Effect of storage on the nitro blue tetrazolium reduction test in dog blood samples

Altres ajuts: acords transformatius de la UABIntroduction: The nitro blue tetrazolium (NBT) reduction test (NBTT) has been used for measuring the metabolic activity of phagocytes of mammals. Activated neutrophils transform NBT into formazan in the cytoplasm. The NBTT can detect the activation of neutrophils in peripheral blood and is used to assess neutrophil function in dogs. However, the NBTT is not used frequently in the clinical setting, as samples should be processed after blood collection. Objective: The aim of this study was to evaluate the effect of storage on NBTT in dog blood samples. Materials and Methods: Residual EDTA blood samples from 22 dogs were included of different ages, breeds, and sex. The buffy coat layer was separated from the blood and incubated with 0.1% NBT. The NBTT was performed at 0, 24, 48, and 72 h after the collection of blood. Blood samples were stored at 4°C until the tests were performed. Blood smears were evaluated by light microscopy, and the NBT reduction rate was reported, which represents the percentage of activated neutrophils. The NBT reduction rate was calculated after counting 300 neutrophils in each slide. Results: The means of NBTT in dog blood samples at 0, 24, 48, and 72 h were 8.3%, 8.5%, 8.7%, and 7.8%, respectively. No significant differences were observed between time points. Conclusions: This study showed that the NBTT can be performed up to 72 h after the collection of canine blood if correctly refrigerated at 4°C. This finding supports the performance of the NBTT in the clinical setting

Are dogs with clinical leishmaniosis co-infected with other vector-borne zoonotic and non-zoonotic pathogens?

Introduction: Canine leishmaniosis (CanL) due to Leishmania infantum is a zoonotic protozoan disease endemic in Spain, where other vector-borne pathogens are quite common (Rickettsia conorii, Ehrlichia canis, Anaplasma spp, Hepatozoon canis, Babesia spp). Dogs are considered the main peridomestic reservoir for L. infantum infection and its clinical manifestation can vary from a total absence of clinical signs and clinicopathological abnormalities to a severe fatal clinical illness. Furthermore, there is evidence that other vector-borne organisms might affect the severity of CanL or mimic its clinical signs and/or clinicopathological abnormalities. The aim of this study was to determine co-infections with other vector-borne pathogens based on serological and molecular techniques in dogs with clinical leishmaniosis living in the Mediterranean basin and to associate them with clinical signs and clinicopathological abnormalities as well as disease severity. Materials and methods: Sixty-two dogs with clinical leishmaniosis and sixteen apparently healthy dogs were tested for Rickettsia conorii, Ehrlichia canis and Anaplasma phagocytophilum by the immunofluorescence antibody test (IFAT) and for Ehrlichia canis, Anaplasma spp, Hepatozoon spp. and Babesia spp. by polymerase chain reaction (PCR). Results: Among the dogs examined by IFAT, the seroprevalences were: 69.3% for R. conorii, 57.3% for E. canis and 44.0% for A. phagocytophilum; while the prevalences found by PCR were: 7.9% for E. canis/Anaplasma, 2.6% for Anaplasma platys and 1.3% for H. canis. Statistical association was found between dogs with clinical leishmaniosis and seroreactivity to R. conorii antigen (P = 0.025; OR = 4.09) and A. phagocytophilum antigen (P = 0.002; OR = 14.34) and being positive to more than one serological or molecular tests (co-infections) (P = 0.013) when compared with healthy dogs. Interestingly, a statistical association was found between the presence of R. conorii, E. canis and A. phagocytophilum antibodies in sick dogs and some clinicopathological abnormalities such as a decrease of albumin and albumin/globulin ratio associated to the presence of R. conorii, A. phagocytophilum and E. canis high antibody titers, and an increase in serum globulins associated to the presence of A. phagocytophilum and E. canis high antibody titers. Furthermore, seroreactivity with A. phagocytophilum antigens was statistically associated with LeishVet clinical stages III and IV. Conclusion: This study demonstrates that dogs with clinical leishmaniosis from the Barcelona and Tarragona area have a higher rate of co-infections with other vector-borne pathogens when compared with healthy controls. Furthermore, positivity to other vector-borne pathogens was associated with more pronounced clinicopathological abnormalities as well as disease severity with canine clinical leishmaniosis

The effects of polyhexamethylene biguanide (PHMB) and TLR agonists alone or as polyplex nanoparticles against Leishmania infantum promastigotes and amastigotes

Dogs are the main reservoir for Leishmania infantum, manifesting from a subclinical to a fatal disease. Limited treatments are available, although new antiparasitics and immunomodulators are pursued. Polyhexamethylene biguanide (PHMB) has a broad antimicrobial spectrum, including antiparasitic activity. Here, we evaluated the potential for Toll-like receptor agonists (TLRa) and PHMB alone, and as polyplex nanoparticles containing PHMB and TLR4 or TLR9 agonists, to selectively kill L. infantum. Susceptibility of L. infantum promastigotes to PHMB, miltefosine, and allopurinol was performed, and the half-maximum inhibitory concentrations (IC50) were determined. Then, DH-82 cells were infected and treated with PHMB alone or combined with TLR4a (MPLA-SM) or TLR9a (CpG ODNs) and allopurinol alone. The IC50 values of L. infantum promastigotes were PHMB (1.495 µM), miltefosine (9.455 µM), and allopurinol (0.124 µM). After infection, treated DH-82 cells displayed a lower percentage (p = 0.0316), intensity (p = 0.0002), and index of infection (p = 0.0022) when compared to non-treated cells. PHMB induced lower percentage of infection alone (p = 0.043), in combination with TLR9a (p = 0.043), and with TLR4a (p = 0.0213). Supernatants were collected and used to measure TNF-α and IL-6 levels. Increased TNF-α was observed after PHMB plus TLR4a, relative to uninfected and infected untreated macrophages (p = 0.043). PHMB combined with TLR4a shows promise as a potential anti-L. infantum drug combination, as well as inducer of proinflammatory response, as demonstrated by decreased infection and increased TNF-α production

Detection of specific antibodies against Leishmania infantum in canine serum and oral transudate using an in-house ELISA

Canine leishmaniosis caused by the protozoan Leishmania infantum is a complex infection due to its variable clinical signs and laboratory findings. Therefore, a broad range of techniques is available for diagnosis. Testing for specific antibodies in serum is the most commonly used technique, although the testing of other body fluids, such as oral transudate (OT), can be an alternative as its collection is non-invasive and testing can be performed by untrained personnel. The aim of this study was to assess and compare the detection of L. infantum -specific antibodies in paired samples of serum and OT collected from apparently healthy dogs and dogs with clinical leishmaniosis using an in-house enyzme-linked immunosorbent assay (ELISA). Serum and OT were collected from 407 dogs, which varied in breed, sex, age, lifestyle and clinical status, by many practicing veterinarians in Spain. The main geographical areas of sampling included Barcelona (n = 110), Mallorca (n = 94), Cadiz (n = 54) and Asturias (n = 47). The majority of infected dogs were apparently healthy (89.9%) while 41 presented clinical signs and/or clinicopathological abnormalities compatible with L. infantum infection and subsequently diagnosed with leishmaniosis (10.1%). An in-house ELISA was performed to quantify the anti- Leishmania antibodies in serum and OT. The L. infantum infection rate determined by the in-house ELISA was 37.1% in serum samples and 32.7% in OT samples. Serum and OT ELISA results showed a positive correlation (Spearman's correlation coefficient r = 0.6687, P < 0.0001). The percent agreement between the serum and OT ELISA results was 84%, while agreement according to Cohen's kappa statistic (κ) was substantial (0.66) when all samples were analyzed. The highest percent agreement (92.1%) between both tests was found in dogs from low endemicity regions and from sick dogs, with both groups presenting almost perfect agreement according to Cohen's κ agreement test (0.84). Few seronegative dogs (n = 23) tested positive by the OT ELISA. The agreement between serum and OT went from almost perfect to moderate when the geographical distribution and clinical status were analyzed. The results of this study demonstrated an almost perfect to moderate agreement between OT and serum samples tested using the in-house ELISA. These results are particularly promising in sick dogs with high antibody levels while the results seem less optimal in apparently healthy dogs with low antibody levels

OIE disease timelines data analysis

PósterThe WAHID (World Animal Health Information Database) interface provides access to the semi-annual reports stating the health condition of the OIE-listed [1] different diseases in each Country. The current study seeks to analyze OIE data in order to: Describe the spatial tendencies of the different diseases. Identify some factors which can determine the presence of this diseases

Toll-like receptors 2, 4 and 7, interferon-gamma and interleukin 10, and programmed death ligand 1 transcripts in skin from dogs of different clinical stages of leishmaniosis

Background: Canine leishmaniosis (CanL) caused by Leishmania infantum can have several dermatological manifestations. The type of immune response elicited against the parasite appears to be at the basis for such clinical variability. Much of the work in CanL has focused on adaptive immune response and there are scarce data on the importance of the innate immune responses. Moreover, few studies have evaluated the immunological response in the cutaneous lesions in dogs naturally infected with L. infantum and with different degrees of disease severity, and no study has compared clinically-lesioned with normal-looking skin. Methods: We determined and compared the transcription of toll like receptors (TLRs) 2, 4 and 7, interferon gamma (IFN-γ), interleukin (IL) 10 and programmed cell death protein ligand (PD-L) 1 by real-time PCR in paired clinically-lesioned and normal-looking skin from 25 diseased dogs (mild disease-stage I (n = 11) and moderate to severe disease-stages II and III (n = 14) as well as in normal-looking skin from healthy dogs (n = 10) from a non-endemic area. We also assessed the association between the transcripts in clinically-lesioned and normal-looking skin of dogs with leishmaniosis with clinicopathological, immunological and parasitological findings. Results: Clinically-lesioned skin from mildly affected dogs was characterized by a significant upregulation of TLR2 (P < 0.0001) and IL-10 (P = 0.021) and downregulation of TLR7 (P = 0.004) when compared with more severely affected dogs. Normal-looking skin of mildly affected dogs was characterized by a significant lower expression of TLR7 (P = 0.003), IFN-γ(P < 0.0001) and PD-L1 (P = 0.001) when compared with more severely affected dogs. TLR2, TLR4, IL-10 and IFN-γupregulation in clinically-lesioned skin was correlated with lower disease severity while TLR7 upregulation was correlated with markers of disease severity. Upregulation of TLR7, IL-10, IFN-γand PD-L1 in normal-looking skin was correlated with disease severity. Conclusions: This study demonstrated different expression profiles of immune genes in clinically-lesioned and normal-looking skin among mildly and more severely affected dogs. These immunological conditions might favor the maintenance and replication of the parasite in the skin of more severely affected dogs

Exploring the Relationship between Neutrophil Activation and Different States of Canine L. infantum Infection : Nitroblue Tetrazolium Test and IFN-γ

This study aimed to understand the role of neutrophils in canine leishmaniosis (CanL) by assessing neutrophil activation and its relationship with different states of Leishmania infantum infection and antibody and IFN-γ production. The results showed that sick dogs in stage I-mild disease had significantly higher neutrophil activation compared to healthy seronegative and seropositive dogs and sick dogs in advanced stages (II, III-IV). Healthy seropositive dogs exhibited higher neutrophil activation compared to all other groups except sick dogs in stage I. Dogs in advanced disease stages (II, III-IV) did not show significant differences in neutrophil activation compared to healthy seronegative dogs. Furthermore, dogs in stage I had significantly higher IFN-γ concentrations compared to healthy seronegative and sick dogs in advanced disease stages. Dogs in stage II showed higher IFN-γ concentrations compared to healthy seronegative dogs, while no significant differences were observed in dogs in stage III-IV. Healthy seropositive dogs had elevated IFN-γ concentrations compared to healthy seronegative dogs and dogs in stage III-IV. These findings indicate that neutrophil activation is predominant in dogs with mild disease and healthy seropositive dogs with an association with potent IFN-γ production. This study aimed to investigate the role of neutrophils in canine leishmaniosis by assessing neutrophil activation and its relationship with different states of L. infantum infection and antibody and IFN-γ production. Dogs were categorized into five groups: healthy-seronegative (n = 25), healthy-seropositive (n = 21), LeishVet-stage I (n = 25), Leishvet-stage II (n = 41), and LeishVet-stage III-IV (n = 16). Results of the nitroblue tetrazolium reduction test (NBT) showed significantly higher neutrophil activation in stage I (median:17.17, range: [7.33-31.50]%) compared to in healthy-seronegative (4.10 [1.20-18.00]%), healthy-seropositive (7.65 [3.98-21.74]%), stage II (6.50 [1.50-28.70]%), and stage III-IV (7.50 [3.00-16.75]%) groups (p < 0.0001). Healthy-seropositive dogs also displayed higher values than all groups except stage I. Stages II and III-IV did not show significant differences compared to healthy-seronegative. Regarding IFN-γ, stage I dogs had higher concentrations (median:127.90, range: [0-3998.00] pg/mL) than healthy-seronegative (0 [0-109.50] pg/mL) (p = 0.0002), stage II (9.00 [0-5086.00] pg/mL) (p = 0.045), and stage III-IV (3.50 [80.00-548.80] pg/mL) (p = 0.02) dogs. Stage II dogs showed increased IFN-γ compared to healthy-seronegative dogs (p = 0.015), while stage III-IV dogs had no significant differences compared to healthy-seronegative dogs (p = 0.12). Healthy-seropositive dogs had elevated IFN-γ concentrations compared to healthy-seronegative dogs (p = 0.001) and dogs in stage III-IV (p = 0.03). In conclusion, neutrophil activation was higher in dogs with mild disease and healthy-seropositive dogs, and a relationship between neutrophil activation and the production of IFN-γ was found

Use of preventive measures and serological screening tools for Leishmania infantum infection in dogs from Europe

Background: There are several screening tools for detecting Leishmania infantum infection in dogs and various preventive measures to protect against it. Some studies have investigated them, but not many have described their current use. The aim of this study was to investigate which preventive measures and serological screening tools for L. infantum infection were employed from 2012 to 2018 in dogs from different endemic European countries. Methods: A set of electronic datasheets was completed for each dog from several veterinary centres. Classification of preventive measures included: (1) repellents, (2) vaccines and (3) immunomodulators. Classification of serological tests included the: (1) direct agglutination test (DAT), (2) enzyme-linked immunosorbent assay (ELISA), (3) indirect immunofluorescence (IFI), (4) rapid tests and (5) other assays. Dogs were also classified depending on their risk of exposure and living area. Results: Information from 3762 dogs was gathered. Preventive measures were applied in 91.5% of dogs and the most frequently used were repellents (86.2%) followed by vaccines (39.8%) and Leisguard® (15.3%). The different types of repellents (collar and spot-on) were used similarly. A combination of a vaccine and repellents was preferred in the high-risk group while the low-risk preferred a combination of Leisguard® and a repellent (Chi-square test: X2 = 88.41, df = 10, P < 0.001). Furthermore, all preventive measures were similarly used through the years except for repellents, which were predicted to have a small increase of use each year. Regarding serological screening tools, the most used were rapid and ELISA tests. Rapid tests, ELISA tests and DAT were used similarly through the years, but a significant change was found in the use of IFI and other assays whose use decreased a little each year. Conclusions: Repellents were the preferred measure, while vaccines and Leisguard® were second-line options. Some dogs were not treated by any measures, which highlights the need for dog owner education. Moreover, there seems to be a preference for rapid tests in the clinical setting to detect specific L. infantum antibodies while ELISA or IFI are less often employed. This underlines an increasing problem, as qualitative rapid tests have a variable diagnostic performance limiting the adequate diagnosis of seropositive dogs in endemic areas. Graphical abstract

Total serum IgD from healthy and sick dogs with leishmaniosis

This study was supported by Spanish ministry grants, Ministerio de Economía y competitividad and Fondo Europeo de Desarrollo Regional (FEDER, EU) (AGL2012‑32498 and AGL2015‑68477).Background: Canine leishmaniosis (CanL) due to Leishmania infantum is characterized by the development of both cellular and humoral immune responses. The dysfunction of T cell-mediated immunity leads to a lack of proliferation of T cells in response to Leishmania antigens with the consequence of parasite dissemination that seems to be related to a T cell exhaustion mediated by regulatory B cells expressing immunoglobulin D (IgD). The aim of this study was to determine and compare the total serum IgD in dogs with clinical leishmaniosis and in clinically healthy dogs. Results: A total of 147 dog sera were studied. All dogs were tested for L. infantum-specific antibodies by quantitative ELISA. Interferon-gamma (IFN-γ) production was also determined by sandwich ELISA after blood stimulation with L. infantum soluble antigen (LSA) or concanavalin A (ConA). The quantification of total IgD was performed using a human IgD sandwich ELISA quantification set. Dogs were classified in three different groups. Group 1 included 40 clinically healthy non-infected dogs, all serologically negative to L. infantum-specific antibodies and non-producers of IFN-γ upon LSA stimulation. Group 2 included 63 clinically healthy infected dogs that were LSA IFN-γ producers (n = 61) and/or IFN-γ non-producers (n = 2) as well as negative to medium seropositive to L. infantum antigen. Finally, Group 3 included 44 dogs with clinical leishmaniosis (IFN-γ producers, n = 23; and IFN-γ non-producers, n = 21) that were negative to highly positive to L. infantum-specific antibodies. No significant differences were observed when the total IgD concentration was compared within groups. Additionally, total IgD of sick IFN-γ producers and IFN-γ non-producers was not significantly different. Finally, total IgD concentration was not statistically related to demographic parameters such as age, sex and breed. Conclusions: The results of this study demonstrated that there were no differences between groups in total serum IgD. Total serum IgD does not appear to be a marker of disease in CanL

Signalment, Immunological and Parasitological Status and Clinicopathological Findings of Leishmania-Seropositive Apparently Healthy Dogs

Canine leishmaniosis caused by Leishmania infantum is a disease with a wide range of clinical manifestations. Epidemiological serosurveys performed in Europe often lack a thorough assessment of clinical health status of studied dogs. The aim of this study was to evaluate signalment, immunological and parasitological status and clinicopathological findings of L. infantum-seropositive apparently healthy dogs (n = 212) living in endemic areas. Routine laboratory tests, endpoint inhouse ELISA to quantify the anti-Leishmania antibodies, blood Leishmania qPCR and IFN-ELISA were performed. All dogs enrolled were L. infantum-seropositive and were classified as healthy (n = 105) or sick (n = 107) according to LeishVet guidelines. The sick group presented a higher proportion of medium to high antibody levels and positive qPCR and lower IFN-concentration compared to the healthy group. Sick dogs were mostly classified in LeishVet stage IIa. Biochemical alterations (98%) were the most common clinicopathological findings, with fewer urinary tract (46%) and hematological (40%) alterations. Apparently healthy L. infantum-seropositive dogs can be classified between truly healthy dogs and sick dogs with clinicopathological findings. Sick dogs presented medium to high seropositivity and parasitemia and low IFN- concentrations, and their most common clinicopathological abnormalities were serum protein alterations followed by proteinuria and lymphopenia