Heavy metal and metalloid concentrations in red deer (Cervus elaphus) and their human health implications from One Health perspective.

The red deer is an ungulate and large game species. The contamination of the ecosystems by metal(loid)s may lead to the exposure of animals (as well as humans) through water and food resources. The direct contact of hunters and wild animal meat consumers with deer carcasses may be a potential contaminant source. This study aimed to determine the metal(loid)s' concentrations in the liver and kidney of red deer from two regions of Portugal (Idanha-a-Nova and Lousã), and to relate these with histopathologic lesions. Thirteen young male deer were submitted to metal(loid) determination (As, Cd, Co, Cr, Cu, Pb, and Zn) by inductively coupled plasma mass spectrophotometry (ICP-MS) and histopathology examination. Renal Cd (8.072 ± 5.766 mg/kg dw) and hepatic Pb (3.824 ± 6.098 mg/kg dw) mean values were high, considering the maximum values for consumption established by the European Commission. The hepatic mean value of Cu was significantly higher in Idanha-a-Nova (150.059 ± 33.321 mg/kg dw), and it is at the Cu toxicity limit considered for ruminants (150 mg/kg). The pollution induced by Panasqueira mines (Castelo Branco) may be a possible explanation for some of the findings, especially the higher values of hepatic Cu and Pb found in Idanha-a-Nova deer. These results have high importance under a One Health perspective, since they have implications in public health, and pose at risk the imbalance of animal populations and ecosystems.

The first full study of heavy metal(loid)s in western-European hedgehogs (Erinaceus europaeus) from Portugal.

The western-European hedgehog (Erinaceus europaeus) is an insectivore with a wide distribution in Portugal and a potential tool for biomonitoring relevant One Health hazards, including heavy metal(loid)s' pollution. The aim of this study was to positively contribute to the current knowledge about the metal(loid) pollution in Portugal. Forty-six hedgehogs (from rescue centres; with known provenance) were necropsied. Sex, age category and weight were determined. Spines, liver and kidney were collected, and metalloid concentrations were determined by inductively coupled plasma mass spectrophotometry (ICP-MS). In general, results did not present alarming metal(loid) concentrations, with the exception of cadmium (Cd) (in the kidneys) and copper (Cu). Hedgehogs from Viana do Castelo and Viseu showed elevated concentrations of arsenic (As) and Castelo Branco presented concerning values of cadmium (Cd). Adult and heavier hedgehogs tended to present higher levels of metal(loid)s. Sex does not seem to significantly affect the metal(loid)s' concentrations. Further analysis would be needed to prioritize areas with detail and allow the application of the necessary mitigation strategies.

Virus as Teratogenic Agents.

Viral infectious diseases are important causes of reproductive disorders, as abortion, fetal mummification, embryonic mortality, stillbirth, and congenital abnormalities in animals and in humans. In this chapter, we provide an overview of some virus, as important agents in teratology.We begin by describing the Zika virus, whose infection in humans had a very significant impact in recent years and has been associated with major health problems worldwide. This virus is a teratogenic agent in humans and has been classified as a public health emergency of international concern (PHEIC).Then, some viruses associated with reproductive abnormalities on animals, which have a significant economic impact on livestock, are described, as bovine herpesvirus, bovine viral diarrhea virus, Schmallenberg virus, Akabane virus, and Aino virus.For all viruses mentioned in this chapter, the teratogenic effects and the congenital malformations associated with fetus and newborn are described, according to the most recent scientific publications.

Updating an Overview of Teratology.

In this chapter, the authors aim to update an overview of the principles of teratology, beginning with the definition of teratology, the critical point at which this process occurs, and some of the most common etiological agents that improve our understanding of teratology.Modern teratology has greatly improved in recent years with advances in new methods in molecular biology, toxicology, animal laboratory science, and genetics, increasing our knowledge of ambient influences. Nevertheless, there is a lot to do to reduce the influence of hazardous intervening agents, whether they target our genetics or not, that can negatively affect pregnancy and induce congenital development disorders, including morphological, biochemical, or behavioral defects.Certain agents might indeed be related to certain defects, but we have not been able to identify the cause of most congenital defects, which highlights the importance of finding and testing out new genetics techniques and conducting laboratory animal science to unravel the etiology and pathogenicity of each congenital defect.

Lifelong exercise training promotes the remodelling of the immune system and prostate signalome in a rat model of prostate carcinogenesis.

This work aimed to understand how lifelong exercise training promotes the remodelling of the immune system and prostate signalome in a rat model of PCa. Fifty-five male Wistar rats were divided into four groups: control sedentary, control exercised, induced PCa sedentary and induced PCa exercised. Exercised animals were trained in a treadmill for 53 weeks. Pca induction consisted on the sequential administration of flutamide, N-methyl-N-nitrosourea and testosterone propionate implants. Serum concentrations of C-reactive protein (CRP) and tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) were not different among groups. Peripheral levels of γδ T cells were higher in Pca exercised group than in the PCa sedentary group (p < 0.05). Exercise training also induced Oestrogen Receptor (ESR1) upregulation and Mitogen-activated Protein Kinase 13 (MAPK13) downregulation, changed the content of the phosphorylated (at Ser-104) form of this receptor (coded by the gene ESR1) and seemed to increase Erα phosphorylation and activity in exercised PCa rats when compared with sedentary PCa rats. Our data highlight the exercise-induced remodelling of peripheral lymphocyte subpopulations and lymphocyte infiltration in prostate tissue. Moreover, exercise training promotes the remodelling prostate signalome in this rat model of prostate carcinogenesis.

Heavy metals and metalloids in wild boars (Sus Scrofa) - a silent but serious public health hazard.

Wild boars (Sus scrofa) are part of the hunting economy and are highly consumed in the Iberian Peninsula, including in the Castile and Leon regions. As zoonotic diseases, chemical pollutants in wild boars' internal tissues should be interpreted as evidence of environmental contamination and a matter of concern for animal, human and ecosystem health; in other words, a One Health concern. Twenty-eight wild boars' livers and kidneys (n = 28) from Castile and Leon were submitted to metal(loid) determination (As, Cd, Co, Cr, Cu, Ni, Pb, and Zn) with inductively coupled plasma mass spectrophotometry (ICP-MS) and histopathological exam. Cd levels, especially in the kidneys (7.063 ± 7.271 mg/kg dw), were the most concerning results, considering the calculated maximum values for consumption (EC No. 915/2023) (2.491 mg/kg dw or 1.0 mg/kg ww). Wild boars with hydropic changes in the liver presented higher concentrations of Ni. Thus, the metal(loid) contamination of wild boar carcasses seems to be a "no trace" but very relevant problem that should raise awareness of a more accurate monitoring program and other strategies to avoid public health consequences.

A spontaneous ovarian teratoma in an FVB/n female mouse: Case report and literature review.

Background: Teratomas are rare types of germ cell neoplasms composed of various differentiated or undifferentiated tissues. Case Description: A 25-week-old female control FVB /n mouse in a 4-week toxicity study presented abdominal distension and poor body condition. It was euthanized, and the necropsy examination revealed a large mass connected to the tip of the right uterine horn, occupying the entire abdominal cavity. Microscopically, this mass showed areas of epidermal differentiation, with laminated keratin and sebaceous glands, differentiation into respiratory and digestive epithelium, cartilage, bone, and extensive areas of differentiation into the nervous tissue, being classified as an ovarian teratoma. Conclusion: As far as authors know, the occurrence of ovarian teratomas in the FVB/n mouse strain has never been previously described.

Feline calicivirus and natural killer cells: A study of its relationship in chronic gingivostomatitis.

Background and Aims: Feline chronic gingivostomatitis (FCGS) is a frequent chronic inflammatory condition in the oral cavity with an etiopathogenesis not completely identified. This study aimed to contribute to the knowledge of FCGS by identifying the presence of feline calicivirus (FCV) antigens and natural killer (NK) cells and comparing them. Materials and Methods: Forty biopsies from the oral mucosa of cats diagnosed with chronic gingivostomatitis were subjected to immunohistochemical techniques to evaluate cells with FCV antigens and NK cells positive for CD56. Results: NK cells were identified in all samples, with an average of 725.3 ± 409.1 cells. Regarding FCV, it was identified in 18 out of 30 samples (60%), with a different number of cells with virus in between the analyzed cases. In all cases, the number of cells infected with FCV was lower than the number of NK cells present in the same samples, but there was no statistical association between them. Conclusion: This preliminary study shows that NK cells are present in gingivostomatitis lesions not exclusively caused by FCV-stimulus, as only 60% of all cases were positive for this virus, but other antigens should be considered in the etiology of FCGS.

In vivo prostate cancer research: Key interspecies prostate anatomical features for translation medicine.

Prostate cancer (PCa) is a prevalent malignancy affecting men worldwide. Animal models play a crucial role in studying PCa pathology and discovering novel approaches to prevent, detect and treat this disease. However, the challenge of translational medicine is the limited reproducibility and inadequate recapitulation of human conditions in animal models. Therefore, a comprehensive understanding of the macroscopic and microscopic anatomy of the prostate gland among distinct animal species is essential for better translating research findings to clinical practice. This review aims to compare and describe the macroscopic and microscopic anatomy of the prostate gland in humans, rats, and dogs, emphasizing the relevant features. Despite the anatomical differences between these species, rats are a valuable model to study human prostate diseases, once they share some features implicated in carcinogenesis in humans. Dogs, on the other hand, are considered the best model for studying PCa due to the development of spontaneous cancer with a higher incidence when compared with other animals and the development of bone metastases. Moreover, the lymphatic system and the sentinel lymph node role and mapping are similar in dogs and humans. However, it is important to recognize that no animal model can directly mimic all aspects of PCa as the human prostate is anatomically different from that of rats and dogs. Therefore, it is essential to analyze and understand the intra- and interspecies variability when translating research findings into clinical practice. This review highlights the importance of a thorough understanding of the anatomical differences between the prostate gland in humans, rats, and dogs when selecting the appropriate animal model for studying PCa.

Mammary Glands of Women, Female Dogs and Female Rats: Similarities and Differences to Be Considered in Breast Cancer Research.

Breast cancer is one of the most common and well-known types of cancer among women worldwide and is the most frequent neoplasm in intact female dogs. Female dogs are considered attractive models or studying spontaneous breast cancer, whereas female rats are currently the most widely used animal models for breast cancer research in the laboratory context. Both female dogs and female rats have contributed to the advancement of scientific knowledge in this field, and, in a "One Health" approach, they have allowed broad understanding of specific biopathological pathways, influence of environmental factors and screening/discovery of candidate therapies. This review aims to clearly showcase the similarities and differences among woman, female dog and female rat concerning to anatomical, physiological and histological features of the mammary gland and breast/mammary cancer epidemiology, in order to better portray breast tumorigenesis, and to ensure appropriate conclusions and extrapolation of results among species. We also discuss the major aspects that stand out in these species. The mammary glands of female dogs and women share structural similarities, especially with respect to the lactiferous ducts and lymphatic drainage. In contrast, female rats have only one lactiferous duct per nipple. A comprehensive comparison between humans and dogs is given a special focus, as these species share several aspects in terms of breast/mammary cancer epidemiology, such as age of onset, hormonal etiology, risk factors, and the clinical course of the disease. Holistically, it is clear that each species has advantages and limitations that researchers must consider during the development of experimental designs and data analysis.

Detection of Abnormal Prion Protein by Immunohistochemistry.

Abnormal prion proteins (PrPSc) are the disease-associated isoform of cellular prion protein and diagnostic markers of transmissible spongiform encephalopathies (TSEs). These neurodegenerative diseases affect humans and several animal species and include scrapie, zoonotic bovine spongiform encephalopathy (BSE), chronic wasting disease of cervids (CWD), and the newly identified camel prion disease (CPD). Diagnosis of TSEs relies on immunodetection of PrPSc by application of both immunohistochemistry (IHC) and western immunoblot methods (WB) on encephalon tissues, namely, the brainstem (obex level). IHC is a widely used method that uses primary antibodies (monoclonal or polyclonal) against antigens of interest in cells of a tissue section. The antibody-antigen binding can be visualized by a color reaction that remains localized in the area of the tissue or cell where the antibody was targeted. As such, in prion diseases, as in other fields of research, the immunohistochemistry techniques are not solely used for diagnostic purposes but also in pathogenesis studies. Such studies involve detecting the PrPSc patterns and types from those previously described to identify the new prion strains. As BSE can infect humans, it is recommended that biosafety laboratory level-3 (BSL-3) facilities and/or practices are used to handle cattle, small ruminants, and cervid samples included in the TSE surveillance. Additionally, containment and prion-dedicated equipment are recommended, whenever possible, to limit contamination. The PrPSc IHC procedure consists of a formic acid epitope-demasking step also acting as a prion inactivation measure, as formalin-fixed and paraffin-embedded tissues used in this technique remain infectious. When interpreting the results, care must be taken to distinguish non-specific immunolabeling from target labeling. For this purpose, it is important to recognize artifacts of immunolabeling obtained in known TSE-negative control animals to differentiate those from specific PrPSc immunolabeling types, which can vary between TSE strains, host species, and prnp genotype, further described herein.

High Levels of Heavy Metal(loid)s Related to Biliary Hyperplasia in Hedgehogs (Erinaceus europaeus).

Heavy metal(loid) pollution of ecosystems is a current One Health problem. The liver is one of the most affected organs in cases of acute or chronic exposure to abnormal amounts of these substances, inducing histopathologic lesions. In order to assess the influence of heavy metal(loids), forty-five European hedgehogs (Erinaceus europaeus) were submitted to necropsy, and liver samples were collected for a routine histopathology exam and metal(loid)s determination (As, Cd, Co, Cr, Cu and Pb) by ICP-MS. Age was estimated during the necropsy exam. Biliary hyperplasia was the most frequent lesion observed (16/45; 35.56%). No statistically significant associations were found between biliary hyperplasia and age or sex. Metal(loid)s' concentrations were higher in animals with biliary hyperplasia (except for As). There was a statistically significant difference for both Cd and Co. For As, Cd and Co, cubs and juveniles animals showed significantly lower concentrations than elder individuals. Only for Pb were significant differences found between females and males. As described in the literature, exposure to metal(loid)s may be a cause of biliary hyperplasia, although further research (including the use of biochemical methods) is needed to support these results. To the authors' knowledge, this is the first report of this association in hedgehogs.

Estimating sequence diversity of prion protein gene (PRNP) in Portuguese populations of two cervid species: red deer and fallow deer.

Among the transmissible spongiform encephalopathies (TSEs), chronic wasting disease (CWD) in cervids is now a rising concern in wildlife within Europe, after the detection of the first case in Norway in 2016, in a wild reindeer and until June 2022 a total of 34 cases were described in Norway, Sweden and Finland. The definite diagnosis is post-mortem, performed in target areas of the brain and lymph nodes. Samples are first screened using a rapid test and, if positive, confirmed by immunohistochemistry and Western immunoblotting. The study of the genetics of the prion protein gene, PRNP, has been proved to be a valuable tool for determining the relative susceptibility to TSEs. In the present study, the exon 3 of PRNP gene of 143 samples from red deer (Cervus elaphus) and fallow deer (Dama dama) of Portugal was analysed. Three single nucleotide polymorphisms (SNPs) were found in red deer - codon A136A, codon T98A, codon Q226E - and no sequence variation was detected in fallow deer. The low genetic diversity found in our samples is compatible with previous studies in Europe. The comparison with results from North America suggests that the free-ranging deer from our study may present susceptibility to CWD, although lack of experimental data and the necessity of continuous survey are necessary to evaluate these populations.

Do Urban Hedgehogs (Erinaceus europaeus) Represent a Relevant Source of Zoonotic Diseases?

Urban fauna is defined as animal species that can live in urban environments. Several species, including the western-European hedgehog (Erinaceus europaeus), have now been identified as part of this urban fauna, becoming permanent residents of parks and gardens in different cities across Europe. Due to the importance that this phenomenon represents for zoonotic disease surveillance, several authors have been conducting zoonotic agents' surveys on hedgehog. The aim of this study is to compare zoonotic diseases' prevalence in hedgehogs in urban environments with those from more rural areas. A systematic review with meta-analysis of twelve studied of zoonotic diseases' (in urban and rural areas of Europe) was therefore conducted for this purpose. Fifteen different zoonoses have been assessed in urban environments and six in rural areas. Anaplasma phagocytophilum was the most prevalent zoonotic agent found in urban habitats (96%). Dermatophytosis shows statistically significant differences between locations (p-value < 0.001), with a higher prevalence in urban Poland (55%; n = 182). Our results suggest further research and a standardized monitoring of different hedgehog populations are essential to understanding the epidemiology of several zoonotic pathogens in different habitat types (urban, rural, natural, industrial, etc.) and preventing possible disease outbreaks.

Appraising Animal Models of Prostate Cancer for Translational Research: Future Directions.

The growing incidence of prostate cancer has prompted a great investment in basic biology and translational studies to develop new therapies. Multiple animal models have been established to study etiological factors, cancer-preventive strategies and the molecular determinants of aggressiveness and metastases. The rat model of prostate cancer induced by chemical carcinogen N-methyl-N-nitrosourea (MNU) and testosterone exposure has become an important tool to study prostatic carcinogenesis and chemopreventive approaches. Over prolonged treatment, this model develops prostatic lesions that closely mimic those observed in human patients. By modifying the experimental conditions, different research groups have been able to induce a vast spectrum of lesions, ranging from early prostatic intraepithelial neoplasia to metastatic cancer. These carefully tuned experimental settings allowed researchers to test lifestyle interventions, and different pharmacological and chemopreventive strategies. However, this model's great flexibility requires careful planning to ensure that the experimental conditions are adequate to obtain the spectrum of lesions intended. The present review addresses such issues, highlighting the value of the rat prostate cancer model and the multiple challenges and opportunities it offers to researchers worldwide.

Wild Boars (Sus scrofa, L. 1758) from Castile and Leon Region (Spain): A Histopathology Survey.

Wild boars are wild ungulates with a wide distribution in Europe, with a relevant role in wildlife and public health. In Spain, high (and sometimes artificial) densities of wild boars are responsible for several health problems. Regular surveys, with hunters' collaboration, are crucial to monitor these diseases. Histological analyses were performed for lung, liver, and kidneys from 72 wild boars (58 from Zamora, 16 from Palencia). Lungs were the most affected organs, mainly revealing parasitic pneumonia (34.7%). Hydropic, vacuolar, and other cellular changes (33.3%) and congestion (16.7%) were found in the liver, and only 30.6% of the wild boars presented no alterations in this organ. Regarding the kidney, non-purulent nephritis (22.2%) was the most common lesion. This study gives an overview of the health status of wild boar populations in Castile and León. Other laboratory analyses are needed to obtain definitive diagnoses of these lesions, reach other conclusions, or apply any mitigation strategies to protect animals' or consumers' health.

Validation of the Rat Model of Prostate Cancer: Correlating Seminal Vesicle Lesions With Dorsolateral Prostate Lesions.

BACKGROUND/AIM: Lesions in the seminal vesicle are described in the most used protocols for prostate cancer (PCa) induction. This study aimed to characterize the lesions of seminal vesicles associated with a protocol of PCa induction in rats to contribute to better characterization of this model. MATERIALS AND METHODS: Forty-five male Wistar Unilever rats were randomly divided into two control groups: CONT1 (n=10) and CONT2 (n=10); and two PCa-induced groups: IND1 (n=10) and IND2 (n=15), sacrificed at 35 and 61 weeks, respectively. Animals from the induced groups were exposed to a multistep protocol for PCa induction. Animals, seminal vesicles and dorsolateral prostate were weighed. Seminal vesicles and dorsolateral prostate were submitted to histopathological and immunohistochemical analysis. RESULTS: Animals in which PCa was induced had a lower mean body weight when compared with the control animals (p<0.05). The relative mean seminal vesicle weight was higher in groups with PCa when compared with control groups (p<0.05). Although the differences were not statistically significant, animals from the IND2 group developed more lesions than animals from the IND1 and CONT2 groups. It is worth noting that the animals from group IND2 developed papillary adenomas and carcinomas in situ, which were not observed in any other group. Similar to observations in seminal vesicles, animals from group IND2 developed more dorsolateral prostate lesions than animals from the IND1 group (p<0.05). CONCLUSION: We observed that the longer the exposure to testosterone was, the greater was the incidence of preneoplastic and neoplastic lesions in both the seminal vesicle and the prostate, suggesting that testosterone exposure affects the spectrum of developed lesions.

Vimentin and Ki-67 immunolabeling in canine gastric carcinomas and their prognostic value.

This study evaluated the expression of vimentin and Ki-67 proliferative index (PI) by immunohistochemistry in 30 canine gastric carcinomas (GCs) and a possible association with clinical and pathological features and patient's survival time. Vimentin immunoreactivity was assessed in neoplastic cells (in primary lesions, emboli, and metastases) and tumor-associated stroma (TAS) of canine GCs. Ki-67 PI was quantified in the neoplastic epithelial component. Vimentin immunolabeling in neoplastic cells was found in 30% of the primary lesions, in 82% of the neoplastic emboli, and in 50% of the metastases; in TAS, it was observed in all cases. A mean of 16% of the TAS was immunolabeled for vimentin. High vimentin immunolabeling in the TAS (>16%) was detected in 40% of cases. The average value of Ki-67 PI was 50%, and 80% of the lesions had Ki-67 PI above 20%. Vimentin immunolabeling in neoplastic cells was more frequent in less-differentiated carcinomas (diffuse [29%] and indeterminate types [75%]) than well-differentiated carcinomas (intestinal type [0%], P = .049). No significant differences were observed in vimentin immunolabeling in the TAS or Ki-67 PI according to histological diagnosis, depth of invasion, presence of neoplastic emboli or metastases. However, vimentin immunolabeling in the TAS was positively correlated with Ki-67 PI (r = .394, P = .031). Furthermore, a moderate negative correlation was observed between Ki-67 PI and survival time (r = -0.540). Our results suggest that vimentin and Ki-67 PI have potential for providing prognostic information in cases of canine GCs.