Seronegative Dogs Research Articles

Serum and urinary monocyte chemoattractant protein-1 as markers of inflammation and renal damage in dogs with naturally occurring leishmaniosis

BackgroundRenal disease in canine leishmaniosis is of great importance owing to increased risk of mortality. In human visceral leishmaniosis, monocyte chemoattractant protein-1 (MCP-1) has been used as a marker of renal damage and inflammation. The purpose of this study was first to determine the serum MCP-1 and urinary MCP-1-to-creatinine ratio (uMCP-1/Cr) in healthy dogs and dogs with leishmaniosis at diagnosis, and second to determine whether these markers can differentiate disease severity at diagnosis.MethodsIn total, 19 healthy seronegative dogs and 38 dogs with leishmaniosis were included in the study. Dogs with leishmaniosis were classified as LeishVet clinical staging and as International Renal Interest Society (IRIS) staging. Serum and urinary MCP-1 concentrations were measured with an enzyme-linked immunosorbent assay. A receiver operating characteristic (ROC) curve determined disease severity at diagnosis between two LeishVet groups (Stage II versus stage III and IV).ResultsDogs in Leishvet stages IIb, III, and IV had a median serum MCP-1 and uMCP-1/Cr concentration higher than healthy dogs (P < 0.0001). No statistical differences were found in serum MCP-1 and uMCP-1/Cr between dogs in LeishVet stage IIa and healthy dogs. The dogs in LeishVet stage IV had significantly higher serum MCP-1 and uMCP-1/Cr compared with the dogs in LeishVet stage IIa (P < 0.0001). Serum MCP-1 and uMCP-1 were significantly higher in dogs in IRIS stage I and II + III + IV compared with healthy dogs. Dogs stage II + III + IV of IRIS had a significantly higher serum MCP-1 compared with dogs in IRIS stage I (P < 0.0001). The area under the ROC curve for serum MCP-1 was 0.78 [95% confidence interval (CI) 0.64–0.93] and for uMCP-1/Cr it was 0.86 (95% CI, 0.74–0.99). The optimal cutoff value for serum MCP-1 and uMCP-1/Cr was 336.85 pg/ml (sensitivity of 79% and specificity of 68%) and 6.89 × 10−7 (sensitivity of 84% and specificity of 79%), respectively.ConclusionsSerum MCP-1 and uMCP-1/Cr are increased in dogs with leishmaniosis compared with healthy dogs, suggesting the presence of inflammation and renal injury. Serum MCP-1 and uMCP-1/Cr were more elevated in the advanced stages of the disease compared with the moderate stages and, therefore, can be markers of the severity of the disease process.Graphical

Evaluation of the toxicogenetic potential of visceral leishmaniasis in naturally infected dogs and salivary samples for definitive disease diagnosis

Visceral leishmaniasis is an infectious and zoonotic disease caused by the protozoan Leishmania infantum chagasi, transmitted through the bite of the sandfly Lutzomyia longipalpis, with dogs serving as the primary reservoir. Given the increasing spread of this disease in dogs and its epidemiological and public health significance, this study aimed to investigate whether salivary samples from naturally infected dogs could serve as a diagnostic tool, attempting to replace invasive conventional methods. Additionally, the study analyzed the potential toxicogenetic effects of the disease. Ten adult dogs, both males and females, intact and neutered, of mixed breed, from Rifaina (SP) and attended at the Veterinary Hospital of the University of Franca, were included. These dogs exhibited various clinical signs suggestive of visceral leishmaniasis, with five of them confirmed through serological and parasitological examinations. Salivary samples were collected using sterile swabs from the five seropositive dogs, placed on glass slides, and stained with hematoxylin-eosin for subsequent analysis of amastigote forms of the protozoan using bright-field microscopy; the results were descriptive. Furthermore, bone marrow samples from the sternum were collected from the five seropositive dogs and compared with those from the five seronegative dogs to investigate the toxicogenetic potential of the disease through the micronucleus test. To assess possible genotoxicity, each slide made from bone marrow was analyzed for 2000 immature erythrocytes (IE), totaling 4000 IE/animal, and those containing micronuclei (MN) were identified (IE-MN). Cytotoxicity was determined by analyzing 1000 erythrocytes/slide, totaling 2000 erythrocytes/dog, and subsequently calculating the ratio of IE/IE+NCE (normochromatic erythrocytes). Toxicogenetic results of seropositive dogs were compared with seronegative dogs and statistically verified by simple analysis of variance (ANOVA). The protozoan Leishmania was not detected in any of the salivary samples from seropositive dogs. The mean frequency of IE-MN in seropositive dogs was statistically higher compared to seronegative dogs (p&lt;0.0001), indicating the genotoxicity of the disease. In two of the five seropositive dogs, a decrease in erythrocyte production suggested cytotoxicity of the disease. Given the established methodology, it can be inferred that simple saliva analysis, without more specific assays, does not detect amastigote forms of the protozoan in dogs seropositive for visceral leishmaniasis and should not replace established diagnostic methods. Additionally, the micronucleus test suggested the genotoxicity and cytotoxicity of the disease.

Seroprevalence of melioidosis and its association with blood profiles and pathogens in sheltered dogs in southern Thailand.

Melioidosis is a notable zoonotic disease in Thailand that can affect both humans and animals. Although dogs are one of the most popular pets worldwide, there is a remarkable lack of information on the prevalence and knowledge of canine melioidosis. This study aimed to estimate the seroprevalence of melioidosis in sheltered dogs and its relationship with the blood profile and blood pathogens. Melioidosis in 156 dogs was analyzed using an indirect hemagglutination assay. Hematology and serum biochemistry tests were performed using an automated system. Blood pathogens (e.g., Ehrlichia, Anaplasma, Hepatozoon, and Babesia) were diagnosed using conventional polymerase chain reaction. The seroprevalence rates of canine melioidosis and blood pathogen infection were 5.77% (9/156) and 50.64% (79/156), respectively. Seropositive dogs generally have higher lymphocyte counts and aspartate aminotransferase levels but lower total white blood cell, neutrophil, and platelet (PLT) counts than seronegative dogs. No statistically significant difference (p > 0.05) was observed between the seropositive and seronegative dogs' hematology and serum biochemistry findings. Neither the correlation between melioidosis and blood pathogen infection nor the association between melioidosis and thrombocytopenia was statistically significant (p > 0.05). Remarkably, dogs that had coinfections with both melioidosis and blood pathogens demonstrated a significantly reduced PLTcount (49,167 ± 7,167) compared with dogs that tested positive for melioidosis but negative for blood pathogens (139,333 ± 29,913) (p < 0.01). In southern Thailand, the prevalence of canine melioidosis was low but the prevalence of blood pathogens was high. Coinfection with blood pathogens can significantly reduce PLT counts, which may have a potentially serious impact. Future research should focus on conducting seroprevalence studies in the general dog population.

Clinical features of chronic kidney disease in dogs with the serological presence of Leptospira spp., Ehrlichia canis, and Anaplasma phagocytophilum

Chronic kidney disease is commonly diagnosed in dogs, and clinical signs may be aggravated when infected agents are involved. In this case report, 33 dogs with chronic kidney disease were clinically evaluated and serologically tested for Leptospira spp., Ehrlichia canis, and Anaplasma phagocytophilum. The seroprevalence for Leptospira spp. was 39.4%. The most frequent serovars found were Pyrogenes, Canicola, Bratislava and Australis, with serological titers between 1:100 to 1:800. Clinical signs included fever, depression, decreased body condition, vomiting and hematuria. Significant laboratory findings were anemia, leukocytosis, thrombocytopenia, increased liver enzymes, urea and creatinine, hyperbilirubinemia and hyperphosphatemia. All leptospira seronegative dogs were positive for one or both monitored homoparasites (i.e., E. canis and A. phagocytophilum); only three leptospira seropositive dogs were positive for one or both hemoparasites. Findings also suggest that endemic hemoparasites of dogs should be monitored in dogs with a kidney condition for a better clinical picture of the patients and therapeutic approach.

Canine Leishmania spp. infection in two distinct foci of visceral and cutaneous leishmaniasis in Tunisia.

Visceral leishmaniasis (VL) and zoonotic cutaneous leishmaniasis (ZCL) caused by Leishmania (L.) infantum and L. major, respectively, are endemic in Tunisia. The aim of the study was to assess canine Leishmania spp. infection prevalence as well as to identify the Leishmania species involved in two well-documented and geographically distinct VL and ZCL foci. One hundred seventy-six dogs were randomly recruited in the VL focus of Sbikha-Zaghouan (n = 100) and the ZCL focus of Echrarda-Nasrallah (n = 76). Physical examination and blood collection were systemically performed. Needle aspiration was done in case of lymph node (LN) enlargement. All sera were tested by ELISA. kDNA RT-PCR was performed on DNA extracts from (i) buffy coats of seropositive dogs and (ii) LN aspirates. Leishmania species identification was done by ITS1 PCR-sequencing. Thirty-three dogs (18.8%) were infected by Leishmania; 30 having anti-Leishmania antibodies and 3 were seronegative dogs with Leishmania DNA in LN aspirates. Prevalence of infection was significantly higher in VL foci than in ZCL foci (27% versus 7.9%, p = 0.002). Leishmania species was identified in 11 dogs and corresponded to L. infantum. Combination of serology and qPCR on LN aspirates seems to be the best option for canine leishmaniasis diagnosis. Infection is more frequent in VL foci and L. infantum is the only identified species.

Canine Toxoplasmosis: Cytokine Profile in Seropositive Stray Dogs

Toxoplasma gondii is a ubiquitous protozoan parasite that may cause devastating conditions in animals, including humans. Dogs are the main host of T. gondii; however, little information is available on the cytokine expression profile in T. gondii–seropositive dogs. The present study aims to evaluate the levels of certain cytokines in serum collected from T. gondii–seropositive stray dogs. With a commercial enzyme-linked immunosorbent assay kit, we investigated the presence of antibodies for T. gondii in stray dogs in Hefei, Anhui, P. R. China, and found 26 of those tested were seropositive. For proinflammatory cytokines, higher levels of interleukin (IL)-6 were detected in the sera of T. gondii–seropositive dogs compared with seronegative dogs. Additionally, the concentration of anti-inflammatory cytokine IL-10 in seropositive dogs was slightly lower.

Serological and Molecular Survey on Domestic Dog Hepadnavirus in Household Dogs, Italy

The discovery of hepadnaviruses in cats (domestic cat hepadnavirus, DCH) and of a DCH-like virus in dogs has raised several questions regarding the role of these viruses in pets, with particular emphasis on their potential impact on animal health and epidemiology, as well as possible zoonotic implications. In this study, by screening an age-stratified collection of 600 canine serum samples for DCH with an ELISA assay based on the recombinant core antigen (DCHCAg), specific antibodies were found with an overall prevalence of 10.0% (60/600), with a higher prevalence in younger and older dogs. By retesting the canine DCHCAbs-positive sera with an ELISA test based on the recombinant surface protein of DCH (DCHSAg), a total of 18 sera (30%, 18/60) also contained IgG anti-DCHSAg. All the sera were also assessed molecularly using either a consensus hepadnavirus PCR or a specific real-time PCR for DCH. Hepadnavirus DNA was detected in four seronegative dogs, with a prevalence rate of 0.7% (4/600). On sequence analysis of the polymerase region amplified with pan-hepadnavirus primers, the amplicons displayed the highest nucleotide identity (97.3-99.6%) to DCH sequences detected in cats and to the domestic dog hepadnavirus recently identified in a canine serum sample from Italy.

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

BackgroundCanine 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).MethodsSerum 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.ResultsThe 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 rs = 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.ConclusionsThe 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.Graphical

Evaluation of five different rapid immunochromatographic tests for canine leishmaniosis in Spain

Canine leishmaniosis is a vector-borne disease caused by Leishmania parasites. Serological methods are the most common tests used for the diagnosis. This study aimed to evaluate and compare different serological commercial immunochromatographic rapid tests available in Spain to detect anti-Leishmania canine antibodies. The immunochromatographic tests were evaluated in different groups of dogs (healthy seronegative dogs (n = 21), naturally-sick dogs with moderate anti-Leishmania antibodies (n = 39), naturally-sick dogs with high anti-Leishmania antibodies (n = 37), dogs with the serological result of other pathogens infection (n = 20) and exposed dogs (n = 33)) admitted to the Veterinary Teaching Hospital of the University of Zaragoza (Spain) according to the clinical information sent with the sample to the laboratory for diagnostic purposes. The serology status was also routinely recorded through an in-house enzyme-linked immunosorbent assay (ELISA) and an in-house indirect immunofluorescence test (IFAT). The qualitative commercial serological immunochromatographic tests used were: FASTest LEISH, Uranotest Leishmania, Uranotest Leishmania 2.0, Speed Leish K, Witness Leishmania, and DFV Test Leishmania. Performance measures analyzed for each test were: sensitivity, specificity, and area under the receiver-operating (ROC) curve. The maximum specificity (1.00) was attained for Uranotest Leishmania and DFT Test Leishmania, followed by FASTest LEISH (0.98), Uranotest Leishmania 2.0 (0.98), Speed Leish K (0.98), and Witness Leishmania (0.95). The maximum sensitivity was attained for FASTest LEISH (1.00), followed by Uranotest 2.0 (0.97), Speed Leish K (0.97), Uranotest (0.96), and the lowest results with Witness (0.84) and DFV Test (0.59). Regarding the ROC curve, the maximum value was attained with the FASTest LEISH (0.99), followed by Uranotest (0.98), Uranotest 2.0 (0.97), Speed Leish K (0.97), Witness (0.90), and the lowest result with DFV Test (0.79). Efforts in the field of diagnosis should focus on establishing a commercial immunochromatographic test with high sensitivity and specificity with a reasonable cost-benefit balance.

Visceral leishmaniasis in a recent transmission region: 27.4% infectivity rate among seronegative dogs.

American visceral leishmaniasis (VL) is a parasitic disease whose main domestic reservoir in the urban environment is dog and is considered one of the most important zoonoses in the context of public health. Serological tests are typically used for the diagnostic screening of the disease. This study aimed to analyse the performance of different methodologies used in the diagnosis of VL in dogs sampled from a recent transmission area. The sample consisted of 52 dogs separated into groups based on the absence and presence of clinical signs of VL. The following serological techniques were carried out: the DPP® rapid test (RT), the ALERE® RT and an RT and immunoenzymatic assay with a recently developed protein (rKDDR-plus). In addition, molecular techniques were carried out with conjunctival swabs, and bone marrow aspirate samples and parasitological samples were obtained directly from bone marrow aspirates. It was concluded that 27.4% of seronegative dogs were infected, but the serological tests, used as screening tests, showed unsatisfactory sensitivity results (average: 51.2%) for dogs without clinical signs. It was suggested that polymerase chain reaction with conjunctival swabbing be used as a screening test for dogs without clinical signs, as this is a non-invasive collection technique with high-sensitivity values.

Brucella suis Seroprevalence and Associated Risk Factors in Dogs in Eastern Australia, 2016 to 2019

Brucella suis is a zoonotic disease of feral pigs that also affects pig hunting dogs, pig hunters, veterinarians and veterinary staff. In recent years the incidence of B. suis in the eastern Australian states of New South Wales (NSW) and Queensland (QLD) has increased. A cross-sectional study was conducted to document the seroprevalence, geographical extent and risk factors for B. suis in dogs at-risk of contracting the disease. Eligible dogs were those that were known to hunt or consume feral pig meat. Dogs were enrolled through private veterinary clinics and/or directly by District Veterinarians in six regions of NSW and QLD. Blood was collected by venepuncture and tested for B. suis antibodies using the Rose Bengal Test (RBT) followed by a Complement Fixation Test (CFT) if they returned a positive RBT. Owners were invited to complete a questionnaire on the dogs' signalment, husbandry including hunting practices and locations, and any clinical signs referable to brucellosis. Of the 317 dogs included in the prevalence survey, 21 were seropositive returning a survey-adjusted true seroprevalence of 9.3 (95% CI 0.45 to 18) B. suis positive dogs per 100 dogs at-risk. True seroprevalence ranged from 0 to 24 B. suis positive dogs per 100 across eastern Australia, with the highest prevalence in central west NSW and southern QLD. Adjusted for other factors, dogs that shared a household with other seropositive dogs and those that traveled away from their home regions to hunt were more likely to be seropositive. Clinical signs at presentation were not predictive of serostatus, with seropositive and seronegative dogs equally likely to present with signs consistent with brucellosis. The results obtained from this study show that B. suis exposure is relatively common in dogs that have contact with feral pigs, with one in 10 testing seropositive. Further studies are needed to understand the progression and risk of transmission from seropositive dogs.

Differential acute-phase protein responses in dogs seropositive or seronegative for Neospora caninum.

Neospora caninum is one of the most prevalent Apicomplexa parasites that causes abortion in cattle, as it infects dogs as its definitive host, causing subclinical disease or active neosporosis, marked by meningoencephalitis, and myopathies with muscle and neuromuscular signs of disease. This study aimed to evaluate the acute phase protein response in dogs seropositive and seronegative for N. caninum. Serum samples of 72 dogs were tested by an immunofluorescence antibody test using N. caninum NC-1 strain, and the study population was divided into four groups: symptomatic - muscular and/or neuromuscular signs - and seropositive (n = 16); symptomatic and seronegative (n = 9); asymptomatic and seropositive (n = 34); and asymptomatic and seronegative (n = 13). C-reactive protein (CRP) was measured via immunoturbidimetric assay and serum haptoglobin (Hp) via hemoglobin-binding capacity assay. In the symptomatic groups, seropositive dogs had higher levels of Hp, but not CRP, while seronegative dogs had higher CRP levels. There was no difference in CRP concentration in asymptomatic dogs. Dogs with neuromuscular signs had higher concentrations for Hp in the group seropositive. Hp concentration did not differ between dogs seropositive and seronegative dogs for each group. Serum Hp and CRP could not sufficiently alone flag subclinical infections. Measurement of CRP and Hp concentrations could be clinically valuable to the diagnosis of neurological diseases, and their relative change may indicate the stage of the infection, although their sole use is not able to support the diagnosis of canine neosporosis. Further studies are encouraged to evaluate the specific dynamics of acute phase proteins in canine neosporosis. Serum samples of 72 dogs were tested by an immunofluorescence antibody test using N. caninum NC-1 strain, and the study population was divided into four groups: (1) dogs with muscular and/or neuromuscular signs and seropositive for N. caninum; (2) dogs with muscular and/or neuromuscular signs and seronegative for N. caninum; (3) dogs seropositive for N. caninum with no neuromuscular signs; and (4) healthy dogs and seronegative for N. caninum. The study evaluated if N. caninum infection could have pathophysiological changes activating the acute phase response and an increase in the concentration of acute phase proteins in serum.

Systemic inflammatory response syndrome in dogs naturally infected with Babesia canis: Association with the parasite load and host factors

The common signs of canine babesiosis caused by an infection with Babesia canis are fever, anorexia, lethargy, pulse alterations, anemia, and occasionally mild icterus. Dogs with these clinical signs can be divided into two groups: those with acute-phase reaction and those with systemic inflammatory response syndrome (SIRS). Factors associated with the occurrence of SIRS in canine babesiosis have not been thoroughly researched. This article outlines a cross-sectional study of 54 client-owned dogs with an acute B. canis infection, and evaluates the differences in age, gender, laboratory findings, parasite load, and seroreactivity against B. canis between the SIRS and the SIRS-free dogs. We have analyzed a complete blood count, serum biochemistry, serum amyloid A, ceruloplasmin, paraoxonase-1, serology, and PCR testing using standard methodologies. The frequency of SIRS among the investigated dogs reached 0.59. Male dogs and those seronegative against B. canis, were more frequent in the SIRS group, whilst age and parasite load could not be associated with the presence of SIRS. Dogs with SIRS had a lower count of total leukocytes, neutrophils, lymphocytes, and monocytes, and a lower concentration of iron and bilirubin compared with SIRS-free dogs. No significant differences in the concentration of acute-phase proteins have been noticed to exist between the groups of dogs. Further, the seronegative dogs had a lower count of lymphocytes and monocytes and a higher parasite load than the seroreactive dogs. Multivariate logistic regression analysis has identified leukopenia (<6 × 109/L) and monocytopenia (<0.2 × 109/L) as independent associates of SIRS in the investigated dogs, thus implying that these routine tests could be used as reliable markers for SIRS.

Myeloid-derived suppressor cell and regulatory T cell frequencies in canine myasthenia gravis: A pilot study

Myasthenia gravis (MG) is a T cell-dependent, B cell-mediated autoimmune disease. Little is known about its cellular pathogenesis in dogs. This study provides the first preliminary assessment of the frequency of myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs) in the peripheral blood of dogs with seropositive generalized MG. No alteration in frequency of either MDSCs or Tregs in dogs with MG was observed when compared to those in either seronegative dogs with diagnoses other than MG, or healthy dogs. A longitudinal study in three dogs with MG revealed no correlation between the relative numbers of either population and the clinical course of disease. Neither the frequency of MDSCs nor of Tregs showed a correlation with anti-AChR antibody titer in dogs with MG. These findings suggest that aberrations in the frequency of either immunosuppressive population do not occur in MG, but they need to be validated in large-scale prospective studies.

Toll-Like Receptors 2, 4, and 7, Interferon-Gamma, Interleukin 10, and Programmed Death Ligand 1 Transcripts in Leishmanin Skin Test-Positive Reactions of Ibizan Hound Dogs.

The leishmanin skin test (LST) is an in vivo technique commonly used to evaluate the Leishmania-specific cellular immune response in dogs. However, information regarding the local immune response in LST-positive reactions is scarce. We examined the pattern of toll-like receptor 2 (TLR2), TLR4, TLR7, interleukin- (IL-) 10, interferon gamma (IFN-γ), and (program death ligand) PD-L1 gene expression in LST-positive reactions and paired normal-looking skin of nine infected Ibizan hound dogs. Healthy skin from ten seronegative dogs from a nonendemic area was analysed as a negative control. Immune gene expressions were examined by quantitative PCR (qPCR) analysis. LST-positive reactions presented significant upregulation of TLR2, TLR4, IL-10, IFN-γ, and PD-L1 and downregulation of TLR7 when compared with healthy skin of seronegative control dogs from a nonendemic area. All transcripts but TLR7 were significantly higher in LST-positive reaction than in paired normal-looking skin of Ibizan hound. The expression profile of immune genes in LST-positive reactions was similar to that previously observed in clinically lesioned skin of mildly diseased dogs with papular dermatitis due to Leishmania infantum infection. This data provide additional support for the important role of TLRs in canine leishmaniosis.

Immunogenicity of a new, inactivated canine adenovirus type 2 vaccine for dogs.

PurposeWe constructed a new canine adenovirus type 2 (CAV-2) vaccine candidate using the recently isolated Korean CAV-2 strain; we termed the vaccine APQA1701-40P and evaluated its safety and immunogenicity in dogs.Materials and MethodsTo generate the anti-CAV-2 vaccine, APQA1701 was passaged 40 times in MDCK cells growing in medium containing 5 mM urea and the virus was inactivated using 0.05% (volume per volume) formaldehyde. Two vaccines were prepared by blending inactivated APQA1701-40P with two different adjuvants; both were intramuscularly injected (twice) into guinea pigs. The safety and immunogenicity of the Cabopol-adjuvanted vaccine were evaluated in seronegative dogs. The humoral responses elicited were measured using an indirect enzyme-linked immunosorbent assay (I-ELISA), and via a virus neutralization assay (VNA).ResultsThe new, inactivated CAV-2 vaccine strain, APQA1701-40P, lacked six amino acids of the E1b-19K protein. In guinea pigs, the Cabopol-adjuvanted vaccine afforded a slightly higher VNA titer and I-ELISA absorbance than an IMS gel-adjuvanted vaccine 4 weeks post-vaccination (p>0.05). Dogs inoculated with the former vaccine developed a significantly higher immune titer than non-vaccinated dogs.ConclusionThe Cabopol-adjuvanted, inactivated CAV-2 vaccine was safe and induced a high VNA titer in dogs.

Sand fly synthetic sex-aggregation pheromone co-located with insecticide reduces the incidence of infection in the canine reservoir of visceral leishmaniasis: A stratified cluster randomised trial.

ObjectiveTo evaluate the efficacy of a synthetic sex-aggregation pheromone of the sand fly vector Lu. longipalpis, co-located with residual insecticide, to reduce the infection incidence of Leishmania infantum in the canine reservoir.MethodsA stratified cluster randomised trial was designed to detect a 50% reduction in canine incident infection after 24 months in 42 recruited clusters, randomly assigned to one of three intervention arms (14 cluster each): synthetic pheromone + insecticide, insecticide-impregnated dog collars, or placebo control. Infection incidence was measured by seroconversion to anti-Leishmania serum antibody, Leishmania parasite detection and canine tissue parasite loads. Changes in relative Lu. longipalpis abundance within households were measured by setting three CDC light traps per household.ResultsA total 1,454 seronegative dogs were followed-up for a median 15.2 (95% C.I.s: 14.6, 16.2) months per cluster. The pheromone + insecticide intervention provided 13% (95% C.I. 0%, 44.0%) protection against anti-Leishmania antibody seroconversion, 52% (95% C.I. 6.2%, 74·9%) against parasite infection, reduced tissue parasite loads by 53% (95% C.I. 5.4%, 76.7%), and reduced household female sand fly abundance by 49% (95% C.I. 8.2%, 71.3%). Variation in the efficacy against seroconversion varied between trial strata. Equivalent protection attributed to the impregnated-collars were 36% (95% C.I. 14.4%, 51.8%), 23% (95% C.I. 0%, 57·5%), 48% (95% C.I. 0%, 73.4%) and 43% (95% C.I. 0%, 67.9%), respectively. Comparison of the two interventions showed no statistically consistent differences in their efficacies; however, the errors were broad for all outcomes. Reductions in sand fly numbers were predominant where insecticide was located (chicken and dog sleeping sites), with no evidence of insecticide-induced repellence onto humans or dogs.ConclusionThe synthetic pheromone co-located with insecticide provides protection particularly against canine L. infantum parasite transmission and sand fly vector abundance. The effect estimates are not dissimilar to those of the insecticide-impregnated collars, which are documented to reduce canine infection incidence, human infection and clinical VL disease incidence, in different global regions. The trialled novel lure-and-kill approach is a low-cost potential vector control tool against ZVL in the Americas.

Geospatial analysis applied to seroepidemiological survey of canine leishmaniosis in east-central Portugal

Despite the high prevalence of canine Leishmania infantum infection in Portugal, significant differences associated with different risk factors can be found between geographically contiguous areas. In this study, a geographical area within the central region of Portugal (municipalities of Proença-a-Nova, Mação and Vila de Rei) was investigated. An epidemiological survey involved the analysis by an indirect enzyme-linked immunosorbent assay of serum samples collected during the anti-rabies vaccination campaign from 282 dogs. Geospatial analysis showed the distribution of geospatial prevalence of leishmaniosis and has delimited two areas (clusters) with a statistically significant higher risk of seropositivity in dogs (p = 0.003 and p = 0.027, for clusters 1 and 2, respectively). The highest seroprevalence (56.0%; CI: 41.2–70.0) was found in Vila de Rei. Five land occupation types showed a possible influence on the geographic distribution of seropositivity, with statistically significant differences between seropositive and seronegative dogs. Land occupied by temporary irrigated crops (p = 0.026), olive groves (p = 0.013), complex cultural systems and parcelling (p = 0.021), open forests, logging and new plantations (p = 0.043) and watercourses (p = 0.012) influenced the geographical distribution of canine Leishmania infection. Seropositive dogs had a greater average area of occupied land (i.e. open forests, logging and new plantations) than the seronegative ones (3.1439 km2 versus 2.5650 km2, respectively; p = 0.043).

Effectiveness of the mass use of deltamethrin-impregnated dog collars for preventing transmission of canine leishmaniasis by Lutzomyia spp.: A cluster randomized controlled trial

Visceral leishmaniasis (VL) is a zoonosis that greatly impacts public health, especially in developing countries. The effectiveness of deltamethrin-impregnated dog collars for preventing transmission of canine leishmaniasis (CanL) by the sand fly Lutzomyia spp. in countries with high incidence of the disease, such as Brazil, remains unknown. A cluster randomized controlled trial was carried out to evaluate the effectiveness of these collars in preventing infection of dogs with Leishmania infantum.Two areas were selected in a municipality endemic for VL in Brazil: an experimental area (EA) and a control area (CA). In both areas, blood samples were collected from dogs for detection of CanL. Seronegative dogs from the EA received collars impregnated with deltamethrin 4%. After six months, the dogs were retested for CanL and the incidence of the disease, the relative risk, and the effectiveness of collaring determined. In the baseline survey, 1020 dogs from the EA and 589 from the CA were examined. The prevalence calculated for EA was 4.41% (CI95% 3.27–5.91), and 7.13% (CI95% 5.25–9.59) for CA. In the second evaluation, 454 dogs from the EA and 292 from the CA were examined. The person-time incidence calculated for EA was 3.51% (CI95% 1.30–7.48), and for CA was 11.81% (CI95% 7.03–18.23). The difference between the calculated incidences was statistically significant (p = 0.009). The relative risk was 0.2972 (CI95% 0.1204-0.7339; p = 0.009), and the efficacy of the proposed measure was estimated in 70.27%.This report evaluated the effectiveness of the mass use of deltamethrin-impregnated dog collars in areas of transmission of VL by Lutzomyia spp. Collaring was able to protect dogs and may represent an effective strategy that could be implemented to control infection in both dogs and humans in Brazil and perhaps other countries where Lutzomyia spp. is the main vector of the disease.

Subcutaneous Immunization of Dogs With Bordetella bronchiseptica Bacterial Ghost Vaccine

The Bordetella species are Gram-negative bacterial pathogens that colonizes mammalian respiratory tract causing respiratory diseases in humans and animals. B. bronchiseptica causes clinical conditions in many mammals including immunocompromised humans. Using the dog model of respiratory infection, it has been shown in this study that a newly developed B. bronchiseptica Bacterial Ghost (BbBG) vaccine exhibited significant protection in the face of a severe pathogenic bacterial challenge in seronegative dogs. The protein E-specific lysis mechanism was used to produce BbBGs. Bacterial Ghosts (BGs) are the empty cell envelope of Gram-negative bacterium. They are genetically processed to form a microscopic hole in their membrane, through which all the cytoplasmic contents are expelled leaving behind intact empty bacterial shells. Due to the intact surface structures of BGs, they offer the safety of inactivated but efficacy of live attenuated vaccines. In this study, seronegative dogs were vaccinated subcutaneously (s/c) with two different doses of a newly developed BbBG vaccine [lower 10∧5 (BbBG – 5) and higher 10∧7 (BbBG – 7)] on day 0 and 21. The animals were challenged (by aerosol) with virulent live B. bronchiseptica strains 41 days after first vaccination. The dogs vaccinated s/c with BbBG – 7 vaccine had significantly lower spontaneous coughing scores (P = 0.0001) than dogs in negative control group. Furthermore, the tested BbBG – 7 vaccine was equivalent to the positive control vaccine Bronchicine CAe in terms of safety and efficacy. For the first time, we report the successful use of liquid formulated BGs vaccines in animal studies. Earlier reported studies using BGs vaccines were performed with resuspended freeze-dried BGs preparations.