Following an episode of sudden lethargy, an 18-month-old female black-capped parrot (Pionites melanocephalus) died while being examined. On gross examination, there was fluid within the coelom, hepatomegaly with yellow colouration and the heart appeared enlarged with pallor throughout the myocardium. On histological examination, cardiomyocytes were swollen with loss of cross striations and contained 6-12-μm diameter intrasarcoplasmic pale grey inclusions of storage material. Cardiomyocytes were occasionally karyomegalic with mitotic figures, lost or replaced by fibrosis and inflammation. Within the liver, there was periportal and centrilobular fibrosis and mild lipid-type vacuolar change with extramedullary haematopoiesis. In the lung, the bronchi and parabronchi had luminal haemorrhage and oedema with hypertrophy of epithelium lining the parabronchi. On transmission electron microscopy, the storage material was non-membrane bound, fibrillar and intrasarcoplasmic and had an occasional homogeneous, electron-dense, central core. The morphological features of this material, combined with the histological findings, are consistent with a diagnosis of polyglucosan storage disease and subsequent heart failure.

Protothecosis is an infectious disease caused by unicellular algae of the genus Prototheca, which are mainly known for causing mastitis in farm animals. However, there are reports of protothecosis in humans and other animals, such as dogs and cats, which develop cutaneous lesions or systemic lesions. Dogs with the systemic form generally develop bloody diarrhoea and blindness and neurological signs may also occur. The condition generally leads to the animal being euthanized due to the unfavourable prognosis. The objective of this work was to report two cases of disseminated protothecosis, one in a Pekingese dog (case 1) and the other in a German Shepherd Dog (case 2). Both animals had a history of chronic diarrhoea with haematochezia, which was unresponsive to various treatments in case 2. Both dogs were euthanized due to their clinical condition and were sent to the Veterinary Pathology Laboratory of the Federal University of Paraná for post-mortem examination. Macroscopically, the lesions in both cases were similar and were characterized by multifocal millimetre-sized white nodulations in the heart, skeletal muscles and intestinal serosa, as well as signs of fibrinonecrotic enterocolitis and lymphoplasmacytic interstitial nephritis. In both cases there was also retinal displacement with lymphoplasmacytic uveitis and chorioretinitis. The cytological and histopathological evaluation of the tissues revealed the presence of structures compatible with Prototheca spp. Polymerase chain reaction identified Prototheca bovis in both cases. This is a rarely reported disease and must be considered as a differential diagnosis in cases of chronic diarrhoea with haematochezia that progress to blindness in dogs.

From January 2000 to December 2023 the Laboratory of Animal Pathology of the Federal University of Campina Grande received 6,218 canine autopsy and biopsy submissions, with six (0.09%) autopsy cases diagnosed as transmissible venereal tumour (TVT) with encephalic metastases. The mean age of affected dogs (four females and two males) was 7 years. Dogs were all from urban areas and were either semidomiciled (four cases) or stray (two cases). Clinical findings consisted of multilobular, ulcerated masses affecting the genital area (five cases) or skin (one case). Neurological signs were reported in three cases and included lethargy, seizures and ataxia. Grossly, encephalic metastases were subdural and extraparenchymal and affected the telencephalic hemispheres (four cases) or the base of the brain (two cases). In all cases, histology revealed a round cell neoplasm arranged in sheets supported by a scant fibrovascular stroma. In the brain, tumours often compressed and occasionally infiltrated the surrounding neuroparenchyma. Neoplastic cells had immunolabelling for vimentin, CD45RA, Iba1 and lysozyme, and no immunolabelling for MHC class II, CD117, CD3, CD79 and MUM1. Encephalic metastases of TVT should be included in the differential diagnosis of nervous system diseases of dogs in endemic areas where TVT is diagnosed in genital or extragenital tissues.

Hypertension is a common condition in older cats, often secondary to chronic kidney disease (CKD). Although the heart is one of the organs damaged by hypertension, the pathology of the feline hypertensive (HT) heart has been poorly studied. The aim of this retrospective study was to describe the gross and microscopic pathology of hearts obtained from cats at post-mortem examination and to compare cats diagnosed with hypertension with cats of similar age and kidney function for which antihypertensive treatment was not deemed clinically necessary. Hearts from 32 cats were examined‒18 from HT and 14 from normotensive (NT) cats. The prevalence of CKD was 72.2% vs. 78.6% in the HT and NT groups, respectively. The time-averaged blood pressure over the longitudinal follow-up from diagnosis was significantly higher in the HT group compared with the NT group (153.4 ± 20.8 vs. 133.9 ± 19.3 mmHg; P = 0.0106), respectively. HT cats, when compared with NT cats, had a thicker left ventricular free wall (7.67 [5.45-9.29] vs. 5.07 [4.72-7.16] mm; P = 0.001) and interventricular septum (6.92 [6.26-7.56] vs. 4.96 [4.15-6.46] mm; P = 0.008) and higher ventricular weight as a percentage of body weight (0.34 [0.29-0.36] vs. 0.28 [0.21-0.31]%; P = 0.02), respectively. Myocardial fibrosis was present in 72% of cases with no significant difference in the prevalence (P = 0.45) or score (P = 0.81) between the HT (1 [0.75-2]; 77.8% scoring one or above) and NT cats (1 [0-2]; 64.3% scoring one or above). Similarly, the population prevalence of myocyte hypertrophy, myofibre disarray and microvascular change was 71.9%, 50% and 43.7%, respectively, and did not differ significantly between groups. These results suggest that age-related cardiac pathology, exacerbated by azotaemic CKD, in cats is very common. The role that hypertension plays in mediating these pathological changes is uncertain.

Mycoplasma phocicerebrale, the causative agent of seal ('speck') finger, a zoonotic disease, is a common commensal in the oral cavity of various seal species. Historically associated with seal hunters, it remains a significant risk for those handling or rehabilitating marine mammals. While primarily known for causing severe cellulitis in humans, M. phocicerebrale can also lead to severe infections in seals, including osteomyelitis and sepsis. Recent studies have revealed the predatory behaviour of grey seals (Halichoerus grypus) on harbour porpoises (Phocoena phocoena). Examination of scarring patterns on stranded animals suggest that some initial predation attempts are unsuccessful, and porpoises escape only to succumb to infection originating from bite wounds. Here we describe two cases in which the isolation of M. phocicerebrale and other bacterial species from chronically infected bite wounds, the lungs and other internal organs of harbour porpoises suggest failed predation attempts by grey seals. Therefore, the impact of delayed, indirect mortality from sympatric predation attempts may be a significant additional and underestimated cause of mortality in harbour porpoises. One of the cases examined in this study involved a harbour porpoise with gross and histological evidence of a failed predation attempt and a fatal systemic M. phocicerebrale infection. These findings highlight the severe consequences of such infections and underscore the importance of understanding the ecological implications of grey seal predation on harbour porpoise populations.

Nocardia spp are ubiquitous, gram-positive, variably acid-fast, branching and beaded filamentous, facultative intracellular bacteria that are resistant to phagocytosis and can cause localized or systemic disease in a variety of mammals, including humans, as well as in birds, fish and reptiles. Seventeen pet domestic ferrets (Mustela putorius furo) were diagnosed with nocardiosis by several methods including cytological evaluation, histopathology, Ziehl-Neelsen staining and polymerase chain reaction (PCR). All except two ferrets were 2 years old or older at the time of clinical presentation. Clinical findings included anorexia, weight loss, lymphadenomegaly, splenomegaly, masses/nodules in internal organs, hindlimb weakness or paresis, vomiting, dyspnoea, central nervous system signs, diarrhoea, abdominal pain and coughing. Anaemia, leucocytosis, neutrophilia, hypoalbuminaemia and hyperglobulinaemia were frequent. All ferrets had granulomatous or pyogranulomatous inflammation involving most commonly the lymph nodes, spleen, lungs, liver and adipose tissue. Intralesional acid-fast, branching filamentous bacilli were detected in 15 of 17 ferrets. PCR for the 16S rRNA gene of Nocardia spp was positive in 13 of 15 ferrets, including the two that were negative by acid-fast staining; of these two ferrets, one had intralesional coronavirus antigen. BLASTn analysis of eight sequences revealed six different Nocardia spp, including N. globerula, N. seriolae and N. donostiensis, that have rarely been reported to cause disease in humans and terrestrial animals. Antibiotic treatment (most commonly trimethoprim-sulfamethoxazole alone or in combination with clarithromycin and marbofloxacin) was followed by a marked improvement in several patients, although relapses were frequent. Six ferrets had concurrent neoplasia as a potential predisposing factor. Nocardiosis should be included in the differential diagnosis of granulomatous/pyogranulomatous inflammation in ferrets, particularly in those over 2 years of age.

Leptospirosis is a prevalent zoonotic disease in dogs. Although it is known that leptospires are primarily harboured in kidney tissues of dogs, it has been reported that they also infect testicular tissue. Leptospira interrogans causes various lesions in canine testicular tissues; however, the pathogenesis has not been clearly explained. In this study, 20 canine testicular tissue samples infected with L. interrogans were investigated for evidence of oxidative DNA damage, inflammation, apoptosis and autophagy. Ten samples of canine testicular tissue that were negative for L. interrogans were used as a positive control group. All tissues were examined by histopathological, immunohistochemistry (IHC) and immunofluorescence (IF) methods. Histopathological examination revealed that testicular tissues infected with L. interrogans had inflammation and oedema, and degeneration and necrosis of spermatocytes. In cases of severe disease, enzyme-linked immunosorbent assay, reverse transcriptase polymer chain reaction and IHC and IF indicated significant increases in levels of TLR4, NF-kB, IL-1β, TNF-α, 8-OHdG, JNK1/3, caspase-8, caspase-3, LC3A and LC3B but lower levels in milder cases. These results indicate that L. interrogans stimulated the immune system through the TLR4/NF-kB/JNK pathway in dog testicular tissues, leading to inflammation and apoptosis. The infection also caused oxidative DNA damage and autophagy.

Persistent subcutaneous granulomas form at the injection site following administration of aluminium oxyhydroxide (AlOOH), a widely used vaccine adjuvant. Small ruminant lentiviruses (SRLVs) can infect macrophages within granulomas induced by commercial AlOOH-based vaccines in sheep. The entry of SRLVs into target cells involves the mannose receptor (MR), while catalytic polypeptide-like 3 protein containing Z1 domain (A3Z1) is considered a restriction factor for lentiviral replication. The objective of this study was to investigate the temporal evolution of AlOOH-induced post-vaccination granulomas in sheep experimentally infected with SRLVs. Twenty-four male lambs underwent two identical vaccination protocols and were challenged with SRLVs. Granulomas were detected in vaccinated groups only and progressively decreased in size. At post-mortem examination, 91.3% of the granulomas were recovered. Fistulas were present in granulomas following the second vaccination protocol. Central necrosis was present in 58.0% of granulomas and was associated with the vaccine used. Orthokeratotic keratin was seen within granulomas in 47.1% of the lambs. Considering all granulomas studied, significantly higher expression of MR was found compared with A3Z1. Differences in MR expression were related to the type of vaccine and the time since vaccination. A3Z1 expression was upregulated in granulomas from the infected groups. Macrophage polarization may influence SRLV infection of granulomas. While SRLV infection does not influence the architecture of post-vaccination granulomas, it may modulate their immune microenvironment. Further studies are needed to elucidate the complex interactions between AlOOH-induced granulomas and SRLV infection in sheep.

Although peritonitis is highly prevalent in cattle, there have been only limited studies on the pathology of this condition. We describe the gross and histological aspects of primary and secondary peritonitis in cattle based on necropsy reports of 46 cases. Twenty-six were female (26/46; 56.5%) and 24 male (24/46; 43.5%), 31 (31/46; 67.4%) beef breed and 15 (15/46; 32.6%) dairy breed. Twenty were 0-12 months old (20/46; 43.5%), nine were 13-36 months old (9/46; 19.6%) and 15 were >36 months old (15/46; 32.6%). Two were of unknown age. Primary peritoneal tuberculosis (PT) was present in 7/46 cases (15.2%) and macroscopic lesions were mainly multifocal (6/7; 85.7%) and characterized by white to yellow, firm nodules of variable size. Histologically, they were characterized by granulomatous inflammation with intralesional acid-alcohol-resistant bacilli. All cases were positive for Mycobacterium tuberculosis complex and negative for Mycobacterium avium on immunohistochemistry. Secondary peritonitis was diagnosed in 39/46 cases (84.8%), caused by organ rupture or perforated ulcer (21/39; 53.9%), traumatic reticuloperitonitis (9/39; 23.1%), post-castration inflammation (7/39; 17.9%) or omphalophlebitis (2/39; 5.1%). Macroscopic lesions in secondary peritonitis were mainly diffuse (31/39; 79.5%) and comprised roughening of the peritoneal surface, which was covered by free white to yellow-orange fibrin strands with a liquid to pasty, opaque, white-grey effusion (30/39; 76.9%). Bacteriological culture of the exudate from 15 secondary peritonitis cases identified mainly Truperella pyogenes (8/15; 53.3%) and Escherichia coli (5/15; 33.3%). This study enhances our understanding of the pathological manifestations of peritonitis in cattle.

Histoplasmosis is a rarely reported clinical disease of equids in North America and is historically attributed to Histoplasma capsulatum var. capsulatum. This report details a case of intestinal histoplasmosis with lymphadenitis in an American Mammoth Jackstock donkey from Mississippi. Clinically, the donkey had chronic wasting, poor appetite, diarrhoea and an intra-abdominal mass detected by ultrasound. Grossly, the distal small and proximal large intestines contained multifocal, raised, tan, nodular, button ulcers along the mucosal surface. The associated colonic and mesenteric lymph nodes were severely enlarged, mottled tan to red and firm, and formed a large multinodular mass within the mesentery. Microscopically, lesions consisted of nodular infiltrates of epithelioid macrophages and multinucleated giant cells laden with numerous yeasts, along with mucosal ulceration and necrosis. Fungal polymerase chain reaction from lesioned intestines and lymph nodes targeting the internal transcribed spacer region yielded a 585 bp sequence that matched identically to two human isolates of Histoplasma mississippiense, one of several cryptic species previously classified together as H. capsulatum var. capsulatum endemic to North America. This case represents the first molecular identification of a Histoplasma species infecting a North American equid and the second report of histoplasmosis in a donkey from North America.

Lymphoid and haematopoietic neoplasms are rare in cetaceans. This report describes intravascular lymphoma in an adult common dolphin that presented with neurological signs prior to stranding. Intravascular lymphoma is a rare neoplasm previously recorded in humans, dogs, a cat and a horse. The condition is characterized by intravascular neoplastic lymphocytic proliferation, the sequelae of which are dependent on the organ affected. Key pathological findings in this case included multifocal cerebrocortical malacia secondary to vascular occlusion associated with intravascular neoplastic lymphocytic proliferation. Immunohistochemistry supported a T-cell phenotype similar to that observed in dogs. The neurological presentation was consistent with that seen in other species in which the central nervous system is involved. The aetiology and pathogenesis have not been established in other species and although no further investigations were made in this common dolphin, this report adds to the short list of species affected by this rare neoplasm.

Astrocytomas are relatively common primary brain tumours of humans and companion animals. In dogs, they represent approximately 17-28% of primary central nervous system tumours. However, extracranial metastasis is extremely rare. This case report describes a grade IV astrocytoma (glioblastoma) in the cerebrum of a young Cane Corso dog with pulmonary metastases. The diagnosis was obtained via histopathological morphology and immunophenotyping, which showed strong positivity for glial fibrillary acidic protein, vimentin and connexin-43. The glioblastoma in this Cane Corso had epithelioid morphology with histological features of malignancy including high mitotic count, microvascular proliferation, serpentine necrosis and subventricular zone involvement. Epithelioid glioblastoma is a rare subtype that has only relatively recently been formally acknowledged in human medicine and it can also pose a diagnostic challenge in veterinary medicine.

Mouse lemurs (Microcebus spp) are small Madagascan strepsirrhine primates increasingly used as an animal model in ageing research. During a period of 10 years, neoplastic disease occurred in 47 grey (Microcebus murinus) and Goodman's (Microcebus lehilahtsara) mouse lemurs from a captive colony in Germany. Approximately half of these tumours appeared histologically as soft tissue tumours (STTs) with a significantly higher proportion of STTs in Goodman's mouse lemurs (87.5%) compared with grey mouse lemurs (38.5%) (P ≤0.025). Most STTs grew subcutaneously in old or senile animals and were commonly located on the trunk, less often on the head and rarely at visceral sites. The majority of STTs were of fibrous or myofibroblastic origin, followed by undifferentiated pleomorphic sarcomas and extraskeletal chondro-osseous neoplasia. Histological grading of malignant STTs revealed all but one as grade II or III, with more than 60% being grade III. Female mouse lemurs of both species were affected significantly more often by grade II and III tumours than males (P = 0.0412). This study gives a comprehensive overview of the spectrum of mesenchymal neoplastic disease in mouse lemurs and highlights some histomorphological characteristics of spontaneous STTs in this small non-human primate species.

Xenarthran mammals of the suborder Folivora are arboreal species that feed mainly on leaves and Cecropia spp, an abundant plant in the Brazilian Amazon region, is favoured. The stomach of sloths represents 20-30% of their body weight and is adapted to gastric fermentation of plant material. These anatomical and physiological adaptations can be altered by many factors when confined to captivity, which favours fatal gastric conditions. The objective of this study was to describe the gastric anatomopathological lesions in three two-toed sloths (Choloepus didactylus) from a wildlife rescue centre. Necropsy revealed wide variation in the thickness of the gastric mucosa, with large areas having a papilliform appearance. Histologically, hyperkeratinization of the stratified squamous epithelium of the aglandular gastric region and infiltration of lymphocytic and plasmacytic cells were seen. The histopathological diagnosis was chronic hyperkeratotic gastritis, which has not previously been reported in sloths. Repeated exposure to dietary antigens, drugs, chemicals, toxins or infectious agents can initiate allergic or immune-mediated responses that can ultimately lead to chronic gastritis in these animals.

Feline infectious peritonitis (FIP) is one of the most important infectious diseases and a significant cause of death in cats. While the pathology of FIP has been well documented, there is little information on the hepatic lesions. The aim of this study was to characterize the macroscopic, microscopic and immunohistochemical patterns of the liver lesions in cats with FIP. A retrospective study was conducted on 131 cats with FIP, of which 59 had histological inflammatory lesions in the liver and were immunopositive for feline coronavirus. Four major gross patterns were defined: perihepatitis (21/59); nodular with perihepatitis (18/59); nodular multifocal (13/59); and diffuse (7/59). Four histological patterns were observed: (1) random pyogranulomas; (2) random multifocal to coalescent caseous necrosis surrounded by pyogranulomatous inflammatory infiltrate, fibrin and cellular debris; (3) perihepatitis; and (4) random multifocal to coalescent caseous necrosis surrounded by neutrophils, macrophages, lymphocytes and plasma cells. Most cats had more than one histological pattern. FIP virus antigen was detected in macrophages in the pyogranulomas and around the necrotic areas. The diagnosis of FIP remains challenging, especially in cats without effusion. The liver can be considered a suitable site for biopsy when feasible.

A young free-ranging black-horned capuchin (Sapajus nigritus) with a history of ataxia, anisocoria, diminished threat and pupil reflexes and increased cranial circumference was referred for evaluation to the Medicine and Wildlife Conservation Section, Federal University of Paraná, Paraná, Brazil. Due to the clinical presentation and radiographic findings of hydrocephalus, euthanasia was performed. Necropsy revealed a dark red mass (1.5 × 1.8 cm) in the region of the third ventricle and marked dilation of the lateral ventricles. Histopathological findings were characterized by proliferation of ciliated round cells with eosinophilic cytoplasm, as well as the formation of ependymal rosettes and pseudorosettes. In some areas the cells contained brown cytoplasmic pigment, which stained with Fontana-Masson. The neoplastic cells were immunoreactive for glial fibrillary acidic protein and negative for pancytokeratin. Considering the anatomopathological and immunohistochemical findings, the diagnosis was ependymoma grade II with melanocytic features, which is the first report of this neoplasm in any member of the genus Sapajus.

Chromatophoromas are neoplasms arising from pigment cells in vertebrates. Iridophoromas are a type of chromatophoroma that are reported in several teleost species. There are multiple case reports of this diagnosis in betta fish (Betta splendens), but all previously reported cases originated from the skin. This is the first report of an intra-ocular iridophoroma. An adult betta fish had buphthalmia of the right eye and enucleation was performed. The fish survived surgery but was found dead 2 days later. The eye and entire body were examined histologically, and the right eye was also examined ultrastructurally. Histologically, the uveal tract of the right eye was unilaterally and markedly expanded by a neoplasm that expanded and obliterated the iris and choroid and regionally invaded the cornea and sclera. The neoplasm was composed of spindle cells that contained pale green, birefringent, crystalline granules (iridophores). Transmission electron microscopy revealed that neoplastic cells contained a single elongated nucleus and one or more thin cytoplasmic bundles of reflecting plates that were oriented parallel to the basal lamina, further confirming the diagnosis of iridophoroma. This is the first reported case of an iridophoroma arising from the uveal tract. Most cases of iridophoromas and other chromatophoromas in fish are reported as benign. However, there are no established histological criteria of malignancy in these neoplasms. Despite the bland cellular morphology, most reported cases of iridophoromas and other chromatophoromas in bettas (including this case) had substantial tissue invasion and/or destruction. This suggests that iridophoromas and chromatophoromas in bettas may have local invasion, consistent with malignancy, despite bland cytological features.

Amyloid-producing ameloblastomas (APAs) are a histological variant of ameloblastoma in which the neoplastic odontogenic epithelial cells are associated with the production of amyloid. This jaw-associated entity has been only rarely described in animals and purportedly does not exist in humans. This report is the first description of an APA in a primate. A 17-year-old, male, captive cynomolgus macaque (Macaca fascicularis) had a slowly growing mass on the left rostral maxillary region that involved the bone. The mass was first seen in 2022 associated with the left maxillary canine tooth (203) and regrew after incomplete excision and extraction of the affected canine tooth. Additional excision was performed in 2024, at which time the mass was 3.8 cm in diameter, roughly spherical, tan and hard. Histologically, the mass was composed of packets, cords, anastomosing trabeculae and plexiform ribbons of odontogenic epithelial cells embedded in abundant fibrovascular stroma. Packets of epithelial cells surrounded an abundant, apical, congophilic amyloid matrix that was variably mineralized. Odontogenic epithelium was immunoreactive to cytokeratin AE1/AE3 and had no immunoreactivity for vimentin. These findings are diagnostic for APA. This is the first report of APA in any primate, human or non-human, raising the question of whether this entity can occur in humans.

An adult, female, mixed breed sow from a commercial breeding unit, with a clinical history of lethargy, poor body condition, hypersalivation, dyspnoea and reddened ears, was submitted for necropsy. Post-mortem examination identified the presence of an intestinal volvulus, which developed around an osseous intra-abdominal mesenteric lesion. Gross pathology and histopathology combined with post-mortem computed tomography (CT) of the mass confirmed the lesion as intra-abdominal heterotopic mesenteric ossification (IHMO), according to criteria applied to human disease. This report highlights a rare case of porcine IHMO, a poorly understood metaplastic condition, comparable to the human disease. We highlight the importance of a combination of CT and histopathology to reach a final diagnosis of the disease. Histopathology and further investigation of similar cases may contribute to a better understanding of the pathogenesis in animals, which is currently poorly understood.

Malignant osteoblasts can have markedly pleomorphic phenotypes and variable amounts of tumour-associated matrix, complicating the ability of pathologists to accurately differentiate osteosarcoma (OSA) from other types of neoplasms using only histopathology. Current immunohistochemical markers for animals have limited sensitivity and specificity in identifying OSA or produce inconsistent results. Immunohistochemistry (IHC) for special AT-rich sequence-binding protein 2 (SATB2) has been used in human medicine to aid in identification of normal and neoplastic osteoblasts, and the objective of this study was to determine whether this marker could also be useful for the diagnosis of canine OSA. Initially, SATB2 IHC was performed on eight samples from cases of well-differentiated canine OSA and on other tumour types for which OSA is a differential diagnosis, as well as on normal tissues, to assess sensitivity and cross-reactivity. Following confirmation that SATB2 is immunoreactive for normal and neoplastic canine osteoblasts and negative in other non-osseous mesenchymal cell types and organs, SATB2 IHC was tested on 123 cases of poorly differentiated malignant neoplasms as part of a panel with other immunohistochemical markers, as appropriate, based on histomorphology and differential diagnoses. The conclusion is that SATB2 IHC is a sensitive and specific marker for identifying canine OSA when used in a panel with other immunohistochemical markers and in conjunction with supportive clinical history.

Parameters for wound age estimation are essential in forensic pathology but difficult to assess. With this retrospective study, wound age parameters in canine skin wounds were assessed with archive material of known age. Haematoxylin and eosin (HE) staining as standard, Prussian blue, Van Gieson (VG) and multiple other special stains were used as well as various immunohistochemistry (IHC) tests. Collagen fibre formation examination included HE staining but also immunolabelling for elastin and collagen I. Collagen fibre formation was further assessed by polarization in HE and VG. HE staining, erythrophagocytosis and presence of haemoglobin decay products in the Prussian blue stained-slides proved to be useful tools in wound age estimation in the early phase of wound repair of less than 5 days. The earliest detection of newly formed collagen was possible in 5-day-old wounds with HE and VG staining. Collagen I reactivity by IHC was weak at this time point, moderate to strong in the lesions older than 10 days and up to 30 days and strong in the lesions older than 3 months. Very slight polarization of collagen fibres was observed at 6 days, becoming stronger in wound lesions of 10-14 days and up to 30 days and reaching the same intensity as normal tissue in a 3-month-old lesion. The morphology of newly formed collagen fibres, utilizing HE and VG staining and polarization as well as collagen I IHC, which proved to be the most important parameters, correlated with distinguishable time points in the available cases of known wound age.