Canine mammary gland disease in New Zealand: a review of samples from 797 dogs.

AIMS: To evaluate the proportions of canine mammary gland lesions submitted to a New Zealand diagnostic laboratory, that were neoplastic vs. non-neoplastic and, among neoplasms, malignant vs. benign, and to determine whether age, reproductive status or breed of dog, or size of the mammary mass were associated with the histological diagnosis. METHODS: Canine mammary gland biopsies submitted between the start of 2012 and the end of 2016 were selected from the surgical biopsy database of IDEXX Laboratories, NZ. For each case, details on age, breed, and reproductive status of the patient were registered as reported by the submitting veterinarians, along with the size (classified as small, medium or large) of the lesion and the histological diagnosis reported by the pathologists. χ2 tests and independent sample t-tests were performed to evaluate associations. RESULTS: Samples (n = 895) were submitted from 797 dogs, of which 673 had mammary neoplasms while 124 had non-neoplastic lesions. Neoplasms composed of a single nodule were found in 591/673 (87.8%) dogs, while 82/673 (12.2%) dogs had multiple nodules. Of the total 771 neoplasms, 432 (56.0%) were histologically malignant, while 339 (44.0%) were benign. Among malignancies, the most common histological sub-types were simple carcinoma (160/771; 20.8%), complex carcinoma (54/771; 7%), and ductal carcinoma (32/771; 4.2%), while benign mixed mammary tumour (128/771, 16.6%) and complex adenoma (105/771; 13.6%) were the most frequently reported benign mammary neoplasms. There was no evidence of a difference in age (p = 0.09) or reproductive status (p = 0.79) of the dog or the size of the mass (p = 0.21) between neoplastic and non-neoplastic lesions. However, neoplastic mammary gland lesions were more frequent in purebred dogs (612/671; 91.2%) than crossbred dogs (61/126; 48.4%; p < 0.001). There was no evidence of a difference in age (p = 0.15) reproductive status (p = 0.36) or breed (p = 0.45) of dog between malignant and benign neoplasms. There was an association between size and histological benign or malignant status of a neoplasm (φ = 0.65, p < 0.001). CONCLUSIONS: Most canine mammary gland samples submitted for examination were neoplastic with slightly more malignant than benign lesions. Masses submitted from purebred dogs were more likely to be neoplastic, while large neoplasms were more likely to be malignant. CLINICAL RELEVANCE: The present findings provide the first description of the distribution of mammary gland lesions in a relatively large number of dogs in New Zealand, representing a preliminary investigation of canine mammary gland diseases in this country.

Low Stromal Mast Cell Density in Canine Mammary Gland Tumours Predicts a Poor Prognosis.

Tumour histological classification and grade are frequently used to predict the prognosis of canine mammary gland tumours. While these techniques provide some information about tumour behaviour, it is currently difficult to predict which tumours will metastasize. Mast cell density has been shown to predict metastasis in human breast cancer. The present study investigated whether the average mast cell density in 10 high-power (×400) microscopical fields (10 HPFs), evaluated by toluidine blue staining, similarly predicted the behaviour of canine mammary gland tumours. Mast cell density was evaluated in 53 canine mammary neoplasms for which the clinical outcome was known. Stromal mast cell density in malignant tumours that had subsequently developed radiographical evidence of metastasis (n = 21) was significantly lower (P <0.001) than in malignant tumours that did not show evidence of metastases (n = 20) or in benign tumours (n = 12). The density of stromal mast cells that best predicted the disease outcome was ≤10/10 HPFs. Eighty-one percent of malignant tumours with ≤10 stromal mast cells/10 HPFs subsequently metastasized, while only 9.5% of malignant tumours with >10 stromal mast cells/10 HPFs developed metastases. There was a positive correlation between stromal mast cell density and survival time (rs = 0.50, P <0.001). These findings suggest that assessing stromal mast cell density using toluidine blue staining may represent an easy to perform and cost-effective histopathological measure that, in conjunction with classification and grading, could better predict the behaviour of canine mammary neoplasms.

Postmortem detection and histopathological features of canine spirocercosis-induced putative esophageal chondrosarcoma.

AIM: The objective of this study was to describe and characterize the postmortem and histopathological findings of putative esophageal chondrosarcoma associated with Spirocerca lupi. MATERIALS AND METHODS: Spirocerca-associated esophageal nodules were collected from 54 dogs at postmortem examination and were stained with hematoxylin and eosin. Of the cases examined, 15 were selected randomly for further investigation, of which 11 were classified as non-neoplastic nodules while 4 had changes reflecting a neoplastic process. RESULTS: In all four neoplastic cases, the wall of the esophageal nodules contained islands and nests of highly proliferative atypical chondroblasts within a cartilaginous matrix. However, there was no statistically significant association between gender (p=0.228), age (p=0.568), and breeds (p>0.05) with the occurrence of spirocercosis. Moreover, all esophageal nodules identified were located near the caudal segment, and their diameters ranged from 1 to 6 cm (4.7±1.5 cm). A number of worms in each nodule varied from 5 to 25 (11.3±5). CONCLUSION: Histopathology and cytology revealed that the wall of the esophageal nodules contained islands and nests of highly proliferative atypical chondroblasts within a cartilaginous matrix, a rare finding, and clinical challenge in spirocercosis.

Phylogenetic relationships and protein modelling revealed two distinct subfamilies of group II HKT genes between crop and model grasses.

Molecular evolution of large protein families in closely related species can provide useful insights on structural functional relationships. Phylogenetic analysis of the grass-specific group II HKT genes identified two distinct subfamilies, I and II. Subfamily II was represented in all species, whereas subfamily I was identified only in the small grain cereals and possibly originated from an ancestral gene duplication post divergence from the coarse grain cereal lineage. The core protein structures were highly analogous despite there being no more than 58% amino acid identity between members of the two subfamilies. Distinctly variable regions in known functional domains, however, indicated functional divergence of the two subfamilies. The subsets of codons residing external to known functional domains predicted signatures of positive Darwinian selection potentially identifying new domains of functional divergence and providing new insights on the structural function and relationships between protein members of the two subfamilies.

A comparative gene analysis with rice identified orthologous group II HKT genes and their association with Na(+) concentration in bread wheat.

BACKGROUND: Although the HKT transporter genes ascertain some of the key determinants of crop salt tolerance mechanisms, the diversity and functional role of group II HKT genes are not clearly understood in bread wheat. The advanced knowledge on rice HKT and whole genome sequence was, therefore, used in comparative gene analysis to identify orthologous wheat group II HKT genes and their role in trait variation under different saline environments. RESULTS: The four group II HKTs in rice identified two orthologous gene families from bread wheat, including the known TaHKT2;1 gene family and a new distinctly different gene family designated as TaHKT2;2. A single copy of TaHKT2;2 was found on each homeologous chromosome arm 7AL, 7BL and 7DL and each gene was expressed in leaf blade, sheath and root tissues under non-stressed and at 200 mM salt stressed conditions. The proteins encoded by genes of the TaHKT2;2 family revealed more than 93% amino acid sequence identity but ≤52% amino acid identity compared to the proteins encoded by TaHKT2;1 family. Specifically, variations in known critical domains predicted functional differences between the two protein families. Similar to orthologous rice genes on chromosome 6L, TaHKT2;1 and TaHKT2;2 genes were located approximately 3 kb apart on wheat chromosomes 7AL, 7BL and 7DL, forming a static syntenic block in the two species. The chromosomal region on 7AL containing TaHKT2;1 7AL-1 co-located with QTL for shoot Na(+) concentration and yield in some saline environments. CONCLUSION: The differences in copy number, genes sequences and encoded proteins between TaHKT2;2 homeologous genes and other group II HKT gene families within and across species likely reflect functional diversity for ion selectivity and transport in plants. Evidence indicated that neither TaHKT2;2 nor TaHKT2;1 were associated with primary root Na(+) uptake but TaHKT2;1 may be associated with trait variation for Na(+) exclusion and yield in some but not all saline environments.

Characterization of the multigene family TaHKT 2;1 in bread wheat and the role of gene members in plant Na(+) and K(+) status.

BACKGROUND: A member of the TaHKT2;1 multigene family was previously identified as a Na(+) transporter with a possible role in root Na(+) uptake. In the present study, the existing full-length cDNA of this member was used as a basis to query the International Wheat Genome Survey Sequence to identify all members of the TaHKT2;1 family. Individual TaHKT2;1 genes were subsequently studied for gene and predicted protein structures, promoter variability, tissue expression and their role in Na(+) and K(+) status of wheat. RESULTS: Six TaHKT2;1 genes were characterized which included four functional genes (TaHKT2;1 7AL-1, TaHKT2;1 7BL-1, TaHKT2;1 7BL-2 and TaHKT2;1 7DL-1) and two pseudogenes (TaHKT2;1 7AL-2 and TaHKT2;1 7AL-3), on chromosomes 7A, 7B and 7D of hexaploid wheat. Variability in protein domains for cation specificity and in cis-regulatory elements for salt response in gene promoters, were identified amongst the functional TaHKT2;1 members. The functional genes were expressed under low and high NaCl conditions in roots and leaf sheaths, but were down regulated in leaf blades. Alternative splicing events were evident in TaHKT2;1 7AL-1. Aneuploid lines null for each functional gene were grown in high NaCl nutrient solution culture to identify potential role of each TaHKT2;1 member. Aneuploid lines null for TaHKT2;1 7AL-1, TaHKT2;1 7BL-1 and TaHKT2;1 7BL-2 showed no difference in Na(+) concentration between Chinese Spring except for higher Na(+) in sheaths. The same aneuploid lines had lower K(+) in roots, sheath and youngest fully expanded leaf but only under high (200 mM) NaCl in the external solution. There was no difference in Na(+) or K(+) concentration for any treatment between aneuploid line null for the TaHKT2;1 7DL-1 gene and Chinese Spring. CONCLUSIONS: TaHKT2;1 is a complex family consisting of pseudogenes and functional members. TaHKT2;1 genes do not have an apparent role in controlling root Na(+) uptake in bread wheat seedlings under experimental conditions in this study, contrary to existing hypotheses. However, TaHKT2;1 genes or, indeed other genes in the same chromosome region on 7AL, are candidates that may control Na(+) transport from root to sheath and regulate K(+) levels in different plant tissues.

Challenges in testing genetically modified crops for potential increases in endogenous allergen expression for safety.

Premarket, genetically modified (GM) plants are assessed for potential risks of food allergy. The major risk would be transfer of a gene encoding an allergen or protein nearly identical to an allergen into a different food source, which can be assessed by specific serum testing. The potential that a newly expressed protein might become an allergen is evaluated based on resistance to digestion in pepsin and abundance in food fractions. If the modified plant is a common allergenic source (e.g. soybean), regulatory guidelines suggest testing for increases in the expression of endogenous allergens. Some regulators request evaluating endogenous allergens for rarely allergenic plants (e.g. maize and rice). Since allergic individuals must avoid foods containing their allergen (e.g. peanut, soybean, maize, or rice), the relevance of the tests is unclear. Furthermore, no acceptance criteria are established and little is known about the natural variation in allergen concentrations in these crops. Our results demonstrate a 15-fold difference in the major maize allergen, lipid transfer protein between nine varieties, and complex variation in IgE binding to various soybean varieties. We question the value of evaluating endogenous allergens in GM plants unless the intent of the modification was production of a hypoallergenic crop.

Establishing objective detection limits for the pepsin digestion assay used in the assessment of genetically modified foods.

RATIONALE: Guidelines for assessing the potential allergenicity of genetically modified (GM) organisms recommend testing the digestibility of the introduced protein by pepsin. Previous studies detailed the digestion procedure but have not described a simple objective measurement of the extent of digestion nor evaluated the impact of variation in pepsin activity. METHODS: Samples of eight proteins were digested by pepsin at pH 1.2 and 2.0 using standard conditions (10,000 U of pepsin activity per mg test protein) as well as 5000 and 20,000 units per mg of test protein. An independent digestion assay of hemoglobin was used to verify pepsin activity for each assay. Digestion was stopped in timed samples between 0.5 and 60 min. Digestion samples and undigested protein (10% and 100%) were separated by SDS-PAGE. Residual stained protein bands were measured by image analysis. RESULTS: The differences in pH and pepsin concentration only had minor effects on digestion of intermediately stable proteins: concanavalin A, ovalbumin, and lysozyme, but not on rapidly digested or stable proteins. CONCLUSIONS: Verification of pepsin activity and measurement of an objective endpoint of digestion (e.g. (90%) should provide more comparable results for the safety assessment of novel food proteins.