all major organs, as well as immunohistochemistry for markers of immune cells and renal tubular injury. In all strains, the gastrointestinal mucosa was the initial site of viral replication beginning at 3 DPI and persisting through the study without associated lesions. In B6 and CD1 mice, viral replication was first detected in renal tubules on 28 and 14 DPI, respectively, and lymphoplasmacytic tubulointerstitial nephritis was first evident on 63 and 49 DPI, respectively. B6 mice displayed the lowest levels of renal viral replication and lesion severity. In contrast, renal viral replication was highest in NSG mice; the virus was first detected on 42 DPI and in association with tubular degeneration from 63 DPI. Electron microscopy on kidney tissues of infected mice revealed parvoviral virions, nuclear replication, and assembly compartments for the first time.

In October 2020, adult male and female NSG (NOD. Cg-Prkdcscid Il2rgtm1Wjl/Sz) mice were reported for diarrhea within a mouse barrier facility. Other immunodeficient strains harboring the SCID (Prkdcscid) or Rag (Ragnull) mutations together with the IL2rg (Il2rgnull) mutation were affected. At its peak, over 20 laboratories in 10/16 (62.5%) barrier rooms were affected. Mortality was rare except in lactating females (≥ P11). Grossly, nonlactating adult female and male mice (n = 16) had mild to moderate, small and large intestinal distension with corresponding individual cell death and sloughing of superficial enterocytes in the cecocolonic mucosa. Lactating NSG dams (n=6) had moderate to severe gastrointestinal distension and/or segmental, dark red to gray, small intestinal discoloration. In addition to the same histologic lesions seen in nonlactating female NSG mice, lactating NSG dams often had severe ulcerative inflammation affecting the jejunum, ileum, cecum, and colon. Traditional ancillary diagnostic tests including aerobic and anaerobic cultures (blood, liver, spleen, and intestines), fecal PCR, and fecal floatation failed to yield a causative organism. Further cohousing and oral gavage studies determined neither immunocompetent CD1 (Crl:CD1 [ICR]) mice nor immunodeficient NOD scid (NOD.Cg-Prkdcscid/J) and Rag2 KO (C57BL/6. Cg-Rag2tm1.1Cgn/J) mice were susceptible to clinical disease. Extensive control barriers were implemented including a veterinary-managed NSG breeding barrier, alterations in husbandry practices, and strategic environmental disinfection, allowing for continuity of experimental studies while avoiding widespread depopulation of the barrier. Subsequent strain-resolved metagenomics and qPCR assay development identified Clostridium cuniculi and its enterotoxin exclusively within diarrheic mice.

Chronic aeromoniasis in turbot (Scophthalmus maximus), caused by Aeromonas salmonicida, provokes cutaneous lesions that occasionally expose dermal tubercles, which may act as unconventional ecological niches. A storm in February 2024 caused severe damage at a turbot farm in Galicia (Northwestern Spain), leaving various tanks uncovered and subsequently exposing fish with chronic aeromoniasis to several days of intense solar irradiation. Afterward, farm staff observed green tufts over the animals. Two affected turbots were submitted for necropsy. Morphological features identified the epibiont as the alga Ulva spp., covered by a biofilm containing A. salmonicida DNA (detected by real-time polymerase chain reaction). The algae did not trigger additional host responses beyond those induced by aeromoniasis, indicating true epibiosis rather than infection. However, algal colonization physically obstructs re-epithelialization and may prolong lesion chronicity, while the associated biofilm could serve as an environmental reservoir for the pathogen. This case illustrates how environmental disruptions can generate unexpected host-pathogen-epibiont interactions. To our knowledge, this is the first report of Ulva epibiosis on fish.

Brucellosis is an infectious disease that affects a wide range of animals, caused by bacteria belonging to 13 species and several biovars within the Brucella genus (B. melitensis, B. abortus, B. suis, B. ovis, B. neotomae, B. canis, B. ceti, B. pinnipedialis, B. microti, B. inopinata, B. papionis, B. vulpis, B. nosferati, and unclassified strains). Brucella spp. infects small ruminants, cattle, swine, dogs, wild mammals, reptiles, amphibians, and fish, and most Brucella spp. have zoonotic potential. Characterization of lesions in animal brucellosis remains paramount in the diagnosis in endemic regions and even for countries free of brucellosis, where passive surveillance plays an indispensable role in upholding and sustaining their status. Pathology also contributes to understanding the pathogenesis and associating emerging pathogenic Brucella spp. with disease in animals. Here, we review the pathology of brucellosis in animals with an emphasis on cross-species transmission of "classical" Brucella spp. to nonpreferential hosts and "atypical" Brucella spp. We conclude that Brucella is an expansive genus identified in numerous animals, but there are still significant gaps in the knowledge of the pathology of brucellosis in unusual hosts and emerging Brucella spp. This is a great opportunity for veterinary pathologists to spearhead advancements in the knowledge of the pathogenesis and diagnosis of Brucella spp. infections. The isolation of a Brucella spp. and the correlation with lesions should follow molecular diagnostics to define the genetic signature of the isolate, providing a better understanding of epidemiology and contributing to the control of brucellosis across animal and human populations.

Canine intestinal T-cell lymphoma (ITCL) may arise from intraepithelial lymphocyte (IEL) subsets expanded in chronic enteropathy (CE). To investigate this potential cell of origin, we performed flow cytometry, immunohistochemistry, and RNA in situ hybridization on IELs using biopsy samples from 62 dogs, including 6 large-cell lymphomas (LCLs), 9 small-cell lymphomas (SCLs), 31 CEs with increased IELs (IEL+CE), and 16 CEs without increased IELs (IEL-CE). IELs in ITCLs were predominantly CD4-CD8α- (LCL, 5/6; SCL, 4/9), and 4/31 IEL+CEs had a higher proportion of CD4-CD8α- IELs than the other CE cases. Forward scatter values of CD4-CD8α- IELs were higher in IEL+CEs compared with IEL-CEs, correlating positively with the Ki-67 index, indicating proliferation of this population. An increase in IELs lacking T-cell receptor expression was observed in 1/4 LCL, 2/5 SCL, and 1/13 IEL+CE cases. Lack of surface CD3 was observed in 2/9 SCL cases. One of 5 LCL cases partially expressed NKp46, whereas the proportion of NKp46+ IELs was low (< 4%) in 32/33 CE cases examined. In addition, 3/6 LCL cases expressed NKp46 mRNA without detectable protein expression. Clonality analysis of isolated IELs yielded at least 1 clonal peak in 1/2 LCLs, 4/7 SCLs, 12/23 IEL+CEs, and 4/13 IEL-CEs, underscoring the limited diagnostic utility on distinguishing ITCL, particularly SCL, from IEL+CE. These findings suggest that most canine ITCLs have a CD4-CD8α- immunophenotype and that unconventional IELs in IEL+CE may be the cells of origin. Moreover, the distinction between ITCL and CE remains challenging, emphasizing the importance of integrated diagnostic approaches.