Association of prognostic features and treatment on survival time of dogs with systemic mastocytosis: A retrospective analysis of 40 dogs.

Systemic mastocytosis is a rare phenomenon, with limited information regarding prognostic features and effective treatment of canine patients with this disease. The objective of this study is to determine the impact of certain features and treatments on dogs with systemic mastocytosis. The medical records of 40 dogs from 4 northeastern US veterinary hospitals, with evidence of systemic mast cell disease, were evaluated retrospectively. Variables analysed with relation to overall survival and prognostic significance included treatment protocol used, substage, presence of a cutaneous or visceral tumour, presence of multiple cutaneous Mast cell tumours, grade of the primary tumour and metastatic site(s). Dogs with metastatic disease confined to distant lymph nodes lived longer than those with circulating mast cells in the blood (P = .001), and those with metastatic disease evident in more than 2 sites had a worse prognosis than those with disease in a single location (P = .005). Additionally, administration of chemotherapeutic agents led to increased survival over prednisone therapy alone (P = .008), with the combination of lomustine, vinblastine and prednisone prolonging survival over the tyrosine kinase inhibitor, toceranib phosphate (P = .002). Presence of mast cells in the blood and/or evidence of disease in more than 2 sites indicate widespread dissemination suggesting their use as negative prognostic features. Furthermore, a chemotherapy protocol including combination lomustine and vinblastine therapy may be more effective than toceranib phosphate for the treatment of dogs with disseminated mast cell disease. Overall, patients with systemic mastocytosis have a grave prognosis and more effective treatment options are needed.

Dynamics of the microbiota found in the vaginas of dairy cows during the transition period: Associations with uterine diseases and reproductive outcome.

We investigated the microbiota found in the vaginas of Holstein dairy cows during the transition period and described the differences in bacterial composition and total bacterial load (TBL) associated with disease and fertility. Vaginal swabs were collected at -7, 0, 3, and 7 d relative to parturition from 111 dairy cows housed on a commercial dairy farm near Ithaca, New York. Microbiota were characterized by next-generation DNA sequencing of the bacterial 16S rRNA gene, and TBL was determined by real-time quantitative PCR. We applied repeated-measures ANOVA to evaluate the associations of uterine disease and related risk factors with the microbiota and TBL. We estimated phylum-specific bacterial load by multiplying the TBL by the relative abundance of each phylum observed in the metagenomics results. We confirmed the validity of this approach for estimating bacterial load by enumerating the number of bacteria in an artificial sample mixed in vitro and in clinical and healthy vaginal samples. Phyla associated with uterine disease and related risk factors were Proteobacteria, Fusobacteria, and Bacteroidetes. Cows with retained placenta and healthy cows had similar TBL at the day of parturition, but at d 7 postpartum, cows with retained placenta showed a significantly higher TBL, mainly driven by higher estimated loads of Fusobacteria and Bacteroidetes. Cows diagnosed with metritis had a significantly higher estimated load of Proteobacteria at d -7 and at calving and higher estimated loads of Fusobacteria in the postpartum samples. Additionally, the estimated load of Bacteroidetes at d 7 postpartum was higher for cows diagnosed with endometritis at 35 days in milk. Higher estimated loads of Fusobacteria and Bacteroidetes were also evident in cows with postpartum fever, in primiparous cows, in cows with assisted parturition, and in cows that gave birth to twins. Our findings demonstrated that microbiota composition and TBL were associated with known periparturient risk factors of uterine diseases and reproductive failure, including parity, assisted parturition, and retained fetal membranes.

The microbiome of bulk tank milk: Characterization and associations with somatic cell count and bacterial count.

Numerous studies have evaluated associations between bacterial groups and milk quality parameters. However, to our knowledge, no research has been published that has analyzed associations between the microbiome and quality parameters of bulk tank milk (BTM). Thus, the aims of this study were to identify the core microbiome of BTM and to examine associations between the microbiome and milk quality parameters. Four hundred seventy-two BTM samples from 19 different dairy farms located in New York State were analyzed by next-generation sequencing and quantitative PCR of the 16S rRNA gene to assess the milk microbiome and measure total bacterial load, respectively. Flow cytometry was used to determine bacterial and somatic cell counts. Heatmaps were constructed and simple linear regressions and response screening analysis were performed. To facilitate data analysis and interpretation of the results, we dichotomized the BTM samples into high (HSCC, >200,000) and low somatic cell count (LSCC, ≤200,000) and into high (HSPC, >3.6) and low log10 SPC (LSPC, ≤3.6). Spoilage-causing, spore-forming, and pathogenic bacteria of importance to the dairy industry were identified in the core microbiome. In addition, the taxa Thermoanaerobacterium and 5-7N15 were identified in the core microbiome; to our knowledge, these genera have not been previously identified in milk samples. Several bacterial genera were encountered in significantly higher relative abundances in the HSCC group when compared with the LSCC group, including Corynebacterium, Streptococcus, Lactobacillus, Coxiella, Arthrobacter, and Lactococcus. Additionally, several bacterial taxa were found in significantly higher relative abundances in the HSPC groups versus the LSPC groups: Acinetobacter, Enterobacteriaceae, Corynebacterium, and Streptococcus. In addition, Streptococcus was highly correlated with HSPC, and this genus was the second most abundant bacterial taxon detected in samples classified as HSCC. Bacterial diversity (Shannon index) was negatively correlated with bacterial load, suggesting that the microbiomes of high-bacterial load BTM samples are dominated by smaller groups of bacterial taxa. In conclusion, the associations described corroborated current knowledge about pathogens and spoilage bacteria in relationship to milk quality, and also indicated that other bacterial taxa should be a focus of further investigations.

The Lactococcus genus as a potential emerging mastitis pathogen group: A report on an outbreak investigation.

The bacterium Lactococcus lactis is widely used in food production and in medical applications, and is considered safe for human and animal use. However, studies have also linked Lactococcus bacteria to infection. For example, certain variants of Lactococcus species have been associated with bovine mastitis (e.g., Lactococcus lactis and Lactococcus garvieae). In this study, we investigated an outbreak of bovine mastitis thought to be associated with Lactococcus bacteria by using microbiological and molecular techniques. We used bacterial isolation, next-generation sequencing, DNA fingerprinting, and other methods to test our hypothesis that Lactococcus microbes were the primary pathogen causing the mastitis outbreak. Twenty-eight Lactococcus isolates were obtained from mastitic milk of 28 dairy cows. The isolates were identified as L. lactis (27 isolates) and L. garvieae (1 isolate). Phylogenetic analysis based on 16S rDNA gene sequence comparison indicated similarity among the L. lactis isolates as well as between the isolates and reference sequences. The DNA fingerprinting analysis based on random amplified polymorphic DNA results of the 27 L. lactis isolates identified different random amplified polymorphic DNA profiles, which suggests they originated from multiple sources. Microbiome analysis determined Lactococcus to be the dominant genus in the majority of the mastitic milk samples, whereas it was found in low relative abundance in healthy milk samples. The Lactococcus genus was detected in all environmental samples tested, and sampling of bulk tank milk corroborated that Lactococcus was not abundant in healthy milk from the same dairy herd. In summary, our findings suggest that Lactococcus bacteria are a potential etiological agent in the mastitis outbreak studied. Further studies should be conducted to understand the importance of Lactococcus, especially L. lactis, as pathogenic microbes in veterinary medicine and food safety.

Effect of an injectable trace mineral supplement containing selenium, copper, zinc, and manganese on immunity, health, and growth of dairy calves.

The objective of this study was to evaluate the effect of 2 subcutaneous injections of a multimineral preparation, each containing 60 mg of zinc, 10mg of manganese, 5mg of selenium, and 15 mg of copper at 3 and 30 d after birth on immunity, health, and growth of dairy calves during the preweaning period. The study was conducted in upstate New York in 2 commercial dairy farms. A total of 790 Holstein heifer calves were randomly allocated at birth into 1 of 2 treatments: trace mineral supplement (TMS) treated or control. Blood samples were collected at 3, 14, and 35 d after birth to evaluate glutathione peroxidase (GPx) activity, superoxide dismutase (SOD) activity, haptoglobin, and neutrophil and monocyte function. Incidence of diseases and average daily gain was evaluated in the first 50 d of life. At 14 d of life, TMS-treated calves had increased neutrophil activity compared with control calves. Moreover, TMS-treated calves had greater GPx activity on d 14 after birth than control calves. The TMS treatment reduced the incidence of diarrhea (TMS=41.7% vs. control=49.7%) and combined incidence of pneumonia or otitis or both (TMS=41.7% vs. control=49.1%). Additionally, GPx was greater for calves diagnosed with otitis at d 35 after birth. However, calves diagnosed with pneumonia had decreased GPx activity at d 35 after birth. Serum SOD and haptoglobin concentrations were not affected by treatment or disease. Moreover, no effects were observed on average daily gain and survivability between TMS-treated and control calves during the preweaning period. Supplementation with trace minerals at 3 and 30 d of life increased neutrophil function and GPx activity and reduced the incidence of health disorders.

The effect of injectable trace minerals (selenium, copper, zinc, and manganese) on peripheral blood leukocyte activity and serum superoxide dismutase activity of lactating Holstein cows.

The objective of this study was to evaluate the effect of subcutaneous supplementation of 300 mg of zinc, 50 mg of manganese, 25 mg of selenium, and 75 mg of copper on peripheral blood leukocyte activity and serum ß-hydroxybutyrate (BHBA) concentrations at 10 ± 2 days in milk (DIM), and on serum superoxide dismutase (SOD) activity during the transition period and subsequent lactation of multiparous Holstein cows. A total of 250 multiparous cows were randomly allocated into one of two treatments groups, namely, trace mineral supplemented (TMS) or control. Cows in the TMS group were injected at 230 and 260 days of gestation, and 35 days postpartum. Serum SOD activity was measured at enrollment, and 10, 60 and 100 DIM. Serum BHBA concentration and leukocyte function were assessed at 10 DIM. Overall serum SOD activity for TMS and control was 16.01 and 12.71 U/mL, respectively. The interaction between treatment and time of serum collection was significant. Additionally, overall serum SOD activity was 12.85 and 14.78 U/mL for cows diagnosed with mastitis and unaffected cows, respectively. Treatment did not affect leukocyte function. For parity >2, TMS cows had lower serum BHBA concentrations than control cows; BHBA concentrations were 0.41 and 0.27 mmol/L for control and TMS cows, respectively. In conclusion, cows diagnosed with mastitis had decreased serum SOD activity, and trace mineral supplementation increased serum SOD activity although leukocyte function was not affected by supplementation.

The effects of drugs, ions, and poly-l-lysine on the excretory system of Schistosoma mansoni

We have been able to label the excretory system of cercariae and all forms of schistosomula, immature and adult worms with the highly fluorescent dye resorufin. We have shown that the accumulation of the resorufin into the excretory tubules and collecting ducts of the male adult worm depends on the presence of extracellular calcium and phosphate ions. In the adult male worms, praziquantel (PZQ) prevents this accumulation in RPMI medium and disperses resorufin from tubules which have been prelabelled. Female worms and all other developmental stages are much less affected either by the presence of calcium and phosphate ions, or the disruption caused by PZQ. The male can inhibit the excretory system in paired female. Fluorescent PZQ localises in the posterior gut (intestine) region of the male adult worm, but not in the excretory system, except for the anionic carboxy fluorescein derivative of PZQ, which may be excreted by this route. All stages of the parasite can recover from damage by PZQ treatment in vitro. The excretory system is highly sensitive to damage to the surface membrane and may be involved in vesicle movement and damage repair processes. In vivo the adult parasite does not recover from PZQ treatment, but what is inhibiting recovery is unknown, but likely to be related to immune effector molecules.

The effects of drugs, ions, and poly-l-lysine on the excretory system of Schistosoma mansoni.

We have been able to label the excretory system of cercariae and all forms of schistosomula, immature and adult worms with the highly fluorescent dye resorufin. We have shown that the accumulation of the resorufin into the excretory tubules and collecting ducts of the male adult worm depends on the presence of extracellular calcium and phosphate ions. In the adult male worms, praziquantel (PZQ) prevents this accumulation in RPMI medium and disperses resorufin from tubules which have been prelabelled. Female worms and all other developmental stages are much less affected either by the presence of calcium and phosphate ions, or the disruption caused by PZQ. The male can inhibit the excretory system in paired female. Fluorescent PZQ localises in the posterior gut (intestine) region of the male adult worm, but not in the excretory system, except for the anionic carboxy fluorescein derivative of PZQ, which may be excreted by this route. All stages of the parasite can recover from damage by PZQ treatment in vitro. The excretory system is highly sensitive to damage to the surface membrane and may be involved in vesicle movement and damage repair processes. In vivo the adult parasite does not recover from PZQ treatment, but what is inhibiting recovery is unknown, but likely to be related to immune effector molecules.

Lateral diffusion of human CD2 wild type and mutants with large deletions in the transmembrane domain.

Integral membrane proteins anchor to the cell surface and span the lipid bilayer by an alpha-helix of 17-30 amino acids, the transmembrane segment. However, little is known about the association of this alpha-helix and the lipid bilayer. In the present study human CD2 molecule was chosen as a model for an integral membrane protein. Truncate forms with transmembrane segments 14 and 12 amino acids long were created by oligonucleotide site-directed mutagenesis. Lateral diffusion revealed that even large deletions in the membrane domain of CD2 do not interfere with its lateral mobility. On the other hand, the fraction of free molecules for diffusion was higher in CD2 protein with transmembrane region 12 amino acids long. These results suggest that deletions in the transmembrane domain can interfere with the stability of the protein within the lipid bilayer.

Altered behaviour of carbohydrate-bound molecules and lipids in areas of the tegument of adult Schistosoma mansoni worms damaged by praziquantel.

By using fluorescent probes the distribution and fluid properties of lipid and saccharide-bound molecules was assessed in the tegument of praziquantel (-) treated Schistosoma mansoni adult male worms. Our results show that higher amounts of glycoproteins and/or glycolipids are exposed in damaged areas of the membrane compared with undamaged ones. At damaged regions these molecules present high lateral diffusion coefficient and mobile fraction values which suggests that after praziquantel(-) treatment they are being shed by the worm into the medium. The lateral diffusion coefficient of the lipid analogue 5'-octadecanoyl aminofluorescein is unchanged in damaged or undamaged areas but the mobile fraction is significantly reduced at damaged areas. The immunological significance of these altered surface properties is discussed.

Effects of culture and praziquantel on membrane fluidity parameters of adult Schistosoma mansoni.

Membrane fluidity parameters, lateral diffusion coefficient and mobile fraction were assessed in male and female Schistosoma mansoni teguments immediately after their recovery from infected mice or after 24 or 48 h of culture. Our results show that male and female teguments have different properties and behave differently when in culture. In general, male worms displayed a tendency to increase their lateral diffusion coefficient while females showed a significant reduction. The effects of praziquantel (-) and praziquantel (+) on the fluidity properties of the worm surface were also studied. It was demonstrated that both enantiomers of the drug interact with the tegument inducing a decrease in the average velocity of lipid molecules. However, it is only the active form which reduces the number of molecules that are able to move. In explanation we propose that praziquantel (-) and praziquantel (+) have different ways of inserting into the tegument and that some of the effects of the drug are mediated by altering membrane fluidity.