Case report: Findings in ovaries development from an aborted equine fetus.

An aborted female foal was submitted for necropsy. During the gross examination, the ovaries were pale, grayish, and enlarged (6 × 5 cm), with a well-developed vascular structure surrounding the external surface; the cut surface of the ovaries showed a brownish parenchyma with white follicular areas mainly localized in the peripheral region. The ovaries were fixed for histological investigations. The histological evaluation of the ovaries showed polygonal-shaped cells with abundant cytoplasm and round or oval nuclei, arranged in cords of single cells. The tissue architecture was characterized by the presence of lobular-like tissues with a central vein. The tissue mimicking hepatocytes was delimited by a mature fibrous tissue and was surrounded by the normal ovarian tissue characterized by germinal epithelium and primordial follicular structures. Based on the histological findings, a diagnosis of bilateral ovarian hamartoma was carried out initially. For a better characterization of the ovarian tissue, the expression of tissue-specific (liver and ovary) markers was investigated using immunohistochemistry. Following the immunohistochemical analysis, the hamartoma diagnosis was excluded. The ovaries exhibited unique characteristics different from those of adult horse ovaries as well as unique morphological features different from other mammalian species. This case report enhances our understanding of ovaries at a later stage of pregnancy and unveils unique characteristics of horse ovaries development, avoiding misdiagnosis with pathological findings, hamartomas, or neoplasia.

Milk fat miRNome changes in response to LPS challenge in Holstein cows.

Mastitis is an inflammatory disease in dairy cows, causing economic losses and reducing animal welfare. In order to contribute for the discovery of early and noninvasive indicators, our objective was to determine the effects of a lipopolysaccharide (LPS) challenge on the microRNA profile (miRNome) of milk fat, using microarray analyses in cows. Cows were fed a lactation diet at ad libitum intake (n = 6). At 27 ± 3 days in milk, cows were injected with 50 µg of LPS Escherichia coli in one healthy rear mammary quarter. Milk samples were collected just before LPS challenge (LPS-) and 6.5 h after LPS challenge (LPS +) from the same cows. Microarray analysis was performed using customized 8 × 60 K ruminant miRNA microarrays to compare LPS- to LPS + miRNome. In silico functional analyses were performed using OmicsNet and Mienturnet software. MiRNome comparison between LPS- and LPS + identified 37 differentially abundant miRNAs (q-value ≤ 0.05). The predicted target genes of the 37 differentially abundant miRNAs are mostly involved in cell life including apoptosis, cell cycle, proliferation and differentiation and in gene expression processes. MiRNome analyses suggest that miRNAs profile is related to the inflammation response of the mammary gland. In conclusion, we demonstrated that milk fat might be an easy and rapid source of miRNAs that are potential indicators of early mastitis in cows.

Case-report: Massive infection by Cysticercus longicollis in a captive Lemur catta from Italy.

An adult male ring-tailed lemur (Lemur catta) from a biopark of northern Italy was submitted to necropsy. A multi-organ parasitic infection was macroscopically evident. Abundant sero-hemorrhagic fluid with larval parasites was present in all cavities. The microscopic evaluation of parasites and the molecular characterization revealed the presence of Cysticercus longicollis (the larval stage of Taenia crassiceps). Histology of liver, lungs, intestine and urinary bladder revealed several larval parasites surrounded by a severe lymphocytic infiltrate, fibrous tissue and hemorrhages. This is the first report of a ring-tailed lemur with an infection of C. longicollis in Italy. The source of infection is still not known however, the discovery of this parasite in a captive lemur poses more attention on the control of parasitic diseases implementing monitoring tests and biosecurity measures.

MALDI-TOF mass spectrometry profiling of bovine skim milk for subclinical mastitis detection.

Introduction: Mastitis is one of most impacting health issues in bovine dairy farming that reduces milk yield and quality, leading to important economic losses. Subclinical forms of the disease are routinely monitored through the measurement of somatic cell count (SCC) and microbiological tests. However, their identification can be tricky, reducing the possibilities of early treatments. In this study, a MALDI-TOF mass spectrometry approach was applied to milk samples collected from cows classified according to the SCC, to identify differences in polypeptide/protein profiles. Materials and methods: Twenty-nine raw milk samples with SCC >200,000 cell/ml (group H) and 91 samples with SCC lower than 200,000 (group L) were randomly collected from 12 dairy farms. Spectral profiles from skim milk were acquired in the positive linear mode within the 4,000-20,000 m/z mass acquisition range. Results and discussion: Based on signal intensity, a total of 24 peaks emerged as significant different between the two groups. The most discriminant signals (4,218.2 and 4,342.98 m/z) presented a ROC curve with AUC values higher than 0.8. Classification algorithms (i.e., quick classifier, genetic algorithm, and supervised neural network) were applied for generating models able to classify new spectra (i.e., samples) into the two classes. Our results support the MALDI-TOF mass spectrometry profiling as a tool to detect mastitic milk samples and to potentially discover biomarkers of the disease. Thanks to its rapidity and low-cost, such method could be associated with the SCC measurement for the early diagnosis of subclinical mastitis.

Amoxicillin and thiamphenicol treatments may influence the co-selection of resistance genes in the chicken gut microbiota.

The aim of this study was to assess the dynamics of microbial communities and antimicrobial resistance genes (ARGs) in the chicken gut following amoxicillin and thiamphenicol treatments and potential co-selection of ARGs. To this purpose, the microbial community composition, using 16S rRNA NGS, and the abundance of ARGs conferring resistance to ß-lactams and phenicols, using qPCRs, were determined. Results revealed that the administered antimicrobials did not significantly reduce the gut microbiota diversity, but changed its composition, with taxa (e.g. Gallibacterium and Megamonas) being enriched after treatment and replacing other bacteria (e.g. Streptococcus and Bifidobacterium). Positive correlations were found between ARGs (e.g. cmlA, blaCMY-2, and blaSHV) and the relative abundance of specific taxa (e.g. Lactobacillus and Subdoligranulum). The selective pressure exerted by both amoxicillin and thiamphenicol resulted in an increased abundance of ARGs conferring resistance to ß-lactams (e.g. blaTEM-1, blaSHV, and blaCTX-M1-like) and phenicols (e.g. floR and cmlA). These findings, together with the co-occurrence of genes conferring resistance to the two antimicrobial classes (e.g. blaTEM-1 and cmlA), suggest a possible interaction among antimicrobials on resistance emergence, possibly due to the presence of mobile genetic elements (MGEs) carrying multiple resistance determinants.

Development of a droplet digital PCR assay to detect illicit glucocorticoid administration in bovine.

Glucocorticoids are often used illegally in food-producing animals for the growth promotion of livestock animals. In accordance to official chemical methods for glucocorticoid detection, an animal is declared as non-compliant when a residue is identified in the sample. Neverthless, growth promoting molecules can often escape identification due to their rapid elimination or due to the use of non-detectable new generation drugs. Therefore, an indirect screening method able to detect the biological effect of long-term administration of low doses of dexamethasone and prednisolone on livestock has been developed to support official methods. As already described, FKBP5 (FKBP prolyl isomerase 5) expression in bovine thymus is regulated by glucocorticoids, and this specific regulation can be exploited in an indirect screening assay. In the present study, male veal calves and young bulls were considered in three different trials in which estradiol, dexamethasone, and prednisolone were administered alone or in combination with Revalor-200 subcutaneous pellets. Thoracic thymus was sampled from all animals and molecular analysis was performed. A duplex droplet digital PCR assay with EvaGreen® was employed to detect the target gene expression using absolute quantification. The developed droplet digital PCR assay was precise, showing intra- and inter-assay mean coefficient of variation values of about 6.16% and 3.17%, respectively. It was also highly specific (100%) with Youden's index of 76.92% and 53.57% applied to veal calves and young bulls, respectively. The lowest detection limit in which the target gene expression level was kept constant, was 0.05 ng/µl of cDNA with 1 copies/µL and 0.5 copies/µL for target and reference gene, respectively. This study establishes the basis for using a digital PCR-based assay as an efficient test to identify animals illegally treated with glucocorticoids.

Assessment of Antimicrobial Effects on Broiler Gut Barrier Through Histopathology and Immunohistochemistry of Tight-Junction Proteins.

In recent years, antimicrobial (AM) use in poultry farming has been attracting attention worldwide mainly due to AM resistance spreading. The role of AM prophylaxis in the modulation of gut microbiota, as well as of gut health, is still not clearly understood. Therefore, this study aimed to investigate the role of different prophylaxis protocols in the modulation of the gut barrier in broilers by applying a histopathological approach. Intestinal tissue samples were collected from a total of 240 male broilers (Ross 306), reared and treated with different AM protocols. Haematoxylin and Eosin (HE) staining and a multiple scoring system were used to evaluate the presence of lesions in ileum, cecum and colon of treated broilers. Moreover, immunohistochemistry (IHC) was performed to assess the expression of claudin-3 and ZO-1 proteins in intestinal tissues. The application of a semi-quantitative scoring system was used in IHC stained samples. HE results revealed that intestinal tissues were mainly characterized by epithelial detachment and fusion of the intestinal villi, but also by the presence of lymphocytic infiltrate in the mucosa and submucosa of AM-treated broilers. However, the IHC approach for the evaluation of claudin-3 and ZO-1 proteins showed that their expression was not affected by the different AM treatments. Nevertheless, the presence of intestinal lesions highlighted by histopathology suggests that AM treatments could harm the gut health of broilers, inducing an inflammatory response and consequent epithelial lesions. In order to clarify the role of AM treatments in the modulation of gut barrier in broilers, further studies are needed.

16S rRNA Sequencing Analysis of the Gut Microbiota in Broiler Chickens Prophylactically Administered with Antimicrobial Agents.

In poultry production, gut microbiota (GM) plays a pivotal role and influences different host functions related to the efficiency of production performances. Antimicrobial (AM) use is one of the main factors affecting GM composition and functions. Although several studies have focused their attention on the role of AMs as growth promoters in the modulation of GM in broilers, the consequences of higher AM concentrations administered during prophylactic treatments need to be better elucidated. For this purpose, 16S rRNA gene sequencing was performed to evaluate the impact of different prophylactic AM protocols on the composition and diversity of the broiler GM. Diversity analysis has shown that AM treatment significantly affects alpha diversity in ileum and beta diversity in both ileum and caecum. In ileal samples, the Enterobacteriaceae family has been shown to be particularly affected by AM treatments. AMs have been demonstrated to affect GM composition in broiler. These findings indicate that withdrawal periods were not enough for the restoral of the original GM. Further studies are needed for a better elucidation of the negative effects caused by an altered GM in broilers.