Time and sequence of emergence of the deciduous dentition in dogs and its applicability for age estimation.

This study investigated the influence of several covariates on the time and sequence of deciduous dentition emergence in puppies. Data were obtained in a longitudinal study, with some cross-sectional observations, of 1001 puppies of 53 dog breeds. A parametric proportional hazards survival model was used to estimate median emergence time and evaluate the effect of the covariates. No significant differences were found between the left and right sides of a puppy's dentition, but differences were statistically significant for the earlier appearance of maxillary incisors and canines and later appearance of maxillary premolars compared with their mandibular counterparts. The tendency for delayed onset and completion of emergence in female compared to male puppies was statistically but not clinically significant. The differences between puppies of breeds of different size or skull type were both statistically and clinically significant, with small and brachycephalic breeds showing later emergence times, longer clinical eruption times and more individual variation. Per quadrant, regardless of dog breed, canines or incisors were usually the first teeth to emerge and fully erupt, followed by premolars in the order Pd3 > 4 > 2. The maxillary canines and incisors usually emerged earlier than mandibular canines. Age estimation standards for breed size groups are presented based on the number of emerged teeth per quadrant. To assess whether a puppy has reached the legally required minimum age of 8 weeks to leave the litter, the best predictive capability using the data from this study is obtained when assessing the emergence status of the deciduous third premolars.

Depletion of Embryonic Macrophages Leads to a Reduction in Angiogenesis in the Ex Ovo Chick Chorioallantoic Membrane Assay.

Macrophages play an important but poorly understood role in angiogenesis. To investigate their role in vessel formation, relevant in vivo models are crucial. Although the chick chorioallantoic membrane (CAM) model has been frequently used as an angiogenesis assay, limited data are available on the involvement of chicken macrophages in this process. Here, we describe a method to deplete macrophages in the ex ovo chick CAM assay by injection of clodronate liposomes and show that this depletion directly affects vascularisation of collagen onplants. Chicken embryos were injected intravenously with either clodronate or phosphate-buffered saline (PBS) liposomes, followed by placement of collagen type I plugs on the CAM to quantify angiogenic ingrowth. Clodronate liposome injection led to a significant 3.4-fold reduction of macrophages compared with control embryos as measured by immunohistochemistry and flow cytometry. Furthermore, analysis of vessel ingrowth into the collagen plugs revealed a significantly lower angiogenic response in macrophage-depleted embryos compared with control embryos, indicating that chicken embryonic macrophages play an essential function in the development of blood vessels. These results demonstrate that the chick CAM assay provides a promising model to investigate the role of macrophages in angiogenesis.

A Study of Carry-Over and Histopathological Effects after Chronic Dietary Intake of Citrinin in Pigs, Broiler Chickens and Laying Hens.

Citrinin (CIT) is a polyketide mycotoxin occurring in a variety of food and feedstuff, among which cereal grains are the most important contaminated source. Pigs and poultry are important livestock animals frequently exposed to mycotoxins, including CIT. Concerns are rising related to the toxic, and especially the potential nephrotoxic, properties of CIT. The purpose of this study was to clarify the histopathological effects on kidneys, liver, jejunum and duodenum of pigs, broiler chickens and laying hens receiving CIT contaminated feed. During 3 weeks, pigs (n = 16) were exposed to feed containing 1 mg CIT/kg feed or to control feed (n = 4), while 2 groups of broiler chickens and laying hens (n = 8 per group) received 0.1 mg CIT/kg feed (lower dose group) and 3 or 3.5 mg CIT/kg feed (higher dose group), respectively, or control feed (n = 4). CIT concentrations were quantified in plasma, kidneys, liver, muscle and eggs using a validated ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method. Kidneys, liver, duodenum and jejunum were evaluated histologically using light microscopy, while the kidneys were further examined using transmission electron microscopy (TEM). Histopathology did not reveal major abnormalities at the given contamination levels. However, a significant increase of swollen and degenerated mitochondria in renal cortical cells from all test groups were observed (p < 0.05). These observations could be related to oxidative stress, which is the major mechanism of CIT toxicity. Residues of CIT were detected in all collected tissues, except for muscle and egg white from layers in the lowest dose group, and egg white from layers in the highest dose group. CIT concentrations in plasma ranged between 0.1 (laying hens in lower dose group) and 20.8 ng/mL (pigs). In tissues, CIT concentrations ranged from 0.6 (muscle) to 20.3 µg/kg (liver) in pigs, while concentrations in chickens ranged from 0.1 (muscle) to 70.2 µg/kg (liver). Carry-over ratios from feed to edible tissues were between 0.1 and 2% in pigs, and between 0.1 and 6.9% in chickens, suggesting a low contribution of pig and poultry tissue-derived products towards the total dietary CIT intake for humans.

PirABVP Toxin Binds to Epithelial Cells of the Digestive Tract and Produce Pathognomonic AHPND Lesions in Germ-Free Brine Shrimp.

Acute hepatopancreatic necrosis disease (AHPND), a newly emergent farmed penaeid shrimp bacterial disease originally known as early mortality syndrome (EMS), is causing havoc in the shrimp industry. The causative agent of AHPND was found to be a specific strain of bacteria, e.g., Vibrio and Shewanella sps., that contains pVA1 plasmid (63-70 kb) encoding the binary PirAVP and PirBVP toxins. The PirABVP and toxins are the primary virulence factors of AHPND-causing bacteria that mediates AHPND and mortality in shrimp. Hence, in this study using a germ-free brine shrimp model system, we evaluated the PirABVP toxin-mediated infection process at cellular level, including toxin attachment and subsequent toxin-induced damage to the digestive tract. The results showed that, PirABVP toxin binds to epithelial cells of the digestive tract of brine shrimp larvae and produces characteristic symptoms of AHPND. In the PirABVP-challenged brine shrimp larvae, shedding or sloughing of enterocytes in the midgut and hindgut regions was regularly visualized, and the intestinal lumen was filled with moderately electron-dense cells of variable shapes and sizes. In addition, the observed cellular debris in the intestinal lumen of the digestive tract was found to be of epithelial cell origin. The detailed morphology of the digestive tract demonstrates further that the PirABVP toxin challenge produces focal to extensive necrosis and damages epithelial cells in the midgut and hindgut regions, resulting in pyknosis, cell vacuolisation, and mitochondrial and rough endoplasmic reticulum (RER) damage to different degrees. Taken together, our study provides substantial evidence that PirABVP toxins bind to the digestive tract of brine shrimp larvae and seem to be responsible for generating characteristic AHPND lesions and damaging enterocytes in the midgut and hindgut regions.

Identification of a microscopically selected microorganism in milk samples.

Identification of unwanted microbial contaminants microscopically observed in food products is challenging due to their low abundance in a complex matrix, quite often containing other microorganisms. Therefore, a selective identification method was developed using laser capture microdissection in combination with direct-captured cell PCR. This procedure was validated with Geobacillus stearothermophilus and further used to identify microbial contaminants present in some industrial milk samples. The microscopically observed contaminants were identified as mainly Methylobacterium species.