Quantification of zolpidem in canine plasma

Problem statement: Zolpidem is a non-benzodiazepine hypnotic agent currently used in human medicine. In contrast to benzodiazepines, zolpidem preferentially binds with the GABAA complex πvariant 1 receptors while poorly interacting with the other πvariant receptor complexes. Recent studies have suggested that ZP may be used to initiate sedation and diminish severe anxiety responses in dogs. The aim of the present study is to develop and validate a new HPLC-FL based method to quantify zolpidem in canine plasma. Approach: Several parameters both in the extraction and in the detection method were evaluated. The applicability of the method was determined by administering zolpidem to one dog. Results: The final mobile phase was acetonitrile: KH2PO4 (15 mM; pH 6.0) 40:60 v/v, with a flow rate of 1 mL min-1 and excitation and emission wave lengths of 254 and 400 nm, respectively. The best extraction solvent was CH2Cl2:Et2O (3:7 v/v), this gave recoveries ranging from 83-95%. The limit of quantification was 1 ng mL-1. The chromatographic runs were specific with no interfering peaks at the retention times of the analyte. The other validation parameters were in agreement with the EMEA. Conclusion/Recommendations: This method (extraction, separation and applied techniques) is simple and effective. This technique may have applications for pharmacokinetic or toxicological studies. © 2012 Science Publications

Study of Macrophage Activity in Cats with FIP and Naturally FCoV-Shedding Healthy Cats

Coronavirus frequently infects humans and animals, showing the ability to recombine and cross over to different species. Cats can be considered a model for studying coronavirus infection, in which feline coronavirus (FCoV) represents a major enteric pathogen related to gastroenteric disease. In this animal, the virus can acquire tropism for macrophage cells, leading to a deadly disease called feline infectious peritonitis (FIP). In this study, monocyte-derived macrophages were isolated by CD14-positive selection in venous whole blood from 26 cats with FIP and 32 FCoV-positive healthy cats. Phagocytosis and respiratory burst activities were investigated and compared between the groups. This is the first study comparing macrophage activity in cats affected by FIP and healthy cats positive for FCoV infection. Our results showed that in cats with FIP, the phagocytic and respiratory burst activities were significantly lower. Our results support the possible role of host immunity in Coronaviridae pathogenesis in cats, supporting future research on the immune defense against this systemic disease

Prevalence of Dog Erythrocyte Antigen 1 in 7, 414 Dogs in Italy

The study aim was to establish the prevalence of DEA 1, the most immunogenic and clinically important blood group in canine blood transfusion, in 7,414 dogs from Italy. The potential sensitization risk following a first transfusion and the acute reaction risk following a second transfusion given without a cross-matching and blood typing test were also calculated. Dogs tested were purebred (4,798) and mongrel (2,616); 38.8% were DEA 1 negative and 61.2% were DEA 1 positive. High prevalence for DEA 1 positive blood type was found in Ariegeois and English Setter, whereas German Shepherd and Boxer had higher DEA 1 negative blood type. Breeds with blood type never reported before included French Brittany Spaniel and Pug showing a high prevalence of DEA 1 positive type, while French Bulldog and West Highland White Terrier were more often DEA 1 negative. Just 48.8% of purebred and 13.9% of mongrel dogs were considered as prospective blood donors based upon their blood type.Most of the breeds had a sensitization risk of 20.0–25.0%. Rottweiler and Ariegeois had less risk of sensitization (9.4 and 4.2%) and the minor risk of an acute transfusional reaction (0.9–0.2%).The prevalence of DEA 1 positive and negative dogs in Italy agrees withmost of the data already reported in the literature

Abnormal findings in haemograms of Dachshund puppies: Presumptive (Immunodeficiency) familial disease

In a litter of seven Dachshund puppies, one subject was stillborn and six presented with diffuse skin infections characterized by dermatitis, abscessation, pustules, crusts and ulcers. Two of the puppies were referred for further evaluation. One male puppy was referred at four months of age and a sister litter mate was referred two months later. A complete blood count, biochemical and histological examination were performed on these puppies to identify the pathologic process. Clinical, biochemical, haematological and histological evaluation of subjects. This report characterizes quantitative and qualitative haematological abnormalities in two puppies that resulted in a diagnosis of dysmyelopoiesis. The existence of a familial immunodeficiency syndrome was speculated. This is the first report of such a syndrome in Dachshund puppie

INTESTINAL MICROBIOTA IN LYMPHOMA: A COMPARISON IN HEALTY DOGS AND DOGS WITH NON HODGKIN LYMPHOMA

Background and objectives Animal models play a key role in understanding the importance of gut microbiome in immune development and composition as well as to reinforce the relationship between the microbiota and health and disease. Intestinal bacteria have been implicated in several types of cancer. Regardless, microbes influence immune cells directly, indirectly, or both, and increased lymphocyte proliferation can lead to a higher chance of aberrant DNA replication. This particularly occurs with some B lymphocytes which are innately vulnerable to genetic instability and activation. Methods We analyzed the microbiome (by using 16S rRNA gene sequencing and qPCR assays) of naturally voided fecal samples from 12 healthy and 12 Non Hodgkin Lymphoma (NHL) dogs in order to evaluate the microbiota composition using a dysbiosis index. An index value greater than 2 indicates dysbiosis, while below 0 indicates normal microbiota. Results. Significant differences were observed when comparing the fecal microbiota structure of all healthy dogs vs NHL dogs (ANOSIM; P<0.05). Specifically, differences were observed for Faecalibacterium (P<0.001) with concentrations higher in healthy vs NHL dogs. The dysbiosis index was significantly lower (p=0.007) in healthy vs NHL dogs (mean, SD: H2.6, 2.0 vs 1.7, 3.2), respectively. Conclusion Interestingly, lower levels of Fecalibacterium prausnitzii were recently found in humans with some chronic colonic conditions as well as colorectal cancer (P < 0.001) compared with healthy subjects. This study showed that NHL have a increased dysbiosis index, indicating dysbiosis. Animal models of cancer can be critical in order to demonstrate a link between the microbiome and carcinogenesis

Fecal microbiota differences in Non-Hodgkin Lymphoma (NHL) affected dogs: preliminary results.

Animal models play an essential role in understanding the importance of gut microbiome in immune development and composition, and play a key role to reinforce the relationship between the microbiome and health and disease [3]. Non-Hodgkin Lymphoma (NHL) is the most common hematopoietic malignancy in dogs, caused by clonal proliferation of lymphocytes in solid organs [2]. Whether microbes influence immune cells directly, indirectly, or both, increased lymphocyte proliferation can lead to a higher chance of aberrant DNA replication, particularly in some B lymphocytes which are innately vulnerable to genetic instability and activation. Oxidative stress caused by intestinal microbiota, either directly or indirectly through the immune system, can also affect tumorigenesis, thus, the microbiota can affect several pathways associated with lymphomagenesis [4]. The optimal responses to cancer therapy require an intact commensal microbiota that mediates its effects, by modulating myeloid derived cell functions in the tumour microenvironment [1]. In our study design we analysed the microbiome (by using 16S rRNA gene 454-pyrosequencing and qPCR assays) of naturally voided fecal samples from 6 healthy dogs, 8 NHL dogs before and 4 NHL (of the eight) dogs after induction phase of chemotherapy (cyclophosphamide, vincristine, and prednisolone) plus probiotics (Sivoy TM). Several statistical significances were observed compared the fecal microbiome of healthy dogs vsNHL dogs before chemotherapy. In particular, differences were observed for Bifidobacteria (p=0.0001), Lactobacillus (p=0.0001), Faecalibacterium (p=0.0005), Bacteroidetes (p=0.0480), and Fusobacterium (p=0.0025), which concentrations were higher in healthy dogs compared to NHL dogs. On the contrary, the concentration of Clostridium perfrigens was greater in NHL dogs compared to healthy dogs (p=0.0326). No statistical differences for total bacteria, Escherichia coli, Blautia, and Ruminococcaceae were found. Microbiome shift (total bacteria, Bifidobacteria, Lactobacillus, Faecalibacterium, Bacteroidetes, Fusobacterium, Escherichia coli, Blautia, Ruminococcaceae, and Clostridium perfrigens) of fecal samples were also compared before and after induction phase of chemotherapy plus probiotics (Sivoy TM probiotic mix Slab51, containing 8 strains of lactic acid bacteria and bifidobacteria dosed at 200 billion per stick) but no statistical significance was found. In order to understand microbiome’s changes in NHL affected dogs treated with standard protocol plus probiotics, a larger number of stool samples before and after treatment, from a greater number of animals, should be investigated. The fact that an increased number of lymphomas are becoming associated with bacterial infections underscores the need for more studies involving microbes and lymphoma and about the use of probiotics to restore normal microbiota in affected dogs

Fluorescence Biomodulation for Canine Interdigital Furunculosis: Updates for Once-Weekly Schedule

Interdigital furunculosis is a common multifactorial, inflammatory disease of the canine interdigital skin in which lesions commonly become secondarily infected. Fluorescence biomodulation (FBM) administered twice weekly has shown to effectively control clinical manifestation as adjunct therapy to systemic antibiotic. Since twice weekly regimen could be unaffordable for some pet owners, the aim of this study was to evaluate the effect of once weekly application of FBM in combination with systemic antibiotic on clinical manifestations of canine interdigital pyoderma, comparing the results to those present in literature. Twelve dogs diagnosed with interdigital pyoderma received antibiotic plus once weekly FBM application. Dogs were scored until complete healing based on global lesion score and neutrophil engulfing bacterial score. The results obtained demonstrated that once weekly application of FBM exerts the same beneficial effect on interdigital furunculosis healing as per twice weekly, indicating that once weekly regimen is well tolerated and is yielding similar results to twice weekly applications

Therapeutic approaches to coronavirus infection according to “One Health” concept

Coronaviridae constantly infect human and animals causing respiratory, gastroenteric or systemic diseases. Over time, these viruses have shown a marked ability to mutate, jumping over the human-animal barrier, thus becoming from enzootic to zoonotic. In the last years, numerous therapeutic protocols have been developed, mainly for severe acute respiratory syndromes in humans. The aim of this review is to summarize drugs or other approaches used in coronavirus infections focusing on different roles of these molecules or bacterial products on viral adhesion and replication or in modulating the host's immune system. Within the "One Health" concept, the study of viral pathogenic role and possible therapeutic approaches in both humans and animals is essential to protect public health

Evaluation of the dog population in two Italian shelters in Central Italy (Marche region) as potential blood donors

Background: In recent years, the field of transfusion medicine for dogs has advanced significantly, becoming a crucial aspect of veterinary clinical practice. Nowadays, blood still remains a fundamental biological source and the welfare and health status of eligible species-specific blood donors are essential for veterinary transfusion medicine. Aim: This study focused on evaluating two shelters in Central Italy (Marche region), located in Tolentino (TS) and in Camerino (CS), in order to assess the potential of the shelter dogs as canine blood donors. Methods: We evaluated a total of 45 dogs from these shelters based on physical (age and size), clinical, behavioural, and blood analysis criteria described in the Italian Ministerial Guideline for Veterinary Transfusion Medicine (2016). Results: At the TS shelter, out of 206 resident dogs, 125 met the donation criteria (60.68%), with 28 (13.59%) selected for the study due to the impossibility to collect the samplings or other exclusion causes. In the CS shelter, of the 149 dogs, 17 (11.41%) were identified as potential blood donors and included in the study. Among these, seven dogs (25%) from TS and five dogs (29.41%) from CS were found to have DEA1 negative blood group. High percentages (TS = 25.24%, CS = 40.27%) of dogs were excluded for seniority and 29.53% in CS for behavioural issues. Notable findings included reduced erythrocyte mean corpuscular volume [mean TS = 63.93 fl; CS = 64.00 fl] and Reticulocyte Hemoglobin [mean TS = 22.39 pg; CS = 21.38 pg]. Additionally, in both shelters’ dogs showed a modest increase in eosinophils levels [mean TS = 1.59 K/μl; CS = 1.02 K/μl]. Conclusion: Shelter dogs can fulfill the blood donation requirements set by the Italian Ministerial Guideline on Veterinary Transfusion Medicine. They are generally in good health and present a low risk of transmitting parasitic diseases; however, many are ineligible for donation due to behavioural pathologies or temperament issues and seniority

INVESTIGATION OF CRP AND OTHER HEMATIC INFLAMMATION MARKERS IN DOGS

The systemic reaction to acute inflammation, also known as acute phase response, induces some hemato-biochemical changes, which can be evidenced in laboratory findings. The C Reactive Protein (CRP) is a main acute phase response protein elective in dogs to detect inflammatory disease. Other blood parameters have been described as useful inflammation markers i.e. Fibrinogen and Leukocytes (1, 2, 3). The aim of this retrospective study was to investigate the CRP values in comparison to Fibrinogen (Fib), Albumin (Alb), and Iron (Fe) values, total White Blood Cell (WBC), Segmented Neutrophil (NeuSeg) and Band Neutrophil (Band) counts, and the occurrence of Toxic Neutrophils (Neu TOX), Activated Monocytes (Mon ATT), and Reactive Lymphocytes (Linf REA) in blood smears. For this purpose, data of 1,837 blood samples was collected over a three-year period (2012-2015). Data collected for each sample included: Fib, Alb, Fe, WBC, NeuSeg, Band, Neu TOX, Mon ATT, Linf REA and CRP, as well as information regarding dog’s age, breed, and gender. Blood samples were divided into 2 groups: "inflammatory";; CRP ≥0.30 mg/dL (#1080) and non-inflammatory;; CRP ≤0.29 mg/dL (#757). The 2 groups were compared using: Chi squared for sex, breed, and age; Relative risk (RR) for age; Spearman Rank correlation test (SRct) for all parameters studied; Multiple regression (MR) to assess the relationship between CRP and other inflammation markers; Receiver Operating Characteristic (ROC) curves for diagnostic accuracy of each parameter in comparison to CRP (MedCalc®, 14.8). Dogs belonging to inflammatory group were significantly older (>7 years old) than those of non-inflammatory group (P<0.05), (RR, 1.38). Low yet significant (p<0.01) correlations between CRP and the other markers were noted using the SRct (R): CRP/Fib, +0.26; CRP/NeuSeg, +0.26; CRP/WBC, +0.24; CRP/Alb, -0.21; CRP/Band, +0.14; CRP/Fe, -0.08; CRP/NeuTOX, +0.23; MonATT, +0.22. On the contrary, the MR analysis did not show any relationship between CRP and other markers (R2: 0.05 for CRP ≥ 0.30 mg/dL;; 0.02 for CRP ≤ 0.29 mg/dL). ROC analysis of the parameters yielded the following results: NeuSeg is a moderately accurate inflammation marker with Area Under the Curve (AUC) of 0.71. The other parameters are less accurate markers of inflammation (AUC) compared to CRP: WBC, 0.70; Fib, 0.67; Alb, 0.64; Fe, 0.64; Band, 0.59. The markers with the best combination of Sensitivity (SS) and Specificity (SP) were: Fib (SS, 52.7; SP, 77.5 for 400 mg/dL cut-off) and Band (SS, 17.6; SP, 98.0 for 0.3 K/μL cut-off). The correlation between CRP and all the parameters studied, except Linf REA, is significant but low because they are affected by many conditions aside from inflammation. None of them is able to predict CRP values. The diagnostic accuracy of each single inflammatory marker is lower in comparison to CRP. In order to increase the diagnostic accuracy of inflammation markers, an evaluation of several parameters simultaneously is warranted, particularly in the absence of CRP measurement