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Brucella ovis infects sheep and causes a clinical or subclinical disease characterized by genital lesions and reduced fertility in rams, placentitis and abortions in ewes, and increased perinatal mortality in lambs. As part of this study, analyses were conducted on a sheep farm near Belgrade (Serbia). Of the serological tests, indirect ELISA was performed. A total of 94 blood sera were analyzed, 33 from rams and 61 from ewes. The results showed 23 (69.7%) positive findings in rams and 2 (3.3%) positive findings in ewes, with an overall prevalence of 25.4% bounded by a 95% confidence interval. Bruce-ladder multiplex PCR and Real time PCR were performed on 19 serologically positive rams and two serologically positive ewes. The results indicate a slightly higher sensitivity of Real time PCR compared to conventional PCR in diagnosing B. ovis from the reproductive tissues of rams. However, the differences in sensitivity between different nucleic acid extraction protocols were not significant. Most rams showed a positive PCR result in only one sample of reproductive tissue, suggesting the recommendation to take multiple samples from each animal. Further research is needed to bring the sensitivity of molecular tests in diagnosing ram epididymitis closer to the sensitivity of serological tests.

Scrapie is an infectious neurodegenerative disease affecting the central nervous system of sheep and goats that belongs to transmissible spongiform encephalopathies. The disease is caused by the accumulation of proteinase-resistant isoform of the prion protein. The sheep predisposition to scrapie is associated with polymorphisms of the PrP gene. Genetic susceptibility to scrapie is mainly related to codons 136, 154, and 171. ARR sheep are strongly scrapie resistant and VRQ genotype is the most susceptible. Many countries have scrapie eradication programs based on using rams with resistant genotype. The eradication program has not yet been implemented in the Republic of Serbia. To examine the genetic makeup of sheep in Serbia related to scrapie, we optimized TaqMan probes of real-time polymerase chain reaction (qPCR) technique for three codons. Blood samples from 100 sheep were analyzed by qPCR and the majority of the examined sheep were AA homozygous for the 136 codon. For codon 154 the most frequent genotype was RR and for codon 171 the most frequent genotype was QQ.

The ageing process is accompanied by numerous changes in the brain of dogs, such as accumulation of amyloid, fibrosis of blood vessel walls and meninges, accumulation of lipofuscin, and the presence of polyglucosan bodies (PGBs), satellitosis and neuronophagia. In this study, the presence of lipofuscin and PGBs in various parts of the brain in dogs of different sexes and ages was examined. For this purpose, brain samples were stained using haematoxylin eosin, modified Ziehl Neelsen and Periodic acid Schiff (PAS) methods. Lipofuscin was visualised by Ziehl Neelsen and PAS methods of specific staining on the same brain tissue segments. Lipofuscin had accumulated in 93% of old (more than 10 years old) dog brains, mostly in neurons of the medulla oblongata. The percentage of age-related lipofuscin pigment in other examined brain tissue segments was lower than in the medulla oblongata. There was a small difference in the percentage of lipofuscin-positive individuals between the two staining methods. The presence of PGBs was established by the PAS method for the vast majority (about 93%) of the old dogs (more than 10 years old), while PGBs were not detected in the group of young dogs (up to 5 years old). However, PGBs occurred in all examined segments of the dog’s brain tissues (for each of the tissue types, from 90% to 93% of dogs were positive for PGBs). The results obtained the oldest dogs (15 years old) harboured PGBs both extracellularly and intracellularly, while in other dogs, only extracellular PGBs were seen. Lipofuscin was accumulated mostly in large neurons of olivary nuclei of the medulla oblongata. PGBs were confirmed in all examined segments of the brain tissue of dogs more than 10 years old. This is one of the numerous indications that old dogs could be a very good animal model for studying the normal ageing process or neurodegenerative diseases.

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Lumpy skin disease (LSD) is a disease of cattle and domestic buffaloes caused by Capripoxvirus which can lead to significant economic losses. Until several years ago it was limited to Africa and the Middle East, but recently it was reported in Turkey (2013), Greece (2015), and in 2016 it spread to Bulgaria, North Macedonia, Albania, Montenegro and Serbia. The aim of this study was to determine the microscopic lesions in the skin of naturally infected animals, highlight their features and compare them to the findings of other authors. Gross lesions in the skin of cattle infected with Lumpy skin disease virus (LSDV) were manifested in the form of skin nodules which were round, raised, clearly circumscribed, firm and randomly distributed in the whole skin including the vulva and udder. Histopathological lesions included degeneration of epithelial cells, hyperkeratosis, spongiosis, and acanthosis present in 85.88%, 81.18%, 84.71%, and 80.0% of samples, respectively. Intracytoplasmic inclusion bodies were noted in keratinocytes of the stratum spinosum in 56.47% of samples, and in hair follicle cells and sebaceous glands epithelial cells in 45.88% of samples. Cutaneous lesions were in the form of inflammatory infiltrate present in the dermis and subcutis in 97.65% of samples. Since there are only a few literature data in this field, this study expands the knowledge relating to morphological alterations in LSD. Based on the characteristic microscopic lesions described in the present study, histopathology should be considered as a very useful method for the diagnosis of LSD.

Peripheral nerve sheath tumors (PNSTs) comprise a heterogeneous group of neoplasms originating from the elements of the nerve sheath. They are divided into two forms: benign and malignant PNST. Both benign and malignant PNSTs are not very common in domestic animals but they are reported in different animal species. Histologically, PNSTs are composed predominantly of spindle cells arranged in bundles, whorls and sheets, with a different number of pleomorphic cells and mitotic figures.

Canine distemper virus (CDV) is a worldwide distributed RNA virus that can cause severe disease in carnivore and non-carnivore species. Red foxes are highly susceptible and may act as a reservoir of the virus. As in other wild species, distemper in red foxes can manifest as acute, systemic and chronic nervous form. In the present study, we detected antibodies against CDV among red foxes in Western Serbia, and analyzed histopathologically and immunohistochemically for CDV nuclear protein antigen (CDV-NP) brain samples derived from seropositive animals. Seroprevalence of CDV antibodies was 36.8%. Histopathological changes included gliosis, neuronal degeneration, satellitosis, mononuclear inflammation, demyelination and presence of inclusion bodies. Immunostaining showed a diffuse presence of CDV-NP antigen, mainly in the cytoplasm of astrocytes and neurons. Results of this work contribute to the opinion that red foxes act as a potential reservoir of CDV and underline the importance of routine vaccination of dogs that could come in close contact with these animals. Potential active surveillance program would give a better insight in the degree of CDV infection in wildlife.

The estrus cycle of bitches is divided into four phases: proestrus, estrus, diestrus and anestrus, during which different morphological changes, and also cyclic changes of estrogen and progesterone receptors are present. Several pathological changes can be differentiated on the endometrium, but one of these is the most important - cystic endometrial hyperplasia, which frequently develops into pyometra. The aim of the present study was to describe morphological characteristics, and expression of estrogen and progesterone receptors on the endometrium of mixedbreed bitches during the different phases of the estrus cycle, cystic endometrial hyperplasia and pyometra. The uterus and ovaries of 36 mixed breed bitches in different phases of the estrus cycle and also with cystic endometrial hyperplasia (CEH) and chronic purulent endometritis - pyometra were examined macroscopically, histopathologically, and immunohistochemically for estrogen receptors (ΕR) and progesterone receptors (PR). During proestrus uterine cells showed a weak reaction for both estrogen and progesterone receptors, but during estrus a large number of uterine cells showed a strong reaction on estrogen receptors and moderate reaction on progesterone receptors. On the contrary, during diestrus the scores for the estrogen receptors decreased, while the progesterone receptors level increased - uterine cells expressed strong reaction for progesterone receptors, and moderate reaction for estrogen receptors. Uterine cells in cystic endometrial hyperplasia expressed a strong reaction for estrogen receptors, and moderate reaction for progesterone receptors, but on the other hand the uterine cells in the uterus with pyometra expressed a moderate to strong reaction for progesterone receptors, and a weak reaction for estrogen receptors. In further investigations it would be interesting to perform quantitative analysis for both estrogen and progesterone receptors during different phases of the estrus cycle and also in the uterus with cystic endometrial hyperplasia and pyometra.

nema

Equine glanders is a severe bacterial disease known since ancient times. Although eradicated in the most part of the world it is now considered re-emerging. Considering very scarce literature data, we used from formalin fixed collection material: nasal septum, lung and skin specimens from naturally infected horses. Tissues were grossly examined and photographed. Tissue samples, after standard processing, were stained with HE, Congo red and Groccot and microscopically examined. Gross changes include nodules and ulcers in the nasal mucosa with granulation and scarring, pyogranulomas in the lung tissue and nodules and ulcers of the skin. Microscopically marked inflammation of affected tissues with neutrophilic and mononuclear infiltration and fibrous tissue proliferation were seen. As a potent zoonotic agent it has been already used as a biological weapon in the past.

An apathetic 14-year-old Hungarian Puli bitch was presented with a seven day history of vaginal discharge and reported ovariohysterectomy (OHE) conducted six years ago. Clinical examination showed elevated body temperature (39.5°C), enlarged abdomen, and vulvar swelling. Despite a reported OHE in the anamnesis, ultrasound examination of the abdomen demonstrated the presence of both ovaries and a large anechogenic zone resembling the uterus. Vaginal cytologic examination and hormonal analysis indicated that the bitch was in the oestrus phase of the cycle. After laparotomy, both intact ovaries and an enlarged uterus with marked adhesions and filled with liquid content were visualised. The cervix was situated in the caudal part of the abdominal cavity and connected to the uterus by only an adhesion stump. After extirpation, uterus and ovarian tissue samples were sent for histological examination. Histological findings of uterus and ovarian tissue samples indicated a diagnosis of mucometra. Obviously, OHE was not conducted professionally, and the procedure performed only prevented conception, but ovaries and uterus continued to be active and caused a serious health disorder after six years. When only a part of the ovaries or uterus is left after OHE, health complications can appear up to ten years later. Our case testifies that even if both ovaries and the entire uterus are left after OHE without communication with cervix and vagina, a bitch can live without noticeable health disorders up to six years. This underlines the importance of lymphatic drainage and resorption processes in the uterus as well as evacuation of uterus content through the vagina.

Gastric dilatation and volvulus is a life-threatening condition characterized by rapid accumulation of food and gases that cause displacement and distension of the stomach. The large and giant, deep-chested breeds of dogs are at higher risk for developing the gastric dilatation and volvulus. Uncommonly, it can also develop in cats, but it is also described in free-range polar bears.

Neoplazme poreklom od mastocita mogu da se jave u dva različita oblika: uobičajeno kao kožni tumori - kutani mastocitomi ili ređe kao sistemska forma neoplastične proliferacije mastocita - sistemska mastocitoza. Svrha ovog istraživanja je da se uporede histološke i citološke karakteristike, ekspresija KIT receptora i prisustvo mutacija c-KIT proto-onkogena u neoplastičnim ćelijama kod pasa sa kutanim mastocitomima i sistemskom mastocitozom. Mikroskopskim pregledom citoloških razmaza svih ispitanih pasa utvrđeno je da se ćelijska populacija sastoji uglavnom od okruglih ćelija koje imaju centralno postavljeno jedro i sitne ljubičaste citoplazmatske granule. Histopatološkim pregledom uzoraka kože pasa sa kutanim mastocitomima i sistemskom mastocitozom zapaženo je umnožavanje mastocita u površinskom i/ili dubokom dermisu. Sličan nalaz uočen je i u isečcima tkiva limfnih čvorova, slezine, jetre, miokarda i bubrega psa sa sistemskom mastocitozom. Kod tri psa sa kutanim mastocitomima visokog stepena maligniteta, kao i kod psa sa sistemskom mastocitozom zapažena je citoplazmatska ekspresija CD117, dok je kod tri psa sa kutanim mastocitomima niskog stepena maligniteta uočena membranska ekspresija CD117. Na osnovu našeg istraživanja, histološke karakteristike i citoplazmatska ekspresija CD117 na neoplastičnim ćelijama su slične kod pasa sa sistemskom mastocitozom i pasa sa kutanim mastocitomima visokog stepena maligniteta. I pored toga, mutacije c-KIT proto-onkogena nisu uočene ni kod pasa sa kutanim mastocitomima, ni kod psa sa sistemskom mastocitozom.

The development of tumours implies a disproportion between proliferation, growth and differentiation, which is accompanied by significant genotypic and phenotypic variations in the diseased. The scenario of multiple oncogenic effects on the inhibition of cell growth, apoptosis and maturation, on the stimulation of proliferation, on cell migration and tissue invasion, is responsible for the occurrence of tumours both in humans and in animals. The qualitative and quantitative determination of biological prognostic factors in the tissue of affected cats and dogs directly indicates a possible tumour metastasizing, and consequently also the prognosis of neoplastic disease. Monitoring of clinical and biological prognostic factors is important for therapy and prediction of tumours in humans and animals. The determination of the proliferative potential of tumour cells by immunohistochemical detection of Ki-67 and PCNA is used in grading certain tumours in cats and dogs. It is also possible immunohistochemically to prove factors of angiogenesis and numerous activating and inhibiting proteins in the tumour tissue. In addition to other histological, clinical and biological parameters, they are significant for the further prognosis and therapy of neoplastic diseases.

Background: Helsmoortel-Van der Aa syndrome is a neurodevelopmental disorder in which patients present with autism, intellectual disability, and frequent extra-neurological features such as feeding and gastrointestinal problems, visual impairments, and cardiac abnormalities. All patients exhibit heterozygous de novo nonsense or frameshift stop mutations in the Activity-Dependent Neuroprotective Protein (ADNP) gene, accounting for a prevalence of 0.2% of all autism cases worldwide. ADNP fulfills an essential chromatin remodeling function during brain development. In this study, we investigated the cerebellum of a died 6-year-old male patient with the c.1676dupA/p.His559Glnfs*3 ADNP mutation., Results: The clinical presentation of the patient was representative of the Helsmoortel-Van der Aa syndrome. During his lifespan, he underwent two liver transplantations after which the child died because of multiple organ failure. An autopsy was performed, and various tissue samples were taken for further analysis. We performed a molecular characterization of the cerebellum, a brain region involved in motor coordination, known for its highest ADNP expression and compared it to an age-matched control subject. Importantly, epigenome-wide analysis of the ADNP cerebellum identified CpG methylation differences and expression of multiple pathways causing neurodevelopmental delay. Interestingly, transcription factor motif enrichment analysis of differentially methylated genes showed that the ADNP binding motif was the most significantly enriched. RNA sequencing of the autopsy brain further identified downregulation of the WNT signaling pathway and autophagy defects as possible causes of neurodevelopmental delay. Ultimately, label-free quantification mass spectrometry identified differentially expressed proteins involved in mitochondrial stress and sirtuin signaling pathways amongst others. Protein-protein interaction analysis further revealed a network including chromatin remodelers (ADNP, SMARCC2, HDAC2 and YY1), autophagy-related proteins (LAMP1, BECN1 and LC3) as well as a key histone deacetylating enzyme SIRT1, involved in mitochondrial energy metabolism. The protein interaction of ADNP with SIRT1 was further biochemically validated through the microtubule-end binding proteins EB1/EB3 by direct co-immunoprecipitation in mouse cerebellum, suggesting important mito-epigenetic crosstalk between chromatin remodeling and mitochondrial energy metabolism linked to autophagy stress responses. This is further supported by mitochondrial activity assays and stainings in patient-derived fibroblasts which suggest mitochondrial dysfunctions in the ADNP deficient human brain., Conclusion: This study forms the baseline clinical and molecular characterization of an ADNP autopsy cerebellum, providing novel insights in the disease mechanisms of the Helsmoortel-Van der Aa syndrome. By combining multi-omic and biochemical approaches, we identified a novel SIRT1-EB1/EB3-ADNP protein complex which may contribute to autophagic flux alterations and impaired mitochondrial metabolism in the Helsmoortel-Van der Aa syndrome and holds promise as a new therapeutic target., (© 2024. The Author(s).)

Given our recent discovery of somatic mutations in autism spectrum disorder (ASD)/intellectual disability (ID) genes in postmortem aged Alzheimer's disease brains correlating with increasing tauopathy, it is important to decipher if tauopathy is underlying brain imaging results of atrophy in ASD/ID children. We concentrated on activity-dependent neuroprotective protein (ADNP), a prevalent autism gene. The unique availability of multiple postmortem brain sections of a 7-year-old male, heterozygous for ADNP de novo mutation c.2244Adup/p.His559Glnfs*3 allowed exploration of tauopathy, reflecting on a general unexplored mechanism. The tested subject exhibited autism, fine motor delays, severe intellectual disability and seizures. The patient died after multiple organ failure following liver transplantation. To compare to other ADNP syndrome mutations, immortalized lymphoblastoid cell lines from three different patients (including ADNP p.Arg216*, p.Lys408Valfs*31, and p.Tyr719* heterozygous dominant mutations) and a control were subjected to RNA-seq. Immunohistochemistry, high-throughput gene expression profiles in numerous postmortem tissues followed. Comparisons to a control brain and to extensive datasets were used. Live cell imaging investigated Tau-microtubule interaction, protecting against tauopathy. Extensive child brain tauopathy paralleled by multiple gene expression changes was discovered. Tauopathy was explained by direct mutation effects on Tau-microtubule interaction and correction by the ADNP active snippet NAP. Significant pathway changes (empirical P value < 0.05) included over 100 genes encompassing neuroactive ligand-receptor and cytokine-cytokine receptor interaction, MAPK and calcium signaling, axon guidance and Wnt signaling pathways. Changes were also seen in steroid biosynthesis genes, suggesting sex differences. Selecting the most affected genes by the ADNP mutations for gene expression analysis, in multiple postmortem tissues, identified Tau (MAPT)-gene-related expression changes compared with extensive normal gene expression (RNA-seq) databases. ADNP showed relatively reduced expression in the ADNP syndrome cerebellum, which was also observed for 25 additional genes (representing >50% of the tested genes), including NLGN1, NLGN2, PAX6, SMARCA4, and SNAP25, converging on nervous system development and tauopathy. NAP provided protection against mutated ADNP disrupted Tau-microtubule association. In conclusion, tauopathy may explain brain-imaging findings in ADNP syndrome children and may provide a new direction for the development of tauopathy protecting drug candidates like NAP in ASD/ID.