Case of mastocytemia and systemic mastocytosis in a dog: ProCyte Dx and Sysmex XT-2000i scattergram findings, morphologic features, and c-kit somatic mutation analysis.

A 10-year-old female Golden Retriever was presented for a recheck after the complete removal of low-grade complex mammary carcinoma. The in-house ProCyte Dx automated counts revealed moderate regenerative anemia and moderate eosinophilia. The ProCyte Dx WBC scattergram showed a cloud in an unusual place parallel and to the right of the monocyte dot plot location. Cells were classified as either monocytes or neutrophils with no clear separation. Complete blood count analysis performed in the laboratory on a Sysmex XT-2000iV analyzer showed moderate regenerative anemia and WBC count within RI; a differential count was not provided by the instrument. On the Sysmex XT-2000iV DIFF scattergram, neutrophil and eosinophil dot plots were present at the respective locations and appeared separated, but the instrument did not provide numerical results. In addition to the normal lymphocyte dot plot location, the second cloud of cells classified as lymphocytes was displayed to the right of the monocyte dot plot area. On the WBC/BASO scattergram, the second population of cells was present above and to the right of the leukocyte cluster. Morphologic assessment of the blood smear detected mastocytemia with 16% poorly granulated and degranulated mast cells. FNAs from the liver and spleen contained large aggregates of poorly granulated mast cells. C-kit somatic mutation screening detected the presence of point mutation S479I in exon 9 of the canine c-KIT gene. This is the first description of abnormal scattergrams from ProCyte Dx and Sysmex XT-2000iV analyzers in a dog with concurrent mastocytemia and systemic mastocytosis, and where cytologic assessments of a blood smear, liver, and spleen, and c-kit somatic mutation analysis were performed.

Use of serum amyloid A in equine medicine and surgery.

Serum amyloid A (SAA) has become an indispensable part of the management of equine patients in general practice and specialized hospital settings. Although several proteins possess acute phase properties in horses, the usefulness of SAA exceeds that of other acute phase proteins. This is due to the highly desirable kinetics of the equine SAA response. SAA concentrations exhibit a rapid and pronounced increase in response to inflammation and a rapid decline after the resolution of inflammation. This facilitates the detection of inflammatory disease and real-time monitoring of inflammatory activity. SAA may be used in all stages of patient management: (1) before diagnosis (to rule in/rule out inflammatory disease), (2) at the time of diagnosis (to assess the severity of inflammation and assist in prognostication), and (3) after diagnosis (to monitor changes in inflammatory activity in response to therapy, with relapse of disease, or with infectious/inflammatory complications). By assessing other acute phase reactants in addition to SAA, clinicians can succinctly stage inflammation. White blood cell counts and serum iron concentration change within hours of an inflammatory insult, SAA within a day, and fibrinogen within 2-3 days; the interrelationship of these markers thus indicates the duration and activity of the inflammatory condition. Much research on the equine SAA response and clinical use has been conducted in the last decade. This is the prerequisite for the evidence-based use of this analyte. However, still today, most published studies involve a fairly low number of horses. To obtain solid evidence for use of SAA, future studies should be designed with larger sample sizes.

Comparison of six microscopic methods and two operators for estimation of white and red blood cells in canine urine sediment.

BACKGROUND: Little information is currently available about the analytical variability of urinalysis. OBJECTIVE: We aimed to compare results obtained by two operators using six microscopic methods in the quantification of urinary leukocytes (WBC) and erythrocytes (RBC). METHODS: Forty urine samples (10 mL) were centrifuged (450g, 5 minutes) and resuspended in 0.5 mL of supernatant. Two operators with different expertise in urinalysis interpreted sediment results using the six methods, obtained by combining the use of microscope slides (Slide) or counting chambers (Chamber) with three different techniques: bright-field (BF) microscopy, phase-contrast (PC) microscopy, and stained sediment (SS) evaluations. The mean WBC and RBC counts from 10 fields (Slide) or squares (Chamber) observed at 400× were used to calculate the difference and agreement between operators and methods. We also estimated the concordance between methods in classifying microhematuric or pyuric samples. RESULTS: Operator 2 counted significantly lower WBC counts using Slide+BF (P = 0.009) and Slide+PC (P = 0.001) than Operator 1, whereas no inter-operator differences were recorded for RBC counts. The concordance between the operators ranged from "good" to "very good." No differences or biases were found for WBC counts among the methods, and concordances were "good" to "very good"; proportional biases were found for RBC counts between Slide+BF vs Slide+SS and Slide+PC vs Slide+SS. Concordance measurements for RBC counts ranged from "good" to "very good." CONCLUSIONS: All methods yielded good reproducibility among operators, although stained SS evaluations allowed better identification of WBC by the inexperienced operator. However, we suspected that the SS preparations affected RBC counts. All other methods yielded reproducible WBC and RBC counts.

Establishment of preliminary reference intervals and cytochemical staining of blood cells in big-bellied seahorses (Hippocampus abdominalis).

BACKGROUND: Despite their popularity, hematology reference intervals (RIs) have not been established in big-bellied seahorses (Hippocampus abdominalis). OBJECTIVE: The objectives of this study were to establish hematologic RIs to compare values between sex in regard to cytochemical staining of blood cells. We also sought to compare white blood cell concentrations using the Natt and Herrick technique vs blood smear estimates. METHODS: Forty-three healthy individuals from the Aquarium du Québec (22 females and 21 males) were included. Normal health status was confirmed by an unremarkable physical examination in five individuals and by necropsy of five other individuals, of which all were excluded from further analyses. Venipuncture was performed from the ventral coccygeal vein in the remaining 33 individuals without anesthesia using heparinized insulin syringes. A blood volume of 0.05 to 0.1 ml was collected to prepare Wright Giemsa-stained blood smears and hematocrits immediately after venipuncture. Whole blood was stored in heparinized Eppendorf tubes to determine red and white blood cell concentrations using the Natt and Herrick technique with a hemocytometer in 10 individuals; these results were compared with blood smear estimates. Additional blood smears were stained with alkaline phosphatase substrate, periodic acid Schiff, and toluidine blue stains. RESULTS: The reference intervals included the packed cell volume (27.4-67.5%), thrombocyte count (19.5-197.7 × 109 /L), and white blood cell (WBC) count (2-54.8 × 109 /L), including neutrophils (1.1-21.3 × 109 /L), lymphocytes (2.7-45.5 × 109 /L), and monocytes (0-2.2 × 109 /L). The WBC hemocytometer counts showed no correlation with blood smear estimates (Spearman's rho = 0.2). There was also no significant difference between the sexes. CONCLUSIONS: These preliminary reference intervals will help assess the health of seahorses.

Hematologic Reference Intervals and Comparison of Natt-Herrick Technique and Smear-Based Leukocyte Estimation in Cockatiels (Nymphicus hollandicus).

Although cockatiels are among the most common avian species maintained as companion animals in the United States, information on standard hematologic reference values for this species is limited. The objectives of this study were to establish hematologic reference intervals (RI) for cockatiels, compare methods using both the Natt-Herrick technique (NHT) and the smear-based estimation technique (SBT), explore age and sex differences in the hematologic findings for this species, and produce the first cockatiel RI for fibrinogen concentration and thrombocyte estimate. Healthy cockatiels (60 males and 60 females, 2-11 years old) from a research colony were included in this study. Blood samples were placed in dipotassium ethylenediaminetetraacetic acid tubes, and erythrocyte counts and thrombocyte estimates were determined via automated analyzer (ADVIA 120) and SBT, respectively. Moreover, leukocyte concentrations were determined using both NHT and SBT to compare these common methods for measuring a complete blood count in cockatiels. Data were analyzed for outliers, distributions, descriptive statistics, and RI via Reference Value Adviser, a set of macroinstructions for Microsoft Excel (Microsoft, Redmond, WA, USA). Lymphocytes were the predominant leukocyte across both methods. According to the NHT, females had significantly higher concentrations of total leukocytes, heterophils, bands, lymphocytes, basophils, and total plasma protein compared with males. Significant inverse polynomial relationships were noted between total leukocyte count and age and lymphocyte counts and age for NHT. Total leukocyte count produced via NHT and SBT were compared using Passing-Bablok and Bland-Altman plots, and no significant constant or proportional biases were found. However, these methods showed wide limits of agreement. While the RI were interchangeable between methods from a clinical standpoint, the same method should be used to assess changes in an individual. The reported RI are uniquely robust given the sample size, balanced sex and age distributions, inclusion criteria, and control over sample collection.

Peripheral blood eosinophilia in dogs: Prevalence and associated diseases.

BACKGROUND: Canine eosinophilia has not been evaluated over the last two decades. As in human local differences, changes in the prevalence and associated diseases over time can be expected. OBJECTIVE: This study aims to determine the prevalence and causes of marked blood eosinophilia in dogs. METHODS: Retrospective study. A total of 317 clinical histories of dogs with an eosinophil concentration > 1.5 × 109 /L (marked eosinophilia) between 2013 and 2017 were evaluated. Patients were allocated to 10 groups according to their major clinical findings. RESULTS: Eosinophilia was present in 1,592 of 10,829 dogs (14.7%); it was mild (0.8-1.49 × 109 /L) in 78.4%, moderate (1.5 - 4.9 × 109 /L) in 20.5% and severe (> 5 × 109 /L) in 1.1% of cases. Rottweilers were overrepresented (16.1%). Of 317 cases with marked eosinophilia, 19.6% had neoplasia, 19.1% gastrointestinal disorders, 13.6% health check, 10.4% endoparasites, 6% respiratory, 5.4% neurologic, 5.4% dermatologic, 4.8% urogenital, 3.2% endocrine disorders and 12.6% miscellaneous. Lymphomas (29%) and mast cell tumours (12.9%) were the most frequent tumours in the neoplasia group. A total of 72.6% of tumour-bearing dogs were older than 8 years, while 63.6% of dogs had endoparasites, and 86% of apparently healthy dogs were younger than 5 years. Eosinophilia was significantly higher in patients with respiratory disorders (p < 0.0146). Leukocytosis was found in 50.2% of cases. CONCLUSION: Malignancy was the most common cause of marked blood eosinophilia in older dogs and endoparasitism in younger dogs. Eosinophilia was common in apparently healthy young dogs and may be related to undiagnosed parasitic infestations.

Etiology and outcome of extreme neutrophilic leukocytosis: A multi-institutional retrospective study of 269 dogs.

BACKGROUND: The magnitude of diagnostic abnormalities can influence the perception of clinical outcome. Extreme neutrophilic leukocytosis (ENL) is an uncommon finding caused by markedly increased granulopoiesis. A lack of recent, large-scale studies limits our understanding of the importance, causation, and prognosis associated with ENL in dogs. HYPOTHESIS/OBJECTIVES: Describe disease categories (DC) identified in dogs with ENL and identify variables associated with survival. We hypothesized that factors including fever, segmented and band neutrophil counts, and DC would be negatively associated with survival. ANIMALS: Two-hundred sixty-nine dogs with ENL (segmented neutrophils ≥50 × 103  cells/µL) presented to the veterinary teaching hospitals at Auburn University (n = 164), the University of Missouri (n = 81), and Oklahoma State University (n = 24) between January 1, 2009 and December 31, 2019. METHODS: Retrospective study. Demographic data and outcome variables including temperature, CBC findings, DC, duration of hospitalization (DOH) and outcome were acquired from the medical record. Statistical analyses included chi-squared and Kruskal-Wallis tests, and Pearson product moment correlations with a P < .05 significance level. RESULTS: Mortality was 41%. Survival differed with DC (P = .002). Mortality was higher (P < .05) in dogs with neoplasia (56.2%) vs immune-mediated disease (20.5%) or tissue damage/necrosis (19%). Weight (P = .001, r = -0.14) and total neutrophil count (P = .04, r = -0.02) were weakly negatively associated with survival whereas DOH was weakly positively associated with survival (P = .03, r = 0.14). CONCLUSIONS AND CLINICAL IMPORTANCE: Mortality in dogs with ENL is high but differed according to DC. Only weak correlations between clinical or clinicopathologic variables and mortality were identified. Extreme neutrophilic leukocytosis should be interpreted in conjunction with the underlying disease process, and not broadly used to predict clinical outcome.

Quantitative and functional dynamics of circulating and endometrial polymorphonuclear leukocytes in healthy peripartum dairy cows.

The aim of the present study was to assess the counts, viability, and functionality of circulating and endometrial polymorphonuclear leukocytes (PMN) isolated from fourteen clinically and metabolically healthy multiparous dairy cows in the peripartum period. For this, blood samples were collected at -5, +9, +21 and + 37 days (d) relative to calving. Cytology samples were collected from the vagina, cervix, and uterus at +9, +21 and + 37 d, using the cytobrush technique. Additional vaginal samples were collected at -5 d. Cytology smears were prepared and the PMN-to-all nucleated cell proportions (PMN%) were calculated. The endometrial cytobrush samples were also used for flow cytometric assessment of endometrial PMN (ePMN) viability and functionality. Functionality tests for circulating PMN (cPMN) included phagocytosis (PC), oxidative burst, and intracellular proteolytic degradation. For ePMN, we evaluated PC only. The effect of day relative to calving on PMN viability and functionality were fitted in linear regression models, accounting for repeated measures. The endometrial PMN% were higher at +9 d (23.5 ± 0.4%; least-squares means ± standard error) and +21 d (8.5 ± 0.3%) than at +37 d (1.4 ± 0.3%). No changes in PMN% were found on either vaginal or cervical cytology along the peripartum period. The cPMN counts were higher pre- (6.2 ± 0.4 x 106/mL) than postpartum (4.9 ± 0.4 x 106/mL). Upon viability analysis, only the percentage of viable cPMN tended to be lower at -5 d (90.1 ± 1.5%) than at +37 d (94.1 ± 1.4%), and no other changes in the percentage of apoptotic and necrotic cPMN, nor in their functionality were found during the peripartum period. Analysis of ePMN viability showed that the percentage of viable ePMN did not change over time. In marked contrast, the percentage of apoptotic ePMN was higher at +9 d (37.8 ± 5.1%) than at +21 d (20.9 ± 5.1%) and +37 d (11.9 ± 5.3%), while the percentage of necrotic ePMN was lower at +9 d (27.0 ± 6.3%) than at +37 d (54.9 ± 6.6%). The percentage of ePMN PC was higher at +9 d (27.5 ± 3.4%) than at +37 d (13.3 ± 4.9%). In conclusion, during the peripartum period ePMN in the healthy postpartum uterus are highly dynamic in terms of counts, viability, and functionality compared to their circulating counterparts.

Stress induced by pre-slaughter farm conditions in pigs / [Estresse induzido pelas condições da fazenda pré-abate em suínos]

Cortisol is a steroid hormone, one of the glucocorticoids, made in the cortex of the adrenal glands and then released into the blood, which transports it in the entire body. Almost every cell contains receptors for cortisol and so cortisol can have lots of different actions depending on which sort of cells it is acting upon. These effects include controlling the body's blood sugar levels and thus regulating metabolism, acting as an anti-inflammatory product, controlling salt and water balance and influencing blood pressure. The study was conducted over a period of 3 months, between March-August 2020, in 2 swine farms in Iasi county, Romania, on a total of 46 pigs, 3 to 4 months old, both males and females, in order to investigate stress levels in finishing facilities. The study revealed higher levels of cortisol while eosinophil counts severely decreased, changes which are associated with a strong reaction to stress for individuals that were housed in finishing facilities.(AU)

O cortisol é um hormônio esteroide, um dos glicocorticoides, produzido no córtex das glândulas suprarrenais e, em seguida, liberado no sangue, que o transporta por todo o corpo. Quase todas as células contêm receptores para o cortisol e, portanto, ele pode ter muitas ações diferentes, dependendo do tipo de célula sobre a qual atua. Esses efeitos incluem controlar os níveis de açúcar no sangue do corpo e, assim, regular o metabolismo, atuando como um produto anti-inflamatório, controlando o equilíbrio de sal e água e influenciando a pressão arterial. O presente estudo foi realizado durante um período de três meses, entre março-agosto de 2020, em duas granjas de suínos no condado de Iasi, Romênia, em um número total de 46 porcos, de três a quatro meses de idade, machos e fêmeas, a fim de investigar níveis de estresse nas instalações de acabamento. O estudo revelou níveis mais elevados de cortisol, enquanto a contagem de eosinófilos diminuiu severamente, mudanças que são consideradas associadas a uma forte reação ao estresse para indivíduos que foram alojados em instalações de acabamento.(AU)

Effects of Season, Location, Species, and Sex on Hematologic and Plasma Biochemical Values and Body Mass in Free-ranging Grebes (Aechmophorus species).

The effects of season, location, species, and sex on body weight and a comprehensive array of blood chemistry and hematology analytes were compared for free-ranging western (Aechmophorus occidentalis) and Clark's (Aechmophorus clarkii) grebes. Birds (n = 56) were collected from Puget Sound, WA, and Monterey Bay and San Francisco Bay, CA, from February 2007 to March 2011. The data supported generalization of observed ranges for most analytes across Aechmophorus grebe metapopulations wintering on the Pacific coast. Notable seasonal and location effects were observed for packed cell volume (winter 6% greater than fall; winter California [CA] 5% greater than Washington [WA]), total white blood cell count (CA 3.57 × 103 cells/µL greater than WA), heterophils (WA 10% greater than CA), lymphocytes (winter 19% greater than fall), heterophil to lymphocyte ratio (fall 5.7 greater than winter), basophils (CA greater than WA), plasma protein (WA about 10 g/L [1.0 g/dL] greater than CA), plasma protein to fibrinogen ratio (winter about 15 greater than fall), potassium (CA 2 mmol/L greater than WA), and liver enzymes (alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase: WA greater than CA). Within California, season had a greater effect on body mass than sex (mean winter weights about 200 g greater than fall), whereas within a season, males weighed only about 80 g more than females, on average. These data give biologists and veterinarians quantitative reference values to better assess health at the individual and metapopulation level.

Inflammation and its association with oxidative stress in dogs with heart failure.

BACKGROUND: Inflammation and oxidative stress can contribute to the development and progression of heart failure. This study aimed to investigate the association between inflammatory and oxidative stress markers in dogs with congestive heart failure (CHF). Associations between the disease severity marker N-terminal pro-B-type natriuretic peptide (NT-proBNP) and markers of inflammation and oxidative stress were also determined. RESULTS: Thirty-seven dogs with cardiovascular diseases (dilated cardiomyopathy, DCM (16 dogs), myxomatous mitral valve disease, MMVD (21 dogs)) and ten healthy dogs were included in this prospective study. The patients were further divided into groups with (26) and without CHF (11). We found a significantly higher serum concentration of C-reactive protein (P = 0.012), white blood cell (P = 0.001), neutrophil (P = 0.001) and monocyte counts (P = 0.001) in patients with CHF compared to control dogs. The concentration of tumor necrosis factor-alpha (TNF-α) was significantly higher in patients with CHF compared to patients without CHF (P = 0.030). No significant difference was found in most of the measured parameters between MMVD and DCM patients, except for glutathione peroxidase (GPX) and NT-proBNP. In patients with CHF, TNF-α correlated positively with malondialdehyde (P = 0.014, r = 0.474) and negatively with GPX (P = 0.026, r = - 0.453), and interleukin-6 correlated negatively with GPX (P = 0.046, r = - 0.412). NT-proBNP correlated positively with malondialdehyde (P = 0.011, r = 0.493). In patients without CHF none of the inflammatory and oxidative stress markers correlated significantly. Furthermore, in the group of all cardiac patients, GPX activity significantly negatively correlated with NT-proBNP (P = 0.050, r = - 0.339) and several markers of inflammation, including TNF-α (P = 0.010, r = - 0.436), interleukin-6 (P = 0.026, r = - 0.382), white blood cell (P = 0.032, r = - 0.369), neutrophil (P = 0.027, r = - 0.379) and monocyte counts (P = 0.024, r = - 0.386). CONCLUSION: Inflammatory and oxidative stress markers are linked in canine CHF patients, but not in patients without CHF. These results suggest complex cross communication between the two biological pathways in advanced stages of CHF.

Impact of preheparinization and sample volume on routine hematology findings in healthy cats.

OBJECTIVES: It can be challenging to collect sufficient blood from feline patients for both a biochemical profile and a complete blood count (CBC). The ability to generate accurate hematologic and biochemical data from a single, small (<2 ml) sample could reduce patient stress and improve clinical efficiency. The objective of this study was to determine the impact of preheparinization and/or sample size on routine hematology findings in cats. METHODS: Blood was collected from 20 healthy cats; measured aliquots were placed directly into tubes containing either EDTA or lithium-heparin (Hep). Within 2 mins, specific volumes were removed from the Hep tubes and placed in additional EDTA tubes. Four distinct sample sizes/types were created from each cat: (1) 1.3 ml EDTA (criterion standard); (2) 0.5 ml EDTA; (3) 1.3 ml Hep + EDTA; and (4) 0.5 ml Hep + EDTA. Three CBCs were performed on each sample using an automated bench-top hematology analyzer. Drops of blood were contemporaneously used to create three air-dried stained slides from each tube. Triplicate results were averaged for statistical analysis; results were compared across all sample types and against the criterion standard. Significance was set at P <0.05. RESULTS: Preheparinization did not significantly impact determinations of erythrocyte number, hematocrit, hemoglobin concentration, mean cell volume and neutrophil count. Platelet counts for the non-traditional samples correlated poorly with the criterion standard, although numbers could be effectively estimated using stained slides. Cell morphology was well preserved across all sample types. CONCLUSIONS AND RELEVANCE: These results indicate that a 0.5 ml preheparinized EDTA blood sample can generate clinically useful hematologic data (excluding platelet count) in cats, using a bench-top analyzer. Our findings support the collection of a single small (<2 ml) sample that can be used for both biochemical and hematologic analyses. Further studies are needed to verify these findings using other hematology machines and in diseased cats.

Expression-based analysis of genes related to single nucleotide polymorphism hits associated with bovine leukemia virus proviral load in Argentinean dairy cattle.

In dairy cattle infected with bovine leukemia virus (BLV), the proviral load (PVL) level is directly related to the viral transmission from infected animals to their healthy herdmates. Two contrasting phenotypic groups can be identified when assessing PVL in peripheral blood of infected cows. A large number of reports point to bovine genetic variants (single nucleotide polymorphisms) as one of the key determinants underlying PVL level. However, biological mechanisms driving BLV PVL profiles and infection progression in cattle have not yet been elucidated. In this study, we evaluated whether a set of candidate genes affecting BLV PVL level according to whole genome association studies are differentially expressed in peripheral blood mononuclear cells derived from phenotypically contrasting groups of BLV-infected cows. During a 10-mo-long sampling scheme, 129 Holstein cows were phenotyped measuring anti-BLV antibody levels, PVL quantification, and white blood cell subpopulation counts. Finally, the expression of 8 genes (BOLA-DRB3, PRRC2A, ABT1, TNF, BAG6, BOLA-A, LY6G5B, and IER3) located within the bovine major histocompatibility complex region harboring whole genome association SNP hits was evaluated in 2 phenotypic groups: high PVL (n = 7) and low PVL (n = 8). The log2 initial fluorescence value (N0) transformed mean expression values for the ABT1 transcription factor were statistically different in high- and low-PVL groups, showing a higher expression of the ABT1 gene in low-PVL cows. The PRRC2A and IER3 genes had a significant positive (correlation coefficient = 0.61) and negative (correlation coefficient = -0.45) correlation with the lymphocyte counts, respectively. Additionally, the relationships between gene expression values and lymphocyte counts were modeled using linear regressions. Lymphocyte levels in infected cows were better explained (coefficient of determination = 0.56) when fitted a multiple linear regression model using both PRRC2A and IER3 expression values as independent variables. The present study showed evidence of differential gene expression between contrasting BLV infection phenotypes. These genes have not been previously related to BLV pathobiology. This valuable information represents a step forward in understanding the BLV biology and the immune response of naturally infected cows under a commercial milk production system. Efforts to elucidate biological mechanisms leading to BLV infection progression in cows are valuable for BLV control programs. Further studies integrating genotypic data, global transcriptome analysis, and BLV progression phenotypes are needed to better understand the BLV-host interaction.

Methyl donor supply to heat stress-challenged polymorphonuclear leukocytes from lactating Holstein cows enhances 1-carbon metabolism, immune response, and cytoprotective gene network abundance.

Mechanisms controlling immune function of dairy cows are dysregulated during heat stress (HS). Methyl donor supply-methionine (Met) and choline (Chol)-positively modulates innate immune function, particularly antioxidant systems of polymorphonuclear leukocytes (PMN). The objective of this study was to investigate the effect of Met and Chol supply in vitro on mRNA abundance of genes related to 1-carbon metabolism, inflammation, and immune function in short-term cultures of PMN isolated from mid-lactating Holstein cows in response to heat challenge. Blood PMN were isolated from 5 Holstein cows (153 ± 5 d postpartum, 34.63 ± 2.73 kg/d of milk production; mean ± SD). The PMN were incubated for 2 h at thermal-neutral (37°C; TN) or heat stress (42°C; HS) temperatures with 3 levels of Chol (0, 400, or 800 µg/mL) or 3 ratios of Lys:Met (Met; 3.6:1, 2.9:1, or 2.4:1). Supernatant concentrations of IL-1ß, IL-6, and tumor necrosis factor-α were measured via bovine-specific ELISA. Fold-changes in mRNA abundance were calculated separately for Chol and Met treatments to obtain the fold-change response at 42°C (HS) relative to 37°C (TN). Data were subjected to ANOVA using PROC MIXED in SAS (SAS Institute Inc., Cary, NC). Orthogonal contrasts were used to determine the linear or quadratic effect of Met and Chol for mRNA fold-change and supernatant cytokine concentrations. Compared with PMN receiving 0 µg of Chol/mL, heat-stressed PMN supplemented with Chol at 400 or 800 µg/mL had greater fold-change in abundance of CBS, CSAD, GSS, GSR, and GPX1. Among genes associated with inflammation and immune function, fold-change in abundance of TLR2, TLR4, IRAK1, IL1B, and IL10 increased with 400 and 800 µg of Chol/mL compared with PMN receiving 0 µg of Chol/mL. Fold-change in abundance of SAHH decreased linearly at increasing levels of Met supply. A linear effect was detected for MPO, NFKB1, and SOD1 due to greater fold-change in abundance when Met was increased to reach Lys:Met ratios of 2.9:1 and 2.4:1. Although increasing Chol supply upregulated BAX, BCL2, and HSP70, increased Met supply only upregulated BAX. Under HS conditions, enhancing PMN supply of Chol to 400 µg/mL effectively increased fold-change in abundance of genes involved in antioxidant production (conferring cellular processes protection from free radicals and reactive oxygen species), inflammatory signaling, and innate immunity. Although similar outcomes were obtained with Met supply at Lys:Met ratios of 2.9:1 and 2.4:1, the response was less pronounced. Both Chol and Met supply enhanced the cytoprotective characteristics of PMN through upregulation of heat shock proteins. Overall, the modulatory effects detected in the present experiment highlight an opportunity to use Met and particularly Chol supplementation during thermal stress.

Translocation of intrauterine-infused bacterial lipopolysaccharides to the mammary gland in dexamethasone-treated goats.

Our previous study showed that intrauterine-infused lipopolysaccharide (LPS) can be translocated to the mammary gland to induce weak inflammation. This study aimed to determine whether dexamethasone treatment facilitated the translocation of LPS from the uterus to the mammary gland to induce a heavy inflammatory response. Sixteen goats were divided into control and LPS groups, subjected to daily dexamethasone administration before saline or LPS infusion. Milk and blood samples were collected before and after LPS infusion to determine the milk yield and somatic cell count (SCC) and blood leucocyte count (BLC), cytokines, antimicrobial peptides and serum amyloid A (SAA) concentrations. Mammary gland tissues were collected from two goats before and 24 hr after LPS infusion for immunohistochemical analysis of LPS. The mean SCC in the LPS group was significantly higher, whereas the milk yield was significantly lower than that in the control group after LPS infusion. The mean BLC in the LPS group was significantly lower than in the control group after LPS infusion. Furthermore, milk concentrations of IL-1ß, S100A8 and lactoferrin were higher in the LPS group than in the control group after infusion. LPS was detected in the connective tissues and inner alveolar spaces of the mammary glands 24 hr after LPS infusion. We concluded that dexamethasone administration facilitated the translocation of intrauterine-infused LPS to the mammary gland, where it induced an inflammatory response. Therefore, LPS translocated from other organs, such as the uterus, can induce heavy inflammation in the mammary gland under immunosuppressive conditions.

Cytobrush cytology patterns of early postpartum dairy cows.

Endometrial cytobrush cytology has been recommended as a reliable method for determining the percentage of polymorphonuclear leukocytes (PMN%) in cattle smears to diagnose cytological endometritis (CE). In this study, the clarity of cytobrush　cytological smears and the influence of different sample evaluation methods (number　and types of cells counted) on CE diagnosis were evaluated. Samples from 28 lactating　Holstein cows were collected weekly between 3 and 7 weeks postpartum. Smear clarity,　based on cell density, quality of cell morphology, and red blood cell contamination,　was significantly poorer at 3 weeks than between 5 and 7 weeks postpartum. Five　different cell counting methods (C100, C200, C300, C400, and C500) were used,　where 100-500 nucleated cells (endometrial epithelial cells, PMN consisting of neutrophils, eosinophils and basophils, lymphocytes, and macrophages) were counted. Agreement of diagnostic results for CE between C300 and C500 and between C400 and C500 was excellent at all observation times. In calculations of the PMN% based on whether the number of lymphocytes and macrophages were or were not excluded in the denominator, exclusion of these cells in the calculations did not affect the diagnosis of CE. While reduced clarity in earlier stage samples might interfere with the accuracy of cytobrush cytology, C300 can be recommended to determine the endometrial PMN%.

Dairy cattle with bovine leukaemia virus RNA show significantly increased leukocyte counts.

Infection with bovine leukaemia virus (BLV), a retrovirus, causes dysfunction of the immune system and can have a marked economic impact on dairy industries due to decreased milk production and reduced lifespan in affected dairy cattle. The presence of proviral DNA has been the major diagnostic indicator of BLV infection. However in the course of BLV infection, the viral genome can be dormant, without detectable gene expression, resulting in limited impact on infected animals. At present, there is limited knowledge regarding haematological indices in dairy cattle that could indicate activation of the BLV genome and suggest reactivated BLV infection. In this study, BLV infection and BLV genome reactivation were evaluated based on the presence of BLV DNA and BLV env gene transcripts, respectively. BLV RNA transcription was confirmed. Among 93 whole blood samples obtained from asymptomatic dairy cattle, the prevalence of BLV proviral DNA and transcripts was 93.5% (n = 87/93) and 83.9% (n = 78/93), respectively. Between groups with and without BLV, the mean counts of white blood cells and lymphocytes in whole blood were significantly associated with the presence of BLV RNA (P < 0.05), but not with BLV proviral DNA. These results shed light on the activation status of the BLV genome and should be taken into account when evaluating the possible impact of BLV on cattle.

Sanitary conditions of the Goliath grouper Epinephelus itajara in captivity on estuarine environment: case report / Condições sanitárias do mero Epinephelus itajara em cativeiro em ambiente estuarino: relato de caso

This study aimed to report the sanitary conditions through the hematological analysis of grouper E. itajara reared in captivity on estuarine conditions. Seven Goliath groupers (1,881.5±1,246.03g) were captured and kept in two tanks located on estuary. After 20 days, fish were collected for morphologic and hemato-physiologic evaluation. Two fish had clinical signs such as hemorrhagic spots and loss of scale due to agonistic behavior. Blood samples were collected, and the hematological parameters (biochemical, erythrogram and leukogram) were determined. Blood cells were characterized by their size, color and shape. Univariate statistic and principal components analysis were used to identify a hematological standard between fish with or without clinical signs. Four leukocyte types were found: lymphocyte, monocyte, neutrophil and basophil. Regardless of the clinical signs the cell morphology did not present any difference among the fish. However, there is a significant correlation between erythrocyte and lactate on fish with clinical signs. Thus, agonistics encountered among the fish is a stressing factor in captivity conditions making it necessary to have adequate management related to the size of fish and stocking density.(AU)

Este estudo avaliou as condições sanitárias do peixe mero E. itajara mantido sob cativeiro, em condições estuarinas, pelas análises hematológica e morfológica. Sete peixes (1.881,5±1.246,03g) foram capturados e mantidos em tanques localizados no estuário. Após 20 dias, os peixes foram coletados para avaliações morfológica e hematológica. Dois peixes tiveram sinais clínicos, como manchas hemorrágicas e perda de escamas devido ao comportamento agonístico entre os peixes. Amostras sanguíneas foram coletadas dos peixes anestesiados com auxílios de seringas umedecidas com EDTA 3%. Determinaram-se os parâmetros hematológicos (bioquímico, eritrograma e leucograma). Células sanguíneas foram caracterizadas por seu tamanho, cor e padrão. Estatística univariada e análises de componentes principais foram usadas para identificar um padrão hematológico entre os peixes com e sem sinais clínicos. Quatro tipos de leucócitos foram encontrados: linfócito, monócito, neutrófilo e basófilo. Os sinais clínicos não apresentaram diferença entre os peixes e a morfologia celular. Contudo, observou-se uma correlação entre os peixes com sinais clínicos e a quantidade de eritrócitos e lactato. Assim, encontros antagônicos entre os peixes são um fator estressante em condições de cativeiro, tornando-se necessário um manejo mais adequado relacionado ao tamanho dos indivíduos e à densidade de estocagem.(AU)

Intrauterine therapy with ozone reduces subclinical endometritis and improves reproductive performance in postpartum dairy cows managed in pasture-based systems.

New postpartum strategies have been developed in dairy cows to ameliorate uterine health and reproductive performance, especially the first service conception rates. This study aimed to assess the effect of intrauterine therapy with ozone (IUTO) in early postpartum on subclinical endometritis prevalence and reproductive parameters in dairy cows under commercial farm conditions. For this purpose, eighty clinically healthy cows with a body condition score between 3.0 and 3.5, from four dairy farms, were randomly allocated into two groups: ozone therapy group (OG, n = 40), which were subjected to IUTO, and control group (CG, n = 40). Content of uterine polymorphonuclear (PMN) leukocytes and subclinical endometritis (SE) percentage were assessed at 35 days after calving by uterine cytology. A second cytology was performed 72 h after IUTO. Reproductive parameters such as interval calving to first service (IFS), number of services per conception (nSC), interval calving to conception (ICC) and first service conception rate (FSCR) were analysed. The second endometrial cytology demonstrated that IUTO reduced (P < 0.01) both PMN (3.7 ± 1.4 vs. 7.6 ± 1.1%) and SE (5.0 vs. 50.0%) percentages compared with CG. Likewise, after ozone treatment, both nSC (2.1 ± 0.3 vs. 3.1 ± 0.2; P < 0.01) and ICC (126.2 ± 9.7 vs. 149.0 ± 9.0; P = 0.0672) decreased, and FSCR increased (50.0 vs. 16.2%; P < 0.01) compared with CG. In conclusion, intrauterine ozone therapy applied at 35 days after calving reduced subclinical endometritis prevalence and improved reproductive performance in postpartum dairy cows managed in a pasture-based system.

Effect of siltuximab, omalizumab, infliximab, pembrolizumab and vedolizumab on selected haematological and biochemical parameters in a pig model.

From the regulatory point of view a strong link between an animal model and human pharmacodynamics of biological drugs is very important to qualify the model as "relevant". Consistent changes in cell population between human physiology and animal model gain value of this model which then can be pharmacodynamically "relevant" from the regulatory point of view. Consequently, the aim of this study was to determine how similar to human observations is the effect of selected biological drugs on blood cells in a pig model. The study was to carry out a comparative analysis of the variability of selected biochemical and hematological parameters of the blood after administration of five different human therapeutic monoclonal antibodies (mAbs) after a single subcutaneous (SC) dose in breeding pigs. The tested drugs were siltuximab (Syl- vant®), omalizumab (Xolair®), infliximab (Inflectra®), pembrolizumab (Keytruda®), and vedoli- zumab (Entyvio®) given in a single 1 mg/kg SC injection. Each of the tested drugs exerted a sig- nificant effect on at least two of the tested parameters three weeks after the administration. Siltuximab significantly influenced 9 of the analyzed parameters. Vedolizumab significantly influenced 8 of the analyzed parameters. Infliximab had the lowest impact of all the tested drugs, as it significantly influenced only two of the analyzed parameters. The study has proved that the impact of mAbs on the analyzed parameters can be significantly extended over time. This requires the monitoring of hematological parameters in the pig model even many weeks after administration of a drug in a relatively small dose.