Determination of neutrophil-to-lymphocyte ratio, platelet-to-lymphocyte ratio and systemic immune-inflammation index in dogs with leptospirosis.

Leptospirosis is a globally distributed zoonosis with multisystemic involvement in canine species, capable of causing a pulmonary hemorrhagic syndrome (LPHS) in the most severe cases. In humans, the neutrophil to lymphocyte ratio (NLR), platelets to lymphocytes (PLR) and systemic immune-inflammation index (SII) have been described as predictors of morbidity and mortality in various pathologies, but no such studies have been developed for canine leptospirosis. Hence, we aimed to assess the usefulness of NLR, PLR and SII in dogs affected with leptospirosis, focusing on those that died or survived after hospitalization, whether or not they developed LPHS. The leptospirosis group was composed by 36 dogs while the control group consisted of 32 healthy dogs. The NLR, associated with inflammation, demonstrated a threefold or greater increase in all leptospirosis groups compared to the control group (median 2.44 ± 1.66) (developing or not LPHS). Dogs that died (median 67.78 ± 158.67), developed LHPS (median 85.17 ± 143.77), or both developed LHPS and died (median 67.78 ± 155,14) had a lower PLR in comparison to the control group (median 101,82 ± 53,75) and the rest of groups, but no statistically significant differences were observed (p > 0.05). The SII was higher in leptospirosis-affected dogs that survived (median 1356,92 ± 2726,29) and statistically significant differences were observed in those who did not develop LPHS (median 1770,41 ± 2630,77; p < 0.05) compared to the control group (median 555,21 ± 313,26). Our data shows that NLR may be used as inflammation indicator, while more studies are needed for PLR and SII in canine leptospirosis.

Spatiotemporal analysis and clinico-epidemiological study for seroprevalence of canine leptospirosis.

Leptospirosis is a worldwide re-emerging zoonotic disease. The study was conducted to estimate the Seroprevalence of canine leptospirosis in a total of 450 dogs, from a total of 97 puppies and 353 adult dogs selected for examination Sampling, started from January to December 2023 in District Kasur in the province Punjab of the country Pakistan. Leptospira IgG ELISA kit manufactured by DRG Instruments GmbH, Germany was used for the screening of canine Leptospira antibodies. Out of 450 tested dogs, 183 dogs (40.67%) were tested positive for Leptospira antibody for the screening of Leptospira antibodies. The estimated Seroprevalence of leptospirosis in various age groups of dogs, were 23.7% (23/97) and 45.3% (160/353), in puppies and adults, respectively (P < 0.05). It was found that out of the sampled dogs, a total of 35/127 (27.6%), 29/100 (29%), 73/130 (56.2%), and 46/93 (49.5%) dogs were tested seropositive for Leptospira antibodies in winter, spring, summer and fall, respectively (P < 0.05).

Improved Antibody Detection for Canine Leptospirosis: ELISAs Modified Using Local Leptospiral Serovar Isolates from Asymptomatic Dogs.

Leptospirosis is a zoonotic disease of significant concern for human and animal health, with domestic animals, including dogs, acting as reservoirs for human infection. Serology is widely used for leptospirosis diagnosis, even though the standard microscopic agglutination test (MAT) using a panel of serovars lacks specificity and can lead to detection limitations in certain regions. In this study, we aimed to develop an antibody detection tool for dogs using an indirect enzyme-linked immunosorbent assay (ELISA) with a set of local serovar isolates, including Paidjan, Dadas, and Mini, to enhance the accuracy of leptospirosis surveillance in our region. The specificity and sensitivity of various antigen preparations, namely leptospiral whole-cell protein (WCP), total membrane protein (TMP), and outer membrane protein (OMP), were assessed using sera from infected and non-infected dogs, as well as negative puppy sera. Leptospirosis diagnosis was supported using a genus-specific nested polymerase chain reaction test on all collected sera. Protein preparations were validated using SDS-PAGE and Western blotting analysis. In the results, the standard MAT failed to detect antibodies in any of the dogs confirmed as being infected using PCR and isolation, highlighting its limitations. In contrast, the OMP-based ELISAs using local isolates of Leptospira serovars gave positive results with sera from all infected dogs, and negative results with sera from all dogs from non-endemic areas. IgG titres of infected and unvaccinated dogs from endemically affected areas were significantly higher than those in non-endemic regions. Using the OMP-based IgG/ELISAs with the local serovar Dadas resulted in higher specificity and lower sensitivity than when using the WCP- and TMP-based IgG/ELISAs. Agreement analysis revealed fair and moderate concordance between OMP-based IgG/ELISAs and PCR results, whereas slight and fair agreement was observed between OMP-based ELISAs and the MAT. Overall, the modified OMP-based IgG/ELISAs, utilising relevant local serovar isolates from dogs, demonstrated improved accuracy in detecting leptospirosis in the study area, overcoming the limitations of the MAT. This study highlights the importance of identifying and incorporating these local circulating serovar isolates into serological techniques for leptospirosis diagnosis and surveillance.

TaqMan real-time PCR for detection of pathogenic Leptospira spp. in canine clinical samples.

Introduction: Canine leptospirosis has always been a differential diagnosis in dogs presenting with clinical signs and blood profiles associated with kidney and/or liver disease. The conventional polymerase chain reaction (PCR) provides diagnoses, but real-time PCR-based tests provide earlier confirmation and determine the severity of infection, especially in the acute stage, allowing early detection for immediate treatment decisions. To our knowledge, real-time PCR has not been routinely adopted for clinical investigation in Malaysia. This study evaluated TaqMan real-time PCR (qPCR) assays diagnosing leptospirosis and compared their applicability to clinical samples from dogs with kidney and/or liver disease against a conventional PCR reference. Material and Methods: The qPCR assays were validated using existing leptospiral isolates. Whole blood and urine samples were analysed using a conventional PCR, LipL32(1) and LipL32(2) qPCRs and a microscopic agglutination test. The sensitivity and specificity of the qPCRs were determined. Results: The LipL32(1) qPCR assay had more diagnostic value than the LipL32(2) qPCR assay. Further evaluation of this assay revealed that it could detect as low as five DNA copies per reaction with high specificity for the tested leptospiral strains. No cross-amplification was observed with other organisms. Analysing the clinical samples, the LipL32(1) qPCR assay had 100.0% sensitivity and >75.0% specificity. Conclusion: The LipL32(1) qPCR assay is sensitive, specific and has the potential to be applied in future studies.

Molecular Epidemiology of Pathogenic Leptospira spp. Infecting Dogs in Latin America.

Canine leptospirosis is a bacterial disease caused by spirochetes of the genus Leptospira. Infections can vary from asymptomatic and chronic infections to clinical acute diseases. The disease is endemic in tropical areas, such as Latin American countries, but a broad understanding of the dynamics of circulation of strains, based on molecular data, has not yet been performed. Based on in silico analyses, the present study aims to analyze the genetic diversity and circulation patterns of haplotypes from pathogenic leptospires infecting dogs in Latin America. DNA sequences were obtained from GenBank platform, curated, and aligned. Genetic distances were calculated, and a maximum likelihood tree and haplotype network were constructed. According to the inclusion criteria adopted, a total of 148 sequences were identified. Most of the records were from Brazil, including sequences from L. interrogans serogroup Icterohaemorrhagiae. Phylogenetic analysis showed a genetically closely related cluster, consisting of a larger haplogroup that includes the reference strain Fiocruz L1-130, known to be the major circulating strain in humans. Moreover, no genetic variations were observed according to clinical history and/or geographical localization. We described the molecular epidemiology of leptospires circulating among dogs in Latin America and demonstrated a very genetically homogeneous group, elucidating its ubiquitous circulation pattern and drawing attention to the important role of dogs in the One Health transmission dynamics of leptospirosis.

Seroepidemiological Analysis of Canine Leptospira Species Infections in Changchun, China.

Leptospirosis is a significant worldwide zoonotic infectious disease that infects a wide range of animals and humans. Leptospira will colonize the animal's urinary and reproductive systems and be excreted with urine, potentially causing a wide range of infections. Dogs are an essential host for Leptospira, and epidemiological investigation studies of leptospirosis must be conducted to clarify the prevalence of leptospirosis and to reduce the risk of transmission to humans. This study aimed to investigate the seroepidemiology of leptospiral infection in dogs from Changchun, China, using Microscopic Agglutination Test (MAT). A total of 1053 canine blood samples were collected and tested by MAT. The positive rate of MAT was approximately 19.1%. The main prevalent Leptospira serogroups were L. Icterohaemorrhagiae (8.1%), L. Canicola (7.6%), L. Australis (5.3%), L. Ballum (4.7%) and L. Pyrogenes (4.2%). No statistically significant difference among different varieties, sexes and sampling seasons (p > 0.05), except the age (p < 0.05). The seropositive rate was much higher in adult and aged dogs than in juvenile dogs. Our results showed the seroprevalence and the prevalent serogroup of Canine leptospirosis in Changchun, China.

Pathogenesis and Genomic Analysis of a Virulent Leptospira Interrogans Serovar Copenhageni Isolated from a Dog with Lethal Infection.

Dogs are highly susceptible to leptospirosis and are a public health concern due to their important role as a source of spreading disease, particularly in urban settings. In this study, we present the pathogenesis, serological characterization, and complete genome sequencing of a virulent Brazilian strain (NEG7) of L. interrogans serovar Copenhageni isolated from the urine of a dog that died due to acute leptospirosis. Clinical investigation showed that the dog was presented with icteric mucous membranes, weakness, dehydration, anorexia, and kidney and liver failures. Necropsy followed by histopathological evaluation revealed lesions compatible with liver and kidney leptospirosis. The leptospires recovered from the urine were further characterized by genome analysis, which confirmed that the isolate belonged to L. interrogans serogroup icterohaemorrhagiae serovar Copenhageni. Multiple bioinformatics tools were used to characterize the genomic features, and comparisons with other available Copenhageni strains were performed. Characterization based on absence of an INDEL in the gene lic12008, associated with phylogenetic and ANI (99.99% identity) analyses, confirmed the genetic relatedness of the isolate with L. interrogans serovar Copenhageni. A better understanding of the diversity of the pathogenic Leptospira isolates could help in identifying genotypes responsible for severe infections. Moreover, it can be used to develop control and prevention strategies for Leptospira serovars associated with particular animal reservoirs.

Leptospirosis with multiple organ dysfunction in a mongoose-scat-detection dog infected with Leptospira interrogans serogroup Hebdomadis, Okinawa, Japan.

A 2-year-old male mongoose-scat-detection dog was diagnosed with leptospirosis by urine PCR. The patient developed acute renal failure, hepatic dysfunction, and disseminated intravascular coagulation. Treatment with antibiotics was administered, including ampicillin and doxycycline, and supportive care management was provided. Seroconversion against serogroup Hebdomadis was observed on day 8. The leptospiral gene flaB was detected only in urine collected on day 1, from which Leptospira interrogans ST329 was identified by multilocus sequence typing using seven housekeeping genes. L. interrogans serogroup Hebdomadis ST329 has been isolated from mongooses and humans in Okinawa, Japan. This patient received early treatment with antibiotics, which may have contributed to the early recovery of renal function and removal of L. interrogans from kidney tissue.

Effect of Vaccination against Leptospira on Shelter Asymptomatic Dogs Following a Long-Term Study.

(1) Background: Vaccination of dogs against leptospirosis is of paramount importance, as they ideally must provide not only long-term protection, but also against the renal carrier state of leptospires. This study assessed the post-vaccine humoral response against Leptospira in naturally exposed dogs and effects on renal carrier status. (2) Methods: A total of 118 dogs were studied for 365 days, separated into Group A (vaccinated, n = 94) and Group B (non-vaccinated, n = 24). Group A was subdivided into three groups: A1 with 32 dogs immunized with the vaccine #1; A2 by 32 dogs with #2; and A3 30 dogs with #3. Serology (MAT and IgG-ELISA) and urinary PCR were conducted. (3) Results: Seroreactivity increased at D15 post-vaccination and, regardless of vaccine brand, remained high up to D180, with antibody switch to IgG after D30. A total of 46.8% of animals from Group A were PCR-positive at least once, in contrast to 75% in Group B, regardless of vaccine brand (p < 0.05; OR: 0.3). (4) Conclusions: All commercial vaccines succeeded at eliciting a long-term IgG-based response and were partially effective at protecting against kidney infection.

Seroprevalence of pathogenic Leptospira spp. in domestic dogs from southern Chile and risk factors associated with different environments.

Leptospirosis is a neglected and severe zoonotic bacterial disease that affects canine populations worldwide, and it is present in animals from different settings, such as urban and rural environments. Therefore, a cross-sectional study was conducted on owned domestic dogs from urban and rural origins in southern Chile. The study aimed: 1) to estimate the true prevalence of pathogenic Leptospira in dogs from urban and rural environments in southern Chile, 2) to determine the serovars circulating in an endemic area of Chile, 3) to assess potential risk factors associated with seropositivity in dogs from urban and rural environments. Blood samples from 706 canines were collected, and the serum was tested with Micro-Agglutination-Test (MAT), using a panel of 13 serovars. A Bayesian approach was applied to estimate True prevalence (TP). In addition, dog owners answered a questionnaire that had prompts regarding dog characteristics and potential risk factors. The effect of these factors on the risk of being infected was analysed using a logistic regression model. The TP for urban dogs was 9.4% and 9.1% for rural animals. However, seroprevalence was higher in dogs with different individual, lifestyle, and habitat characteristics regarding their origin. For example, in urban animals, those living in places with rodents roaming near houses and dogs with rodent hunting habits had decreased risk for the seropositivity in MAT. On the other hand, in rural dogs, those of a recognized breed, with contact with cattle and sheep, living with more than one cat at home and living in places close to natural water sources have more risk to seropositivity in MAT. The presence of anti-Leptospira antibodies is widespread in pet dogs in urban and rural environments in southern Chile, and individual, lifestyle and habitat characteristics of the animals, according to the origin, are associated with the seropositivity in MAT. Therefore, the adoption of preventive measures is urgent, in addition to increase the awareness either in public health institutions, practitioners and companion animals' owners, considering the zoonotic potential of Leptospira infection and the close contact between people and their pets.

Carrier Status of Leptospira spp. in Healthy Companion Dogs in Sri Lanka.

Dogs are asymptomatic chronic carriers of Leptospira spp. and excrete these bacteria in their urine, resulting in environmental contamination and potentially leading to zoonotic transmission. Although a previous study in Sri Lanka detected anti-Leptospira antibodies in companion dogs, the urinary shedding of Leptospira spp. and the Leptospira species and serogroups prevalent in them remain unclear. Thus, the current study identified the prevalent Leptospira serogroups and the carrier status of Leptospira spp. in apparently healthy, client-owned dogs in the Kandy District of Sri Lanka. Serum and urine samples were collected from 96 unvaccinated and 82 vaccinated dogs. Anti-Leptospira antibodies and Leptospira DNA in urine were detected using the microscopic agglutination test (MAT) and nested PCR that targeted the pathogenic leptospiral gene, flaB. The flaB sequences were compared with those of Leptospira spp. using the public databases. MAT detected anti-leptospiral antibodies in 15.6% (15/96) of the unvaccinated dogs, and the reactive serogroups were observed to be Sejroe (11.5%), Canicola (2.1%), Icterohemorrhagiae (1.0%), and Javanica (1.0%). Furthermore, MAT results revealed that 11.0% (9/82) of the vaccinated dogs tested positive for the anti leptospira antibodies and the only reactive serogroup was Sejroe. Leptospira DNA was detected in 15.6% (15/96) and 15.9% (13/82) of urine samples collected from unvaccinated and vaccinated dogs, respectively, and phylogenetic analysis revealed that the animals were infected with L. borgpetersenii, L. interrogans, L. kmetyi, and L. weilii. The L. interrogans sequence detected in the canine sample was identical to the one that was previously reported in a human sample from the Kandy District. This study demonstrated that both unvaccinated and vaccinated dogs excrete various pathogenic Leptospira spp. in their urine, suggesting that they may play an important role in environmental contamination that poses a health risk to the dog owners and the general public.

Evaluation of the diagnostic potential and DIVA capability of recombinant LigBCon1-5 protein of Leptospira interrogans serovar Pomona in canine leptospirosis.

BACKGROUND: Canine leptospirosis is a serious public health concern. AIMS: This study aims to investigate the feasibility of conserved first to fifth domains of recombinant Leptospira immunoglobulin like protein B antigen (rLigBCon1-5) as a serodiagnostic marker for detecting canine leptospirosis. METHODS: A total of 340 unvaccinated canine serum samples were screened using microscopic agglutination test (MAT) and rLigBCon1-5 based immunoglobulin G (IgG) indirect-enzyme-linked immunosorbent assay (I-ELISA). Further, 60 vaccinated canine sera were screened using MAT and rLigBCon1-5 based latex agglutination test (LAT). RESULTS: Microscopic agglutination test results revealed seropositivity of 28.6%. The relative sensitivity, specificity, and accuracy of IgG I-ELISA in comparison to MAT were 100%, 96.0%, and 97.2%, respectively. Out of 60 vaccinated sera, 46 sera reacted with MAT alone, and eight sera reacted by both tests, while six sera were non-reactive with both tests. Anti-LigB antibodies were detected in eight canine sera by rLigBCon1-5 based LAT. In five LAT reactive sera, agglutinins of locally circulating Leptospira serovars Grippotyphosa (n=4) and Australis (n=1) were detected. In three LAT reactive sera, agglutinins against Icterohaemorrhagiae (n=3) produced due to natural infection were present. CONCLUSION: Immunoglobulin G based indirect ELISA assay (IgG I-ELISA) can be employed as an alternative test instead of MAT. rLigBCon1-5 based LAT detected anti-LigB antibodies in eight vaccinated sera where the vaccine failure occurred partially or totally due to the limited efficacy spectrum of Nobivac® RL and cold chain breakage. This vaccine could not provide cross-protection against locally circulating Leptospira serovars. The recombinant LigBCon1-5 antigen based LAT possesses capability of differentiating infected from vaccinated individuals (DIVA capability) when employed as a pen-side test for detecting canine leptospirosis.

Persistent High Leptospiral Shedding by Asymptomatic Dogs in Endemic Areas Triggers a Serious Public Health Concern.

(1) Background: Dogs are known as hosts of Leptospira interrogans and can spread this bacterium to the environment. Although Canicola is responsible for determining chronic disease in dogs, when affected by incidental serogroups such as Icterohaemorrhagiae, acute disease may occur with a predominance of clinical signs with hepatic and renal changes. In endemic areas, it is a serious public health problem. Thus, this study aims to estimate the incidence and duration of elimination of leptospires in the urine of dogs, taking another step from a previous study of our group, by a longitudinal, long-term and molecular approach. (2) Methods: A total of 125 dogs without apparent symptoms of leptospirosis were included in the study. The dogs were all PCR-negative and seronegative at the beginning of the study. Blood samples were collected for hematological examinations and urine for amplification of the lipL32 gene by PCR at five different time points during one year. (3) Results: Out of the 125 dogs, 62 became lipL32 PCR-positive (48.8% (95% CI, 47.9-49.7%)) at some point during the study, distributed as follows: at day 0, all negative; day 90, 18/125 (14.4% (95% CI, 13.5-15.3%)); day 180, 18/125 (14.4% (95% CI, 13.5-15.3%)); day 270, 12/125 (9.6% (95% CI, 8.7-10.5%)); and day 365, 14/125 (11.2% (95% CI, 10.3-12.1%)). Out of the 62 amplicons, 22 were sequenced, targeting a short region of secY gene. Of these, 20 (90.9%) were identical to the L. interrogans serovar Icterohaemorrhagiae, while two (9.1%) were Leptospira noguchii. (4) Conclusions: The fact that the leptospires of the Icterohaemorrhagiae serogroup were characterized was unexpected, since the animals remained clinically asymptomatic during the study. The fact that asymptomatic dogs shed leptospires is not new, but the extent of this fact and the characterized strain is impressive, with an impact on public health that cannot be overlooked.

Meta-analysis of risk factors for canine leptospirosis.

Leptospirosis is one of the most widespread zoonotic diseases, with more than one million human cases reported worldwide every year. Dogs could develop infections that range from asymptomatic to severe, and shed leptospires with their urine. Given their close contact with humans, dogs may act both as epidemiological links or as sentinels of pathogenic leptospires in the environment. The aims of our study were to quantitatively summarize the overall prevalence of leptospiral antibodies and to identify factors associated with the probabilities of infection. We searched the electronic databases Scopus, PubMed, PMC and ScienceDirect for observational studies on canine leptospirosis published between 1989 and December 2019 and written in English, Spanish or Portuguese. We fitted a series of multilevel random effects meta-analysis models to estimate the prevalence of antibodies against Leptospira for different types of dogs, health statuses, diagnostic tests, geographic regions and income categories of the countries. We also fitted a number of random effects meta-analysis models to estimate the pooled odds-ratio of factors associated with canine leptospirosis. After removing duplicates and articles not meeting selection criteria, a total of 130 studies in 91 articles were included in this work. We found lower seroprevalence estimates in North America countries (P<0.001) and other high income countries (P<0.001). We also found higher probabilities of leptospiral infection in adult (P=0.017), male dogs with access to the streets (P<0.001). Identifying the profile of dogs that are more exposed to leptospirosis could be useful in the design of public health strategies for the prevention and control of leptospirosis.

Identification of Serogroups Australis and Icterohaemorrhagiae in Two Dogs with a Severe Form of Acute Leptospirosis in Italy.

Leptospirosis is an infectious disease that causes serious illness in dogs. For this reason, epidemiological and clinical studies focusing on disease characterization are widely advocated. The aim of this study was to characterize the leptospires identified in dogs with confirmed symptomatic acute leptospirosis. Leptospira spp. DNA detected in urine, blood, or both samples from nine infected dogs was analyzed using the multi-locus sequence typing (MLST) technique. Leptospires from two dogs were successfully typed: one was identified as belonging to Sequence Type (ST) 17 and one to ST198, both within the L. interrogans species, serogroups Icterohaemorrhagiae and Australis, respectively. Based on the results of routine serologic tests, antibodies reactive toward these serogroups are commonly revealed in dogs in Italy. This study provides the first molecular analysis that identifies infecting Leptospira directly on DNA from biological samples of dogs, showing that serogroup Australis can lead to a severe clinical presentation of leptospirosis in infected dogs.

Leptospira seroprevalence in owned dogs from Spain.

Leptospira is a widespread zoonosis that has been linked to transmission between dogs and humans. The main purposes were to determine the seroprevalence of anti-Leptospira serum antibody and to identify the most common serovars in dogs in Spain. This is a cross-sectional study with 1,310 records of canine Leptospira testing data from Spain since 2015 to 2017. Inclusion criteria were individual cases with MAT test results for 8 serovars (Bratislava, Icterohaemorrhagiae, Australis, Pomona, Grippotyphosa, Autumnalis, Canicola and Saxkoebing) and to have the zip code data. Three hundred and thirty-eight samples (25.8%; 95%CI 23.6-28.4) were seropositive (≥1:100). According to geographic areas, North had the highest seroprevalence (38.0%; 95%CI 28.9-47.1) followed by South (29.4%; 95%CI 20.1-38.8), Center (28.6%; 95%CI 24.3-33.0), Mediterranean (22.3%; 95%CI 19.1-25.6) and Northwest (22.2%; 95%CI 7.9-36.4). Seropositivity (MAT ≥1:100) was most common to serovars Icterohaemorrhagiae (19.4%; 95%CI 17.2-21.5) and Bratislava (8.5%; 95%CI 7.0-10.0), followed by Grippotyphosa (7.2%; 95%CI 5.8-8.6), Australis (6.4%; 95%CI 5.0-7.7), Autumnalis (5.0%; 95%CI 3.8-6.2), Pomona (4.5%; 95%CI 3.3-5.6), Canicola (3.4%; 95%CI 2.4-4.4) and Saxkoebing (0.8%; 95%CI 0.3-1.3). An association was found between positivity (MAT ≥1:100) and males (P = 0.003) and dogs that were 6 years old or older were at higher risk of exposure (P = 0.001; OR 4.61; 95%CI 1.86-11.43). This study has shown that dogs in Spain are commonly exposed to Leptospira infection and points out the necessity to control the prevalence of this severe widespread zoonosis in dogs and humans.

Diagnosis of canine leptospirosis: evaluation of two PCR assays in comparison with the microagglutination test / Diagnóstico de leptospirose canina: avaliação de dois ensaios de PCR em comparação com o teste de microaglutinação

Canine leptospirosis is definitely diagnosed by demonstrating seroconversion in paired serum samples from the acute and convalescent period by the microagglutination test (MAT). However, the application of a polymerase chain reaction (PCR) assay can provide earlier confirmation of suspected cases. The objective of this study was to evaluate two PCR assays used in diagnosis of human leptospirosis (lipL32 real-time PCR and rrs conventional PCR) in cultured microorganisms and experimentally contaminated samples (whole blood, serum, urine), and investigate their applicability in clinical samples from dogs with presumptive diagnosis of leptospirosis by using the MAT as a reference. The analytical sensitivity of the lipL32 real-time PCR was 1 genome equivalent per reaction, whereas that for the rrs conventional PCR was 10 genome equivalents per reaction. Both assays amplified the pathogenic strains but were negative when evaluating the DNA of other microorganisms that may be present in clinical samples. The lipL32 real-time PCR detected 100 bacteria/mL in whole blood samples, 1000 bacteria/mL in serum samples and 10 bacteria/mL in urine samples, whereas the rrs conventional PCR detected 1000 bacteria/mL in whole blood and serum samples and 100 bacteria/mL in urine samples. Seven out of the 51 samples from dogs with presumptive diagnosis of leptospirosis were considered as confirmed cases. ThelipL32 real-time PCR detected positive results in six of the seven confirmed cases, whereas the rrs conventional PCR detected four. The PCR assays evaluated proved to be useful diagnostic tools in the confirmation of canine leptospirosis when used together with the MAT.(AU)

O diagnóstico definitivo da leptospirose canina é geralmente realizado demonstrando a seroconversão em amostras do paciente no período agudo e de convalescença por serologia. No entanto, a aplicação de técnicas de PCR pode contribuir para a confirmação de casos suspeitos num período de tempo mais curto. O objetivo deste estudo foi avaliar dois ensaios de PCR publicados em humanos (PCR-lipL32 em tempo real e PCR-rrs convencional) em culturas puras e em amostras de sangue com anticoagulante, soro e urina experimentalmente contaminados. Posteriormente, investigamos a utilidade de ambos os ensaios de PCR em amostras clínicas de cães com suspeita de leptospirose tomando a técnica de microaglutinação (MAT) como referência. A sensibilidade analítica foi de 1 e 10 genoma equivalente por reação para PCR-lipL32 em tempo real e para PCR-rrs convencional, respectivamente. Ambos os ensaios amplificaram corretamente as 14 estirpes patogênicas, mas foram negativos para avaliar o ADN de outros microrganismos que poderiam estar presentes em amostras clinicas. Em nas amostras experimentalmente contaminadas PCR-LipL32 em tempo real detectou 100 bactérias/mL em sangue total, 1000 bactérias/mL em soro e 10 bactérias/mL em urina. Enquanto o PCR-rrs convencional detectou 1000 bactérias/mL em sangue total e soro e 100 bactérias/mL na urina. Dos 51 cães suspeitos, sete foram considerados casos confirmados pela MAT. O PCR-lipL 32 em tempo real detectou seis dos sete casos confirmados, enquanto o PCR-rrs convencional foi positivo em quatro deles. As técnicas de PCR avaliadas provaram ser uma ferramenta de diagnóstico útil na confirmação de casos clínicos caninos quando utilizados em conjunto com a técnica MAT.(AU)

Prevalence and risk factors to Leptospira sp. infection in dogs attended at veterinary clinics in João Pessoa, Paraíba State, Northeastern Brazil / Prevalência e fatores de risco da infecção por Leptospira sp. em cães atendidos em clínicas veterinárias em João Pessoa, Paraíba, Nordeste do Brasil

This study investigated the prevalence and risk factors associated with Leptospira sp. in dogs attended at veterinary clinics in the city of João Pessoa, State of Paraíba, Northeast Brazil. A total of 384 blood samples from dogs from 34 veterinary clinics were used from April 2015 to May 2016. The serological Leptospira sp diagnosis was carried out through Microscopic Agglutination Test (MAT), using a collection of 20 pathogenic antigens and adopting a 1:100 dilution as cutoff point. An epidemiological questionnaire was applied to the animal's owners to obtain the information used in risk factors analysis. The prevalence of seropositive animals was 11.7% (45/384), with reactions for serogroups Icterohaemorrhagiae (62.3%), Grippotyphosa (22.2%), Canicola (13.3%), Djasiman 2%) and Pomona (2.2%). The following risk factors were identified: age from 49 to 72 months (odds ratio = 2.74); Age > 72 months (odds ratio = 3.22); and monthly cleaning of the environment where the animals were kept (odds ratio = 10.70). We concluded that dogs attended at veterinary clinics in João Pessoa are exposed to Leptospira sp infection, with predominance of serogroups kept by wild or synanthropic animals, and suggest a monthly periodic environment cleaning where the animals are kept.(AU)

O presente trabalho investigou a prevalência e os fatores de risco associados à infecção por Leptospira sp. em cães atendidos em clínicas veterinárias da cidade de João Pessoa, Estado da Paraíba, Nordeste do Brasil. Foram utilizadas 384 amostras sanguíneas de cães provenientes de 34 clínicas veterinárias no período de abril de 2015 a maio de 2016. O diagnóstico da infecção por Leptospira sp. foi realizado com o emprego reação de Soroaglutinação Microscópica (SAM), utilizando uma coleção com 20 antígenos patogênicos e adotando como ponto de corte a diluição 1:100. Foi aplicado um questionário epidemiológico aos proprietários dos animais para obtenção de dados a serem utilizados na análise de fatores de risco. A prevalência de animais soropositivos foi de 11,7% (45/384), com reações para os sorogrupos Icterohaemorrhagiae (62,3%), Grippotyphosa (22,2%), Canicola (13,3%), Djasiman (2,2%) e Pomona (2,2%). Foram identificados os seguintes fatores de risco: idade entre 49 e 72 meses (odds ratio = 2,74), idade maior que 72 meses (odds ratio = 3,22), e limpeza mensal do ambiente onde os animais permanecem (odds ratio = 10,70). Conclui-se que cães atendidos em clínicas veterinárias de João Pessoa estão expostos à infecção por Leptospira sp., com predominância de sorogrupos mantidos por animais selvagens, e foi sugerida a realização da limpeza periódica do ambiente ocupado pelos animais.(AU)

Evaluation of 3 Serological Tests for Early Detection Of Leptospira-specific Antibodies in Experimentally Infected Dogs.

BACKGROUND: Leptospirosis in dogs is a disease of global importance. Early detection and appropriate therapeutic intervention are necessary to resolve infection and prevent zoonotic transmission. However, its diagnosis is hindered by nonspecific clinical signs and lack of rapid diagnostic tests of early infection. Recently, 2 rapid point-of-care tests (WITNESS Lepto [WITNESS Lepto, Zoetis LLC, Kalamazoo, MI, USA] and SNAP Lepto [SNAP Lepto, IDEXX Laboratories, Westbrook, ME, USA]) for detection of Leptospira-specific antibodies in canine sera were developed. HYPOTHESIS: Immunoglobulin M-based WITNESS Lepto containing multiple detection antigens can detect Leptospira-specific antibodies to common leptospiral serovars earlier in the course of infection as compared to microscopic agglutination test (MAT) and SNAP Lepto. ANIMALS: Four groups of 8 6- to 8-month-old male Beagle dogs were used. METHODS: Thirty-two healthy seronegative dogs were inoculated experimentally with serovars Canicola, Grippotyphosa, Icterohaemorrhagiae, and Pomona (8 dogs/serovar). Acute-phase sera were collected at regular intervals and monitored for Leptospira-specific antibodies by WITNESS Lepto, MAT, and SNAP Lepto. RESULTS: Seroconversion was detected in all dogs by day 10 by WITNESS Lepto and in 30 of 32 dogs by day 14 by MAT. The SNAP Lepto test detected seroconversion in 3 dogs during the 2 weeks postchallenge. CONCLUSIONS: Immunoglobulin M-based WITNESS Lepto detected immune responses specific to multiple leptospiral serovars early in the course of infection and identified seroconversion in all animals earlier than did the gold standard MAT. The SNAP Lepto test displayed considerably lower and inconsistent performance during the study period. At the point-of-care, WITNESS Lepto should be the test of choice for rapid and reliable screening of acutely ill dogs suspected to have leptospirosis.

Development of indirect enzyme-linked immunosorbent assay for diagnosis of canine leptospirosis.

AIM: This study was taken up to develop an indirect enzyme-linked immunosorbent assay (i-ELISA) for screening antibodies against Leptospira spp. in canines. MATERIALS AND METHODS: An i-ELISA was developed using outer membrane protein extracted from Leptospira interrogans serovar canicola used for coating the well with concentration of 0.5 µg/µl. A total of 250 serum samples from clinically affected and apparently healthy dogs were collected along with relevant epidemiological data at Teaching Veterinary Clinical Complex, Veterinary College and Research Institute, Namakkal, and subjected to i-ELISA. RESULTS: Out of 250 sera samples, 140 (56.00%) were found to be positive by i-ELISA. All the sera samples were subjected to microagglutination test (MAT) with panel of 12 different serovars. A total of 71 (28.40%) sera samples were positivity to MAT excluding the sera samples positive to L. interrogans serovars canicola and icterohaemorrhagiae in vaccinated dogs. Sensitivity and specificity of i-ELISA were higher in compared with MAT was 91.54% and 58.10%, respectively. CONCLUSION: An indirect ELISA developed for the detection of canine antileptospiral antibodies proved to be highly sensitive, rapid and easy to perform and overcome the drawbacks of MAT.